CN115960268A - A kind of collagen-cono peptide fusion protein and its preparation method and application - Google Patents

Abstract

The invention provides a collagen-conus peptide fusion protein and a preparation method and application thereof, wherein the collagen-conus peptide fusion protein comprises a collagen fragment and a conus peptide fragment which are sequentially connected from an N end to a C end, the amino acid sequence of the collagen fragment is shown as SEQ ID No.2, and the amino acid sequence of the conus peptide fragment is shown as SEQ ID No. 3. The collagen-conotoxin fusion protein is a product obtained by coupling a collagen fragment with an amino acid sequence of a conotoxin fragment, the sequence is shown as SEQ ID No.1, the collagen-conotoxin fusion protein has biological activity functions of collagen and conotoxin, the defect that the short-time wrinkle removing effect of recombinant collagen is not obvious and the defect that the conotoxin does not have a long-acting repairing function are overcome in function, and the product can be widely applied to medicines and cosmetics.

Description

Collagen-conopeptide fusion protein and preparation method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and specifically relates to a collagen-conopeptide fusion protein and a preparation method and application thereof.

Background Art

Protein is the main bearer of human life activities, an important component of human tissue, and the main raw material for human tissue metabolism, renewal and repair. The role of protein in the human body also includes transporting substances, participating in the body's immunity, providing energy and regulating hormones, etc. Among them, collagen is the most abundant protein in the human body, accounting for about 25-33% of the total protein in the body. According to statistics, collagen accounts for about 5% of the body weight, about 75% of the skin weight, and 90% of the total volume of the skin. In connective tissue, collagen accounts for about 80%. It can be said that collagen exists in most tissues of the human body. Its function is to maintain the morphology and structure of the skin and tissues and organs, and it plays an indispensable role in the normal functioning of the human body.

Collagen is an extracellular protein, a fibrous protein twisted into a spiral shape by three peptide chains. Collagen contains a G-X-Y repeating sequence, also called a collagen domain, where X and Y can be any amino acid other than Gly, where X is often proline and Y is often hydroxyproline. Hydroxyproline residues can stabilize collagen molecules by forming intramolecular hydrogen bonds. The three α-peptide chains are wound into a "rope-like" triple helix structure in a parallel, right-handed spiral form by van der Waals forces, hydrogen bonds, and covalent cross-linking, so it has a very high tensile strength.

Collagen, as one of the most abundant proteins in the human body, ensures the stability and strength of body tissues by forming a support network on the cell structure. Over time, the fibers can be damaged, which, among other things, can cause the skin to develop undesirable wrinkles. Studies have shown that when subjects ingest collagen, damaged fibers can be replaced by new ones, so collagen helps to restore and improve tissue structure. It has been shown that collagen has shown antioxidant activity, hypotensive activity, lipid-lowering activity, and repair activity on damaged skin. In addition, this property of collagen has a dual role in the skin: first, it provides the basic elements for the formation of elastic and collagen fibers, and second, it acts as a ligand or binding receptor for fibroblasts to stimulate the previously mentioned components and hyaluronic acid.

Compared with traditional collagen extraction methods, recombinant human collagen is a product of genetic engineering. Human protein avoids immune rejection reactions, greatly improves biocompatibility and biosafety, lacks animal-derived pathogenic microbial contamination, and avoids drastic measures such as strong acids, strong alkalis, and high temperatures during the production process. This not only reduces the degree of environmental pollution during the production process, but the gentler production method also greatly ensures the activity of collagen.

Cono peptides, also known as arginine/lysine peptides, are a special type of polypeptide venom secreted by cone snails through their venom ducts, mainly used for predation and defense. There are currently more than 2,000 known cono peptides. These small peptide toxins are generally composed of 10-46 amino acids, are rich in disulfide bonds, and have a special structure. They can specifically act on receptors or ion channels, thereby paralyzing muscles and relaxing them, helping to relieve wrinkles in a shorter period of time, and retaining 5% of the neuromuscular current conduction in the muscles, so they can provide a very natural wrinkle removal effect.

However, the disadvantage of cono peptides is that they are relatively expensive, so how to reduce the amount of cono peptides without affecting the efficacy has become a topic. The following are the amounts of cono peptides used in several patents: "A cosmetic composition, facial mask and preparation method with anti-wrinkle effect" discloses a composition containing cono peptides, and the cono peptide content in the most preferred composition is 0.8%. "A composition containing peptides and its application" discloses a composition containing cono peptides, and the cono peptide content in the most preferred composition is 6%. The content of cono peptides in the "Formula for a multi-functional eye mask with both instant and long-lasting effects and its preparation method" ranges from 1% to 5%. The present invention will use new technology to further reduce the amount of cono peptides used.

There are two main methods for synthesizing peptides: biosynthesis and chemical synthesis. Chemical synthesis is widely used in the synthesis of short peptides (the number of amino acids is ≤ 10). Although the chemical synthesis process is mature and easy to industrialize, for longer peptides (the number of amino acids is greater than 10), the chemical synthesis method also faces the disadvantage of a sharp increase in production costs. Such long peptides are often difficult to mass produce in industry. Therefore, it is of great application value to provide a low-cost preparation method that can be mass-produced, as well as a collagen-cono peptide fusion protein with high biological activity, immediate anti-wrinkle and long-term repair.

Summary of the invention

The present invention provides a collagen-conopeptide fusion protein and a preparation method and application thereof. The collagen-conopeptide fusion protein (SEQ ID NO.1) is a product obtained by coupling the amino acid sequence of a collagen fragment (SEQ ID NO.2) and a conopeptide fragment (SEQ ID NO.3).

In view of the shortcomings of the prior art, the collagen-conopeptide fusion protein (YLT-ColI) of the present invention is a product obtained by coupling the amino acid sequence of the collagen fragment and the conopeptide fragment, which has the biological activity functions of collagen and conopeptide and can be widely used in medicines and cosmetics. At the same time, the present invention provides a preparation method and composition of the collagen-conopeptide fusion protein with instant anti-wrinkle and long-term repair.

FIG2 is an exemplary diagram of a skin anti-wrinkle test using the composition provided by the present invention. It can be seen that after using the composition having instant anti-wrinkle and long-lasting repair effects, the area ratio of wrinkles is reduced, the skin becomes smooth, and an immediate anti-wrinkle effect is achieved.

In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

In a first aspect, the present invention provides a collagen-conopeptide fusion protein, wherein the collagen-conopeptide fusion protein comprises a collagen fragment and a conopeptide fragment connected sequentially from the N-terminus to the C-terminus;

The amino acid sequence of the collagen fragment is shown in SEQ ID NO.2;

The amino acid sequence of the conopeptide fragment is shown in SEQ ID NO.3.

The collagen-conopeptide fusion protein of the present invention has the advantages of both collagen and conopeptide, and has better effects in short-term wrinkle removal and long-term repair.

Preferably, the collagen-conopeptide fusion protein further comprises a HIS tag fragment.

Preferably, the amino acid sequence of the collagen-conopeptide fusion protein is as shown in SEQ ID NO.1:

SEQ ID NO.1 (amino acid sequence of collagen-conopeptide fusion protein):

EAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKG AAGEPGKAGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGERGGGGGSCCRAHDRCW KSSCGKPGNCCGE.

SEQ ID NO.2 (amino acid sequence of collagen fragment):

EAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKG AAGEPGKAGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGER.

SEQ ID NO.3 (amino acid sequence of conopeptide fragment):

CCRAHDRCWKSSCGKPGNCCGE.

In a second aspect, the present invention provides a nucleic acid molecule encoding the collagen-conopeptide fusion protein described in the first aspect.

Preferably, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO.4.

SEQ ID NO.4:

gaagctggtctgccgggtgctaaaggtctgaccggttctccgggttctccgggtccggacggtaaaaccggtccgccgggtccggctggtcaggacggtcgtccgggtccgccgggtccgccgggtgctcgtggtcaggctggtgttatgggtttcccgggtccgaaaggtgctgctggtgaacc gggtaaagctggtgaacgtggtgttccgggtccgccgggtgctgttggtccggctggtaaagacggtgaagctggtgctcagggtccgccgggtccggctggtccggctggtgaacgtggtggtggtggtggttcttgctgccgtgctcacgaccgatgctggaaatcttcttgcggtaaaccg ggtaactgctgcggtgaataa.

In a third aspect, the present invention provides an expression vector comprising at least one copy of the nucleic acid molecule described in the second aspect.

In a fourth aspect, the present invention provides a recombinant cell, wherein the recombinant cell expresses the collagen-conopeptide fusion protein described in the first aspect.

Preferably, the nucleic acid molecule described in the second aspect is integrated into the genome of the recombinant cell;

Preferably, the recombinant cell contains the expression vector described in the third aspect.

In a fifth aspect, the present invention provides a method for preparing the collagen-cono peptide fusion protein described in the first aspect, the preparation method comprising the following steps: constructing an expression vector containing a nucleic acid molecule encoding the collagen-cono peptide fusion protein described in the first aspect, transferring the expression vector into a recipient cell, and screening a positive strain containing the expression vector; culturing the positive strain containing the expression vector, inducing expression, disrupting the bacteria, and purifying to obtain the collagen-cono peptide fusion protein.

The present invention utilizes a gene recombination method to connect the sequences of collagen fragments and conopeptide fragments in series, and after expression and purification by an Escherichia coli host, the obtained product can be widely used in the pharmaceutical and cosmetic industries. The preparation method described in the present invention is suitable for the production of collagen-conopeptide fusion protein, and the method can effectively reduce the production cost of the fusion protein, improve the production efficiency, and also provide another biological approach for the production of the fusion protein.

The target product expressed by the preparation method of the present invention is often present in a soluble form in Escherichia coli, has a high yield, and does not need to undergo an enzyme cleavage step after purification; the product has both collagen function and conopeptide function, and functionally makes up for the defects of collagen's lack of obvious short-term wrinkle removal effect and conopeptide's lack of long-term repair function.

Preferably, the recipient cell comprises Escherichia coli, the recipient cell comprises Escherichia coli, and the Escherichia coli comprises DH5α, BL21 (DE3) or BL21 STAR (DE3).

The preparation method of the present invention is adopted to culture the positive strain containing the expression vector, induce expression, break the bacteria, and purify to obtain the collagen-conopeptide fusion protein.

Preferably, the method of inducing expression comprises IPTG induction;

Preferably, the temperature for inducing expression is 36-38°C (for example, 36°C, 36.5°C, 37°C, 37.5°C or 38°C, etc.).

The rotation speed of the induced expression in the present invention is 120-320rpm (for example, it can be 120rpm, 140rpm, 160rpm, 180rpm, 200rpm, 220rpm, 240rpm, 260rpm, 280rpm, 300rpm, or 320rpm, etc.), and when the growth OD grows to the range of 0.6-0.8, IPTG solution is added to induce the product, and the induced expression time is 4-6h (for example, it can be 4h, 4.5h, 5h, 5.5h or 6h, etc.).

The final concentration of IPTG used in the IPTG induction of the present invention is 0.5-1.5 mM, for example, it can be 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM or 1.5 mM, etc.

Preferably, the bacterial cell disruption is carried out by the following steps: resuspending the bacterial cell with a bacterial disruption buffer, performing high-pressure homogenization until the bacterial solution is clear and transparent, centrifuging the bacterial solution, and collecting the supernatant and filtering it.

Preferably, the lysis buffer comprises, by molar concentration, 10-50 mM PB, 100-300 mM NaCl, and the solvent is water.

The concentration of PB in the lysis buffer of the present invention is 10-50mM, for example, it can be 10mM, 20mM, 30mM, 40mM or 50mM, etc.; the concentration of NaCl is 100-300mM, for example, it can be 100mM, 120mM, 140mM, 160mM, 180mM, 200mM, 220mM, 240mM, 260mM, 280mM or 300mM, etc.

The volume ratio of the lysis buffer to the bacteria in the present invention is (1-50):1, for example, it can be 1:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1 or 50:1.

The pressure of the high-pressure homogenization in the present invention is 600-1000 bar, for example, it can be 600 bar, 700 bar, 800 bar, 900 bar or 1000 bar.

The centrifugal speed of the present invention is 12000-16000rpm, for example, it can be 12000rpm, 13000rpm, 14000rpm, 15000rpm or 16000rpm, etc., and the centrifugal time is 40-60min, for example, it can be 40min, 45min, 50min, 55min or 60min, etc.

Preferably, the purification method comprises nickel ion column affinity chromatography.

Preferably, the nickel ion column affinity chromatography comprises the following steps: after the filtered bacterial liquid supernatant is adsorbed on the nickel ion column, the pre-elution buffer of the nickel ion column affinity chromatography with increasing imidazole concentration is used for primary and secondary washing, and then the elution buffer with increasing imidazole concentration is used for primary and secondary elution.

Preferably, the imidazole concentration in the pre-elution buffer used in the first wash is 40-60 mM, for example, 40 mM, 45 mM, 50 mM, 55 mM or 60 mM.

The pre-elution buffer used for the primary washing in the present invention includes, by molar concentration, 10-50 mM PB (for example, 10 mM, 20 mM, 30 mM, 40 mM or 50 mM, etc.); 100-300 mM NaCl (for example, 100 mM, 120 mM, 140 mM, 160 mM, 180 mM, 200 mM, 220 mM, 240 mM, 260 mM, 280 mM or 300 mM, etc.); 40-60 mM imidazole (for example, 40 mM, 45 mM, 50 mM, 55 mM or 60 mM, etc.); and the solvent is water.

Preferably, the imidazole concentration in the pre-elution buffer used in the secondary washing is 90-110 mM, for example, 90 mM, 95 mM, 100 mM, 105 mM or 110 mM.

The pre-elution buffer used in the secondary washing of the present invention includes, by molar concentration, 10-50mM PB (for example, 10mM, 20mM, 30mM, 40mM or 50mM, etc.); 100-300mM NaCl (for example, 100mM, 120mM, 140mM, 160mM, 180mM, 200mM, 220mM, 240mM, 260mM, 280mM or 300mM, etc.); 90-110mM imidazole (for example, 90mM, 95mM, 100mM, 105mM or 110mM, etc.); and the solvent is water.

Preferably, the imidazole concentration in the elution buffer used in the primary elution is 150-250 mM, for example, it can be 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM or 250 mM, etc.

The elution buffer used for the primary elution in the present invention includes, by molar concentration, 10-50mM PB (for example, 10mM, 20mM, 30mM, 40mM or 50mM, etc.); 100-300mM NaCl (for example, 100mM, 120mM, 140mM, 160mM, 180mM, 200mM, 220mM, 240mM, 260mM, 280mM or 300mM, etc.); 150-250mM imidazole (for example, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, 210mM, 220mM, 230mM, 240mM or 250mM, etc.); and the solvent is water.

Preferably, the imidazole concentration in the elution buffer used in the secondary elution is 450-550 mM, for example, it can be 450 mM, 460 mM, 470 mM, 480 mM, 490 mM, 500 mM, 510 mM, 520 mM, 530 mM, 540 mM or 550 mM, etc.

The elution buffer used in the secondary elution of the present invention includes, by molar concentration, 10-50mM PB (for example, 10mM, 20mM, 30mM, 40mM or 50mM, etc.); 100-300mM NaCl (for example, 100mM, 120mM, 140mM, 160mM, 180mM, 200mM, 220mM, 240mM, 260mM, 280mM or 300mM, etc.); 450-550mM imidazole (for example, 450mM, 460mM, 470mM, 480mM, 490mM, 500mM, 510mM, 520mM, 530mM, 540mM or 550mM, etc.); and the solvent is water.

Preferably, the purification further comprises a step of desalting using a G25 gel column.

As a preferred technical solution of the present invention, the preparation method comprises the following steps:

(1) connecting the nucleic acid molecule encoding the collagen-conopeptide fusion protein into a plasmid vector to construct an expression vector, transforming the expression vector into Escherichia coli, screening positive strains containing the expression vector, and obtaining a genetically engineered Escherichia coli strain;

(2) fermenting and culturing the genetically engineered Escherichia coli strain, inducing protein expression with IPTG, and collecting the cells by centrifugation, wherein the induction expression temperature is 36-38° C.;

(3) resuspending the bacterial solution with a lysis buffer, wherein the lysis buffer comprises: 10-50 mM PB, 100-300 mM NaCl, and the solvent is water; performing high pressure homogenization until the bacterial solution is clear and transparent, centrifuging the bacterial solution, taking the supernatant and filtering;

(4) After the filtered bacterial supernatant is adsorbed on a nickel ion column, a nickel ion column affinity chromatography pre-elution buffer with increasing imidazole concentration is used for primary and secondary elutions in sequence; the imidazole concentration in the pre-elution buffer used for the primary wash is 40-60 mM imidazole; the imidazole concentration in the pre-elution buffer used for the secondary wash is 90-110 mM imidazole; and an elution buffer with increasing imidazole concentration is used for primary and secondary elutions; the imidazole concentration in the elution buffer used for the primary elution is 150-250 mM imidazole; the imidazole concentration in the elution buffer used for the secondary elution is 450-550 mM imidazole; after purification, a G25 gel column is used for desalting to obtain the collagen-conopeptide fusion protein.

In a sixth aspect, the present invention provides a composition having immediate anti-wrinkle and long-lasting repair effects, wherein the composition contains the collagen-conopeptide fusion protein described in the first aspect.

Preferably, the composition comprises, by weight: 0.01-1 part of collagen-conopeptide fusion protein, 0.01-1 part of carnosine, 0.01-1 part of phenoxyethanol, 0.01-0.1 part of ethylhexylglycerol, 2-10 parts of glycerol, 0.3-3 parts of betaine and 80-100 parts of water.

The weight proportion of collagen-conopeptide fusion protein in the composition of the present invention is 0.01-1 part, for example, it can be 0.01 part, 0.02 part, 0.04 part, 0.06 part, 0.08 part, 0.1 part, 0.2 part, 0.4 part, 0.6 part, 0.8 part or 1 part, etc.; the weight proportion of carnosine is 0.01-1 part, 0.01 part, 0.02 part, 0.04 part, 0.06 part, 0.08 part, 0.1 part, 0.2 part, 0.4 part, 0.6 part, 0.8 part or 1 part, etc.; the weight proportion of phenoxyethanol is 0.01-1 part, for example, it can be 0.01 part, 0.05 part, 0.1 part, 0.15 part, 0.2 part, 0.3 part, 0.4 part, 0.5 part, 0.6 part, 0.7 part, 0.8 part, 0.9 part or 1 The weight portion of ethylhexylglycerin is 0.01-0.1 parts, for example, it can be 0.01 parts, 0.02 parts, 0.03 parts, 0.04 parts, 0.05 parts, 0.06 parts, 0.07 parts, 0.08 parts, 0.09 parts or 0.1 parts, etc.; the weight portion of glycerol is 2-10 parts, for example, it can be 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, etc. parts or 10 parts; the weight parts of betaine are 0.3-3 parts, 0.3 parts, 0.5 parts, 0.8 parts, 1 parts, 1.3 parts, 1.5 parts, 1.8 parts, 2 parts, 2.3 parts, 2.5 parts or 3 parts, etc.; the weight parts of water are 80-100 parts, for example, 80 parts, 82 parts, 85 parts, 88 parts, 90 parts, 92 parts, 95 parts, 98 parts or 100 parts, etc.

In the composition with instant anti-wrinkle and long-term repair effects of the present invention, the collagen-conopeptide fusion protein functionally makes up for the defects of the recombinant collagen in that the short-term wrinkle removal effect is not obvious and the conopeptide does not have a long-term repair function. Among them, collagen can promote cell adhesion and proliferation through integrins; conopeptide can reduce wrinkles by blocking the current conduction of neuromuscular, especially the NAV1.4 channel of muscle, to achieve the effect of wrinkle removal. Carnosine, as an antioxidant, can prevent the collagen-conopeptide fusion protein from being oxidized; at the same time, carnosine has a good anti-glycation and anti-aging effect, can promote the synthesis of skin collagen, and play a synergistic role with the collagen-conopeptide fusion protein. Phenoxyethanol, as a preservative, can inhibit the growth of microorganisms, effectively reduce the degradation of protein by microorganisms, ensure the long-term stability of the system, and then help to improve the preservation effect of protein. Both ethylhexylglycerin and glycerin act as system thickeners to increase the viscosity of the system and maintain the stability of the system. They can play a protective role around the preserved protein and reduce the possibility of protein degradation, thereby preventing the precipitation of collagen-conopeptide fusion protein. Betaine acts as a moisturizer to keep the skin surface moisturized, forming a hydration film, and coordinating the collagen-conopeptide fusion protein and carnosine to be absorbed by the skin.

In a seventh aspect, the present invention provides the use of the collagen-conopeptide fusion protein described in the first aspect and/or the composition with immediate anti-wrinkle and long-lasting repair effects described in the sixth aspect in medicines and cosmetics.

The numerical range described in the present invention not only includes the point values listed above, but also includes any point values between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range.

Compared with the prior art, the present invention has the following beneficial effects:

(1) For collagen-conopeptide fusion protein, this method can effectively reduce the production cost of the fusion protein, improve the production efficiency, and also provide another biological pathway for the production of fusion protein.

(2) The target fusion protein expressed by the preparation method of the present invention exists in a soluble form in Escherichia coli with high yield. The product does not need to undergo an enzymatic cleavage step after purification. The product has both collagen function and cono peptide function, and functionally makes up for the defects of recombinant collagen in that the short-term wrinkle removal effect is not obvious and cono peptide does not have a long-term repair function.

(3) The composition with immediate anti-wrinkle and long-term repair effects of the present invention, wherein the collagen-conopeptide fusion protein can promote cell adhesion and proliferation through integrin, and the conopeptide fragment can reduce wrinkles by blocking the current conduction of nerve muscles, especially the NAV1.4 channel of muscles, so as to achieve the effect of wrinkle removal. Carnosine, as an antioxidant, can prevent the collagen-conopeptide fusion protein from being oxidized. At the same time, carnosine has a strong anti-glycation and anti-aging effect, can promote the synthesis of skin collagen, and play a synergistic role with the collagen-conopeptide fusion protein.

(4) The composition with instant anti-wrinkle and long-term repair effects of the present invention, wherein the collagen fused with cono peptide is human collagen. Since human collagen has better compatibility with the human body, the fusion protein containing cono peptide fragments is more easily absorbed by the human body, which reduces the minimum effective amount of cono peptide, so less cono peptide can be used to achieve the same effect.

Description of the drawings:

FIG. 1 is the SDS-PAGE gel electrophoresis result of the collagen-conopeptide fusion protein in Example 1 (the box in FIG. 1 is the target protein and its corresponding protein molecular weight standard).

FIG. 2 is an exemplary diagram of the skin anti-wrinkle test of the composition in Application Example 1.

Specific implementation method:

The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only used to help understand the present invention and should not be regarded as specific limitations of the present invention.

If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be obtained through regular channels.

The sources of the experimental materials used in the embodiments, comparative examples, application examples and comparative application examples of the present invention are shown in Table 1:

Table 1

Components factory Part Number pET28a vector Laboratory storage / BL21 STAR (DE3) cells Laboratory storage / IPTG Suzhou Tianke KI90541 NI column Lanxiao Biology A4013203 G25 gel column Borgron AG0063 Phenoxyethanol Thor Specialty Chemicals (Zhenjiang) Co., Ltd. 122-99-6 Ethylhexylglycerin Thor Specialty Chemicals (Zhenjiang) Co., Ltd. 70445-33-9 glycerin Kuala Lumpur Kepong Oil and Fat Chemicals Co., Ltd. 56-81-5 Betaine Dezhou Onlida Biotechnology Co., Ltd. /

Example 1

This embodiment provides a method for preparing a collagen-conopeptide fusion protein, the preparation method comprising the following steps:

(1) Host bacteria construction

The sequences of the collagen fragment and the cono peptide fragment were concatenated in a 1:1 manner. The concatenated nucleotide sequence was shown in SEQ ID NO.4. A 6×HIS tag was added to the N-terminus. The amino acid sequence was constructed into a pET28a vector after Escherichia coli codon optimization. The constructed vector was introduced into the Escherichia coli host BL21 STAR (DE3) cells.

(2) Strain expression

Pick a single clone of recombinant E. coli, transfer it to 50mL LB medium, add kanamycin with a final concentration of 50μg/mL, and culture it overnight at 37℃ and 220rpm. On the second day, draw the bacterial liquid from the seed liquid, inoculate it into fresh LB medium at a ratio of 1:100, and culture it at 37℃ and 220rpm. When the growth OD grows to the range of 0.6-0.8, add IPTG solution with a final concentration of 1mM to induce the product. The induction temperature is 37℃, the induction speed is 220rpm, and the E. coli is induced to express for 5h and then centrifuged to collect the bacteria.

(3) Bacteria fragmentation

Take an appropriate amount of bacteria, resuspend them with lysis buffer and bacteria at a volume ratio of 10:1, the lysis buffer is an aqueous solution of 20mM PB+200mM NaCl, pH 7.4, and use a high-pressure homogenizer to break the bacteria three times at 800bar until the bacterial solution is clear and transparent, and it can be observed under a microscope that the bacteria are completely broken; centrifuge the bacterial solution at 14000rpm for 50min, collect the supernatant as the bacterial cell breaking supernatant, and filter the bacterial solution with a 0.45μm filter membrane.

(4) Purification

After the bacterial supernatant was adsorbed by a nickel ion column (NI column), it was washed with 20mM PB+200mM NaCl+50mM imidazole, pH 7.4 aqueous solution and 20mM PB+200mM NaCl+100mM imidazole, pH 7.4 aqueous solution, and then eluted with 20mM PB+200mM NaCl+200mM imidazole, pH 7.4 aqueous solution and 20mM PB+200mM NaCl+500mM imidazole, pH 7.4 aqueous solution. The eluate was further desalted with a G25 gel column to obtain a collagen-conopeptide fusion protein, the amino acid sequence of which is shown in SEQ ID NO.1.

(5) Protein identification and preservation

Take an appropriate amount of protein solution for SDS-PAGE detection, and use the grayscale scanning method to determine the proportion of YLT-ColI protein (collagen-cono peptide fusion protein) in the entire lane, which is the purity. The gel electrophoresis results of collagen-cono peptide fusion protein are shown in Figure 1: Lane M: protein molecular weight marker, sample volume 5μL; Lane 1: sample mixture, sample volume 10μL; Lane 2: flow-through, sample volume 10μL; Lane 3: eluent 1 (eluent of the first wash), sample volume 10μL; Lane 4: eluent 2 (eluent of the second wash), sample volume 10μL; Lane 5: eluent 3 (eluent of the first elution), sample volume 10μL; Lane 6: eluent 4 (eluent of the second elution), sample volume 10μL. Lanes 1-6 show the purification process of YLT-ColI protein, and lanes 5-6 show the purified protein with a purity of 90%. The fusion protein is frozen at low temperature (-20 to -80°C) for a long time, or the fusion protein is freeze-dried into powder.

Example 2

This embodiment provides a method for preparing a collagen-conopeptide fusion protein, the preparation method comprising the following steps:

(1) Host bacteria construction

Same as Example 1.

(2) Strain expression

Pick a single clone of recombinant E. coli, transfer it to 50mL LB medium, add kanamycin with a final concentration of 45μg/mL, and culture it overnight at 37℃ and 170rpm. On the second day, draw the bacterial solution from the seed solution and inoculate it into fresh LB medium at a ratio of 1:100. Culture it under the same conditions at 37℃ and 170rpm. When the growth OD grows to the range of 0.6-0.8, add IPTG solution with a final concentration of 0.7mM to induce the product. The induction temperature is 37℃ and the induction speed is 170rpm. After the E. coli is induced to express for 4.5h, the bacteria are collected by centrifugation.

(3) Bacteria fragmentation

Take an appropriate amount of bacteria, resuspend them with lysis buffer and bacteria at a volume ratio of 10:1, the lysis buffer is an aqueous solution of 30mMPB+150mM NaCl, pH 7.4, and use a high-pressure homogenizer to break the bacteria three times at 700bar until the bacterial solution is clear and transparent, and it can be observed under a microscope that the bacteria are completely broken; centrifuge the bacterial solution at 13000rpm for 45min, collect the supernatant as the bacterial disruption supernatant, and filter the bacterial solution with a 0.45μm filter membrane.

(4) Purification

After the bacterial supernatant was adsorbed by the NI column, it was washed with 30mM PB+150mM NaCl+55mM imidazole, pH 7.4 aqueous solution and 30mM PB+150mM NaCl+105mM imidazole, pH 7.4 aqueous solution, and then eluted with 30mM PB+150mM NaCl+220mM imidazole, pH 7.4 aqueous solution and 30mM PB+150mM NaCl+520mM imidazole, pH 7.4 aqueous solution. The eluate was further desalted with a G25 gel column to obtain a collagen-conopeptide fusion protein, the amino acid sequence of which is shown in SEQ ID NO.1.

(5) Protein identification and preservation

Same as Example 1.

Example 3

This embodiment provides a method for preparing a collagen-conopeptide fusion protein, the preparation method comprising the following steps:

(1) Host bacteria construction

Same as Example 1.

(2) Strain expression

Pick a single clone of recombinant E. coli, transfer it to 50mL LB medium, add kanamycin with a final concentration of 55μg/mL, and culture it overnight at 37℃ and 270rpm. On the second day, draw the bacterial solution from the seed solution and inoculate it into fresh LB medium at a ratio of 1:100. Culture it under the same conditions at 37℃ and 270rpm. When the growth OD grows to the range of 0.6-0.8, add IPTG solution with a final concentration of 1.3mM to induce the product. The induction temperature is 37℃ and the speed of induction is 270rpm. After the E. coli is induced to express for 5.5h, the bacteria are collected by centrifugation.

(3) Bacteria fragmentation

Take an appropriate amount of bacteria, resuspend them with lysis buffer and bacteria at a volume ratio of 10:1, the lysis buffer is an aqueous solution of 10mMPB+250mM NaCl, pH 7.4, and use a high-pressure homogenizer to break the bacteria three times at 900bar until the bacterial solution is clear and transparent, and it can be observed under a microscope that the bacteria are completely broken; centrifuge the bacterial solution at 15000rpm for 55min, collect the supernatant as the bacterial supernatant, and filter the bacterial solution with a 0.45μm filter membrane.

(4) Purification

After the bacterial supernatant was adsorbed by the NI column, it was washed with 10mM PB+250mM NaCl+45mM imidazole, pH 7.4 aqueous solution and 10mM PB+250mM NaCl+95mM imidazole, pH 7.4 aqueous solution, and then eluted with 10mM PB+250mM NaCl+180mM imidazole, pH 7.4 aqueous solution and 10mM PB+250mM NaCl+480mM imidazole, pH 7.4 aqueous solution. The eluate was further desalted with a G25 gel column to obtain a collagen-conopeptide fusion protein, the amino acid sequence of which is shown in SEQ ID NO.1.

(5) Protein identification and preservation

Same as Example 1.

Example 4

This embodiment provides a method for preparing a collagen-conopeptide fusion protein, the preparation method comprising the following steps:

(1) Host bacteria construction

Same as Example 1.

(2) Strain expression

Pick a single clone of recombinant E. coli, transfer it to 50mL LB medium, add kanamycin with a final concentration of 40μg/mL, and culture it overnight at 36℃ and 120rpm. On the second day, draw the bacterial solution from the seed solution and inoculate it into fresh LB medium at a ratio of 1:100, culture it at 36℃ and 120rpm, and when the growth OD grows to the range of 0.6-0.8, add IPTG solution with a final concentration of 0.5mM to induce the product. The induction temperature is 36℃, the induction speed is 120rpm, and the E. coli is induced to express for 4h and then centrifuged to collect the bacteria.

(3) Bacteria fragmentation

Take an appropriate amount of bacteria, resuspend them with lysis buffer and bacteria at a volume ratio of 10:1, the lysis buffer is an aqueous solution of 40mMPB+100mM NaCl, pH 7.4, and use a high-pressure homogenizer to break the bacteria three times at 600bar until the bacterial solution is clear and transparent, and it can be observed under a microscope that the bacteria are completely broken; centrifuge the bacterial solution at 12000rpm for 40min, collect the supernatant as the bacterial disruption supernatant, and filter the bacterial solution with a 0.45μm filter membrane.

(4) Purification

After the bacterial supernatant was adsorbed by the NI column, it was washed with 40mM PB+100mM NaCl+40mM imidazole, pH 7.4 aqueous solution and 40mM PB+100mM NaCl+90mM imidazole, pH 7.4 aqueous solution, and then eluted with 40mM PB+100mM NaCl+150mM imidazole, pH 7.4 aqueous solution and 40mM PB+100mM NaCl+450mM imidazole, pH 7.4 aqueous solution. The eluate was further desalted with a G25 gel column to obtain a collagen-conopeptide fusion protein, the amino acid sequence of which is shown in SEQ ID NO.1.

(5) Protein identification and preservation

Same as Example 1.

Example 5

This embodiment provides a method for preparing a collagen-conopeptide fusion protein, the preparation method comprising the following steps:

(1) Host bacteria construction

Same as Example 1.

(2) Strain expression

Pick a single clone of recombinant E. coli, transfer it to 50mL LB medium, add kanamycin with a final concentration of 60μg/mL, and culture it overnight at 38℃ and 320rpm. On the second day, draw the bacterial liquid from the seed liquid, inoculate it into fresh LB medium at a ratio of 1:100, and culture it at 38℃ and 320rpm. When the growth OD grows to the range of 0.6-0.8, add IPTG solution with a final concentration of 1.5mM to induce the product. The induction temperature is 38℃, the induction speed is 320rpm, and the E. coli is induced for 6h before centrifugation to collect the bacteria.

(3) Bacteria fragmentation

Take an appropriate amount of bacteria, resuspend them with lysis buffer and bacteria at a volume ratio of 10:1, the lysis buffer is an aqueous solution of 50mMPB+300mM NaCl, pH 7.4, and use a high-pressure homogenizer to break the bacteria three times at 1000bar until the bacterial solution is clear and transparent, and it can be observed under a microscope that the bacteria are completely broken; centrifuge the bacterial solution at 16000rpm for 60min, collect the supernatant as the bacterial disruption supernatant, and filter the bacterial solution with a 0.45μm filter membrane.

(4) Purification

After the bacterial supernatant was adsorbed by the NI column, it was washed with 50mM PB+300mM NaCl+60mM imidazole, pH 7.4 aqueous solution and 50mM PB+300mM NaCl+110mM imidazole, pH 7.4 aqueous solution, and then eluted with 50mM PB+300mM NaCl+250mM imidazole, pH 7.4 aqueous solution and 50mM PB+300mM NaCl+550mM imidazole, pH 7.4 aqueous solution. The eluate was further desalted with a G25 gel column to obtain a collagen-conopeptide fusion protein, the amino acid sequence of which is shown in SEQ ID NO.1.

(5) Protein identification and preservation

Same as Example 1.

Example 6

This embodiment provides a method for preparing a collagen-cono peptide fusion protein, wherein the amino acid sequence of the collagen-cono peptide fusion protein is consistent with that in Example 1. The only difference between this embodiment and Example 1 is that in the method for preparing the recombinant human collagen, in the strain expression in step (2), the induction expression conditions include: the induction treatment temperature is 35°C.

Example 7

This embodiment provides a method for preparing a collagen-cono peptide fusion protein, wherein the amino acid sequence of the collagen-cono peptide fusion protein is consistent with that in Example 1, and the difference between this embodiment and Example 1 is only that, in the method for preparing the recombinant human collagen, in the strain expression in step (2), the induction expression conditions are as follows: the induction treatment temperature is 39°C.

Example 8

This embodiment provides a method for preparing a collagen-cono peptide fusion protein, wherein the amino acid sequence of the collagen-cono peptide fusion protein is consistent with that in Example 1, and the only difference between this embodiment and Example 1 is that in the method for preparing the recombinant human collagen, in the bacterial cell disruption step (3), the molar concentration of PB in the bacterial disruption buffer is 5 mM.

Example 9

This embodiment provides a method for preparing a collagen-cono peptide fusion protein, wherein the amino acid sequence of the collagen-cono peptide fusion protein is consistent with that in Example 1. The only difference between this embodiment and Example 1 is that in the method for preparing the recombinant human collagen, in the bacterial cell disruption step (3), the molar concentration of PB in the bacterial disruption buffer is 60 mM.

Example 10

This embodiment provides a method for preparing a collagen-cono peptide fusion protein, wherein the amino acid sequence of the collagen-cono peptide fusion protein is consistent with that in Example 1, and the only difference between this embodiment and Example 1 is that in the method for preparing the recombinant human collagen, in the bacterial cell disruption step (3), the molar concentration of NaCl in the bacterial disruption buffer is 50 mM.

Embodiment 11

This embodiment provides a method for preparing a collagen-cono peptide fusion protein, wherein the amino acid sequence of the collagen-cono peptide fusion protein is consistent with that in Example 1, and the only difference between this embodiment and Example 1 is that in the method for preparing the recombinant human collagen, in the bacterial cell disruption step (3), the molar concentration of NaCl in the bacterial disruption buffer is 350 mM.

Example 12

This embodiment provides a method for preparing a collagen-cono peptide fusion protein. The amino acid sequence of the collagen-cono peptide fusion protein is consistent with that in Example 1. The only difference between this embodiment and Example 1 is that, during the purification process in step (4), the molar concentrations of imidazole in the pre-elution buffer used for the first and second washings are 20 mM and 70 mM, respectively.

Example 13

This embodiment provides a method for preparing a collagen-cono peptide fusion protein. The amino acid sequence of the collagen-cono peptide fusion protein is consistent with that in Example 1. The only difference between this embodiment and Example 1 is that, during the purification process in step (4), the molar concentrations of imidazole in the pre-elution buffer used in the first and second washings are 80 mM and 130 mM, respectively.

Embodiment 14

This embodiment provides a method for preparing a collagen-cono peptide fusion protein, wherein the amino acid sequence of the collagen-cono peptide fusion protein is consistent with that in Example 1. The only difference between this embodiment and Example 1 is that, during the purification process in step (4), the molar concentrations of imidazole in the elution buffer used in the first and second elutions are 100 mM and 400 mM, respectively.

Embodiment 15

This embodiment provides a method for preparing a collagen-cono peptide fusion protein, wherein the amino acid sequence of the collagen-cono peptide fusion protein is consistent with that in Example 1. The only difference between this embodiment and Example 1 is that, during the purification process in step (4), the molar concentrations of imidazole in the elution buffer used in the first and second elutions are 300 mM and 600 mM, respectively.

The quality of the collagen-conopeptide fusion protein prepared by the preparation method in Example 1-15 was tested, and the test results are shown in Table 2.

Table 2

sample purity sample purity Example 1 91.8% Example 9 77.6% Example 2 90.1% Example 10 81.1% Example 3 89.8% Embodiment 11 85.3% Example 4 85.7% Example 12 70.9% Example 5 83.9% Example 13 79.2% Example 6 69.4% Embodiment 14 79.6% Example 7 71.1% Embodiment 15 71.8% Example 8 75.6% / /

It can be seen from Table 2 that the purity of the recombinant human collagen obtained by the preparation methods in Examples 1-15 varies, and the recombinant human collagen obtained in Example 1 has the highest purity.

By comparing Example 1 with Examples 6-7, it can be seen that when the induced temperature exceeds the original range, the purity of the obtained product will decrease.

By comparing Example 1 with Examples 8-11, it can be seen that when the concentrations of PB and NaCl in the buffer system change, the purity of the obtained product will be affected.

By comparing Example 1 with Examples 12-15, it can be seen that when the imidazole concentration in the impurity washing process is too low, the impurities cannot be effectively eluted; when the imidazole concentration in the impurity washing process is too high, it will cause product loss. When the imidazole concentration in the elution process is too low, part of the product is wasted because it is not eluted; when the imidazole concentration in the elution process is too high, a large amount of impurities are also eluted at the same time, resulting in a decrease in purity.

Comparative Example 1

This comparative example provides a human collagen protein ColI, the amino acid sequence of which is shown in SEQ ID NO.2.

Comparative Example 2

This comparative example provides a commercially available collagen protein Ctrl-Col, whose amino acid sequence is different from that of SEQ ID NO.2.

Comparative Example 3

This comparative example provides a cono peptide, and the amino acid sequence of the cono peptide (YLT) is shown in SEQ ID NO.3.

Comparative Example 4

This comparative example provides another conopeptide, and the amino acid sequence of the conopeptide (YLT2) is shown in SEQ ID NO.5.

SEQ ID NO. 5: GDCKPCMHPDCRFNPGRCR.

Comparative Example 5

This comparative example provides another collagen-cono peptide fusion protein (YLT2-ColI), wherein the amino acid sequence of the cono peptide (YLT2) is shown in SEQ ID NO.5, and the amino acid sequence of the collagen (ColI) is shown in SEQ ID NO.2. The difference between this comparative example and Example 1 is that the cono peptide (YLT2) replaces the cono peptide (YLT) in the fusion protein of Example 1.

Comparative Example 6

This comparative example provides a mixture of collagen ColI and conopeptide (YLT), wherein the amino acid sequence of the collagen ColI is shown as SEQ ID NO.2, and the amino acid sequence of the conopeptide is shown as SEQ ID NO.3.

Application Example 1

This application example provides a composition with instant anti-wrinkle effect and long-term skin repairing effect, wherein the composition contains the collagen-conopeptide fusion protein described in Example 1, and the composition comprises, by weight: 0.1 parts of collagen-conopeptide fusion protein, 0.1 parts of carnosine, 0.4 parts of phenoxyethanol, 0.05 parts of ethylhexylglycerin, 5 parts of glycerin, 1 part of betaine and 93.35 parts of water.

The preparation method of the composition with instant anti-wrinkle effect and long-term skin repairing function is as follows: collagen-conopeptide fusion protein, carnosine, phenoxyethanol, ethylhexylglycerin, glycerin, betaine and water are mixed in sequence to obtain the composition with instant anti-wrinkle effect and long-term skin repairing function.

Application Example 2

This application example provides a composition with instant anti-wrinkle effect and long-term skin repair effect, wherein the composition contains the collagen-conopeptide fusion protein described in Example 1, and the composition comprises, by weight: 0.05 parts of collagen-conopeptide fusion protein, 0.05 parts of carnosine, 0.2 parts of phenoxyethanol, 0.03 parts of ethylhexylglycerin, 3 parts of glycerin, 0.7 parts of betaine and 95.97 parts of water.

The preparation method of the composition having instant anti-wrinkle effect and capable of long-term skin repair is the same as that of Application Example 1.

Application Example 3

This application example provides a composition with instant anti-wrinkle effect and long-term skin repairing effect, wherein the composition contains the collagen-conopeptide fusion protein described in Example 1, and the composition comprises, by weight: 0.5 parts of collagen-conopeptide fusion protein, 0.5 parts of carnosine, 0.7 parts of phenoxyethanol, 0.07 parts of ethylhexylglycerin, 7 parts of glycerin, 2 parts of betaine and 89.23 parts of water.

The preparation method of the composition having instant anti-wrinkle effect and capable of long-term skin repair is the same as that of Application Example 1.

Application Example 4

This application example provides a composition with instant anti-wrinkle effect and long-term skin repairing effect, wherein the composition contains the collagen-conopeptide fusion protein described in Example 1, and the composition comprises, by weight: 0.01 parts of collagen-conopeptide fusion protein, 0.01 parts of carnosine, 0.01 parts of phenoxyethanol, 0.01 parts of ethylhexylglycerin, 2 parts of glycerin, 0.3 parts of betaine and 97.66 parts of water.

The preparation method of the composition having instant anti-wrinkle effect and capable of long-term skin repair is the same as that of Application Example 1.

Application Example 5

This application example provides a composition with instant anti-wrinkle effect and long-term skin repairing effect, wherein the composition contains the collagen-conopeptide fusion protein described in Example 1, and the composition comprises, by weight: 1 part of collagen-conopeptide fusion protein, 1 part of carnosine, 1 part of phenoxyethanol, 0.1 part of ethylhexylglycerin, 10 parts of glycerin, 3 parts of betaine and 83.9 parts of water.

The preparation method of the composition having instant anti-wrinkle effect and capable of long-term skin repair is the same as that of Application Example 1.

Application Example 6

This application example provides a composition with instant anti-wrinkle effect and long-term skin repair effect, wherein the composition contains the collagen-conopeptide fusion protein described in Example 1, and the composition comprises, by weight: 0.2 parts of collagen-conopeptide fusion protein, 0 parts of carnosine, 0.4 parts of phenoxyethanol, 0.05 parts of ethylhexylglycerin, 5 parts of glycerin, 1 part of betaine and 93.35 parts of water.

The preparation method of the composition having instant anti-wrinkle effect and capable of long-term skin repair is the same as that of Application Example 1.

Application Example 7

This application example provides a composition with instant anti-wrinkle effect and long-term skin repairing effect, wherein the composition contains the collagen-conopeptide fusion protein described in Example 1, and the composition comprises, by weight: 0 parts of collagen-conopeptide fusion protein, 0.2 parts of carnosine, 0.4 parts of phenoxyethanol, 0.05 parts of ethylhexylglycerin, 5 parts of glycerin, 1 part of betaine and 93.35 parts of water.

The preparation method of the composition having instant anti-wrinkle effect and capable of long-term skin repair is the same as that of Application Example 1.

Comparative application example 1

This comparative application example provides a composition with skin care effect. The difference between the composition and application example 1 is that the collagen ColI provided in comparative example 1 replaces the collagen-conopeptide fusion protein in application example 1.

Comparative Application Example 2

This comparative application example provides a composition with skin care effects. The difference between the composition and application example 1 is that the collagen Ctrl-Col provided in comparative example 2 replaces the collagen-conopeptide fusion protein in application example 1.

Comparative Application Example 3

This comparative application example provides a composition with skin care effect. The difference between the composition and application example 1 is that the cono peptide (YLT) provided in comparative example 3 replaces the collagen-cono peptide fusion protein in application example 1.

Comparative Application Example 4

This comparative application example provides a composition with skin care effects, and the difference between the composition and application example 1 is only that the cono peptide 2 (YLT2) provided in comparative example 4 replaces the collagen-cono peptide fusion protein in application example 1.

Comparative Application Example 5

This comparative application example provides a composition with skin care effect. The difference between the composition and application example 1 is that the collagen-conopeptide 2 fusion protein (YLT2-ColI) provided in comparative example 5 replaces the collagen-conopeptide fusion protein in application example 1.

Comparative Application Example 6

This comparative application example provides a composition with skin care effect, and the difference between the composition and Application Example 1 is only that the collagen (ColI) and cono peptide (YLT) provided in Comparative Example 6 replaces the collagen-cono peptide fusion protein in Application Example 1.

Test Example 1 Detection of relative expression of zebrafish collagen and elastin

The relative expression levels of zebrafish collagen and elastin were detected for the fusion proteins in Examples 1-5 and Comparative Examples 1-6.

1. Detection of relative expression of zebrafish collagen

Skin growth, repair, nutrition, elasticity and tension are all related to collagen. Its loss will reduce the smoothness of the skin and produce wrinkles. In tetrapods, type I collagen is a trimer, mainly composed of two α peptide chains and one α2 chain, encoded by col1a1a and col1a2 genes, respectively, and performs collagen-related biological functions in connective tissue and bone. There are three type I collagen genes in zebrafish, colIa1a, col1a1b and col1a2 encoding α1(I), α2(I) and α3(I) chains, respectively. Therefore, by detecting the relative expression levels of colIa1a or (and) col1a1b or (and) col1a2 genes, it can be shown whether the sample has a repair effect.

Experimental methods:

The highest concentration in the experiment should not be higher than the NOEC value determined in the preliminary experiment. According to the experimental requirements, the concentration of the test substance is diluted in equal proportions.

According to the experimental requirements, a sufficient number of normally developed zebrafish fry at 96 hpf were pre-screened and randomly assigned to 6-well plates, 30 per well, and 3 wells per concentration group. The buffer water in the 6-well plate was removed without harming the fry, and 3 mL of the test solution of different concentrations was added. Buffer water was added to the blank control group. The culture plate was covered and incubated in a biochemical incubator at 28.5°C ± 0.5°C in the dark until 120 hpf (incubation time was 24 h).

According to the instructions of the RNA rapid extraction kit, the total RNA of each group of zebrafish was extracted, and the total RNA was measured using an ultra-micro UV spectrophotometer, requiring the absorbance ratio of each experimental group at 260nm and 280nm to be in the range of 1.90-2.20. The extracted RNA was synthesized into cDNA according to the instructions of the reverse transcription kit for later use.

The SYBR Green fluorescent dye kit was used to spot the plate and configure a 20μL reaction system. It contains 10μL SYBRGreen Mix, 1μL upstream and downstream primers, 2μL DNA template, and 7μL DEPC water. The reaction solution was added to the reaction wells, and then the PCR reaction program was run on the fluorescent quantitative PCR instrument. The specific operating parameters are as follows: 94℃, 30s; 94℃, 5s, 61℃, 35s; 98℃, 10s; 65℃, 60s; 98℃, 1s; 40 cycles.

The Ct values of qPCR detection of the internal reference gene (β-actin) and target gene col1a1a in each group were recorded.

The test results of zebrafish collagen Col1a1a expression are shown in Table 3:

Table 3

Samples to be tested Relative expression of Col1a1a Samples to be tested Relative expression of Col1a1a Blank control 1.0 Comparative Example 1 1.36 Example 1 1.79 Comparative Example 2 1.31 Example 2 1.74 Comparative Example 3 1.24 Example 3 1.75 Comparative Example 4 1.20 Example 4 1.70 Comparative Example 5 1.49 Example 5 1.71 Comparative Example 6 1.62

2. Detection of relative expression of zebrafish elastin

Elastin is the main component of elastic fibers in skin tissue, which can provide structural support for the skin and protect the skin from aging and sagging. Elastin is composed of two types of short peptides arranged alternately. Eln1 and eln2 are responsible for encoding different peptides of elastin. The two genes are jointly responsible for regulating the expression level of elastin, making the skin firm and elastic. Zebrafish have elastin (eln1, eln2) genes similar to those of humans. Therefore, by detecting the relative expression of eln1 or (and) eln2 genes, it can be shown whether the sample has anti-wrinkle and firming effects.

Experimental methods:

The highest concentration in the experiment should not be higher than the NOEC value determined in the preliminary experiment. According to the experimental requirements, the concentration of the test substance is diluted in equal proportions.

According to the experimental requirements, a sufficient number of normally developed zebrafish fry at 96 hpf were pre-screened and randomly assigned to 6-well plates, 30 per well, and 3 wells per concentration group. The buffer water in the 6-well plate was removed without harming the fry, and 3 mL of the test solution of different concentrations was added. The blank control group was added with buffer water, and the non-water-soluble samples required a solvent control group. Cover the culture plate and incubate in a biochemical incubator at 28.5°C ± 0.5°C in the dark until 120 hpf (incubation time is 24 hours).

According to the instructions of the RNA rapid extraction kit, the total RNA of each group of zebrafish was extracted, and the total RNA was measured using an ultra-micro UV spectrophotometer, requiring the absorbance ratio of each experimental group at 260nm and 280nm to be in the range of 1.90-2.20. The extracted RNA was synthesized into cDNA according to the instructions of the reverse transcription kit for later use.

The SYBR Green fluorescent dye kit was used to spot the plate and configure a 20μL reaction system. It contains 10μL SYBRGreen Mix, 1μL upstream and downstream primers, 2μL DNA template, and 7μL DEPC water. The reaction solution was added to the reaction wells, and then the PCR reaction program was run on the fluorescent quantitative PCR instrument. The specific operating parameters are as follows: 94℃, 30s; 94℃, 5s, 61℃, 35s; 98℃, 10s; 65℃, 60s; 98℃, 1s; 40 cycles.

Record the Ct value of qPCR detection of each group of internal reference gene (β-actin) and target gene eln1.

The test results of zebrafish collagen eln1 expression are shown in Table 4:

Table 4

Samples to be tested Relative expression of eln1 Samples to be tested Relative expression of eln1 Blank control 1.0 Comparative Example 1 1.21 Example 1 1.61 Comparative Example 2 1.19 Example 2 1.50 Comparative Example 3 1.25 Example 3 1.47 Comparative Example 4 1.23 Example 4 1.43 Comparative Example 5 1.38 Example 5 1.46 Comparative Example 6 1.40

It can be seen from Tables 3 and 4 that the collagen-conopeptide fusion protein provided by Examples 1-5 can significantly increase the relative expression levels of collagen col1a1a gene and elastin eln1 gene compared with the blank control group and other comparative examples. Therefore, the collagen-conopeptide fusion protein has the effects of repairing and anti-wrinkle and firming.

Test Case 2: Safety Assessment

30 volunteers who meet the requirements, aged 18-60 years old, 24 females and 6 males, were selected to test the fusion proteins obtained in Application Examples 1-7 and the compositions obtained in Comparative Application Examples 1-6. 0.025 mL of the test sample solution was taken respectively and placed in the small chamber of the spot tester, and no treatment was performed on the control well. The spot tester with the test sample was applied to the inner side of the forearm of the subject with a low-allergenic tape, and the palm of the hand was gently pressed to evenly apply it to the skin, and waited for 24 hours. After removing the spot tester for 30 minutes, 24 hours, and 48 hours (after the indentation disappears), the skin reaction was observed according to the standards in Table 5.

Table 5

The results show that application examples 1-5 are all negative reactions, indicating that the safety of the composition and its derivative products involved in the present invention is guaranteed, and there are no adverse reactions such as skin irritation and sensitization (except for people who are prone to allergies or people who are allergic to this product). The experimental results of the safety assessment are shown in Table 6:

Table 6

From the results in Table 6, it can be seen that the safety of the compositions provided in Application Examples 1-5 is guaranteed, and there are no adverse reactions such as skin irritation and sensitization (except for people who are prone to allergies or are allergic to this product).

Test Example 3 Skin Elasticity Test

Under the guidance of technicians, the test subjects used the compositions provided in Application Examples 1-7 and Comparative Application Examples 1-6, and then used a skin elasticity tester (Cutometer MPA580) to measure the change in the elasticity value _R2 of the applied part.

Test group: 39 people aged 30-60 with wrinkles, including 12 males and 27 females, with healthy skin.

The test environment temperature is 21℃±1℃, humidity is 50±10%, and real-time dynamic monitoring is performed. Before the test, the subject cleans the test area with a designated cleaning product, rinses it with clean water, and then wipes it dry with a dandruff-free absorbent paper towel. After cleaning the test area, the subject sits quietly for 30 minutes in a standard test environment, cannot drink water or beverages, stays relaxed, and avoids touching the test area.

(1) Take the compositions provided in Application Examples 1-7 and Comparative Application Examples 1-6, and test each sample on three subjects, three times for each subject.

(2) Select three experimental sites on the inner side of the subject's arm.

(3) The subjects applied the sample on the inner part of their arms once in the morning and once in the evening. During the experiment, the subjects were not allowed to apply any other cosmetics on the experimental area.

(4) After the subjects used the composition for 14 and 28 days, the elasticity value R ₂ of the applied area was measured and compared with the skin elasticity value (blank) of the skin not using the composition.

(5) The numerical values measured at each experimental site of the subject are counted, and the changing patterns of skin elasticity are analyzed to evaluate the changes in skin elasticity.

The _R2 value of the skin measures the overall elasticity of the skin. The closer it is to 1, the better the skin elasticity. The test results of the _R2 value are shown in Table 7:

Table 7

The calculation formula of the rate of change is:

Change rate = (R _{2 (D14 or D28)} - R _{2 (D0)} ) / R _{2 (D0)} × 100%

As can be seen from Table 7, the compositions in Application Examples 1-7 all have a certain degree of improvement in skin elasticity, and the composition provided in Application Example 1 has a significantly better improvement in skin elasticity than other Application Examples and Comparative Application Examples. As can be seen from Application Example 1 and Comparative Application Example 6, the effect of the fusion protein of conopeptide and collagen is better than the mixture of conopeptide and collagen. The effect of Application Example 1 is better than that of Application Examples 6 and 7, indicating that the fusion protein of conopeptide and collagen may have a synergistic effect with carnosine, resulting in a slight improvement in skin elasticity.

Test Example 4 Skin Barrier Repair Test

39 volunteers were recruited, aged between 30 and 60, including 12 males and 27 females, to test the repairing effects of Application Examples 1-7 and Comparative Application Examples 1-6, respectively. Each sample was tested on 3 subjects, and each person was tested three times. Three experimental sites were selected on the inner side of the subjects' arms, and 0.5% sodium dodecyl sulfate (SLS) solution was repeatedly applied to the skin to form a chemical damage model. Then, 50 μg/mL of the cream prepared in Application Examples 1-7 and Comparative Application Examples 1-6 was used to apply to the damaged skin. After continuous application for 14 and 28 days, the changes in the transepidermal water loss value (TEWL) of the skin were recorded. The results are shown in Table 8:

The calculation formula of the rate of change is:

Change rate = (TEWL _{(day n)} -TEWL _{(day 0)} ) / TEWL _{(day 0)} × 100%

Table 8

The results in Table 8 show that under natural conditions, the skin barrier of the acute injury model will be naturally repaired, and the transepidermal water loss value will gradually decrease. Application Examples 1-5 show that the use of the collagen-conopeptide fusion protein provided by the present invention will accelerate the repair process of the skin barrier. From Application Examples 1, 6, and 7, it can be seen that the collagen-conopeptide fusion protein in the composition produces a synergistic effect with carnosine, and its effect is better than that of the simple collagen-conopeptide fusion protein (Application Example 6) or carnosine (Application Example 7).

By comparing Application Example 1 (YLT-ColI) with Comparative Application Example 6 (a mixture of ColI and YLT) in Tables 7 and 8, it can be seen that the anti-wrinkle and repair effects of Application Example 1 after 14 days and 28 days of use are better than those of the ordinary mixture of Comparative Application Example 6, indicating that the fusion protein of cono peptide and collagen is more effective than the mixture of cono peptide and collagen.

As can be seen from Tables 7 and 8, Application Example 1 is the most preferred embodiment of the present invention. Under the premise of ensuring the efficacy of immediate anti-wrinkle and long-term repair, the amount of collagen-conopeptide fusion protein (YLT-ColI) used is only 0.1%, and the actual amount of conopeptide used is lower than that of most other products on the market. This is because human collagen has good compatibility with the human body, so the use of human collagen and conopeptide can make conopeptide more easily absorbed, thereby reducing the actual amount of conopeptide used.

Test Example 5 Skin Anti-wrinkle Test

Test instruments: Canfield Visia7 facial image analyzer from the United States, Miravex Antera-3D skin imaging analyzer from Ireland.

A total of 39 subjects, aged between 30 and 60, including 12 males and 27 females, were tested on the compositions provided in Application Examples 1 to 7 and Comparative Application Examples 1 to 6. The subjects' faces were first rinsed with clean water and then dried with a non-chip absorbent paper towel. The subjects then sat quietly in a standard environment (temperature 20° C.-22° C., humidity 40%-60%) for 30 minutes.

Test method: Before using the composition provided in Application Examples 1-7 and Comparative Application Examples 1-6, the percentage of facial wrinkle area of the subject was measured as the initial value, recorded as T0. After sitting for 15 minutes and 30 minutes using the composition provided in Application Examples 1-7 and Comparative Application Examples 1-6, the percentage of facial wrinkle area of the subject was measured, recorded as T15 and T30. The test results are shown in Table 9:

The calculation formula of the rate of change is:

T15 change rate = (T15-T0)/T0×100%

T30 change rate = (T30-T0)/T0×100%

Table 9

As shown in Table 9, within 30 minutes after using different compositions, the wrinkle area ratio decreased to varying degrees, and the effects of Application Examples 1-5 were better than the others. By comparing Application Example 1 with Comparative Application Example 6, it can be seen that the YLT-ColI fusion protein provided by Application Example 1 is better than the mixture of cono peptide and collagen in Comparative Application Example 6, indicating that the fusion protein of cono peptide and collagen can further enhance its instant anti-wrinkle effect.

By comparison, it can be seen that the collagen-cono peptide fusion protein (YLT-ColI) described in Application Examples 1-7 has an immediate anti-wrinkle effect and has the effect of long-term skin repair; after replacing the sequence, its anti-wrinkle and repair functions will be reduced and cannot achieve the best effect; compared with collagen or cono peptide alone, the fusion protein has the advantages of both. The collagen fragment can promote cell adhesion and proliferation through integrins, and the cono peptide fragment can reduce wrinkles by blocking the current conduction of neuromuscular, especially the NAV1.4 channel of muscle, to achieve the effect of wrinkle removal; compared with the solution of simply mixing collagen and cono peptide, the collagen-cono peptide fusion protein functionally makes up for the defects of recombinant collagen's lack of obvious short-term wrinkle removal effect and cono peptide's lack of long-term repair function.

In summary, the present application provides a collagen-conopeptide fusion protein and a preparation method thereof. The preparation method described in the present invention can reduce the expression of the target product in Escherichia coli, which is often present in a soluble form, has a high yield, and the product does not need to undergo an enzymatic cleavage step after purification. The obtained collagen-conopeptide fusion protein has both collagen function and conopeptide function, and functionally makes up for the defect that recombinant collagen has no obvious short-term wrinkle removal effect and the defect that conop peptide does not have a long-term repair function.

The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention are within the protection scope and disclosure scope of the present invention.

Claims (10)

1. A collagen-conopeptide fusion protein, characterized in that the collagen-conopeptide fusion protein comprises a collagen fragment and a conopeptide fragment connected sequentially from the N-terminus to the C-terminus; The amino acid sequence of the collagen fragment is shown in SEQ ID NO.2; The amino acid sequence of the conopeptide fragment is shown in SEQ ID NO.3. 2. The collagen-conopeptide fusion protein according to claim 1, characterized in that the collagen-conopeptide fusion protein further comprises a HIS tag fragment; Preferably, the amino acid sequence of the collagen-conopeptide fusion protein is as shown in SEQ ID NO.1. 3. A nucleic acid molecule, characterized in that the nucleic acid molecule encodes the collagen-conopeptide fusion protein according to claim 1 or 2; Preferably, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO.4. 4. An expression vector, characterized in that the expression vector contains at least one copy of the nucleic acid molecule according to claim 3. 5. A recombinant cell, characterized in that the recombinant cell expresses the collagen-conopeptide fusion protein according to claim 1 or 2; Preferably, the nucleic acid molecule of claim 3 is integrated into the genome of the recombinant cell; Preferably, the recombinant cell contains the expression vector according to claim 4. 6. A method for preparing the collagen-conopeptide fusion protein according to claim 1 or 2, characterized in that the preparation method comprises: constructing an expression vector containing a nucleic acid molecule encoding the collagen-conopeptide fusion protein according to claim 1 or 2, transferring the expression vector into a recipient cell, and screening a positive strain containing the expression vector; culturing the positive strain containing the expression vector, inducing expression, breaking the bacterial cells, and purifying to obtain the collagen-conopeptide fusion protein. 7. The method for preparing the collagen-conopeptide fusion protein according to claim 6, characterized in that the receptor cell comprises Escherichia coli, and the Escherichia coli comprises DH5α, BL21 or BL21 STAR; Preferably, the method of inducing expression comprises IPTG induction; Preferably, the temperature for inducing expression is 36-38°C; Preferably, the bacterial cell disruption is performed by the following steps: resuspending the bacterial cell with a bacterial disruption buffer, performing high-pressure homogenization until the bacterial solution is clear and transparent, centrifuging the bacterial solution, taking the supernatant and filtering; Preferably, the bacteriolysis buffer comprises, by molar concentration, 10-50 mM PB, 100-300 mM NaCl, and the solvent is water; Preferably, the purification method comprises nickel ion column affinity chromatography; Preferably, the nickel ion column affinity chromatography comprises the following steps: after the filtered bacterial liquid supernatant is adsorbed on a nickel ion column, the supernatant is washed once and twice using a nickel ion column affinity chromatography pre-elution buffer with increasing imidazole concentrations, and then eluted once and twice using an elution buffer with increasing imidazole concentrations; Preferably, the imidazole concentration in the pre-elution buffer used in the first wash is 40-60 mM; Preferably, the imidazole concentration in the pre-elution buffer used in the secondary wash is 90-110 mM; Preferably, the imidazole concentration in the elution buffer used in the first elution is 150-250 mM; Preferably, the imidazole concentration in the elution buffer used in the secondary elution is 450-550 mM; Preferably, the purification further comprises a step of desalting using a G25 gel column. 8. A composition with immediate anti-wrinkle and long-lasting repair effects, characterized in that the composition contains the collagen-conopeptide fusion protein according to claim 1 or 2. 9. The composition according to claim 8, characterized in that the composition comprises, by weight: 0.01-1 part of collagen-conopeptide fusion protein, 0.01-1 part of carnosine, 0.01-1 part of phenoxyethanol, 0.01-0.1 part of ethylhexylglycerol, 2-10 parts of glycerol, 0.3-3 parts of betaine and 80-100 parts of water. 10. Use of the collagen-conopeptide fusion protein according to claim 1 or 2 and/or the composition with instant anti-wrinkle effect according to claim 8 or 9 in medicines and cosmetics.

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