CN119798414A - Recombinant human type XXIII collagen, its production method and use - Google Patents

Abstract

This patent application discloses recombinant human type XXIII collagen, its production method and use. The amino acid sequence of the recombinant human type XXIII collagen is shown in any one of SEQ ID No: 1 to 7, wherein SEQ ID No: 1 to 6 are truncated proteins of natural human type XXIII collagen, and SEQ ID No: 7 is a recombinant human type XXIII collagen formed by splicing the above truncated proteins in a specific order. The cell adhesion promoting effect of the recombinant human type XXIII collagen is better than or equivalent to that of commercially available recombinant human collagen, and its amino acid sequence is 100% homologous to that of natural human type XXIII collagen and has no immunogenicity.

Description

Recombinant human XXIII type collagen, and production method and application thereof

Technical Field

The invention belongs to the technical field of synthetic biology, and relates to recombinant human XXIII type collagen, a production method and application thereof.

Background

Collagen is the most abundant protein found in mammals, and the 28 types of collagen found at present can be divided into four major classes, fibrillar collagen, reticular collagen, fibril related collagens with discontinuous triple helices (FACITs), and membrane related collagens with discontinuous triple helices (MACITs).

Type XXIII collagen is a novel transmembrane collagen found in recent years, is a type II transmembrane protein, has high similarity to type XIII and type XXV collagens in structure, comprises three triple helix collagen domains, is provided with four non-collagenous domains on two sides, and is located in the NC1 domain. The size of the human XXIII type collagen transcript is 3067bp, the human XXIII type collagen transcript encodes 540 amino acid sequences, the anchoring and cell-indicated protein size is 75KD, and the human XXIII type collagen transcript can be cleaved by furin to generate a 60KD soluble protein.

As a newly discovered collagen, few studies on XXIII type collagen function have been conducted, and the current studies have focused on it as a marker of various metastatic tumors. There is essentially no study to construct functional human XXIII-type recombinant collagen by means of truncation, splicing, etc.

Disclosure of Invention

In view of the technical problems existing in the prior art, the invention aims to provide a truncated protein of natural human XXIII type collagen with good biological activity and recombinant human XXIII type collagen with better biological activity constructed by splicing the truncated proteins.

The inventors found 6 truncated proteins of natural human XXIII type collagen with good cell adhesion promoting and procoagulant activities (comparable to commercially available recombinant human collagen) and spliced them in a specific manner to construct recombinant human XXIII type collagen with better cell adhesion promoting and procoagulant activities (superior to commercially available recombinant human collagen).

Namely, the technical scheme of the invention comprises the following steps:

1. a recombinant human XXIII type collagen has an amino acid sequence shown in any one of SEQ ID No. 1-7.

2. Nucleic acid encoding recombinant human XXIII type collagen of item 1.

3. An expression vector comprising the nucleic acid of item 2.

4 A host cell into which the expression vector of item 3 has been introduced. The host cell may be a prokaryotic cell or a eukaryotic cell, including bacterial hosts such as Escherichia coli, bacillus subtilis, bacillus licheniformis, etc., eukaryotic hosts such as Pichia pastoris, saccharomyces cerevisiae, animal cells, plant cells, etc., preferably Escherichia coli and Pichia pastoris, more preferably Pichia pastoris.

5. A method of producing the recombinant human XXIII type collagen of item 1, comprising the steps of culturing the host cell of item 4, expressing the recombinant human XXIII type collagen of item 1, and collecting a culture comprising the recombinant human XXIII type collagen. The expression may be one or a combination of constitutive expression and inducible expression. Wherein the inducer for inducing expression can be IPTG, beta-galactoside, methanol, ethanol, etc.

6. The production method according to item 5, further comprising the step of subjecting the culture to separation and purification to thereby obtain the purified recombinant human XXIII type collagen.

7. The production method according to item 6, wherein the separation and purification are performed by one or more of salting out, chromatography, affinity chromatography, acid-base precipitation, membrane separation, preferably a combination of chromatography and membrane separation or a combination of ion exchange chromatography and membrane separation.

8 Use of recombinant human XXIII-type collagen according to item 1 in the preparation of a tissue engineering material.

9. The use according to item 8, wherein the tissue engineering material is selected from hemostatic sponges, subcutaneous fillers, artificial bones, artificial skin, orally absorbable biofilms, bone implants, bone repair scaffolds.

The beneficial effects of the invention include:

1) The amino acid sequence of the recombinant human XXIII type collagen is 100% homologous with the natural amino acid sequence, and has no immunogenicity.

2) The recombinant human XXIII type collagen can be expressed in high yield in a commercial pichia pastoris expression system. The expression system has post-translational modification, is easy to secrete and express with high efficiency, and is convenient for subsequent purification due to few endogenous secreted proteins.

3) The recombinant human XXIII type collagen provided by the invention has cell adhesion equivalent to or better than commercial recombinant human collagen, and can achieve the application purpose.

Drawings

FIG. 1 is a diagram showing SDS-PAGE detection of recombinant human XXIII type collagen 1-6 expression.

FIG. 2 is a diagram showing SDS-PAGE detection of recombinant human XXIII type collagen 7 expression.

Detailed Description

Example 1 preparation of recombinant human XXIII-type collagen in Pichia pastoris expression System

Experimental method

1) Preparation of recombinant human XXIII-type collagen expression vector

According to the amino acid sequence shown in SEQ ID No. 1-SEQ ID No. 7, codon optimization of a yeast expression system is carried out to obtain a target gene sequence, the obtained target gene sequence is entrusted to the engineering and biotechnology Co-Ltd for gene synthesis, and the synthesized gene is connected into pPicZ alpha A plasmid to obtain a recombinant human XXIII type collagen expression vector pPicZ alpha A-XXIII 1-7. It should be noted that, SEQ ID No. 1-6 is truncated protein of natural human XXIII type collagen (referred to as recombinant human XXIII type collagen 1-6), and SEQ ID No. 7 is recombinant human XXIII type collagen (referred to as recombinant human XXIII type collagen 7) formed by splicing the truncated proteins according to a specific sequence.

2) Preparation of recombinant human XXIII-type collagen Yeast expression Strain

And linearizing pPicZ alpha A-XXIII 1-7 by using Pme I, respectively converting the competent pichia pastoris X-33, and screening transformants by taking bleomycin resistance as a screening mark to obtain the corresponding recombinant human XXIII type collagen yeast expression strain.

3) Inducible expression of recombinant human XXIII-type collagen

(1) Preparation of primary seed, wherein single colony is selected from the plate and added into 5ml YPD liquid culture medium (1% yeast extract, 2% peptone and 2% glucose), and cultured at 30deg.C and 200rpm for 48h for activation;

(2) Secondary seed preparation, namely transferring the seed to 200ml YPD liquid culture medium with an inoculum size of 1%, culturing the seed at 30 ℃ and 200rpm for 24 hours, and taking the seed as seed in a tank;

(3) Preparing a BSM culture medium 3L in a 5L fermentation tank, sterilizing for 20min at 121 ℃, cooling to 30 ℃, adjusting the pH to 5.0 by ammonia water, and adding the secondary seeds prepared in the step (2) into the fermentation tank in a flame inoculation mode for fermentation culture;

(4) Culturing until OD ₆₀₀ is 50, starting to supplement 50% glycerol, stopping supplementing until OD ₆₀₀ reaches 120, and starting to add methanol for induction when dissolved oxygen rebounds to 100%;

(5) DO is controlled to be not lower than 30% in the induction process, pH is about 5.0, fermentation is finished after induction is carried out for 40 hours, culture solution is taken and centrifuged for 10min under the condition of 8000rpm, supernatant is collected, and protein purity and concentration are detected by an SDS-PAGE method.

4) Purification of recombinant human XXIII collagen

Decolorizing the collected supernatant by a hollow fiber column, purifying by ion exchange and molecular sieve, and freeze-drying after vacuum freezing and desalting to obtain the purified recombinant human XXIII collagen 1-7.

Second, result

The detection result of the SDS-PAGE method on the supernatant after fermentation shows that the corresponding recombinant human XXIII collagen 1-7 is successfully expressed, and the yield and purity are high. The results of protein electrophoresis are shown in FIGS. 1 and 2.

EXAMPLE 2 detection of recombinant human XXIII-type collagen adhesion Activity

Experimental method

Cell adhesion promotion experiments were performed using various recombinant human XXIII collagen, commercially available recombinant human collagen and bovine serum albumin (purchased from beijing solebao) as described in example 1 on HSF (human skin fibroblasts) cells. Human HSF cells were inoculated into a sterile 96-well plate, 200. Mu.L of medium per well, a blank control group was added with PBS alone, the groups were washed 3 times with PBS after standing at 37℃for 2 hours, the non-adherent cells were washed off, 100. Mu.L of culture solution and 50. Mu.L of MTT solution were added per well, shaking was performed uniformly, the mixture was aspirated after incubation in an incubator for 4 hours, 150. Mu.L of DMSO was added to dissolve purple formazan crystals, shaking was performed for 10 minutes, and the absorbance value per well was measured at a detection wavelength of 570nm in an microplate reader.

Cell adhesion rate= (experimental OD-blank OD) ×100%/blank.

Second, result

Recombinant human XXIII collagen, commercially available recombinant human collagen and bovine serum albumin pro-HSF cell adhesion data are shown in table 1:

TABLE 1 adhesion promotion values of adhesion promotion to skin fibroblasts at different concentrations of different proteins

As can be seen from the results in Table 1, the recombinant human XXIII-type collagens 1 to 6 are substantially equivalent to the commercially available recombinant human collagen in terms of cell adhesion promoting effect, but the cell adhesion promoting effect of the spliced protein, i.e., recombinant XXIII-type collagen 7, is stronger than that of the commercially available protein, i.e., recombinant human XXIII-type collagen 7, and has better cell adhesion promoting ability.

EXAMPLE 3 in vivo clotting assay of recombinant human XXIII-type collagen

Preparation of recombinant human XXIII-type collagen sponge

Accurately weighing refined TG enzyme (0.005%), dispersing in a certain volume of purified water, fully dissolving, weighing recombinant human XXIII collagen 7 (2%) prepared in example 1, stirring at room temperature (25 ℃) in a stirrer until fully dissolving, standing for defoaming, filling and dispersing in a mould, standing in a refrigerator at 2-8 ℃ for 48 hours, crosslinking, and freeze-drying the crosslinked collagen sponge in a freeze dryer to obtain the collagen sponge.

In vivo clotting assay for recombinant human XXIII collagen

60 Kunming mice (purchased from the department of medicine, university of Western An traffic, laboratory animal center) were taken. A blank group (distilled water), a commercially available collagen sponge group (tin-free Bedset bioengineering Co., ltd.), and a recombinant human XXIII type collagen sponge group were set, and the collagen sponge thickness was 5mm.

10 Kunming mice, male and female halves, per treatment group. Sterilizing the scalpel with medical alcohol and flame, making an incision on the inner side of the thigh of the mouse with the scalpel, sucking the blood with gauze after the blood flows out, applying the collagen sponge to the wound rapidly, cutting the size of the sponge according to 2 times of the wound of the mouse, vertically applying a weight of 20g, timing, observing the wound every 20s, stopping timing after no blood seepage, and obtaining the time required for hemostasis. The blank group was sprayed (kept sprayed within 30 seconds) with distilled water, and the clotting time and clotting process were observed. Comparison of the clotting effects of the three groups is shown in table 2 below:

TABLE 2 comparison of coagulation Effect of recombinant human XXIII-type collagen sponge

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Sequence information:

SEQ ID No:1:GRRGKPGRRGDPGPPGQSGRDGYPGPLGLDGKPGLPGPKGEKGAPG DFGPRGDQGQD

SEQ ID No:2:GAAGPPGPPGPPGARGPPGDTGKDGPRGAQGPAGPKGEPGQDGEM GPKGPPGPKGEPGVPGKK

SEQ ID No:3:QPGPPGPKGEPGSMGPRGENGVDGAPGPKGEPGHRGTDGAAGPRG APGLKGEQGD

SEQ ID No:4:GPPGPQGPPGPPGIPGAKGELGLPGAPGIDGEKGPKGQKGDPGEPGP AGLKGEAGEMGLS

SEQ ID No:5:GPPGPPGPPGPMGLQGIQGPKGLDGAKGEKGASGERGPSGLPGPVGP PG

SEQ ID No:6:GLPGTKGEKGRPGEPGLDGFPGPRGEKGDRSERGEKGERGVPGRKG VKGQKGEPGPPGLD

SEQ ID No:7：GRRGKPGRRGDPGPPGQSGRDGYPGPLGLDGKPGLPGPKGEKGAPG DFGPRGDQGQDGAAGPPGPPGPPGARGPPGDTGKDGPRGAQGPAGPKGEPGQDGEMGPKGPPGPKGEPGVPGKKQPGPPGPKGEPGSMGPRGENGVDGAPGPKGEPGHRGTDGAAGPRGAPGLKGEQGDGPPGPQGPPGPPGIPGAKGELGLPGAPGIDGEKGPKGQKGDPGEPGPAGLKGEAGEMGLSGPPGPPGPPGPMGLQGIQGPKGLDGAKGEKGASGERGPSGLPGPVGPPGGLPGTKGEKGRPGEPGLDGFPGPRGEKGDRSERGEKGERGVPGRKGVKGQKGEPGPPGLD.

Claims (10)

1. A recombinant human XXIII type collagen has an amino acid sequence shown in SEQ ID No. 7.

2. A recombinant human XXIII type collagen has an amino acid sequence shown in any one of SEQ ID No. 1-6.

3. A nucleic acid encoding the recombinant human XXIII type collagen of claim 1 or 2.

4. An expression vector comprising the nucleic acid of claim 3.

5. A host cell into which the expression vector of claim 4 has been introduced, preferably Pichia pastoris.

6. A method of producing the recombinant human XXIII type collagen of claim 1 or 2, comprising the steps of culturing the host cell of claim 4 to express the recombinant human XXIII type collagen of claim 1, and collecting a culture comprising the recombinant human XXIII type collagen.

7. The production method according to claim 6, further comprising the step of subjecting the culture to separation and purification, thereby obtaining purified recombinant human XXIII type collagen.

8. The production method according to claim 7, wherein the separation and purification are performed by one or more of salting out, chromatography, affinity chromatography, acid-base precipitation, and membrane separation, preferably by a combination of chromatography and membrane separation or by a combination of ion exchange chromatography and membrane separation.

9. Use of recombinant human XXIII type collagen according to claim 1 or 2 in the preparation of tissue engineering material.

10. The use according to claim 9, wherein the tissue engineering material is selected from hemostatic sponges, subcutaneous fillers, artificial bone, artificial skin, orally absorbable biological membranes, bone implants, bone repair scaffolds.