CN107141339A - antibacterial peptide and its preparation method and application - Google Patents

Abstract

The present invention provides a kind of antibacterial peptide and its preparation method and application.The amino acid sequence of antibacterial peptide of the present invention is：Tyr‑Ile‑Pro‑Gln‑Pro‑Arg‑Pro‑Pro‑His‑Pro‑Arg‑Leu.The preparation method of the antibacterial peptide includes：Cation-exchange chromatography, hydrophobic chromatography and size exclusion chromatography are carried out to the zymotic fluid containing the antibacterial peptide, the antibacterial peptide is obtained.The antiseptic peptide stability of the present invention is good, and antibacterial activity is high.

Description

Antimicrobial peptide and its preparation method and application

technical field

The invention relates to a polypeptide, in particular to an antibacterial peptide and its preparation method and application.

Background technique

Antimicrobial peptides are a class of polypeptide substances with antibacterial activity. At present, a variety of proteins and polypeptides with antibacterial effects have been found in different animal tissues, which are a class of broad-spectrum antibacterial active polypeptides that exist in organisms and are also important components of the innate immune system. These antimicrobial peptides have the characteristics of strong antibacterial ability, wide antibacterial spectrum, many types, wide range of options, and the target strains are not easy to produce resistance mutations. At the same time, in view of the fact that many microorganisms have developed resistance to antibiotics, antimicrobial peptides are expected to become a new generation of antimicrobial peptides. Clinical antibacterial drugs, and have broad application prospects in cosmetics, biological pesticides, animal feed additives, natural food preservatives, animal and plant disease resistance genetic engineering, etc.

Honey bee peptide (apidaecin, referred to as AP) is a kind of proline-rich polypeptide derived from Hymenoptera insects. It was first isolated from the lymph fluid of bees, generally containing 16-19 amino acid residues, and it is effective for a variety of leather Lamb-negative bacteria, especially Enterobacteriaceae, have a strong killing effect, but have no effect on Gram-positive bacteria including Lactococcus lactis, and have no toxic effect on eukaryotic cells. Due to the low content of honeybee peptide in insects, people are increasingly trying to express honeybee peptide in different expression systems. However, antimicrobial peptides have problems such as low gene expression, easy degradation of expression products, low activity of expression products, and easy toxicity of antimicrobial peptides to host bacteria in the expression of foreign genes. Therefore, an antimicrobial peptide with good stability and high antibacterial activity is desired.

Contents of the invention

The invention provides an antibacterial peptide and its preparation method and application. The antibacterial peptide has good stability and high antibacterial activity.

The present invention provides an antibacterial peptide, the amino acid sequence of which is: Tyr-Ile-Pro-Gln-Pro-Arg-Pro-Pro-His-Pro-Arg-Leu (SEQ ID NO: 1).

The preparation method of the antimicrobial peptide having the above-mentioned amino acid sequence is not strictly limited in the present invention, it can be obtained by chemical synthesis, and can also be obtained by fermenting the strain containing the above-mentioned antimicrobial peptide expression vector; The sequence is easily constructed to obtain the above-mentioned antimicrobial peptide expression vector, and then the bacterial strain containing the above-mentioned antimicrobial peptide expression vector is obtained and fermented.

The present invention also provides a preparation method of the antimicrobial peptide, comprising: performing cation exchange chromatography, hydrophobic chromatography and size exclusion chromatography on the fermentation broth containing the above antimicrobial peptide to obtain the antimicrobial peptide.

The present invention does not impose strict restrictions on the preparation method of the above-mentioned fermentation broth, as long as the above-mentioned antimicrobial peptide can be obtained through fermentation. In one embodiment, the preparation method of the fermentation broth may include: fermenting the Bacillus subtilis containing the antimicrobial peptide expression vector to obtain the fermentation broth.

Specifically, the recombinant Bacillus subtilis disclosed in the Chinese invention patent with publication number CN 105671069 A can be transformed to obtain the strain containing the above-mentioned antimicrobial peptide expression vector; the transformation method is not strictly limited, and ultraviolet mutagenesis can be used. Physical mutagenesis methods, chemical mutagenesis methods such as mutagen mutagenesis, genetic engineering methods such as site-directed mutagenesis, etc.

In the present invention, the order of cation exchange chromatography, hydrophobic chromatography and size exclusion chromatography is not strictly limited, and it is preferred to perform cation exchange chromatography, hydrophobic chromatography and size exclusion chromatography in sequence.

The present invention uses a chromatography system composed of cation exchange chromatography, hydrophobic chromatography, and size exclusion chromatography to remove miscellaneous proteins in the fermentation broth that have charge difference, hydrophobicity difference, and size difference from the target antimicrobial peptide, respectively. Other substances, so as to achieve the effect of purification; the purified antimicrobial peptides can be combined with HPLC-MS, so that the further purified antimicrobial peptides and their structural information can be obtained. The above method not only separates and purifies antimicrobial peptides scientifically and reasonably, but also provides a basis for the detection of antimicrobial peptides by utilizing the characteristics of antimicrobial peptides and the characteristic differences between different laminate methods.

Among them, cation exchange chromatography utilizes the different affinity between the exchangeable ions on the ion exchanger (i.e. the exchange medium) and the various ions separated in the surrounding medium, that is, uses the charged groups of the ion exchanger to absorb the fermentation broth The ions or ionic compounds of opposite charge in the medium, the adsorbed substance is then eluted by being replaced by other ions with the same type of charge, because various ions or ionic compounds have different binding forces to the ion exchanger, so the elution The rates are different to form a chromatographic layer. Because ion exchange is reversible, it can reach equilibrium at an extremely fast rate, so ion exchange chromatography has the advantages of high sensitivity, repeatability, good selectivity, and fast analysis speed.

In a specific scheme of the present invention, the exchange medium of the cation exchange chromatography is Capto S; the exchange medium is a chromatographic medium built on a novel high-flow rate agar matrix platform, which combines high mechanical and high dynamic loading The properties of large amount and rapid mass transfer enable rapid purification to meet the requirements of flexible process design and maximum cost-effectiveness. Compared with the medium without dextran-modified backbone, the binding capacity of the exchange medium is greatly increased.

The present invention does not strictly limit the specific separation conditions of the cation exchange chromatography, which may be conventional separation conditions in the art. Specifically, the equilibrium solution may be a phosphate buffer solution with a pH value of 5-6; in addition, a 0.03-0.12 mol/L phosphate buffer solution with a pH value of 5-6 may be used as the eluent for elution.

In the present invention, hydrophobic chromatography utilizes the hydrophobic group of the polypeptide/protein and the hydrophilic group to separate, and the surface of the polypeptide/protein usually has a hydrophobic group, which can be separated from the hydrophobic carrier at a high salt concentration. Will bind; in general, the higher the ionic strength (salt concentration), the stronger the hydrophobic bond formed. In a specific solution of the present invention, the hydrophobic medium of the hydrophobic chromatography is phenyl-agarose gel, which can better separate target antimicrobial peptides.

The present invention does not strictly limit the specific separation conditions of the hydrophobic chromatography, which may be conventional separation conditions in the art. Specifically, the balance solution can be a phosphate buffer solution with a pH value of 5-6 and a concentration of 0.2mol/L; in addition, a phosphate buffer solution with a pH value of 0.02-0.05mol/L and a pH value of 5-6 can be used as the eluent Perform gradient elution.

Preferably, the elution is carried out by reducing the salt concentration, for example, using 0.05 mol/L, 0.04 mol/L, 0.03 mol/L, 0.02 mol/L, 0.01 mol/L phosphate with a pH value of 5-6 in sequence buffer solution for gradient elution. During elution, the salt concentration of the eluent is gradually reduced, so that substances with different hydrophobicities can be eluted one by one, so that the target antimicrobial peptide can be separated from other hydrophilic proteins and other substances. In particular, hydrophobic chromatography followed by cation exchange chromatography can make the two chromatography complementary, thus facilitating the separation of substances that are difficult or impossible to separate by cation exchange chromatography.

In the present invention, size exclusion chromatography is separated based on the different sizes of molecules, which is a non-adsorption form of chromatography, so the composition of the buffer will not directly affect the separation results, and the antimicrobial peptides of interest can be used in a wide range of Separation of substances is carried out at pH and ionic strength. Size exclusion chromatography provides flexible selection of suitable chromatographic conditions to meet the needs of further purification.

In a specific solution of the present invention, the filler of the size exclusion chromatography is Sephadex G-100. The present invention does not strictly limit the specific separation conditions of the size exclusion chromatography, which may be conventional separation conditions in the art. Specifically, a 0.02-0.08 mol/L phosphate buffer solution with a pH value of 5-6 can be used as the balance solution and the eluent.

The present invention also provides an antibacterial method, which is carried out by using the above-mentioned antibacterial peptide. The above-mentioned antibacterial peptide has a strong killing effect on various Gram-negative bacteria, especially Enterobacteriaceae, not only has strong antibacterial activity, but also has good stability, which is beneficial to practical popularization and application.

Description of drawings

Fig. 1 is the HPLC chromatogram of the antimicrobial peptide prepared by the embodiment of the present invention 1;

Fig. 2 is the mass spectrogram of the antibacterial peptide prepared in Example 1 of the present invention.

detailed description

In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and embodiments of the present invention. Obviously, the described embodiments are the Some, but not all, embodiments are invented. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

The raw material that each embodiment adopts:

Recombinant Bacillus subtilis strain: prepared according to the method disclosed in the Chinese invention patent with publication number CN 105671069 A;

Swap media: Capto S, purchased from GE Healthcare;

Hydrophobic medium: phenyl‐agarose gel, purchased from Beijing Solarbio Technology Co., Ltd. (Solarbio);

Size exclusion chromatography filler: Sephadex G-100, purchased from Shanghai Yubo Biotechnology Co., Ltd. (Pharmacia);

Antimicrobial peptide (SEQ ID NO: 1): synthesized by ABI 433A automatic peptide synthesizer.

Example 1

1. Strain modification

Ultraviolet mutagenesis was carried out to the recombinant Bacillus subtilis strain by conventional methods, and the strain with a larger inhibition zone (hereinafter referred to as the mutant strain) was screened for future use.

A large number of Salmonella and a small amount of the above-mentioned mutant strains are mixed and cultivated. After the number of viable bacteria of the mutant strains increases and stabilizes, a small amount of bacterial suspension is inoculated into the pure culture of Salmonella, and the mixed culture is repeated. Under selection, a new strain of Bacillus subtilis with stronger antibacterial activity is produced.

A genetic stability test was carried out on the above-mentioned new strain of Bacillus subtilis to obtain a genetically stable new strain of Bacillus subtilis (hereinafter referred to as a modified strain).

2. Fermentation

The above modified strains were inoculated into LB medium, cultured with shaking at 37°C and 250r/min for 22 hours, centrifuged at 10000r/min for 10min to collect the supernatant of the fermentation broth, and set aside.

3. Separation and purification

Carry out cation exchange chromatography, hydrophobic chromatography and size exclusion chromatography sequentially on the supernatant of the above fermentation broth, the specific steps are:

1. Cation exchange chromatography

Use Capto S as the exchange medium to carry out cation exchange chromatography on the supernatant of the above fermentation broth. After equilibrating with a phosphate buffer solution of pH5.8, the sample is loaded at a speed of 2BV/h, and then 0.06mol/L, pH5.8 The phosphate buffer solution was used for elution, and the eluate was collected every 5 minutes, and the antibacterial test was carried out by the double-layer agar perforation diffusion method, and the eluate with the inhibition zone was collected.

2. Hydrophobic chromatography

The eluate collected above was subjected to hydrophobic chromatography using phenyl-agarose gel as a hydrophobic medium, and after equilibrating with a 0.2mol/L, pH5.8 phosphate buffer solution, the sample was loaded at a speed of 2BV/h. Then use 0.05mol/L, 0.04mol/L, 0.03mol/L, 0.02mol/L, 0.01mol/L phosphate buffer solution of pH5.8 for gradient elution, collect the eluate every 5min and carry out For the antibacterial test, collect the eluate with the antibacterial zone.

3. Size exclusion chromatography

The eluate collected above was subjected to size exclusion chromatography using Sephadex G‐100 as a filler, in which the pH5.8 phosphate buffer solution was used to equilibrate, and the sample was loaded at a speed of 2BV/h, followed by pH5. .8 phosphate buffer solution for elution, collect the eluate once every 5min and carry out the antibacterial test, collect the eluate with the bacteriostatic zone, concentrate by ultrafiltration, dry, and freeze-dry in a conventional manner to obtain purified Antimicrobial peptides, sealed and preserved.

4. HPLC-MS analysis and detection

Adopt Agilent 1260 Infinity Binary Liquid Chromatography System (purchased from Agilent Company) to analyze and detect the antimicrobial peptides prepared above, wherein: the chromatographic column is an Agilent ZORBAX 300 StableBond (300SB) C8 HPLC chromatographic column (purchased from Agilent Company); Phase A: 0.1% trifluoroacetic acid aqueous solution; mobile phase B: 80% acetonitrile aqueous solution; column temperature: 25°C, detection wavelength: 280nm; maximum pressure: 200bar; time: 65min; flow rate: 0.700mL/min; Quantity: 20ug, the analysis gradient is shown in Table 1, and the test results are shown in Figure 1.

Table 1 HPLC analysis gradient

time/min 0 3.00 10.00 40.00 55.00 57.00 65.00 A(%) 95.0 95.0 91.0 83.0 70.0 95.0 95.0 B(%) 5.0 5.0 9.0 17.0 30.0 5.0 5.0

Mass spectrometry detection conditions: sheath gas flow (arb): 15; auxiliary gas flow (arb): 5; thermal spray voltage (kV): 4.7; capillary temperature (°C): 275; capillary voltage (V): 49; sleeve lens Compensation voltage (V): 120; test results are shown in Figure 2.

The results show that: the amino acid sequence of the antimicrobial peptide prepared above is: Tyr-Ile-Pro-Gln-Pro-Arg-Pro-Pro-His-Pro-Arg-Leu (SEQ ID NO: 1).

Example 2

In this example, the antimicrobial peptide is synthesized by chemical synthesis, wherein the ABI 433A automatic peptide synthesizer is used for synthesis. The amino acid sequence of the antimicrobial peptide is: Tyr‐Ile‐Pro‐Gln‐Pro‐Arg‐Pro‐Pro‐Pro‐His‐Pro‐Pro Arg-Leu (SEQ ID NO: 1).

Comparative example 1

Using the recombinant Bacillus subtilis strain prepared according to the method disclosed in the Chinese invention patent with publication number CN 105671069 A as a control, the recombinant Bacillus subtilis strain was fermented, separated and purified, and analyzed and detected by HPLC-MS referring to the method of Example 1.

The results showed that: the antimicrobial peptide produced by the fermentation of the recombinant Bacillus subtilis strain was honeybee peptide.

Test Example 1 Antibacterial Test

Antibacterial tests were carried out on the antibacterial peptides of Example 1, Example 2 and Comparative Example 1 by standard agar hole diffusion method.

The antimicrobial peptides of Example 1, Example 2 and Comparative Example 1 were formulated into antimicrobial peptide solutions of the same concentration respectively, with Escherichia coli K88, Staphylococcus aureus, Salmonella, Enterococcus faecalis and Streptococcus faecalis as experimental strains, Mix the culture solution of each experimental strain with 25mL of LB solid medium at 55°C and spread the plate until it solidifies, then punch holes with a sterilized puncher (diameter 5mm), and add each antimicrobial peptide solution dropwise into the holes , cultivated at 37°C for 12 hours, measured the diameter (mm) of each inhibition zone, and calculated the bactericidal activity (X) and bactericidal titer (U/mL) based on the diameter of the inhibition zone, the calculation formula is as follows:

Bactericidal activity (X) = (inhibition zone diameter-2.3mm)/2

Bactericidal potency (U/mL) = 2X × 1000

Observe the size of the inhibition zone, and the results are shown in Table 2.

The antibacterial activity of each antibacterial peptide of table 2

From the results in Table 1, it can be seen that the antibacterial activity of the antimicrobial peptide of the present invention is significantly higher than that of honeybee peptide.

Test 2 Stability Test

The antimicrobial peptides of Example 1, Example 2 and Comparative Example 1 were stored continuously for 6 months at room temperature (28-32° C.), and antibacterial tests were carried out regularly (monthly) according to the method of Test Example 1. After 6 months of storage, The antibacterial results are shown in Table 3.

The stability of each antimicrobial peptide of table 3

In Table 3, "/" means not detected.

The results of the stability test show that the conventional honeybee peptide basically has no antibacterial activity after being stored at room temperature for 1 month, while the antibacterial peptide of the present invention only drops by about 10% after being continuously stored at room temperature for 6 months, which shows that this The invented antimicrobial peptide has good stability.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention. scope.

<110> Gansu Oliver Biotechnology Group Co., Ltd.

<120> Antimicrobial peptide and its preparation method and application

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 12

<212> PRT

<213> Artificial sequence

<400> 1

Tyr Ile Pro Gln Pro Arg Pro Pro His Pro Arg Leu

1 5 10

Claims (10)

1. a kind of antibacterial peptide, it is characterised in that amino acid sequence is：Tyr‐Ile‐Pro‐Gln‐Pro‐Arg‐Pro‐Pro‐His‐ Pro‐Arg‐Leu。

2. a kind of preparation method of antibacterial peptide, it is characterised in that including：To the zymotic fluid containing antibacterial peptide described in claim 1 Cation-exchange chromatography, hydrophobic chromatography and size exclusion chromatography are carried out, the antibacterial peptide is obtained.

3. preparation method according to claim 2, it is characterised in that the preparation method of the zymotic fluid includes：To containing The bacillus subtilis of the peptide expression carrier is fermented, and obtains the zymotic fluid.

4. the preparation method according to Claims 2 or 3, it is characterised in that the exchange media of the cation-exchange chromatography For Capto S.

5. preparation method according to claim 4, it is characterised in that when carrying out the cation-exchange chromatography, is used 0.03-0.12mol/L, pH value are eluted for 5-6 phosphate buffer solution as eluent.

6. the preparation method according to Claims 2 or 3, it is characterised in that the hydrophobic medium of the hydrophobic chromatography be phenyl- Ago-Gel.

7. preparation method according to claim 6, it is characterised in that when carrying out the hydrophobic chromatography, using 0.02- 0.05mol/L, pH value carry out gradient elution for 5-6 phosphate buffer solution as eluent.

8. the preparation method according to Claims 2 or 3, it is characterised in that the filler of the size exclusion chromatography is poly- for Portugal Sugared gel G-100.

9. preparation method according to claim 8, it is characterised in that when carrying out the hydrophobic chromatography, using 0.02- 0.08mol/L, pH value are used as equilibrium liquid and eluent for 5-6 phosphate buffer solution.

10. a kind of antibiotic method, it is characterised in that carried out using the antibacterial peptide described in claim 1.