Method for prokaryotic expression of antibacterial peptide

Abstract

The invention relates to the technical field of gene engineering, in particular to a method for prokaryotic expression of antibacterial peptide. According to the method provided by the invention, the antibacterial peptide gene is connected to the 3'end or the 5 'end of the fusion tag gene for fusion expression, and the antibacterial activity of the original antibacterial peptide is eliminated by forming a large amount of protein in an inclusion body state, so that host bacteria cannot be killed. By utilizing the method provided by the invention, a large amount of antibacterial peptide can be prepared in a prokaryotic expression system, the method is simple and easy to implement, and the obtained antibacterial peptide has an obvious in-vitro antibacterial effect after enzyme digestion and purification.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a method for prokaryotic expression of antimicrobial peptides. Background Art

[0002] At present, due to the irregular use of traditional antibiotics in clinical treatment, pathogenic microorganisms such as bacteria and fungi are developing strong resistance to antibiotics at an alarming rate. In particular, the emergence of multi-drug resistant bacteria and super-resistant bacteria often become popular worldwide in a very short period of time, posing a threat to human life safety and causing huge losses in life safety and economy. Antimicrobial peptides are a class of small molecule polypeptides produced by organisms to resist the invasion of exogenous pathogenic microorganisms. Compared with traditional antibiotics, they have the advantages of small molecular weight, broad antibacterial spectrum, strong thermal stability, and unique antibacterial mechanism. The use of antimicrobial peptides to replace traditional antibiotics is of great significance to the development of modern medicine and animal husbandry, and is expected to completely solve the international problems of drug residues and bacterial resistance. So far, thousands of antimicrobial peptides have been isolated and identified. Antimicrobial peptides are generally composed of 10-60 amino acid residues, with a net charge of +2-+6, strong cationicity (pI 8.9-10.7), and a molecular weight of about 2000-7000Da.

[0003] The sources and preparation of antimicrobial peptides can be roughly divided into three ways: natural biological extraction, chemical synthesis and genetic engineering preparation. However, the extraction of natural products is easily restricted by sources, especially antimicrobial peptides from rare animal and plant resources, the extraction is more susceptible to restrictions on the source of raw materials. The production cost of chemical synthesis is high, and a variety of highly toxic reagents (hydrofluoric acid, potassium cyanide, etc.) need to be used in large quantities, and the pollution problem of the "three wastes" generated to the environment is also difficult to solve. The methods of genetic engineering and fermentation engineering are not restricted by sources and should be pollution-free, but due to the antibacterial activity of antimicrobial peptides themselves, prokaryotic expression is very technically challenging. It is toxic to the host bacteria during the expression process, and because the antimicrobial peptides themselves have a small molecular weight, they are easily degraded by various endogenous proteases of the host bacteria, etc. The conventional solution is to co-express with a fusion tag, but there are the following technical problems: the fusion tag protein is too large, the solubility-promoting effect is poor, and the expression product is sheared inefficiently. What is more serious is that the antimicrobial peptides cannot show their original antibacterial activity after enzymatic cleavage and purification. Although the expression is successful, the antimicrobial peptides have lost their activity and still cannot be used for industrial applications.

[0004] Therefore, how to find a suitable fusion tag for antimicrobial peptides, which can not only achieve non-toxic and efficient expression of antimicrobial peptides in host bacteria, but also improve the yield, and after selecting a suitable enzyme cutting / cleavage method, targeted renaturation to obtain high-purity antimicrobial peptides with good antimicrobial activity is the problem that the present invention is concerned with. The present invention explores the fusion tags of various antimicrobial peptides and studies the method of efficient prokaryotic expression of antimicrobial peptides. Summary of the invention

[0005] In order to solve the above technical problems, the present invention adopts the inclusion body renaturation technology of antimicrobial peptides to inhibit the toxic effects on host cells, so that the antimicrobial peptides are successfully expressed in large quantities in Escherichia coli. The antimicrobial peptides finally obtained still have good activity and show obvious in vitro antibacterial effect.

[0006] Specifically, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for prokaryotic expression of antimicrobial peptides, the method for prokaryotic expression of antimicrobial peptides is characterized in that the antimicrobial peptide gene is connected to the 3' end or 5' end of a fusion tag gene for fusion expression.

[0007] Preferably, the amino acid sequence of the antimicrobial peptide is shown as SEQ ID No.3 and / or SEQ ID No.5.

[0008] Preferably, the fusion tag is β2 microglobulin, whose amino acid sequence is shown in SEQ ID No.1.

[0009] Considering that the market is in urgent need of a production process for efficiently preparing highly active antimicrobial peptides suitable for modern industrial production systems, the present invention provides a method for prokaryotic expression of antimicrobial peptides. The method is to connect the antimicrobial peptide gene to the 3' end or 5' end of the fusion tag gene for fusion expression. The prokaryotic expression of antimicrobial peptides uses β2 microglobulin (whose amino acid sequence is shown in SEQ ID No.1) as an inclusion body fusion tag, which can help the antimicrobial peptide to be expressed in large quantities in Escherichia coli. At the same time, the present invention optimizes the codons preferred by Escherichia coli based on the β2 microglobulin gene, and the optimized nucleotide sequence is shown in SEQ ID No.2. The present invention connects the antimicrobial peptide gene to the 3' end or 5' end of the auxiliary protein expression tag, i.e., the β2 microglobulin gene, for fusion expression. The β2 microglobulin gene is used as an inclusion body fusion tag, so that the antimicrobial peptide can be expressed in large quantities in Escherichia coli into an inclusion body state. The antimicrobial peptide in the inclusion body state will not directly kill the host bacteria, so that the fusion protein containing the antimicrobial peptide can be obtained continuously and in large quantities, thereby achieving an increase in yield and efficiency.

[0010] Furthermore, the present invention preferably adds a histidine tag or other purification tags between the fusion tag gene and the antimicrobial peptide gene to help purify the antimicrobial peptide.

[0011] Furthermore, the present invention preferably adds a protease cleavage site between the fusion tag gene and the antimicrobial peptide gene to assist in the purification of the antimicrobial peptide.

[0012] Adding the above-mentioned enzyme cleavage sites and purification tags is helpful for the separation and purification of antimicrobial peptides. The present invention can subsequently cut the antimicrobial peptides from the fusion protein and release them for later purification by enzyme cleavage and renaturation methods.

[0013] The method provided by the present invention utilizes the Escherichia coli recombinant expression inclusion body system to prepare antimicrobial peptides on a large scale, which helps to solve the drug resistance problem of traditional antibiotics and the threat of super bacteria to humans. It is suitable for industrial and large-scale production, can truly be market-oriented, and benefit mankind.

[0014] In a second aspect, the present invention provides a fusion protein, the amino acid sequence of which is shown in SEQ ID No.7 or SEQ ID No.9.

[0015] In a third aspect, the present invention provides a polynucleotide, the nucleotide sequence of which is a polynucleotide as shown in SEQ ID No.8 or SEQ ID No.10.

[0016] In a fourth aspect, the present invention provides an expression vector comprising the polynucleotide.

[0017] In a fifth aspect, the present invention provides a host cell comprising the expression vector and expressing the polynucleotide.

[0018] Preferably, the host cell is Escherichia coli or yeast.

[0019] In a sixth aspect, the present invention provides the use of the polynucleotide, or the expression vector, or the host cell in producing antimicrobial peptides.

[0020] Beneficial effects: The present invention provides a method for prokaryotic expression of antimicrobial peptides, using β2 microglobulin as an inclusion body fusion tag, and forming a large number of proteins in the inclusion body state when fused with the antimicrobial peptide. This fusion protein eliminates the antimicrobial activity of the original antimicrobial peptide during expression and is non-toxic to the host bacteria, thereby achieving efficient expression in the host bacteria. Afterwards, a high concentration of hydroxylamine solution is added to the purified inclusion body denatured protein to lyse the target protein, release the free antimicrobial peptide, and then dialysis refolding purification is used to activate the original antimicrobial effect of the antimicrobial peptide. The method provided by the present invention is used to prepare a large number of antimicrobial peptides in a prokaryotic expression system, and the method is simple and easy to implement. The obtained antimicrobial peptides still maintain antimicrobial activity after enzyme digestion and purification, and have obvious in vitro antibacterial effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0022] Figure 1 A map and schematic diagram of the construction method for B2MG-HNG-GL18 to enter the pET-26b expression vector.

[0023] Figure 2 A map and schematic diagram of the construction method for B2MG-HNG-GK25 to enter the pET-26b expression vector.

[0024] Figure 3 The figure is the gel electrophoresis result of the inclusion body protein expressed by B2MG-HNG-GL18 in the example.

[0025] Figure 4 The figure is the gel electrophoresis result of the inclusion body protein expressed by B2MG-HNG-GK25 in the example.

[0026] Figure 5 The antibacterial test results of the GL18 antimicrobial peptide prepared in the example (Escherichia coli and Staphylococcus aureus, with kanamycin as the control) are shown.

[0027] Figure 6 The antibacterial test results of the GK25 antimicrobial peptide prepared in the example (Escherichia coli and Staphylococcus aureus, with kanamycin as the control) are shown.

[0028] Figure 7 The results of the plate antibacterial test of the antimicrobial peptides prepared in the examples are shown in FIG. DETAILED DESCRIPTION

[0029] β2-microglobulin (beta-2-microglobulin, also known as β2-microglobulin, β2-MG or B2MG) is an endogenous low molecular weight serum protein. As a component of the HLA (human leukocyte antigen) molecule in the human body, it has important value in the diagnosis and monitoring of certain diseases. It can reflect the glomerular filtration index, be used for monitoring blood system diseases, and can also evaluate the activity of autoimmune diseases.

[0030] The present invention creatively fuses β2 microglobulin with antimicrobial peptides to successfully achieve a fusion protein in the form of inclusion bodies. This fusion protein does not have the antimicrobial activity of the original antimicrobial peptide, so it will not kill the host bacteria. After purification, a high concentration of hydroxylamine solution is added to lyse the target protein, releasing a separate antimicrobial peptide, which still has a good antibacterial effect. This method can be used for the large-scale preparation of a variety of antimicrobial peptides in a prokaryotic expression system. The amino acid sequence of this preferred B2MG fusion tag is shown in SEQ ID No. 1, and its codon-optimized base sequence for Escherichia coli expression is shown in SEQ ID No. 2.

[0031] The present invention also provides sequences of two successful antimicrobial peptides, GL18 and GK25. Both antimicrobial peptides can be co-expressed with B2MG to form inclusion bodies, and highly active antimicrobial peptides can be obtained by enzyme digestion and purification. The amino acid sequence of GL18 is shown in SEQ ID No.3, and the base sequence is shown in SEQ ID No.4. The amino acid sequence of GK25 is shown in SEQ ID No.5, and the base sequence is shown in SEQ ID No.6.

[0032] The present invention also provides sequences of fusion proteins of B2MG+antimicrobial peptides containing purification tags and purification cleavage sites, B2MG-HNG-GL18 and B2MG-HNG-GK25, which are used for prokaryotic expression of GL18 antimicrobial peptides and GK25 antimicrobial peptides, respectively, and the genes both contain 6 histone repeat tag sequences and NG sequences for hydroxylamine cleavage cleavage sites. The amino acid sequence of the fusion protein of B2MG-HNG-GL18 is shown in SEQ ID No.7, and the base sequence is shown in SEQ ID No.8. The amino acid sequence of the fusion protein of B2MG-HNG-GK25 is shown in SEQ ID No.9, and the base sequence is shown in SEQ ID No.10.

[0033] The present invention also provides a specific method for prokaryotic expression of antimicrobial peptides, which comprises the following steps: The gene of B2MG-HNG-GL18 or B2MG-HNG-GK25 is synthesized to construct a recombinant expression vector, such as a commonly used PET series prokaryotic expression vector. The recombinant expression vector is then transformed into an expression strain, such as BL21, and IPTG-induced expression is performed, and the bacteria with high expression of the fusion protein are collected by centrifugation. After the bacteria are broken, the precipitated inclusion body components are collected by centrifugation, washed several times with a buffer, and then the inclusion bodies are dissolved with a protein denaturing solution, such as a urea solution. The fusion protein is then purified by Ni column affinity chromatography to obtain the purified denatured protein B2MG-HNG-GL18 or B2MG-HNG-GK25.

[0034] In a denatured state, a high concentration of hydroxylamine solution is added to lyse the target protein, releasing a single antimicrobial peptide GL18 or GK25. The released antimicrobial peptide can be renatured by dialysis, limiting dilution, and the like. The renatured antimicrobial peptide can be recovered from the mixed solution by conventional methods in the art, such as ion exchange chromatography, extraction, spray drying, evaporation, precipitation, and the like. The purified polypeptide can be measured for concentration by the BCA method, for purity by liquid chromatography, and for accurate molecular weight by mass spectrometry.

[0035] In the present invention, the antimicrobial peptide fused with the B2MG protein can be directly expressed as a fusion protein in inclusion bodies, and the specific steps can be carried out by conventional methods in the art. For example, the production can be carried out by the following steps: (1) construction of genetically engineered Escherichia coli; (2) fermentation and culture of genetically engineered Escherichia coli; (3) induction and expression of the fusion protein; and (4) purification of the fusion protein and cleavage of the antimicrobial peptide.

[0036] In step (1), the construction of the genetically engineered Escherichia coli bacteria can be carried out as follows: using the PCR method to perform codon optimization and splicing recombination on the DNA fragment of the B2MG-antimicrobial peptide fusion protein, and finally obtaining the target gene fragment; inserting the obtained target gene fragment into the PET-26b expression vector to obtain a recombinant expression plasmid; transferring the recombinant expression plasmid into the Escherichia coli competent cell BL21 (DE3), and screening to obtain positive Escherichia coli genetically engineered bacteria.

[0037] In steps (2) and (3), the fermentation culture of genetically engineered Escherichia coli and the induction and expression of B2MG-antimicrobial peptide fusion protein can be carried out as follows: select a single colony of genetically engineered Escherichia coli selected from the LB plate for culture; inoculate the bacterial solution into 2×YT medium for amplification culture; add IPTG for induction and continue culturing, and collect the bacterial cells by centrifugation.

[0038] In step (4), the purification and cleavage of the B2MG-antimicrobial peptide fusion protein can be performed as follows: resuspend the bacteria with phosphate buffer, break the bacteria and collect the inclusion bodies by centrifugation; dissolve the inclusion bodies, and then use the NI-NTA affinity column to bind the B2MG-antimicrobial peptide fusion protein, rinse the impurities, and elute the target protein. The fusion protein is cleaved by hydroxylamine cleavage to release the free antimicrobial peptide. The released antimicrobial peptide can be further purified and renatured by dialysis, and then tested for activity or freeze-dried for later use.

[0039] The antimicrobial peptide expressed by the present invention can achieve efficient expression of the antimicrobial peptide in a prokaryotic expression system through the inclusion body renaturation technology, and can also be purified by a simple method and renatured by the polypeptide, which is convenient for industrial scale-up. The obtained antimicrobial peptide has good antimicrobial activity and significant in vitro antibacterial effect, and has obvious inhibitory effect on Escherichia coli and Staphylococcus aureus.

[0040] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0042] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0043] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0044] In the present invention, the host cell can be a eukaryotic cell, such as fungi and yeast, or a prokaryotic cell, such as Escherichia coli. It should be understood that those skilled in the art can replace the above-mentioned Escherichia coli strain as a host cell by using other expression strains.

[0045] In the present invention, the specific sequences involved are as follows: β2-microglobulin amino acid sequence beta-2-microglobulin (B2MG), SEQ ID No. 1: MGIQRTPKIPPVSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM.

[0046] β2-microglobulin amino acid sequence beta-2-microglobulin (B2MG), SEQ ID No. 2: ATGGGCATCCACGGTACTCCAAAGATTCCGGTTCCCTCACGTCATCCAGCAGAGAATGGAAAGTCAAATTTCCTGAATTGCTATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTACTGAAGAATGGAGAGAGAATTGAAAAAGT GGAGCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTTTCTATCTCTTGTACTACACTGAATTCACCCCCACTGAAAAAGATGAGTATGCCTGCCGTGTGAACCATGTGACTTTGTCACAGCCCAAGATAGTTAAGTGGGATCGAGACATG.

[0047] GL18 amino acid sequence SEQ ID No.3: GNNRPVYIPQPRPPHPRL.

[0048] GL18 base sequence SEQ ID No.4: GGCAATAATCGGCCAGTATATATTCCACAACCAAGACCCCCTCCATCCGCGTCTA.

[0049] GK25 amino acid sequence, SEQ ID No.5: GLLSNVAGLLKQFAKGGVNAVLNPK.

[0050] GK25 base sequence SEQ ID No.6: GGACTCCTTTCTAATGTCGCAGGTCTTTTGAAACAATTTGCAAAAGGTGGTGTAAATGCTGTATTGAACCCAAAA.

[0051] Fusion protein B2MG-HNG-GL18 amino acid sequence SEQ ID No.7: MGIQRTPKIPPVPSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMPKSGSHHHHHHNGNNRPVYIPQPRPPHPRL.

[0052] Fusion protein B2MG-HNG-GL18 base sequence SEQ ID No.8: ATGGGCATCCACGGTACTCCAAAGATTCCGGTTCCCTCACGTCATCCAGCAGAGAATGGAAAGTCAAATTTCCTGAATTGCTATGTGTCTGGGTTTCCATCCATCCGACATTGAAGTTGACTTACTGAAGAATGGAGAGAGAATTGAAAAAGTGGAGCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTTTCTATCT CTTGTACTACACTGAATTCACCCCCACTGAAAAAGATGAGTATGCCTGCCGTGTGAACCATGTGACTTTGTCACAGCCCAAGATAGTTAAGTGGGATCGAGACATGCCAAAGTCTGGTTCTCATCACCACCATCATCATAACGGCAATAATCGGCCAGTATATATTCCACAACCAAGACCCCCTCCATCCGCGTCTA.

[0053] B2MG-HNG-GK25 amino acid sequence SEQ ID No.9: MGIQRTPKIPPVPSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMPKSGSHHHHHHNGLLSNVAGLLKQFAKGGVNAVLNPK.

[0054] B2MG-HNG-GK25 base sequence SEQ ID No.10: ATGGGCATCCACGGTACTCCAAAGATTCCGGTTCCCTCACGTCATCCAGCAGAGAATGGAAAGTCAAATTTCCTGAATTGCTATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTACTGAAGAATGGAGAGAGAATTGAAAAAGTGGAGCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTTTCTATCTCTTGTACTAC ACTGAATTCACCCCCACTGAAAAAGATGAGTATGCCTGCCGTGTGAACCATGTGACTTTGTCACAGCCCAAGATAGTTAAGTGGGATCGAGACATGCCAAAGTCTGGTTCTCATCACCACCATCATCATAACGGACTCCTTTCTAATGTCGCAGGTCTTTTGAAACAATTTGCAAAAGGTGGTGTAAATGCTGTATTGAACCCAAAA.

[0055] Example 1 This example provides a method for preparing and purifying the GL18 antimicrobial peptide.

[0056] The amino acid sequence of the B2MG-GL18 antimicrobial peptide fusion protein B2MG-HNG-GL18 used in this example is shown in SEQ ID No.7; the corresponding base sequence is shown in SEQ ID No.8, and codon optimization has been performed for the codons of Escherichia coli.

[0057] In this example, the B2MG-HNG-GL18 gene sequence is 393 bp in length. Shanghai Huajin Biotechnology Co., Ltd. was commissioned to synthesize the gene fragment, and the synthesized gene fragment was inserted into the PET-26b expression vector through the restriction sites of BamH I (NEB Company, Catalog Number: R0136L) and Xho I (NEB Company, Catalog Number: R0146L).

[0058] In this example, the constructed expression plasmid was transformed into Escherichia coli competent cells BL21 (DE3) (Merck). The specific process is as follows: 1: Take 1µl of the plasmid and add it to 100µl of E. coli competent cells BL21 (DE3) and place it on ice for 30 minutes.

[0059] 2: Heat shock the mixture in a 42°C water bath for 90 seconds, then quickly place on ice for 2 minutes.

[0060] 3: Add 600 µl of LB without resistance to the mixture and incubate at 37°C, 220 rpm for 1 h.

[0061] 4: Take 200µl of the bacterial solution and spread it evenly on an LB plate containing ampicillin resistance (10g / L peptone, 5g / L yeast extract, 10g / L sodium chloride, 15g / L agar, 100µg / ml kanamycin).

[0062] 5: Place the plate upside down in a 37°C incubator for about 20 hours until clearly visible colonies appear.

[0063] In this embodiment, a monoclonal colony was picked from the transformed LB plate and cultured in 10 ml LB (containing 100 μg / ml kanamycin) medium at 37°C and 220 rpm for 12-16 hours, and then transferred to 2×YT medium (16 g / L peptone, 10 g / L yeast extract, 5 g / L sodium chloride) at a ratio of 1:100 for expansion culture, and cultured at 37°C and 220 rpm for about 3 hours. When the OD600 of the bacterial solution was 0.4-0.6, a final concentration of 0.5 mM IPTG (Sigma, catalog number: I5502-1G) was added to induce expression, and the induction conditions were 18°C ​​and 180 rpm for 20 hours. Finally, the bacterial cells were collected by centrifugation and stored at -20°C or immediately entered the next step of purification.

[0064] In this example, about 50 ml of phosphate buffer (pH 7.8) (40 mM sodium dihydrogen phosphate, 500 mM sodium chloride) was used to resuspend (1 L) the bacterial pellet, and the bacteria were lysed using a high-pressure lysis instrument (Xinzhi Biotechnology) and then centrifuged at 13,000 rpm for 30 min to fully separate the soluble protein from the inclusion bodies.

[0065] In this example, the inclusion bodies were washed twice with 30 ml of phosphate buffer (pH 7.8) (40 mM sodium dihydrogen phosphate, 500 mM sodium chloride), centrifuged at 13000 rpm for 30 min, and the washed inclusion bodies were collected. The inclusion bodies were fully dissolved with 20 ml of denaturing buffer (8 M Urea, 40 mM NaH2PO3, 500 mM NaCl, pH 7.8). The fusion protein was collected in the supernatant after centrifugation at 13000 rpm for 30 min.

[0066] In this example, the fusion protein was purified using a Ni column. The Ni-NTA (Qiagen, Cat. No. 30210) affinity column was equilibrated with a binding buffer (8M Urea, 40mM NaH2PO3, 500mM NaCl, pH 7.8). Then the fusion protein supernatant was added and incubated at 4°C for 0.5-1h to allow the target recombinant protein to fully bind to the column material. Then, the impurities were rinsed with 200ml of a washing buffer (10mM imidazole, 8M Urea, 40mM NaH2PO3, 500mM NaCl, pH 7.8) containing 10mM imidazole (Sigma). Finally, 20ml of an elution buffer (250mM imidazole, 8M Urea, 40mM NaH2PO3, 500mM NaCl, pH 7.8) containing 250mM imidazole was added to elute the fusion protein. The resulting product can be analyzed for concentration and purity by SDS-PAGE electrophoresis. The specific process is as follows: take 40µl of protein solution, add 10µl of 5× protein loading buffer (250mM Tris-HCl (pH: 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol), boil in 100℃ boiling water for 10min, then add 10µl to each well of SDS-PAGE protein gel, run at 80V for 2h, use Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) for protein staining for 20min, and then use protein decolorizing solution (10% acetic acid, 5% ethanol) for decolorization.

[0067] In this embodiment, the fusion protein is cleaved by hydroxylamine cleavage to release the free GL18 antimicrobial peptide. The fusion protein solution of B2MG-HNG-GL18 is mixed with hydroxylamine lysis solution (4M hydroxylamine, pH 9.0) in a volume ratio of 1:1, adjusted to pH 9.0, and closed in a 45-degree water bath environment. The reaction can be completed in about 4 to 6 hours. Hydroxylamine solution can specifically cut the Asn-Gly site inside the protein in a weakly alkaline environment, which can just release the GL18 antimicrobial peptide in the fusion protein. The progress of the enzyme cleavage reaction was detected by SDS-PAGE electrophoresis.

[0068] The GL18 antimicrobial peptide released in this embodiment can be renatured and purified by dialysis, and then subjected to activity detection or freeze-dried for later use. The specific process is: transfer the GL18 antimicrobial peptide to a dialysis tape with a molecular weight cutoff of 1 kDa, place it in a distilled water solution with a volume of 100 times the dialysate, dialyze for 24 hours in a 4°C environment, continue stirring, and change the distilled water every 4 hours. Then collect the solution in the dialysis bag, centrifuge and take the supernatant, which is the purified GL18 antimicrobial peptide.

[0069] Example 2 This example provides a method for preparing and purifying the GK25 antimicrobial peptide.

[0070] The amino acid sequence of the B2MG-GK25 antimicrobial peptide fusion protein B2MG-HNG-GK25 used in this example is shown in SEQ ID No. 9, and its corresponding base sequence is shown in SEQ ID No. 10, which has been codon-optimized for the codons of Escherichia coli.

[0071] In this example, the B2MG-HNG-GK25 gene sequence is 414 bp in length. According to the optimized base sequence as shown in SEQ ID No. 10, Shanghai Huajin Biotechnology Co., Ltd. was commissioned to synthesize the gene fragment, and the synthesized gene fragment was inserted into the PET-26b expression vector through the restriction sites of BamH I (NEB Company, Catalog No.: R0136L) and Xho I (NEB Company, Catalog No.: R0146L).

[0072] In this example, the constructed expression plasmid was transformed into Escherichia coli competent cells BL21 (DE3) (Merck). The specific process is as follows: 1: Take 1µl of the plasmid and add it to 100µl of E. coli competent cells BL21 (DE3) and place it on ice for 30 minutes.

[0073] 2: Heat shock the mixture in a 42°C water bath for 90 seconds, then quickly place on ice for 2 minutes.

[0074] 3: Add 600 µl of LB without resistance to the mixture and incubate at 37°C, 220 rpm for 1 h.

[0075] 4: Take 200µl of the bacterial solution and spread it evenly on an LB plate containing kanamycin sulfate resistance (10g / L peptone, 5g / L yeast extract, 10g / L sodium chloride, 15g / L agar, 100µg / ml kanamycin).

[0076] 5: Place the plate upside down in a 37°C incubator for about 20 hours until clearly visible colonies appear.

[0077] In this embodiment, a monoclonal colony was picked from the transformed LB plate and cultured in 10 ml LB (containing 100 μg / ml kanamycin) medium at 37°C and 220 rpm for 12-16 hours, and then transferred to 2×YT medium (16 g / L peptone, 10 g / L yeast extract, 5 g / L sodium chloride) at a ratio of 1:100 for expansion culture, and cultured at 37°C and 220 rpm for about 3 hours. When the OD600 of the bacterial solution was 0.4-0.6, a final concentration of 0.5 mM IPTG (Sigma, catalog number: I5502-1G) was added to induce expression, and the induction conditions were 18°C ​​and 180 rpm for 20 hours. Finally, the bacterial cells were collected by centrifugation and stored at -20°C or immediately entered the next step of purification.

[0078] In this example, about 50 ml of phosphate buffer (pH 7.8) (40 mM sodium dihydrogen phosphate, 500 mM sodium chloride) was used to resuspend (1 L) the bacterial pellet, and the bacteria were lysed using a high-pressure lysis instrument (Xinzhi Biotechnology) and then centrifuged at 13,000 rpm for 30 min to fully separate the soluble protein from the inclusion bodies.

[0079] In this example, the inclusion bodies were washed twice with 30 ml of phosphate buffer (pH 7.8) (40 mM sodium dihydrogen phosphate, 500 mM sodium chloride), centrifuged at 13000 rpm for 30 min, and the washed inclusion bodies were collected. The inclusion bodies were fully dissolved with 20 ml of denaturing buffer (8 M Urea, 40 mM NaH2PO3, 500 mM NaCl, pH 7.8). The fusion protein was collected in the supernatant after centrifugation at 13000 rpm for 30 min.

[0080] In this example, the fusion protein was purified using a Ni column. The Ni-NTA (Qiagen, Cat. No. 30210) affinity column was equilibrated with a binding buffer (8M Urea, 40mM NaH2PO3, 500mM NaCl, pH 7.8). Then the fusion protein supernatant was added and incubated at 4°C for 0.5-1h to allow the target recombinant protein to fully bind to the column material. Then, the impurities were rinsed with 200ml of a washing buffer (10mM imidazole, 8M Urea, 40mM NaH2PO3, 500mM NaCl, pH 7.8) containing 10mM imidazole (Sigma). Finally, 20ml of an elution buffer (250mM imidazole, 8M Urea, 40mM NaH2PO3, 500mM NaCl, pH 7.8) containing 250mM imidazole was added to elute the fusion protein. The resulting product can be analyzed for concentration and purity by SDS-PAGE electrophoresis. The specific process is as follows: take 40µl of protein solution, add 10µl of 5× protein loading buffer (250mM Tris-HCl (pH: 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol), boil in 100℃ boiling water for 10min, then add 10µl to each well of SDS-PAGE protein gel, run at 80V for 2h, use Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) for protein staining for 20min, and then use protein decolorizing solution (10% acetic acid, 5% ethanol) for decolorization.

[0081] In this embodiment, the fusion protein is cleaved by hydroxylamine cleavage to release the free GK25 antimicrobial peptide. The fusion protein solution of B2MG-HNG-GK25 is mixed with hydroxylamine lysis solution (4M hydroxylamine, pH 9.0) in a volume ratio of 1:1, adjusted to pH 9.0, and closed in a 45-degree water bath environment. The reaction can be completed in about 4 to 6 hours. Hydroxylamine solution can specifically cut the Asn-Gly site inside the protein in a weakly alkaline environment, which can just release the GK25 antimicrobial peptide in the fusion protein. The progress of the enzyme cleavage reaction was detected by SDS-PAGE electrophoresis.

[0082] The GK25 antimicrobial peptide released in this embodiment can be renatured and purified by dialysis, and then tested for activity or freeze-dried for later use. The specific process is: transfer the GK25 antimicrobial peptide to a dialysis tape with a molecular weight cutoff of 1 kDa, place it in a distilled water solution with a volume of 100 times the dialysate, dialyze for 24 hours in a 4°C environment, continue stirring, and change the distilled water every 4 hours. Then collect the solution in the dialysis bag, take the supernatant after centrifugation, and it is the purified GK25 antimicrobial peptide.

[0083] Figure 3-Figure 4The gel electrophoresis results of the inclusion body protein expressed by B2MG-HNG-GL18 in the example and the gel electrophoresis results of the inclusion body protein expressed by B2MG-HNG-GK25 in the example are given respectively. The electrophoresis results show that the corresponding two inclusion body proteins are expressed.

[0084] Experimental Example 1 Since the antimicrobial peptides inhibited their antimicrobial activity during the inclusion body expression process, the antimicrobial activity of the antimicrobial peptides often could not be activated during the subsequent refolding and purification process. The present invention successfully verified that GL18 and GK25 still maintained good antimicrobial activity after the above steps, and showed good effects in the inhibition experiments against Escherichia coli and Staphylococcus aureus. The verification experiments also further proved the superiority and feasibility of the present invention.

[0085] In this experimental example, the antibacterial activity of the GL18 antimicrobial peptide prepared in the above-mentioned Example 1 and the GK25 antimicrobial peptide prepared in Example 2 was tested.

[0086] The experimental contents of this experimental example are as follows: (1) Thaw the frozen strain and inoculate it into 100 μl solid culture medium. Pick a single clone at 37°C overnight. Select a single clone colony and inoculate it into a 20 ml liquid culture medium (LB) flask. Place it on a shaker at 180 rpm and incubate it at 37°C for 16 hours as a bacterial stock solution.

[0087] (2) Blank control group: Take 1 ml of bacterial stock solution, add 5 ml of liquid culture medium LB, mix well, and culture at 37°C for 4 h on a shaker. Measure the OD value at 600 nm.

[0088] (3) Antimicrobial peptide (GL18 group, GK25 group) experimental group: 1 ml of bacterial stock solution was taken, and the corresponding antimicrobial peptide was added at a final concentration of 50 ug / ml. The mixture was mixed evenly, and cultured at 37°C for 4 h on a shaker. The OD value at 600 nm was measured.

[0089] (4) Antibiotic control group: 1 ml of bacterial stock solution was taken, and antibiotics were added at a final concentration of 50 μg / ml. The mixture was mixed well, and cultured on a shaker at 37°C for 4 h. The OD value at 600 nm was measured.

[0090] Inhibition rate formula = (blank group bacterial solution OD value - experimental group bacterial solution OD value) / blank group bacterial solution OD value.

[0091] The plate antibacterial test is as follows: several plates are laid according to the E. coli culture, and the sample positions are marked; water is dropped as a blank group, antimicrobial peptides are dropped as an experimental group, and kanamycin is dropped as a control group, and cultured overnight. Finally, it can be observed that the antimicrobial peptides GL18 and GK25 have obvious empty plaques, indicating good antibacterial effects.

[0092] Figure 5 The antibacterial test results of GL18 antimicrobial peptide obtained in Example 1 (Escherichia coli and Staphylococcus aureus, with kanamycin as the control). Figure 5 The results show that at a concentration of 50ug / ml, the inhibition rate of GL18 against Escherichia coli is 97%, and the inhibition rate against Staphylococcus aureus is 92.8%.

[0093] Figure 6 The antibacterial test results of GK25 antimicrobial peptide obtained in Example 2 (Escherichia coli and Staphylococcus aureus, with kanamycin as the control). Figure 6 The results show that at a concentration of 50ug / ml, the inhibition rate of GK25 against Escherichia coli is 97%, and the inhibition rate against Staphylococcus aureus is 96.4%.

[0094] Figure 7 The figures are the plate antibacterial test diagrams of the GL18 antimicrobial peptide obtained in Example 1 and the GK25 antimicrobial peptide obtained in Example 2. Figure 7 It can be seen that against E. coli, GL18 and GK25 both showed large plaques, which were similar to the effect of kanamycin.

[0095] The above experiments successfully verified that the GL18 and GK25 antimicrobial peptides prepared by the present invention still maintained good antimicrobial activity after denaturation and renaturation, and showed good effects in the inhibition experiments against Escherichia coli and Staphylococcus aureus, and also had good antimicrobial effects against other strains. The verification experiment fully demonstrated the industrial applicability of the present invention, which has good industrial value and industrial value, and is conducive to the large-scale production and preparation of antimicrobial peptides.

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for prokaryotic expression of antimicrobial peptides, characterized in that: The antimicrobial peptide gene is connected to the 3' end or 5' end of the fusion tag gene for fusion expression.

2. The method for prokaryotic expression of antimicrobial peptides according to claim 1, characterized in that: The amino acid sequence of the antimicrobial peptide is shown in SEQ ID No.3 and / or SEQ ID No.

5.

3. The method for expressing antimicrobial peptides according to claim 1 or 2, characterized in that: The fusion tag is β2 microglobulin, and its amino acid sequence is shown in SEQ ID No.

1.

4. The method for prokaryotic expression of antimicrobial peptides according to any one of claims 1 to 3, characterized in that: A purification tag is added between the fusion tag gene and the antimicrobial peptide gene.

5. The method for prokaryotic expression of antimicrobial peptides according to any one of claims 1 to 4, characterized in that: A protease cleavage site is added between the fusion tag gene and the antimicrobial peptide gene.

6. A fusion protein, characterized in that Its amino acid sequence is shown in SEQ ID No.7 or SEQ ID No.

9.

7. A polynucleotide, characterized in that Its nucleotide sequence is shown as SEQ ID No.8 or SEQ ID No.

10.

8. An expression vector, characterized in that Comprising the polynucleotide according to claim 7.

9. A host cell, characterized in that Comprising the expression vector of claim 8 and expressing the polynucleotide of claim 7; Preferably, the host cell is Escherichia coli or yeast.

10. Use of the polynucleotide according to claim 7, or the expression vector according to claim 8, or the host cell according to claim 9 in producing antimicrobial peptides.

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