Multi-copy expression method and application of lactic acid bacteria antibacterial peptide LAB4

Abstract

The invention relates to the technical field of molecular biology and protein engineering, in particular to a lactic acid bacteria antibacterial peptide LABA4 multi-copy expression method and application. The invention aims to provide a prokaryotic expression method of an antibacterial peptide LAB4 tandem gene based on a self-assembled peptide and application, so that the problems of toxicity to a host, low expression quantity and non-ideal product activity in a large intestine expression system are solved, the antibacterial peptide is efficiently expressed in escherichia coli, and the antibacterial activity is effectively improved. Particularly, the characteristics of specific cutting and no amino acid residue of enterokinase are utilized, modified recombinant enterokinase is added between LAB4 monomers to serve as an interval restriction enzyme cutting site, a gene shuffling technology is utilized to design and construct three-six copy tandem recombinant plasmids of LAB4, the self-cleavage characteristic of intein is utilized, and the recombinant enterokinase recombinant plasmids of LAB4 can be used for preparing the recombinant enterokinase recombinant plasmids of LAB4, so that the recombinant enterokinase recombinant plasmids of LAB4 can be used for preparing the recombinant enterokinase recombinant plasmids of LAB4. An optimized cspA cold shock promoter in a pCold I vector is adopted, and high-efficiency expression of tandem LAB4 in an escherichia coli expression system is realized through 15 DEG C low-temperature induction precise regulation and control.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology and protein engineering technology, and in particular to a method and application of expressing multiple copies of lactic acid bacteria antimicrobial peptide LABA4. Background Art

[0002] Small molecule peptides have low molecular weight, and most of them have low expression levels in cells and low stability. They are easily degraded by the host cell's own proteases. At the same time, some toxic peptide products will directly affect the growth of host cells themselves or even kill host cells. These limiting factors directly restrict the production of small molecule peptide products using microbial cell factories. The multi-copy gene construction strategy uses molecular biology tools to construct a concatemer of the target gene in the host cell, which can not only increase the yield of the target protein, but also alleviate the toxicity problem caused by single copy expression to a certain extent. However, the random or semi-random insertion of the directed concatenation method is prone to self-circularization and non-specific connection inside the plasmid; the traditional PCR amplification concatenation method often faces the problems of low efficiency, high error rate and fragment length limitation for amplification concatenation; the same tail enzyme method has low construction efficiency, cumbersome operation steps, and it is impossible to obtain a large number of copies in a short time, making it difficult to achieve a high uniformity of expression level. The traditional multi-copy gene construction technology still has many shortcomings in ensuring high efficiency, short cycle and maintaining long-term stability.

[0003] Inteins are naturally occurring protein sequences that are used to generate self-cleaving protein elements and can be combined with conventional affinity tags to produce effective self-cleaving affinity tags. Mxe GyrA inteins are mini inteins that can achieve N-terminal cleavage under the conditions of thiol compounds, the most commonly used reagent being dithiothreitol (DTT). The target protein is connected to the self-assembling peptide through an intein Mxe GyrA, and the target protein can be released into the soluble part through intein-mediated cleavage. This highly specific cleavage reaction allows the affinity tag to be removed without adding expensive proteases, while preventing unnecessary protein cleavage. ELK16 can be efficiently expressed in Escherichia coli cells and self-aggregate to form "active aggregates". The target protein and ELK16 protein sequence are fused, and the aggregation peptide can drive it into aggregates, so that the fusion protein forms active inclusion bodies in the cell. There is no need for complex and expensive techniques such as metal affinity chromatography for purification. High-purity fusion proteins can be obtained by cell disruption and simple centrifugation.

[0004] The efficient expression of recombinant proteins is a core challenge in the field of biomanufacturing. Traditional induction systems are often limited by host toxicity inhibition or abnormal product aggregation. The pCold Ⅰ cold shock expression vector provides an innovative solution to this problem through the precise regulation mechanism of the temperature-sensitive cspA promoter. The cold shock protein (CspA) activates the transcription complex by specifically binding to the promoter region, driving the efficient expression of the target gene. This dynamic regulation not only significantly improves the survival rate of the host, but also reduces the accumulation of metabolic byproducts, significantly optimizing the process stability of bacterial fermentation.

[0005] As natural immune defense molecules, the application of multi-copy gene construction strategy for antimicrobial peptides is a key technical challenge in constructing vectors for efficient heterologous expression. In view of the shortcomings of existing multi-copy gene expression technology in terms of gene stability, cumbersome operation and high cost, we urgently need to develop a new multi-copy gene expression platform that can not only achieve fast and efficient gene concatenation, but also achieve site-specific integration, while ensuring high expression efficiency and reducing application costs. This is of great significance in improving the precise insertion efficiency of the multi-copy strategy and reducing the risk of random integration. Summary of the invention

[0006] The antimicrobial peptide LAB4 has a broad-spectrum antibacterial activity, but its molecular weight is low, its intracellular stability is poor, and it has toxic effects on the host during expression, so there is a problem of low expression. In response to the above problems, the present invention integrates functional self-assembling peptide tags and multi-copy tandem technology, and uses cold shock promoters for low-temperature regulation to explore efficient and low-toxic antimicrobial peptide expression and purification strategies. By optimizing the cutting conditions and purification process, the yield and purity of antimicrobial peptides are increased, the toxicity to host cells is reduced, and the antibacterial activity is enhanced, providing a new gene prokaryotic expression strategy for the industrial production and clinical application of antimicrobial peptides.

[0007] The object of the present invention is to provide a method and application of prokaryotic expression of antimicrobial peptide LAB4 tandem genes based on self-assembling peptides, solve the problems of toxicity to the host, low expression level and unsatisfactory product activity in the large intestine expression system, enable the antimicrobial peptide to be efficiently expressed in Escherichia coli, and effectively improve the antibacterial activity. In particular, the present invention utilizes the characteristics of specific cleavage and no amino acid residue of enterokinase, adds modified recombinant enterokinase between LAB4 monomers as an interval enzyme cleavage site, and designs and constructs three to six copies of LAB4 tandem recombinant plasmids using the "gene shuffle" technology, utilizes the self-cleavage characteristics of the intein, and uses the optimized cspA cold shock promoter in the pCold Ⅰ vector, and achieves efficient expression of the tandem LAB4 in the Escherichia coli expression system through precise regulation by 15°C low temperature induction.

[0008] The technical solution of the present invention to achieve the above object is as follows: The present invention provides a method for improving LAB4 expression, which integrates three major technical modules: (1) inserting recombinant enterokinase (hereinafter referred to as RE) between LAB4 monomers, designing specific recognition sites, and amplifying LAB4 and recombinant enterokinase by PCR to obtain random fragments spliced ​​with the N-terminus, the C-terminus, and both the N-terminus and the C-terminus, and forming a library with these random fragments. When the gene fragment spliced ​​with both the N-terminus and the C-terminus is amplified, the gene fragment spliced ​​with the N-terminus and the C-terminus is used as an intermediate fragment to obtain concatemers with various copy numbers through one homologous recombination. In this way, the concatemer expression framework with the optimal copy number is selected to achieve cleavage without amino acid residues, breaking through the limitations of single-copy expression systems and traditional multi-copy gene technology; (2) The synergistic effect of the intein elements is designed to connect the assembly peptide fusion genes of the self-cleaving peptide Mxe GyrA, the linker peptide PTLinker and the aggregation peptide ELK16 to achieve product purification with a high cleavage recovery rate; (3) A cold shock expression system is used to reduce the toxicity of antimicrobial peptides to the host through temperature regulation, and the assembly peptide tag and the multi-copy antimicrobial peptide gene are integrated and inserted into the multiple cloning site of the pCold I expression vector to construct a recombinant expression plasmid.

[0009] One of the technical solutions provided by the present invention is an antimicrobial peptide LAB4 multi-copy expression vector, which is obtained by enzyme-cutting and ligating the coding genes of LAB4-R, R-LAB4-R, R-LAB4, and Mxe GyrA-PT Linker-ELK16 fragments with the pColdⅠ vector; The LAB4-R is a gene recombinant fragment obtained by connecting recombinant enterokinase to the C-terminus of the antimicrobial peptide LAB4; The R-LAB4 is a gene recombinant fragment obtained by connecting recombinant enterokinase to the N-terminus of the antimicrobial peptide LAB4; The R-LAB4-R is a gene recombinant fragment obtained by connecting recombinant enterokinase to the N-terminus and C-terminus of the antimicrobial peptide LAB4 respectively; The Mxe GyrA-PT Linker-ELK16 is an assembled peptide fusion gene of the self-cleaving peptide Mxe GyrA, the connecting peptide PT Linker and the aggregating peptide ELK16; Furthermore, the amino acid sequence of LAB4 is shown in the sequence listing as SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2; the amino acid sequence of the recombinant enterokinase is shown in SEQ ID NO.3, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.4; the amino acid sequence of the assembly peptide Mxe GyrA-PT Linker-ELK16 is shown in SEQ ID NO.5, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.6; Furthermore, the above antimicrobial peptide LAB4 multi-copy expression vector was E.Coli After expression in DH5α competent medium, recombinant plasmids pCold Ⅰ-3CLAB4-RE-M16 (LAB4 3-copy tandem expression plasmid), pCold Ⅰ-4CLAB4-RE-M16 (LAB4 4-copy tandem expression plasmid), pCold Ⅰ-5CLAB4-RE-M16 (LAB4 5-copy tandem expression plasmid), and pCold Ⅰ-6CLAB4-RE-M16 (LAB4 6-copy tandem expression plasmid) were obtained by colony PCR and / or sequencing. The above multi-copy plasmids were collectively named pCold Ⅰ-nCLAB4-RE-M16.

[0010] The second technical solution provided by the present invention is a method for simultaneously obtaining multi-copy concatemers of the antimicrobial peptide LAB4, wherein the multi-copy expression vector pCold Ⅰ-nCLAB4-RE-M16 obtained in the first technical solution is expressed in a host; the host includes but is not limited to Escherichia coli, preferably Escherichia coli BL21.

[0011] The third technical solution provided by the present invention is a method for preparing an antimicrobial peptide LAB4 monomer, wherein the multi-copy expression vector pCold Ⅰ-nCLAB4-RE-M16 described in the second technical solution is subjected to 15°C low-temperature induction expression in a host, and then bacterial cells are isolated and obtained, and DTT is added to the precipitate after the bacterial cells are crushed to cut Mxe GyrA-PT Linker-ELK16, and the supernatant is collected by centrifugation to release nCLAB4-RE, and then enterokinase is added to cut the recombinant enterokinase from the fusion protein to release a single target peptide LAB4; Preferably, after pCold I-6CLAB4-RE-M16 is expressed in a host, 6CLAB4-RE is obtained by cleavage with DTT, and then a single target peptide LAB4 is obtained by cleavage with enterokinase.

[0012] The beneficial effects of the present invention compared with the prior art are as follows: 1. The present invention transforms the original strain BL21-pCold Ⅰ-LAB4 through the "gene shuffling" technology, and constructs a tandem expression framework of three to six copies of the antimicrobial peptide LAB4, among which the six-copy tandem has the best effect and can achieve efficient expression of the antimicrobial peptide LAB4.

[0013] 2. The present invention utilizes the self-cleavage property of the assembled peptide to release the monomeric antimicrobial peptide LAB4 from the active aggregates, thereby avoiding the cumbersome elution steps and label removal operations in the protein purification process, and can achieve one-step purification to obtain the monomeric protein.

[0014] 3. The present invention adopts the optimized cspA cold shock promoter in the pCold Ⅰ vector, realizes precise expression regulation through low-temperature induction at 15°C, reduces the effect of toxic proteins in the non-induced stage, significantly alleviates the toxic pressure of antimicrobial peptides on the early growth stage of the host, overcomes the problems of high production cost and low extraction yield of the antimicrobial peptide LAB4, and compared with the single-copy expression of the antimicrobial peptide LAB4, the expression yield of the six-copy concatemer is the highest, which is 6.62 times the expression amount of the original strain.

[0015] 4. The antimicrobial peptide prepared by the present invention has a stronger inhibitory effect on Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. Compared with the single-copy expression of the antimicrobial peptide LAB4, the six-copy concatemer has the best antibacterial effect, which is 1.53 times that of the original strain, which is an unexpected technical effect.

[0016] The transformation method of the recombinant strain is easy to prepare and has a high expression level, which significantly reduces production costs and improves production efficiency. At the same time, the inhibitory activity of the target protein against a variety of multidrug-resistant pathogens is verified, which is conducive to the application of the antimicrobial peptide LAB4 in antibacterial agents or bactericides. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the construction scheme of multiple tandem expression vectors constructed using the "gene shuffling" technology.

[0018] Figure 2 A PCR verification diagram of a recombinant colony for the "gene shuffling" technology Among them, lane M is the DNA standard marker.

[0019] Figure 3 This is the detection diagram of nCLAB4-RE-M16 recombinant protein (n=3, 4, 5, 6) Wherein, M: 180 kDa prestained protein marker; C: crude extract of BL21-pCold Ⅰ empty-loaded strain cells; Lane S: supernatant of cell lysis centrifugation; Lane P: cell lysis precipitate. Figure 4 Characterization of the growth level of the recombinant strain per unit volume of culture medium Control: BL21-pCold Ⅰ; A: BL21-pCold Ⅰ-LAB4 (unmodified original bacteria); CnA: concatemers with n copies (n=3, 4, 5, 6).

[0020] Figure 5 Optimization of the cutting time for pCold Ⅰ-6CLAB4-RE-M16 Wherein, M: 180 kDa prestained protein marker, DTT cleavage: DTT cleavage treated sample, centrifugation: DTT cleavage and centrifugation treated sample; S: supernatant; P: precipitate.

[0021] Figure 6 Ultrafiltration dialysis treatment test diagram of pCold Ⅰ-6CLAB4-RE-M16 Wherein, M: 40 kDa pre-stained protein marker; 1: ultrafiltration dialysis filtrate; 2: ultrafiltration dialysis collection fluid.

[0022] Figure 7 Characterization of the antibacterial activity of the cleaved and purified antimicrobial peptide LAB4 against Gram-negative and Gram-positive bacteria Among them, a, c: antibacterial effect on Escherichia coli ATCC25922; b, d: antibacterial effect on Staphylococcus aureus ATCC25923; In the figure, 1: purified sample of six-copy fusion protein; 2: purified sample of five-copy fusion protein; 3: purified sample of four-copy fusion protein; 4: purified sample of three-copy fusion protein; C: BL21-pCold Ⅰ-LAB4 cell lysis supernatant; 5: Tris-HCl solution; 6: DTT cutting solution. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of this patent more clear, this patent is further described in detail in combination with specific embodiments. It should be understood that the specific embodiments described here are only used to explain this patent and are not used to limit the present invention.

[0024] Some of the amplification primers involved in the embodiments of the present invention are as follows: LAB4-R upstream amplification primer F1 (SEQ ID NO.7): 5'-TAAGAAGGAGATATACCATGGATGGCAACCCCGCATAAAT-3'; LAB4-R downstream amplification primer R2 (SEQ ID NO.8): 5'-TTTATCATCATCATCTTTGATTTTGGTGTGGGTGGATCAG-3'; R-LAB4-R upstream amplification primer F2 (SEQ ID NO.9): 5'-AAAGATGATGATGATAAAGCAACCCCGCATAAATATCTG-3'; R-LAB4-R downstream amplification primer R2 (SEQ ID NO.10): 5'-TTTATCATCATCATCTTTGATTTTGGTGTGGGTGGATCAG-3'; R-LAB4 upstream amplification primer F2 (SEQ ID NO.11): 5'-AAAGATGATGATGATAAAGCAACCCCGCATAAATATCTG-3'; R-LAB4 downstream amplification primer R1 (SEQ ID NO.12): 5'-TGCGGCCGCAAGCTTGTCGACTCAGATTTTGGTGTGGTGGATCA-3'; Mxe GyrA-PT Linker-ELK16 upstream amplification primer MF (SEQ ID NO.13): 5'-GATCCACCACACCAAAATCTGCATCACCGGCGACGCC-3'; Mxe GyrA-PT Linker-ELK16 downstream amplification primer MR (SEQ ID NO.14): 5'-CTATCTAGACTGCAGGTCGACCAGTCATTTCAGTTTCAGTTCCAGTTCC-3'; pCold Ⅰ universal upstream amplification primer pCold Ⅰ-F (SEQ ID NO.15): 5′-ACGCCATATCGCCGAAAGG-3′; pCold Ⅰ universal downstream amplification primer pCold Ⅰ-R (SEQ ID NO.16): 5′-GGCAGGGATCTTAGATTCTG-3′; The antimicrobial peptide LAB4 of the present invention is derived from lactic acid bacteria ( lactic acid bacteria ), whose amino acid sequence is shown in the sequence listing SEQ ID NO.1: HMATPHKYLLLVFETLFTVTAFRSAVTALLKALRDLIEALKRKKHRCMIDYTTDTSTYVILIHHTKI Its nucleotide sequence is shown in the sequence table SEQ ID NO.2: CATATGGCAACCCCGCATAAATATCTGCTGCTGGTTTTCGAAACCCTGTTCACCGTTACCGCGTTTCGTTCTGCAGTTACCGCATTACTGAAAGCGTTACGCGATCTGATTGAAGCGCTGAAACGCAAAAAACACCGCTGCATGATCGATTACACCACCGATACCAGCACCTACGTTATCCTGATCCACCACACCAAAATC The recombinant enterokinase provided by the present invention is synthesized by Beijing Qingke Biotechnology Co., Ltd., and its amino acid sequence is shown in SEQ ID NO.3 of the sequence list: KDDDDK; its nucleotide sequence is shown in SEQ ID NO.4 of the sequence list: AAAGATGATGATGATAAA.

[0025] The Mxe GyrA-PT Linker-ELK16 assembly peptide provided by the present invention is synthesized by Beijing Qingke Biotechnology Co., Ltd., and its amino acid sequence is shown in the sequence table SEQ ID NO.5: CITGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAG FARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHAPTPPTTPTPPTTPTPTPLELELKLKLELELKLK; Its nucleotide sequence is shown in the sequence table SEQ ID NO.6: TGCATCACCGGCGACGCCTTAGTGGCACTGCCGGAAGGCGAAAGCGTGCGTATTGCCGATATCGTTCCGGGTGCACGCCCTAACAGCGATAATGCAATCGACCTGAAAGTGTTAGATCGCCACGGCAATCCTGTTCTGGCCGATCGCCTGTTTCACAGTGGCGAACATCCGGT GTACACAGTGCGCACCGTGGAAGGTCTGCGCGTGACCGGCACAGCAAATCACCCGCTGCTGTGTTTAGTGGACGTTGCAGGCGTGCCTACACTGCTGTGGAAGCTGATCGATGAAATCAAGCCGGGTGACTACGCAGTGATTCAGCGCAGTGCCTTCAGCGTTGATTGCGCCG GTTTTGCACGTGGCAAACCGGAATTTGCACCGACCACCTACACCGTGGGTGTGCCGGGCCTGGTTCGTTTCCTGGAAGCACACCATCGTGATCCGGACGCACAGGCCATTGCCGATGAGCTGACCGACGGCCGCTTTTACTATGCCAAAGTTGCCAGCGTGACAGATGCAGGT GTGCAGCCGGTTTACAGTTTACGCGTGGATACCGCCGATCACGCATTCATTACCAACGGCTTCGTTAGCCATGCCCCGACCCCACCGACCACGCCAACGCCACCAACCACCCCAACCCCGACGCCGCTGGAACTGGAACTGAAACTGAAACTGGAACTGGAACTGAAACTGAAA The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions, such as those described in the Molecular Cloning Experiment Guide (Fourth Edition) (Science Press, 2017).

[0026] Example 1 Construction of BL21-pCold I-nCLAB4-RE-M16 engineered bacteria by "gene shuffling" method The specific steps are: 1. According to Figure 1The vector was constructed and designed as shown. The Escherichia coli plasmid pCold Ⅰ plasmid was used as a template, double digested with Nde Ⅰ and Sal Ⅰ, and the digested plasmid was cut and recovered by gel cutting. The LAB4 gene (SEQ ID NO.2) was designed with CE Design for seamless cloning primers. During the design, the nucleic acid sequence of the RE gene (SEQ ID NO.4) was used as the upstream / downstream fragments of the primers and a part of the homology arm of the vector and the target gene. The primers F1 / R2, F2 / R2, and F2 / R1 were used to amplify LAB4 as a template by touchdown PCR, respectively, so that the enterokinase cleavage site was integrated into the N-terminus, both ends of the N-terminus and the C-terminus of the LAB4 fragment, and the three fragments were purified and recovered to obtain R-LAB4, R-LAB4-R, and LAB4-R. The Mxe GyrA-PTLinker-ELK16 gene (SEQ ID NO. 6, referred to as M-16) was used as a template to design seamless cloning primers (MF / MR) using CE Design, and the target gene was amplified by PCR and purified and recovered. The target gene fragments LAB4-R, R-LAB4-R, R-LAB4, Mxe GyrA-PT Linker-ELK16 and pCold Ⅰ digested vector were reacted at 50°C for 30 min using ClonExpress one-step ligase, and the ligation products were chemically transformed into Escherichia coli. E.Coli DH5α competent cells were cultured at 37°C overnight. The universal primers pCold Ⅰ-F / pCold Ⅰ-R of pCold Ⅰ were used as the upstream and downstream primers for colony PCR verification. Single clones with larger colonies after overnight culture were selected for colony PCR using verification primers.

[0027] The results are as follows Figure 2 As shown, one homologous recombination can screen out transformants with multiple copy numbers. The parts circled in red in the figure are three, four, five, and six copies of concatemers, respectively. The correct transformants were transferred to plasmids and the screened plasmids were sent to a gene sequencing company for sequencing. The correctly sequenced plasmids were the recombinant plasmids pCold Ⅰ-3CLAB4-RE-M16 (LAB4 3-copy concatemer expression plasmid), pCold Ⅰ-4CLAB4-RE-M16 (LAB4 4-copy concatemer expression plasmid), pCold Ⅰ-5CLAB4-RE-M16 (LAB4 5-copy concatemer expression plasmid), pCold Ⅰ-6CLAB4-RE-M16 (LAB4 6-copy concatemer expression plasmid). The above multi-copy plasmids were collectively named pCold Ⅰ-nCLAB4-RE-M16.

[0028] Table 1 Ultra One Step Cloning Connection System

[0029] 2. The E. coli plasmid pCold Ⅰ plasmid was used as a template, and double digested with Nde Ⅰ and Sal Ⅰ, and the digested plasmid was cut and recovered by gel cutting. The LAB4 gene (SEQ ID NO.2) was designed with CE Design for seamless cloning primers, amplified by PCR, and the amplified fragment was cut and recovered by gel cutting. The recovered fragment was reacted with the pCold Ⅰ digested vector at 50℃ for 30 minutes using ClonExpress one-step ligase, and the ligation product was chemically transformed into E. coli. E.Coli DH5α competent cells were cultured at 37°C overnight. The universal primers pCold Ⅰ-F / pCold Ⅰ-R of pCold Ⅰ were used as upstream and downstream primers for colony PCR verification. Single clones with larger colonies after overnight culture were selected for colony PCR using verification primers. The correct transformants were transferred to plasmids, and the selected plasmids were sent to a gene sequencing company for sequencing. The correctly sequenced plasmid was the recombinant plasmid pCold Ⅰ-LAB4.

[0030] 3. Heat shock pCold Ⅰ-LAB4 and pCold Ⅰ-nCLAB4-RE-M16 into 100 μL E.Coli BL21(DE3) competent cells were plated on LB solid plate medium containing ampicillin and cultured overnight at 37°C. The universal primers pCold Ⅰ-F / pCold Ⅰ-R of pCold Ⅰ were used as upstream and downstream primers for colony PCR verification. Single clones with larger colonies after overnight culture were selected and colony PCR amplification was performed with verification primers to screen positive clones, thereby obtaining the engineered strains BL21-pCold Ⅰ-LAB4 and BL21-pCold Ⅰ-nCLAB4-RE-M16.

[0031] Example 2 Identification of heterologous induction expression of antimicrobial peptides mediated by assembly tag and optimization of induction conditions in tandem engineered bacteria 1. Pick a single colony of the three-zone line of the engineering strain BL21-pCold Ⅰ-nCLAB4-RE-M16, a single colony of the three-zone line of the BL21-pCold Ⅰ empty vector as a negative control, and a single colony of the three-zone line of the BL21-pCold Ⅰ-LAB4 as a positive control and inoculate them into LB medium. Incubate at 37°C for 12-16 h to activate the strain. Inoculate the activated bacterial solution into fresh LB medium (containing 100 μg / mL ampicillin) at a 1% inoculum and incubate at 37°C until OD 600 After the expression level reached 0.6-0.8, IPTG (isopropyl-β-D-thiogalactoside) inducer was added at a final concentration of 0.2 mM, and the expression was induced at 15°C and 180 rpm for 16 h.

[0032] 2. After the induction expression is completed, measure the OD of the expressed bacterial solution 600 The cells were centrifuged at 12000 rpm for 10 min at room temperature to collect the cells, discard the supernatant and collect the cells. 20 mM Tris-HCl solution was added for every 10 OD to suspend the cells collected above, resuspended and washed 2-3 times, and then the cells were broken with liquid nitrogen. After the cells were broken, 20 μL was taken to prepare the whole liquid sample after the breakage, and then centrifuged at 12000 rpm for 2 min to obtain the supernatant and precipitate after the breakage, and 20 μL of the supernatant and precipitate samples after the breakage were taken for sample preparation.

[0033] 3. Add 5 μL of 5× loading buffer to the prepared sample, boil it in a boiling water bath for 10 min, centrifuge it at 12000 rpm for 2 min, collect the supernatant and perform 15% SDS-PAGE electrophoresis. The obtained product is used for Tris-tricine electrophoresis and Coomassie brilliant blue staining.

[0034] The protein identification results are as follows Figure 3 As shown in the figure, M: Marker; S: cell lysis supernatant; P: cell lysis precipitate; nC-M16: lysate after cell disruption and centrifugation after induced expression of BL21-pCold Ⅰ-nCLAB4-RE-M16. The target protein bands after induction (circled in red) are approximately located at 51.2, 59.4, 67.6, and 75.8 kDa, respectively. According to the theoretical values ​​of LAB4, M16, and RE, which are 7.6, 21.3, and 0.8 kDa, respectively, it can be seen that the target band size is consistent with the theoretical value. This shows that 3CLAB4-RE-M16, 4CLAB4-RE-M16, 5CLAB4-RE-M16, and 6CLAB4-RE-M16 fusion proteins were successfully expressed in E. coli cells, and most of them exist in the precipitate in the form of active aggregates.

[0035] The results of quantitative protein analysis by BCA protein quantification and Image J grayscale scanning analysis are shown in Table 2. The recombinant protein content of 6CLAB4-RE-M16 was the highest, accounting for 28.7% of its total protein content, and the fusion protein concentration reached 7.55 mg / L. The six-copy strain was selected as the optimal strain for subsequent optimization.

[0036] Table 2 Protein expression results and 10 mL enzyme digestion system

[0037] Example 3 Assembly tag-mediated heterologous expression and purification of antimicrobial peptides in tandem engineered bacteria 1. Bacteria collection: The protein heterologous induced expression is the same as the protein induced expression method in Example 2 (Step 1 in Example 2). Samples are taken every 2 hours to measure the absorbance at a wavelength of 600 nm, with the culture time as the horizontal axis and OD 600 The value is the vertical axis and the growth curve is drawn.

[0038] The results are as follows Figure 4 As shown in Figure 2, BL21-pCold Ⅰ-nCLAB4-RE-M16 significantly inhibited the toxicity of antimicrobial peptides to the host, compared with the strain BL21-pCold Ⅰ-LAB4 (i.e. Figure 4 The curve marked as A in the figure), the final OD 600 The expression of the 5-mercaptoethanol was increased to 1.704, an increase of 72.24%, and its growth level was significantly improved. After the induction of expression, the cells were collected by centrifugation at 12000 rpm for 10 min at room temperature, and the supernatant was discarded and the cells were collected. 1 mL of 20 mM Tris-HCl solution was added for every 10 OD to suspend the collected cells, and the cells were resuspended and washed 1-2 times.

[0039] 2. DTT cleavage release of fusion protein: Collect the fusion protein aggregated in the precipitate after elution and centrifugation, add 40 mM DTT to the precipitate sample, and take samples 4h, 8h, and 12h after the cleavage reaction at 4°C. Prepare samples for SDS-PAGE electrophoresis verification. The results are as follows Figure 5 As shown in the figure, after 12 h of cutting, the cutting reached the maximum value, and the protein band of the assembled peptide M16 was located at 21.3 kDa, which was consistent with the theoretical value. Therefore, 12 h of cutting was selected as the optimal cutting time. A 0.5% (v / v) acetic acid solution was added to the solution after the shearing was completed, the suspension was uniform, and the solution was allowed to stand at room temperature for 10 min. The 6CLAB4-RE released in the supernatant was collected by centrifugation at 12000 rpm for 5 min.

[0040] 3. Enterokinase cleavage of fusion protein: Collect the fusion protein released into the supernatant after centrifugation, mix the fusion protein with enterokinase at a ratio of 20 U / mg, incubate at 37°C for 12 h in 10 μL of 500 mM Tris-HCl, pH 8.0 10 mM CaCl2 and 1% Tween-20 (v / v) buffer system to remove the recombinant enterokinase from the fusion protein and release a single target peptide.

[0041] Example 4 Analysis of Monomer Expression Amounts after Cleavage and Purification of Antimicrobial Peptide Heterologous Expression in Tandem Engineering Bacteria Mediated by Assembly Tags The target peptide monomer LAB4 collected in Example 3 was subjected to two centrifugal ultrafiltrations at 4°C to remove impurities using ultrafiltration tubes with cutoffs of 10 kDa and 5 kDa, and dialyzed completely overnight with 20 mM Tris-HCl buffer. The treated filtrate and the collected solution were taken to determine the sample protein concentration using a BCA protein concentration determination kit, and 80 μL was taken for sample preparation. 5 μL of 5× loading buffer was added to the prepared sample, boiled in a boiling water bath for 10 min, centrifuged at 12000 rpm for 2 min, and the supernatant was collected for 16.5% Tricine SDS-PAGE electrophoresis detection. The results are as follows: Figure 6 As shown, the target peptide LAB4 was released in the dialyzed fluid, and its size was consistent with the theoretical relative molecular weight (7.6 kDa).

[0042] The BCA protein quantification method and Image J gel analysis software were used to perform grayscale scanning and quantitative analysis on the target bands, and then the total protein concentration, recombinant protein content in the reconstituted precipitate after centrifugation of the shear solution, and the antimicrobial peptide content in the ultrafiltration dialysis collection fluid were calculated.

[0043] Cutting recovery rate (%) =

[0044] Combined with the calculation results of Examples 3 and 4, it can be seen that the content of antimicrobial peptide LAB4 expressed by the six-copy recombinant strain after cutting and purification reached 5.43 mg / L. After grayscale analysis and calculation, it can be seen that the cutting recovery rate of LAB4 purified by this method is as high as 75.69%, compared with the LAB4 monomer expressed by BL21-pCold Ⅰ-LAB4 of 0.82 mg / L. The intein-mediated tandem expression strategy significantly improves the yield of antimicrobial peptides heterologously expressed in the E. coli system.

[0045] Example 5 Analysis of antibacterial activity of antimicrobial peptide monomers after cleavage and purification Determination of samples: The antimicrobial peptide powder expressed by the six-copy recombinant strain purified in Example 4 and the antimicrobial peptide powder expressed by the single-copy strain BL21-pCold Ⅰ-LAB4 were reconstituted with 0.03 g / ml sterile water and the antibacterial activity was determined; 1. Oxford cup antibacterial experiment (1) Experimental S. aureus and E. coli As indicator bacteria, pathogenic strains of Escherichia coli (ATCC25922) and Staphylococcus aureus (ATCC25923) were taken out of the -80°C refrigerator and inoculated into LB agar plates in three zones at 37°C overnight; (2) Pick a single colony and inoculate it into 5 mL LB liquid medium. Incubate overnight at 37°C and 180 rpm in a shaking incubator. (3) Place four Oxford cups evenly on the LB agar plate and dilute the bacterial solution obtained in step (2) to a concentration of 10 6 CFU / mL and then poured into the plate, and after solidification, 200 μL of antimicrobial peptide reconstitution reagent was added; (4) Incubate overnight at 37°C. Observe, measure, and record the size of the inhibition zone the next day. The results are as follows: Figure 7 And as shown in Table 1.

[0046] Table 3 Statistics of transparent circle sizes of each plate

[0047] like Figure 7 As shown, the antimicrobial peptide LAB4 released from the supernatant of the fermentation broth of BL21-pCold Ⅰ-nCLAB4-RE-M16 after screening and identification had an antibacterial effect on both positive bacteria and negative bacteria indicator bacteria (a: antibacterial effect on Escherichia coli ATCC25922; b: antibacterial effect on Staphylococcus aureus ATCC25923; 1: antimicrobial peptide sample obtained by cleavage and purification of six-copy recombinant protein; 2: antimicrobial peptide sample obtained by cleavage and purification of five-copy recombinant protein; 3: antimicrobial peptide sample obtained by cleavage and purification of four-copy recombinant protein; 4: antimicrobial peptide sample obtained by cleavage and purification of three-copy recombinant protein; 5: Tris-HCl solution; 6: DTT cleavage solution; C is an antimicrobial peptide sample obtained from a single-copy original strain), and the antimicrobial peptide prepared by the six-copy tandem recombinant strain mediated by the intein showed the best antibacterial activity against Staphylococcus aureus ATCC25923, with an inhibition zone diameter of up to 31.56 mm, which was 1.53 times that of the original antimicrobial peptide LAB4 before transformation, and the antibacterial activity was significantly improved by 52.69%, and no antibacterial activity was detected in Tris-HCl solution and DTT cutting solution.

[0048] These data indicate that the recombinant strain BL21-pCold Ⅰ-6CLAB4-RE has excellent broad-spectrum antibacterial ability. Through tandem technology and antimicrobial peptide fusion expression, the recombinant enterokinase cleavage site is introduced into the target molecule to obtain multiple target products in one step, which not only reduces the cost, but also solves the problem that small molecule peptides are not easy to express in large quantities.

[0049] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the attached claims.

Claims

1. An antimicrobial peptide LAB4 multi-copy expression vector, characterized in that: The expression vector is obtained by enzyme-cutting and ligating the coding genes of LAB4-R, R-LAB4-R, R-LAB4, and Mxe GyrA-PT Linker-ELK16 fragments with the pCold Ⅰ vector; The LAB4-R is a gene recombinant fragment obtained by connecting recombinant enterokinase to the C-terminus of the antimicrobial peptide LAB4; The R-LAB4 is a gene recombinant fragment obtained by connecting recombinant enterokinase to the N-terminus of the antimicrobial peptide LAB4; The R-LAB4-R is a gene recombinant fragment obtained by connecting recombinant enterokinase to the N-terminus and C-terminus of the antimicrobial peptide LAB4 respectively; The Mxe GyrA-PT Linker-ELK16 is an assembled peptide fusion gene of the self-cleaving peptide Mxe GyrA, the connecting peptide PT Linker and the aggregating peptide ELK16.

2. The antimicrobial peptide LAB4 multi-copy expression vector according to claim 1, characterized in that: The amino acid sequence of LAB4 is shown in SEQ ID NO.1 in the sequence listing; the amino acid sequence of the recombinant enterokinase is shown in SEQ ID NO.3; and the amino acid sequence of the assembly peptide Mxe GyrA-PT Linker-ELK16 is shown in SEQ ID NO.

5.

3. The multi-copy expression vector of antimicrobial peptide LAB4 according to claim 1, characterized in that: The antimicrobial peptide LAB4 multi-copy expression vector was E.Coli After expression in DH5α competent state, colony PCR and / or sequencing were performed to obtain the recombinant plasmid LAB4's 3-copy tandem expression plasmid pCold Ⅰ-3CLAB4-RE-M16, LAB4's 4-copy tandem expression plasmid pCold Ⅰ-4CLAB4-RE-M16, LAB4's 5-copy tandem expression plasmid pCold Ⅰ-5CLAB4-RE-M16, and LAB4's 6-copy tandem expression plasmid pCold Ⅰ-6CLAB4-RE-M16. The above multi-copy plasmids were collectively named pCold Ⅰ-nCLAB4-RE-M16.

4. A method for simultaneously obtaining multiple copies of antimicrobial peptide LAB4 concatemers, characterized in that: The method is to express the multi-copy expression vector pCold Ⅰ-nCLAB4-RE-M16 described in claim 3 in a host.

5. A method for preparing an antimicrobial peptide LAB4 monomer, characterized in that: The method comprises the following steps: subjecting the multi-copy expression vector pCold Ⅰ-nCLAB4-RE-M16 described in claim 3 to low-temperature induction expression at 15°C in a host, isolating and obtaining bacterial cells, adding DTT to the precipitate after the bacterial cells are crushed to cut Mxe GyrA-PT Linker-ELK16, collecting the released nCLAB4-RE by centrifugation, and then adding enterokinase to cut the recombinant enterokinase from the fusion protein to release the single target peptide LAB4.

6. A method for preparing an antimicrobial peptide LAB4 monomer according to claim 5, characterized in that: After pCold Ⅰ-6CLAB4-RE-M16 was expressed in a host, 6CLAB4-RE was obtained by DTT cleavage, and then the single target peptide LAB4 was obtained by enterokinase cleavage.

7. The antimicrobial peptide LAB4 prepared by the method of claim 5.

8. Use of the antimicrobial peptide LAB4 according to claim 7 in inhibiting pathogenic bacteria.

9. The use according to claim 8, characterized in that The pathogenic bacteria include Escherichia coli and Staphylococcus aureus.

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