Secrete-Expression Of Antimicrobial Peptide Bactenecin7 In Lactococcus Lactis And The Construction Of Its Homologous Recombination Vector

BackgroundIntestinal bacterial infection is a common gastrointestinal disease. The vast majorities of intestinal infections caused by Gram-negative bacteria usually leed to diarrhea, abdominal pain, fever, even give rise to systemic infection. Current treatment of bacterial infection is antibiotics. However, with the unreasonable application of antibiotics, oral antibiotics abuse, as well as an increase in applications, the drug resistance of the digestive tract pathogens has become an increasingly serious problem. For the treatment of intestinal infection with drug-resistant bacteria, the development of new antimicrobial agents and its application become particularly important.Antimicrobial peptides are a class of peptides with antibacterial activity, and play an important role in the innate immunity of organism. Antimicrobial peptides have broad-spectrum, high-effective antibacterial activity and without cross-resistance with other kinds of antimicrobial agents, and not induced the emerge of drug-resistant strains. The study have found that the antimicrobial peptide Bactenecin7 (Bac7) have an efficient antimicrobial activity to Gram-negative bacteria and have no cross-resistance with other antimicrobial agents, and the drug-resistance is hardly induced. Besides the direct bactericidal role, Bac7 have the effect of neutralize endotoxin, anti-inflammation, immune induction, and tissue repair. Obviously antimicrobial peptide Bac7 has many advantages in the treatment of bacterial infection, is expected to become a new generation of antibacterial drugs to treatment of bacterial intestinal infection.PurposesTo construct the expression vector of Bac7-gene and secreted-expression of Bac7 in Lactococcus lactis MG1363 (L. lactis MG1363), this study provides an experimental and theoretical basis for safetly and reasonable treatment of gastro-intestinal infections caused by Gram-negative bacteria. At the same time, construct a homologous recombination vector, which could integrate the Bac7 gene to the chromosome of L. lactis MG1363. This work laid the foundation for further study on the use of genetically modified L. lactis MG1363 to treat the intestinal infection.Methods1. Construction of the secreted-expression vectorAccording to the Bac7 protein sequence in Genbank and the codon preferences of L. lactis MG1363, the Bac7 DNA sequences and its sequence of related regulatory elements were design and chemical synthesis. With Over-lap extension PCR method, these sequences were spliced and cloned into the expression vector pMG36e.2. Prepare the rabbit polyclonal antibody of Bac7Using the software DNASTAR to forecast the Bac7 epitope (N-terminal 20 amino acids), the epitope was chemical synthesized. Then rabbit was immunized with the synthesized peptide coupled with KLH protein, the antiserum from rabbit was purified with ammonium sulfate salting, and its purity and titer were detected with SDS-PAGE and ELISA.3. Electro-transformation of L. lactis MG1363Electro-transform method was appled to transform the recombinant plasmid into L. lactis MG1363. PCR and double-enzyme restriction digestion methods were adopted to identify the recombinant.4. Identify Bac7 proteinRT-PCR was used to determine Bac7 gene at the transcriptional level, and Bac7 protein was indentified with Tricine-SDS-PAGE and Western blot.5. Analysis the biological activity of Bac7 proteinThe Bac7 protein in the supernatant was concentrated by ultra-filtrate centrifugation method, and was tested its antibacterial activities against E. coli and Enterobacter cloacae by agar diffusion method.6. Construction of the homologous recombination vector With the genome DAN of L. lactis MG1363 as a template, the upstream (1 050bp) and downstream (1 010bp) sequences of the ThyA gene were amplified with PCR method and cloned into the vector pMUTIN4 sequentially.Results1. The target gene was spliced by over-lap extension PCR and inserted it to the vector pMG36e. According to the results of double-enzyme digestion and DNA sequencing, the recombinant vector was constructed successfully.2. The rabbit anti-Bac7 polyclonal antibody was successfully prepared and purified. The results of SDS-PAGE and ELISA showed that the prepared antibody had a high purity, and 1:10 000 titers.3. The optimal parameters of electro-transformation were determined as below: voltage 10kV/cm, resistance 200?, capacitance 25μF, time 5.0ms.The recombinant plasmid was electro-transformed into L. lactis MG1363 successfully. RT-PCR, Western blot results showed that Bac7 gene could secrete-expressed in L. lactis MG1363.4. The results of agar diffusion test proved that Bac7 could effectively inhibit the grouth of E. coli and Enterobacter cloacae in vitro.5. Through the identification of PCR and double-enzyme digestion, the sequences of target gene were consistent with the expected. It is testified that homologous recombination vector was constructed successfully.Conclusions1. Recombinant L. lactis MG1363 could secret-express antimicrobial peptide Bac7 and the Bac7 has strong antibacterial activity against E. coli and Enterobacter cloacae in vitro.2. The polyclonal antibody of Bac7 has been prepared successfully.3. Homologous recombination vector pThy-Bac7 was successfully constructed, which lays the foundation for integrated-expression of Bac7 gene in L. lactis MG1363.