Cloning Of Bacteriocin Lac-B23 By Lactobacillus Plantarum And Its Expression In Escherichia Coli

Antimicrobial peptides attract much attention for their potential for being a substitute for food preservatives,they can be commercialized provided that we can get sufficient quantity of highly purified antimicrobial peptides,heterologous expression of antimicrobial peptides is the most economical way.In this study,we used Lac-B23 preservated in our laboratory with a good activity as the source,the plantaricin was successfully expressed in Arctic Express E.coli,then optimized the induction conditions and fermentation conditions to increase the production of antimicrobial peptides. We got the following findings by a series of experiments.In this study,we chose Lac-B23 as the experimental strain,screened primers which can obtain a single clear band by rapid screening methods based on PCR technology.We verified the results by repeating the PCR experiments,recycling the target band,and determinated the target band using blast sequence comparison,it had a close relationship with activity.Puc57 was used as the cloning vector,we successfully connect the target gene with cloning vector.Once connected,we obtained target gene by double digestion and gel extraction.Double cut the expression vector,using T4 enzyme connect target gene and p Cold,then transformed into Arctic Express E.coli,IPTG was selected as the inducing agent,so the antimicrobial peptides structure gene can be successfully expressed in Arctic Express E.coli. Meanwhile,we chose the factors which have significant impact on the expression of antimicrobial peptide,namely induction temperature,induction point on cell growth,induction time and IPTG concentration in the post-induction phase.The results showed that induction point on cell growth was the most significant factor,when IPTG concentration was 0.6mmol/L,the maximum antibacterial activity was obtained.Low temperature could reduce the formation of inclusion effectively,15℃ was the optimum temperature.The impact of induction time was not significant,but reached a maximum after 16 h.The addition of carbon source,nitrogen source and metal ions were beneficial to recombinant expression of antimicrobial peptide activity.In this study,the screening of carbon source and nitrogen source found that the antibacterial activity had increased significantly when the concemtration of glucose was 15g/L,the impact of yeast extract was not significant.Calcium ions significantly inhibited antibacterial activity of recombinant antimicrobial peptide,Magnesium ions and Copper ions were not significant.However,with the changes in Potassium ions concentration, the antibacterial activity changed significantly.In addition,fermentation time h ad a significant effect on the antibacterial activity of recombinant antimicrobial peptides.We chose glucose,KH2PO4,fermentation time as the factors in response surface experiment.As a result,the antimicrobial activity reached maximum when the fermentation time is 15.75 h,the concentration of Potassium ions is 4.88g/L,the concentration of glucose is 15.10g/L,the maximum can reach to 1.64±0.05 cm.