Construction Of Recombinant Lactobacillus Casei Expressing Antimicrobial Peptides PF1and PR39and Detection Of Biological Activity

Antimicrobial peptides is a class of biologically active substances generated in the host innateimmune defense system, it can identify and destroy when bacteria and fungi infect animal body,butit doesn’t have toxicity for the eukaryotic cells. It plays an important role in the the animal innateimmune and non-specific immune process.With the reference template of the gene of PR39and PF1publish on GeneBank, according tothe codon bias of Lactobacillus casei,synthesizing the gene of PF1-PR39. Base on primers P3andP4to amplify PR39gene. The PR39gene connect expression vector plasmid pCold, transferred toE. coli BL21(DE3) named the recombinant strain pCold-PR39/BL21(DE3); We induced therecombinant strain by IPTG and optimize expression conditions，About51kDa expression proteinin the secretary expression system was detected by Western blot; Antibacterial test showedantimicrobial peptides PR39has significant antimicrobial effect on Escherichia coli and Salmonella,diameter of antibacterial circle is5.7mm and4.9mm; There is no effect on Staphylococcus aureusand Lactobacillus casei.The gene of PR39and PF1was cloned into the Lactobacillus casei secretory expression vectorand constructed cell secretary expression system pPG612-PR39/L.casei393and pPG612-PF1-PR39/L.casei393. The recombinant strain was induced by1%lactose. About36kDa,30kDa and6kDa expression protein in the secretary expression system was detected by Western blot. Inantibacterial activity assay, the expressed PR39and PF1-PR39protein showed obviouslyantibacterial activity, then the recombinant protein PF1-PR39better than PR39. The time dynamiccurve method indicated that the induced supernatant of pPG612-PR39/L.casei393and pPG612-PF1-PR39/L.casei393had effect on Gram-negative bacteria Escherichia coli, Salmonella, andwhile the growth of Staphylococcus aureus was not inhibited.We investigate the recombinant protein PF1-PR39and PR39whether could promote animalgrowth, totally36female BALB/c mice of SPF were selected and assigned into4groups with9ineach group randomly, medium group, pPG612/L.casei393group, pPG612-PR39/L.casei393groupand pPG612-PF1-PR39/L.casei393group. The mice were fed with1×109recombinant strains28d.At0d,28d, feces of each group were collected and measured mices on mices growth promotingeffectinitial weight measured mices the efficiency of feed utilization. After28d detect each group ofmice to test peripheral blood lymphocytespercentage, number of white blood cells and spleen index; In sterile environment, take2cm of mice jejunum and duodenum for the sections by SEM;Analysis the diversity of the microbial community structure of each group of mice feces fed withrecombinant strain before and after by PCR-DGGE; At the same time, the K88strains poison attackexperiment was carried out, to detect the protective effect of recombinant strain.The experimental results show that the ratio of feed to gain of pPG612-PR39/L.casei393andpPG612-PF1-PR39/L.casei393is40.200±0.78and42.576±0.9, compared with control group, thedifference is extremely significan（tP<0.01）. The peripheral blood lymphocytes percentage, numberof white blood cells and spleen index of pPG612-PR39/L.casei393and pPG612-PF1-PR39/L.casei393compared with control group, the difference is significant（P<0.05）; The SEM sections ofintestinal villi, jejunum and duodenum villous height, crypt depth and V/C of the mice, comparedwith that of control group, the difference is extremely significant（P<0.01）; The pPG612-PR39/L.casei393and pPG612-PF1-PR39/L.casei393groups compared with control group in the DGGEmaps, for experiment groups, shannon-wiener is unchanged, richness and evenness is reduced. Theresult of protective effect show that recombinant antimicrobial peptides can cut down mortalityK88cause significantly.These results suggest that the recombinant expression system for Lactobacillus casei with apotential value, the recombinant strain would not only have a beneficial nature of lactic acidbacteria, but also play a biological function of antimicrobial peptides. All these works established agood foundation for further study on the lactic acid bacteria as a delivery system and thedevelopment of new type of animal feed.