Molecular Cloning Of Adhesion Protein Gene From L. Plantarumzj316and Expression Of Heterologous Protein In E.Coli

Lactobacillus is the advantage microbial flora in the intestinal of human and animal, the beneficial lactic acid bacteria in organism can regulate microecological balance of intestinal canal in human and animal, restrain and kill pathogenic bacteria, simultaneously, enhance immunity role. Adhesion to the host intestinal is the premise of Lactic acid bacteria exert all kinds of physiological function. The study of adhesion mechanism of lactic acid bacteria has important significance to the development and application of probiotics.In this study, Lactobacillus plantarum ZJ316is preserved in our laboratory, which isolated from infant fecal samples and produce bacteriocin. we took the microbial adhesive hydrocarbon method (MATH) to survey and evaluate the surface property of the three lactobacillus (Lactobacillus plantarum ZJ316, Lactobacillus plantarum ZJ22, Enterococcus faecium ZJ19) including hydrophobic property, surface charge and the polymerization ability and the Sacchaomyces cerevisiae agglutination experiment to make clear of lactic acid bacteria carbohydrate specificity. The results showed that the highest value of hydrophobicity of Lactobacillus plantarum ZJ316is83.67±1.13. Yeast agglutination experiments showed that the value of Saccharomyces cerevisiae agglutination was32and the three lactobacillus which could also agglutinate cells of Saccharomyces cerevisiae to different levels. When added in the D-mannose and methyl alpha-D-mannose glucoside to the experimental groupt, the ability of Saccharomyces cerevisiae agglutination declined. It illustrated that the adhesion of Lactobacillus plantarum ZJ316associated with mannose in specific adhesion.According to Lactobacillus plantarum WCFS1,whose adhesion protein genes sequence (1062815) and (1062646) of surface adhesion protein Slp2588, mucous adhesion protein Muc2486for primer designing, we carried on the adhesion gene cloning and sequencing. Sequencing results:The length of Gene fragment slp2588A is1618bp and The length of gene fragment muc2486A length is3018bp. Similarity comparison in the NCBI website with BLAST software, the result showed that the similarity of gene fragment slp2588A and gene slp2588in Lactobacillus plantarum WCFS1was98%; Similarity comparison in the NCBI website with BLAST software, the result showed that the similarity of gene fragment muc2486A and gene muc2486in Lactobacillus plantarum WCFS1was99%. Therefore, we considered there are surface adhesion protein Slp2588and mucus adhesion protein Muc2486gene sequence in the ZJ316genome.Restructuring the same sticky end purpose fragment (slp2588A, muc2486A) and plasmid vector (pET-28a) in vitro to construct prokaryotic expression vector. We transform the recombinant vector into BL21(DE3), IPTG induced expression, SDS-polyacrylamide gel showed that there were purpose protein banding. Collecting recombinant bacterias and ultrasonic crushing centrifugal, we found that the purpose protein in precipitation. It illustrated that he purpose protein was expressed in form of IB with no activity, needing renaturation to separation and purification. We found the ability of Saccharomyces cerevisiae agglutination of two purposes protein inclusion body is stronger. It Showed that the adhesive protein (surface adhesion protein Slp2588and mucous adhesion protein Muc2486) heterologous expression has played an important role in adhesion of Lactobacillus plantarum ZJ316.