The Research On Cloning And Expression Of Silent Gene SlpB From L.Acidophilus NCFM And The Structure And Function Of S-layer Protein

L.acidophilus is one of the important members of Lactobacillus,which plays an important role in the adhesion of host gut cells,competing with pathogens for adhesion sites and regulating immune function.As an adhesion factor,more and more scholars pay attention to S-layer protein.With the rapid development of genetic engineering technology,researchers began to study genes of S-layer protein.L.acidophilus has another S-layer protein gene slpB,which is silent.There are few reports about the gene slpB and further exploration is needed for whether it has adhesion function.The main methods and results of our research were as follows:1.The slpB gene was successfully cloned from the whole genome of L.acidophilus with the size of nearly 1300 bp,which was consistent with the size of slpB gene in NCBI.The recombinant plasmid pET-28a-slpB was constructed and verified by double enzyme digestion.The target fragment of about 1300 bp and the fragment of 5400 bp were obtained,which was consistent with the predicted value.The result of sequence was correct with NCBI database.2.The recombinant plasmid pET-28a-slpB was expressed and the supernatant of the target protein was higher than the precipitate.The optimal induction conditions were obtained by the control variable method:induction temperature 37°C,IPTG final concentration 1 mM and induction time 14 h.The His-tag fusion protein was successfully expressed and purified by detection of Western-blot and SDS-PAGE.The purified protein was dialyzed and concentrated,and the protein concentration was 0.6 mg/mL by the Bradford method.3.The proteins of L.acidophilus were extracted and purified.The protein extracted from the surface of L.acidophilus was named as SA,and the protein expressed by silent gene slpB in E.coli was named as SB.The-sheet content of SA was slightly higher than that of SB,indicating a more stable structure than SB.The secondary structure table showed that the amino acids at both ends of the two proteins had a higher consistency.Both proteins had good tolerance to pepsin and were less tolerant to trypsin.More SB could bind to the cell walls of L.bulgaricus and L.lactis.SB was superior to SA in the cell binding ability of E.coli and S.aureus.SA was superior to SB in the adhesion to Caco-2 cells.In this study,the silent gene slpB was expressed,and the structure and function of S-layer protein were analyzed and determined,which provided a theoretical basis for the development and utilization of S-layer protein in the future.