Prokaryotic Expression Of S-layer Protein Of Lactobacillus Brevis And Preliminary Identification Of Its Adhesion In Vitro

S-layer protein (SLP) is lattice-like structure of archaebacteria and eubacteria surface which is composed of the single-molecule protein or glycoprotein subunits. It is located in the cell wall or cell membrane outer, and interacts with non-covalent bond. Hydrogen bonds of S-layer protein are usually destroyed with high concentrations of denaturing agents or dissociation agents (such as urea, guanidine hydrochloride, lithium chloride, a metal chelating agent, and cation replacement, etc.,), so that the S-layer protein depolymerization into monomers and dissolved out. When the denaturant is removed, S-layer protein subunits are able to assembled into highly regular lattice structure again in a variety of matrix, which is called the effects of self-assembly. Currently, Studies on S-layer proteins of lactic acid bacterium are more focused on its adhesion property. The S-layer protein can mediate the lactic acid bacteria adhered to the surfaces of various host cells. S-layer protein of Lactobacillus has many advantages, such as highly regular lattice structure, significant adhesion ability and self-assembly property, etc. It is widely used in nano-biotechnology, bio-bionics, bio-medical, as well as effective expression or secretion expression of heterologous protein or polypeptide. In a word, it has huge development potential and bright application prospects.Sip gene was subcloned by PCR from the plasmid of pMD19T-slp which has already been fully constructed in our laboratory. The Slp gene was then inserted into the prokaryotic expression vector of pGEX-4T-3, to make the expression construct of pGEX-slp. Recombinant fusion protein GST-SLP was expressed by IPTG induction, and identified by SDS-PAGE and Western blot. Then the protein was purified by LiCl solution, and the adhesive property of purified vitro protein GST-SLP was identified by the method of ELISA and IFA. These experiments lay a foundation for further study of the biological characteristics of the S-layer protein and the development of genetically engineered vaccine of lactic acid bacteria live vector. The results of this study are as follows:1. The prokaryotic expression vector of pGEX-slp was successfully constructed, and the recombinant fusion protein GST-SLP was gained by IPTG induction. Then the expression protein was identified by SDS-PAGE and Western blot. The tests results all proved that the presence of the fusion protein GST-SLP which had a molecular weight of approximately71kDa. It is consistent with the theoretical value. 2. Recombinant fusion protein was purified by LiCl solution. The SDS-PAGE results showed a dominant protein which had a molecular weight of approximately71kDa. The concentration of purified recombinant fusion protein GST-SLP was approximately1.1mg/mL, which can meet the experiment qualification of identification of adhesion property in vitro.3. Adhesion property in vitro of recombinant fusion protein GST-SLP was identified by ELISA. The results preliminary suggested that it was a positive reaction. Moreover, when the concentration of the protein was high,it could strongly adhere to the surface of Lactococcus lactis NZ9000in vitro. While the concentration was low, it could weakly adhere to its surface.4. Adhesion property in vitro of recombinant fusion protein GST-SLP was further identified by IFA. The results demonstrated that the protein could adhere to the surface of Lactococcus lactis NZ9000and Lactobacillus casei in vitro in certain circumstances.