Expression And Purification Of Ace Recombinant Protein Of Enterococcus Faecalis And Characterization Of Its Adherence

Objective:This study was carried out to construct a recombinant vector containting the gene encoding Ace A domain of Enterococcus faecalis (E.faecalis), then express a fusion protein in E.coli M15 and purify it, and the immunocompetence of the recombinant protein was analyzed. To identify its role in adhesion of E.faecalis, and to lay the foundation for development of quick diagnostic kit applying to detect E.faecalis.Methods:The Ace A domain was amplified by PCR from the genome of E.faecalis JH2-2, subcloned into the expression vector pQE30 to generate recombinant plasmid pQE30/Ace, then expressed in E.coli M15, and analyzed by SDS-PAGE and Western blot. The fusion protein was purified with Ni-NTA affinity chromatography, and the protein concentration was determined by the BCA protein assay kit. Newzealand rabbits were immunized with the fusion protein and antibodies to rAce in sera were detected to evaluate the immunocompetence of rAce. Then the adhesion and inhibition assay were carried out with the FITC-labeled E.faecalis JH2-2, and the results were observed by IFM.Results:The size of PCR amplification product was about 930bp. Restriction enzyme digestion analysis and sequencing showed that the inserted target gene was ace, and it had 100% similarity with gene reported by GenBank, the results of BLAST confirmed that the sequence was ace. A soluble fusion protein with molecular weight about 37kDa was attained after expression and purification. Specific response were elicited after introducing rAce in Newzealand rabbit and the specific antibody titer was above 1:16 000 detected by indirect ELISA. The result of Western blot showed that rAce could be recognized by the 6×His monoclonal antibody, and Ace extracted from E.faecalis JH2-2 cell wall could be recognized by the antibodies to rAce in sera. E.faecalis JH2-2 was able to adhere to typeⅠcollagen and HeLa cells. Antibodies to Ace A domain were showed to inhibit the binding of E.faecalis JH2-2 grown at 46℃to typeⅠcollagen and HeLa cells, and their dilutions had negative correlation with inhibition.Conclusion1. Prokaryotic expression vector pQE30/Ace was constructed successfully and Ace with molecular weight near 37kDa was well attained.2. The rAce showed excellent immunongenicity and can efficiently induce the humoral responses in Newzealand rabbits, and also showed excellent immun- oreactivity, and was expected to be used in quick diagnosis of E.faecalis.3. Ace is a adhesive protein of E.faecalis correlated with pathogenicity.