Cloning And Prokaryotic Expression As Well As Bioinformatics Analysis Of Virulence Factors GelE Of Enterococcus Faecalis From Goose

Objective:To clone and construct the prokaryotic expression vector of gelE gene of Enterococcus faecalis from goose and expressed in E.coli BL21which is the engineering bacteria. Bioinformatics analysis of gelatinase protein.Method:1According to the base sequence of gelatinase gene of Enterococcus faecalis in NCBI database GenBank accession number:D85393.1, used Primer Premier5and Oligo6software to design a couple of specific primers;To extract the whole DNA from the isolates of goose as the amplification temolet;PCR amplification, recycled the fragment after agarose gel electrophoresis isolated the product;Juncted the fragment and pMD18-T cloning vector then transformed into DH5oc competence cell; Screened the positive clones;Then extracted the plasmid DNA, and the recombinant plasmid identificated by single and double restriction enzyme and PCR, then sent it to the company to be sequenced.2Restriction enzyme the recombinant pMD18-T-gelE plasmid which was cloned successfully, recycled the fragment,and juncted the fragment with pET28a expressing vector,constructed the Pet-28a-gelE recombined plasmid and transformed into BL21competence cell; Screened the positive strains of pET-28a-gelE/BL21which was successful cloned, comfirmation identification by protein electrophoresis SDS-PAGE after IPTG induced expression of gelatinase protein.3To analysis composition, molecular mass,titration curve and election point of the expression gelatinase protein by used bioinformatics software, predicted the primary structure of glycosylation sites and so on, according to the comprehensive analysis of its plasticity, hydrophilicity, antigenicity index, surface accessibility, β-turns and random coil, prediction for the potential antigen epitope.Result:In this test, gelE gene of Enterococcus faecalis from goose was successfully cloned, nucleotide sequence of it is1770bp, encoding590amino acids. pET-28a-gelE/BL21expression vector was successfully constructed, used SDS-PAGE identified after IPTG induced expression of gelatinase protein, the expressed protein size was64.9KD, consistent with the expected size.Some amino acid of the gelatinase protein of Enterococcus faecalis from goose, their hydrophilicity index≥0、AI≥0、 surface accessibility≥1, and it had β-turns and random coil, it was the potential antigen epitope in B cell of gelE protein.Conclusion:It was successful to clone of gelE Gene of Enterococcus Faecalis from Goose, to express of the gelE protein, to predict of gelatinase protein of potential antigen epitope in B cell.