Objective To construct an expression vector for the Hemolysin E gene of Escherichia Coli,to induce the expression of the target protein in order to lay the foundation for the regulation of Hemolysin E expression by genetic engineering and the development of clinical diagnostic immunoreagents.Methods The E.coli Hemolysin E gene sequence(NC_000913.3)retrieved from NCBI database was synthesized through the PCR-based Accurate Synthesis(PAS)method.The full-length splicing primers were designed to synthesize target genes and the recombinant plasmid pCZN1-hlyE was successfully constructed.The constructed pCZN1-hlyE recombinant plasmid was transferred into E.coli TOP10.After identification,the expression of Hemilysin E gene was induced with propyl-β-D-thiogalactoside(IPTG)to generate the Hemolysin E virulence protein.Results By SDS-PAGE gel electrophoresis and Western blot analysis,the molecular weight of the target protein Hemolysin E was 28.72 kDa,which was consistent with the molecular weight of the protein obtained by DNAMAN software analysis of the nucleic acid sequence of the Hemolysin E gene.Conclusion The induced expression of Hemolysin E protein is present in the supernatant of bacterial lysate and purified to prepare recombinant protein.
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