Objective: To construct the expression vector of Vibrio cholerae ctxA gene, and realize the Vibrio cholerae ctxA gene to express in E. coli, and lay a basis for future research on the immunogenicity and immunoadjuvant.
Methods: The ctxA gene, an cholera toxin subunit gene (ctxA) of Vibrio cholerae, was obtained by polymerase chain reaction (PCR) from DNA of Vibrio cholerae, and cloned into prokaryotic expressed vector pET32a(+) with thioredoxin (Trx) gene. The recombinant plasmid (pET-ctxA) was analyzed to identify with restriction-endonuclease digestion, PCR and DNA sequencing analysis. And then the pET-ctxA was transferred into E. coli strain BL21 (DE3) for transformation. The ctxA expression of pET-ctxA was induced with isopropy-beta-D-thiogalactoside (IPTG) and the fused protein Trx-CTA was examined by SDS-PAGE and Western blot techniques.
Results: Restriction endonuclease digestion, PCR and DNA sequencing analyses showed that the ctxA gene of 787 bp was amplified from Vibrio cholerae DNA, and the recombinant plasmid pET-ctxA was successfully constructed, and the ctxA expression in prokaryotic cell was detected by SDS-PAGE and Western blot techniques.
Conclusion: The ctxA gene of Vibrio cholerae, in fused protein form with Trx, got a high expression in E. coli.
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