Expression and Identification of Human Alpha Fetoprotein in E. coli
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Abstract:
In this study, through the application of genetic engineering techniques, the prokaryotic expression vector pET22b-AFP of full-length human alpha-fetoprotein AFP gene was recombined, constructed, encoded, and then transformed into E. coli host strain BL21 (DE3) to induce the expression of the target protein AFP. AFP coding sequence was amplified by polymerase chain reaction (PCR), and then cloned into the prokaryotic expression vector pET-22b containing His-tag. The constructed vector, verified by restriction endonuclease digestion and DNA sequencing, was then transformed into E. coli host strain BL21 (DE3) for expression by using IPTG-inducible promoters. Different concentrations of IPTG and different temperatures were optimized to induce the expression of target protein. Expression products were identified by SDS-PAGE electrophoresis and Western Blot. The results showed that AFP gene fragments were obtained by PCR amplification. By double enzyme digestion and gene sequencing methods, it was confirmed that the recombinant plasmid was constructed successfully. Expression product was revealed in the vicinity of the 66 kDa band by using SDS-PAGE and Western Blot., which was consistent with the expected value indicating that the recombinant AFP protein succeeded in expressing.
