Cloning of p21~ ( waf1)gene and its high expression in E.coli JM109

Objective To construct the pronucleus expression vector of human p21~ waf1and express it in E.coli strain of JM109. Methods The total RNA was isolated from human hepatocytes, and p21~ waf1 was cloned by RT-PCR, the PCR product was ligated with PGEM-T vector and sequenced. Then the target sequence was inserted into an temperature-sensitive expression vector pBV220 and expressed in E.coli JM109.The recombinant protein was identified by immunoblot. Results DNA sequencing confirmed that the sequence of cloned p21~ waf1 was identical to the published sequence. The recombinant plasmid pBV220/p21~ waf1 was transformed into E.coli JM109 and over- expressed during 42℃ induction. After induction for 5 h, the expression products showed a high expression level of recombinant protein with 21 000, and was more than 38% of the total bacterial protein. Western blotting analysis indicated that the the recombinant protein could react specifically with anti-P21~ waf1 antibody. Conclusion The p21~ waf1 expression vector is constructed successfully, and the induced-protein is confirmed as P21~ waf1 protein by detection.