p21^(waf1)基因的克隆及在大肠杆菌中的高效表达

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

目的 :构建人 p2 1waf1 基因的原核表达载体并在大肠杆菌 JM10 9中高效表达。方法 :从人肝细胞中分离总 RNA,经逆转录多聚酶链反应 (RT- PCR)得到 p2 1waf1 全基因。用原核细胞表达载体构建了 p BV 2 2 0 / p2 1waf1 重组质粒 ,经 DNA测序确认后 ,将其转化到大肠杆菌 JM10 9进行表达、初步纯化。结果 :SDS- PAGE凝胶电泳分离显示在分子量 2 10 0 0处有一诱导蛋白带 ,薄层扫描显示表达产物在诱导 5 h时可达细菌总蛋白的 38% ,Westernblotting证明表达产物与抗人 P2 1waf1 单克隆抗体有特异性免疫反应。结论 :成功构建出 p2 1waf1 表达载体 ,诱导产生蛋白经检测证实为 P2 1waf1 蛋白。

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.