人源性热休克蛋白70在大肠杆菌中的表达及鉴定

EXPRESSION AND IDENTIFICATION OF HEAT SHOCK PROTEIN 70 IN E.COLI

为在大肠杆菌中表达人源性热休克蛋白 70 (HSP70 )的基因 ,以质粒为模板 ,采用PCR方法进行扩增 ,将PCR产物进行琼脂糖凝胶电泳 ,回收大小约 1 96kb的片段 ;回收片段经T A克隆法克隆到pUCm T载体上 ,并进行DNA测序 ;将目的基因片段从pUCm T载体上酶切后 ,亚克隆到表达载体pET 2 1a(+)上 ;将重组质粒pET 2 1a(+) /HSP转化大肠杆菌 ,经IPTG诱导后 ,收集细菌 ,菌体裂解后进行SDS PAGE及Westernblot检测。结果表明 ,应用PCR方法扩增出约 1 96kb的目的片段 ,序列测定结果证实 ,扩增的HSP70基因序列与GeneBank中HSP70cDNA序列一致 ;经EcoRⅠ+XhoⅠ酶切及PCR鉴定证实 ,HSP70基因成功地克隆到原核表达载体pET 2 1a(+)上 ;转入重组质粒的大肠杆菌经IPTG诱导后 ,进行SDS PAGE ,发现在分子量为 72 0 0 0处有表达量明显增多的蛋白条带 ,Westernblot证实其为目的条带。

To express a gene of heat shock protein 70(HSP70) in E.coli , HSP70 gene was amplified by PCR. The PCR products were cloned into pUCm T vector and were sequenced; The HSP70 gene was subcloned into vector pET 21a(+) and expressed in E.coli. SDS PAGE and Western blot were employed to identify the expression of the HSP70 gene. Results showed that a fragment about 1 96kb was amplified by PCR. Sequence analysis revealed that the sequence of HSP70 was correct. The HSP70 gene was cloned into pET 21a(+) identified by enzyme digestion and PCR. SDS PAGE and Western blot showed that a M 72 000 protein was expressed and could be recognized by anti HSP70 antibody. Therefore,HSP70 gene has been successfully expressed in E.coli .