中文摘要：

目的构建不同激酶活性的hPAK6原核表达载体并诱导和鉴定其融合蛋白表达。方法野生型、激酶活型和激酶死型pcDNA3.1-PAK6经EcoRⅠ和XhoⅠ双酶切后,克隆至GST融合表达载体pGEX-5X-1,在E.coli BL21中诱导不同型GST-hPAK6融合蛋白表达,并经免疫印迹鉴定结果。结果野生型、激酶活型和激酶死型hPAK6编码序列已被克隆至GST融合表达载体pGEX-5X-1,双酶切及测序鉴定正确,并在E.coli BL21中获得了诱导表达,蛋白的分子量均为101 kDa,免疫印迹检测到了融合蛋白表达。结论成功构建不同激酶活性的hPAK6基因原核表达载体,并分别诱导表达出GST-PAK6融合蛋白。

英文摘要：

Objective To construct various kinase activity types of prokatyotic expression vector of PAK6 （p21-activated kinase ）gene and identify their recombinant protein expression induced by isopro-pyl-1-thio-b-Dgalactopyranoside （IPTG）. Methods wild type, kinase active and kinase dead type pcDNA3.1-PAK6 were digested by EcoR Ⅰ and Xho Ⅰ ,and they were cloned into pGEX-SX-1. The expressions of various types GST-hPAK6 fusion proteins were induced by IPTG and identified by Western blot. Results The wild type,kinase active and kinase dead types coding sequences of hPAK6 gene were cloned into the pGEX-SX-1 plasmid which was transformed into E. cob BI21 ,identified by double enzymes digestion and sequencing. The expression of GST-hPAK6 fusion protein was induced by [PTG, and the molecular weight of protein was 101 kDa. Conclusion The various typese of FAK6 recombinant prokaryotic plasmids were successfully constructed into pGEX-5X-1. The expression of GST-hPAK6 fusion proteins were induced by IVFG and identified.