人p21活化激酶6基因截短区域的GST标签原核表达质粒的构建及其重组蛋白的表达

Construction of GST-tagged prokaryotic expression plasmids of human PAK6 gene and expressions of their recombinant proteins

目的:构建人p21活化激酶6(PAK6)的各个截短区域原核表达质粒,并诱导和鉴定其融合蛋白的表达,为探讨PAK6基因的生物学功能提供依据。方法:以真核表达质粒pcDNA3.1-GFP-PAK6为模板,PCR扩增出PAK6基因的各个截短片段。所获得的各截短片段经EcoRⅠ/XhoⅠ双酶切后克隆至GST标签的原核表达载体pGEX-5X-1中。将构建的PAK6各截短质粒转化入E.coli BL21中,采用IPTG诱导PAK6基因截短融合蛋白表达,采用Western blotting法鉴定PAK6基因截短融合蛋白的表达。结果:EcoRⅠ/XhoⅠ双酶切后得到与预期大小相符的载体片段(5 000bp)和PAK6 1-55(165bp)、PAK6 56-210(465bp)、PAK6 211-410(600bp)及PAK6 385-681(891bp)4个片段。Western blotting检测,PAK6各截短区域质粒GST-PAK6截短融合蛋白相对分子质量分别为32 000、43 000、48 000和60 000。结论:成功构建PAK6基因各个截短区域GST标签原核表达质粒并表达其重组蛋白。

Objective To construct the prokaryotic expression plasmids of truncated regions of human p21-activated kinase 6（PAK6） to induce and identify their recombinant proteins,and to provide basis for discussing the biological function of PAK6 gene.Methods The eukaryotic expression plasmid pcDNA3.1-GFP-PAK6 was used as the template,and the truncated segments of PAK6 gene were amplified by PCR method.The truncated segments were cloned into GST-tagged prokaryotic expression vector pGEX-5X-1 after double enzyme digestion of EcoRⅠ/XhoⅠ.The truncated plasmids were transfected into E.coli BL21 and the PAK6 gene truncated fusion proteins were induced by IPTG and verified by Western blotting method.Results The vector fragment（5 000 bp） and PAK6 1-55（165 bp）,PAK6 56-210（465 bp）,PAK6 211-410（600 bp）,and PAK6 385-681（891 bp） vector fragments being consistent with the expected fragments were obtained after double enzyme digestion of EcoRⅠ/XhoⅠ.The Western blotting results showed that the relative molecular mass of GST-PAK6 truncated fusion proteins of PAK6 truncated region plasmids were 32 000,43 000,48 000,and 60 000,respectively.Conclusion GST-tagged prokaryotic expression plasmids of different truncated regions of PAK6 gene are constructed and their recombinant proteins are expressed successfully.