野生型和失活型PAK1基因自我抑制域的GST标签真核表达载体的构建及表达

Construction and expression of wild type and inactive type of GST-tagged eukaryotic expression vectors of PAK1 gene autoinhibitory domain

目的:构建不同活性的人p21活化激酶1(hPAK1)自我抑制域(AID)谷胱甘肽(GST)标签真核表达载体并鉴定其融合蛋白表达,以探讨PAK1AID的功能及肿瘤治疗的靶向意义。方法:以pcDNA3.1HisC-PAK1全长质粒为模板,利用PCR扩增野生型(WT)PAK1AID片段,再以此片段为模板采用大引物法扩增其突变体L107F片段,双酶切克隆至GST融合的真核表达载体pEBG。将质粒转染至工具细胞HEK293中,并经免疫印迹鉴定GST融合蛋白的表达。结果:PCR扩增出约200bp大小的野生型和失活型PAK1AID片段,双酶切得到与预期大小相符的载体与PAK1AID片段,野生型与失活型pEBG-PAK1在工具细胞HEK293中表达,蛋白的相对分子质量均为33 000。结论:成功构建野生型和失活型PAK1基因AID的GST标签真核表达载体,并表达出不同活性的GST-PAK1AID的融合蛋白。

Objective To construct the GST-tagged eukaryotic expression vectors of human p21-activated kinase 1 （hPAK1） autoinhibitory domain（AID） with different activities and identify the expressions of their recombinant proteins, and to explore the function of PAK1 AID and molecular targeting role in tumor therapy. Methods The pcDNA3.1HisC-PAK1 full length plasmid was used as template, and the fragment of wild PAK1 AID was amplified with PCR, and the fragment of PAK1 L107F was amplified with long primer mutant method. The PCR fragments were double-digested and cloned into pEBG. The wild and inactive types of pEBG-PAK1 were transfected into HEK293 cells and their expressions were identified by Western blotting. Results The wild and in active PAK1 AID fragments with 200 bp were obtained by PCR. 5 000 bp vector band and 200 bp PAK1 AID band were obtained by double-digestion. The wild and inactive types of pEBG-PAK1 were expressed in HEK293 cells, and the molecular weight of the protein was 33 000. Conclusion The wild and inactive types of PAK1 AID eukaryotic expression vectors are successfully constructed. The expressions of wild and inactive types of PAK1 AID proteins are identified.