人PAK4基因真核表达载体的构建及其表达和定位
首发时间:2009-01-12
摘要:目的:构建hPAK4的真核表达载体并鉴定PAK4在真核细胞中的表达和定位。方法:利用原核表达载体pGEX-5X-1-PAK4为模板,采用PCR法,进行人PAK4编码序列的扩增,构建pEGFP-C1-PAK4真核表达载体。将pCAN2-MYC-PAK4质粒瞬时转染到HEK293细胞中,利用Western blot方法检测PAK4的表达,并瞬时转染pEGFP-C1-PAK4到胃癌SGC-7901细胞中,用激光扫描共聚焦显微镜观察PAK4的定位。结果:(1)hPAK4编码序列已被克隆至pEGFP-C1真核表达载体中;(2)用MYC抗体鉴定了MYC-PAK4融合蛋白的表达,分子量为68KD;(3)转染pEGFP-C1-PAK4后,在胞浆内可见明亮的绿色荧光。结论:成功地构建了PAK4的真核表达载体pEGFP-C1-PAK4,同时鉴定了MYC-PAK4融合蛋白的表达,证明在胃癌SGC-7901细胞中PAK4主要定位在胞浆,为进一步研究PAK4的生物学特性及其信号转导通路奠定了基础。
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Construction of Human PAK4 Gene Eukaryotic Expression Vector and its Expression and Localization
Abstract:Objective: To construct a GFP-PAK4 fusion gene eukaryotic expression recombinant vector, identify the expression of PAK4 in eukaryotic cells and analyze its subcellular location. Methods: Using the prokaryotic expression vector pGEX-5X-1-PAK4 as template, we obtained human PAK4 coding sequence by PCR amplification and cloned it into the pEGFP-C1 eukaryotic expression vector. HEK293 cells were transiently transfected with plasmid pCAN2-MYC-PAK4 and the expression of PAK4 was analyzed by Western blot.The comfirmed recombinant plasmid pEGFP-C1-PAK4 was then transiently transfected into gastric cancer SGC-7901cells and the subcellular localization of PAK4 was analyzed using laser scanning confocal microscope. Results: (1)The coding sequence of hPAK4 was cloned into pEGFP-C1 eukaryotic expression vector. (2) The expression of MYC-PAK4 fusion protein was identified by the antibody against MYC. (3) Green fluorescence light was seen in the cytoplasm in SGC-7901 cells after transfection of pEGFP-C1-PAK4 plasmid. Conclusion: The eukaryotic expression plasmid pEGFP-C1-PAK4 was successfully constructed. The expression of MYC-PAK4 fusion protein was identified. PAK4 mainly localized in the cytoplasm of gastric cancer SGC-7901 cells. This study provides the basis for research on biological features and its signal transduction pathways of PAK4.
Keywords: PAK4 Green fluorescent protein fusion protein transfection
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