【摘要】 目的构建真核表达载体pEGFP-PAK4,利用它鉴定p21活化激酶4(PAK4)在胃癌细胞中的定位,并检测外源性和内源性PAK4在胃癌细胞中的表达。方法以pCAN2-MYC-PAK4为模板,采用PCR法进行人PAK4编码序列的扩增,并将其克隆至真核表达载体pEGFP-C1中。将pCAN2-MYC-PAK4质粒瞬时转染到胃癌BGC-823细胞中,用Western blot方法检测MYC-PAK4融合蛋白的表达;并用Western blot方法检测不同胃癌细胞系中内源性PAK4的表达;瞬时转染pEGFP-PAK4到胃癌BGC-823细胞中,用激光扫描共聚焦显微镜观察PAK4的定位。结果(1)hPAK4编码序列被成功克隆至真核表达载体pEGFP-C1中;(2)证实了MYC-PAK4融合蛋白及内源性PAK4在胃癌细胞中的表达;(3)转染pEGFP-PAK4后,在胃癌BGC-823细胞胞质内可见明亮的绿色荧光。结论成功地构建了pEGFP-PAK4载体,证实了外源性和内源性PAK4的表达,证明在胃癌BGC-823细胞中PAK4主要定位于细胞质,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。更多还原

【Abstract】 Objective To construct an eukaryotic expression vector pEGFP-PAK4 and identify the expression and localization of extraneous and endogenous PAK4 in gastric cancer cells.Methods Using pCAN2-MYC-PAK4 as template,we obtained human PAK4 coding sequence by PCR amplification and cloned it into the eukaryotic expression vector pEGFP-C1.BGC-823 cells were transiently transfected with plasmid pCAN2-MYC-PAK4 and the expression of MYC-PAK4 fusion protein was analyzed by Western blot.The expression of endogenous PAK4 in different gastric cancer cells was also examined by Western blot.pEGFP-PAK4 was then transiently transfected into gastric cancer BGC-823 cells to determine the subcellular localization of PAK4 by 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 and endogenous PAK4 in gastric cancer cells were identified;(3)Green fluorescence was observed in the cytoplasm of gastric cancer BGC-823 cells after transfection of pEGFP-PAK4 plasmid.Conclusion The plasmid pEGFP-PAK4 was successfully constructed.The expression of extraneous and endogenous PAK4 was identified.PAK4 was mainly localized in the cytoplasm of gastric cancer BGC-823 cells.This study provides the basis for research on biological features and signal transduction pathways of PAK4.更多还原