PAK2真核表达载体的构建及在胃癌细胞内的表达与定位

Construction of the eukaryotic expressing vector of human PAK2 gene and the expression and location in gastric cancer cells

目的构建PAK2真核表达载体及证实在胃癌细胞内的表达与定位。方法提取HeLa细胞的mRNA,反转录为cDNA。PCR扩增PAK2全长编码基因,克隆至pcDNA3.1A表达载体中。将构建的重组质粒测序并转染到胃癌细胞SGC-7901中,Western blot检测蛋白表达,免疫荧光检测PAK2在胃癌细胞内的定位。结果 hPAK2全长基因序列克隆到了真核表达载体pcDNA3.1A中,酶切鉴定片段为2 000 bp。Western blot检测到重组质粒蛋白表达,分子量约为75 kDa。免疫荧光检测pcDNA3.1A-PAK2在胃癌细胞内的定位以细胞浆为主,细胞核内少许。结论成功构建hPAK2全长基因真核表达载体,重组质粒主要定位于胃癌细胞浆。

Objective To construct an eukaryotic expressing vector of human PAK2 gene and identify its recombinant plasmid expression and location in the gastric cancer ceils. Methods Total mRNA was extracted from HeLa cells, cDNA was formed by reverse transcription.The PAK2 coding sequence was amplified by polymerase chain reaction （PCR）metbod and cloned into pcDNA3.1A vector. After the target region was sequenced, the plasmid was transfected into SGC-7901 cell lines. The expression of the pcDNA3.1A-PAK2 recombinant plasmid in SGC-7901 cells was proved by Western blot. The location of the plasmid in the cells was observed by using laser scanning confocal microscopy. Results hPAK2 was successfully constructed into pcDNA3.1A expressing vector. The length of the fragment was about 2000 bp, identified by restruction enzyme digestion. The expression of pcDNA3.1A-PAK2 recombinant plasmid in SGC-7901 cells was detecteded by Western blot, with a molecular weight of about 75 kDa.The plasmids were localized more in the cytoplasm, less in the nucleus. Conclusion The hPAK2 full length sequence was successfully cloned into eukaryotic expressing vector, the recombinant plasmids were localized mainly in the cytoplasm.