真核绿色荧光蛋白表达载体pEGFP-C1/PAK-1的构建及其在结直肠癌SW480细胞内的表达

Construction of a eukaryotic green fluorescent protein expression vector carrying the PAK-1 gene(pEGFP-C1/PAK1) and its expression in SW480 cells

目的:构建重组p21-activated kinase-1(PAK1)基因绿色荧光蛋白表达载体pEGFP-C1/PAK1,并转染入结直肠癌细胞SW480中表达.方法:在南方医科大学附属南方医院消化研究所实验室,从人类结直肠癌细胞株SW620细胞提取总RNA,经逆转录聚合酶链式反应获得人PAK1 cDNA片段,经过限制性内切酶进行酶切,T4连接酶进行连接,将目的基因克隆至真核绿色荧光蛋白表达载体pEGFP-C1上,然后转染结直肠癌细胞株SW480,观察其在细胞中表达.结果:重组载体经限制性内切酶酶切鉴定和DNA序列分析验证,显示插入载体的序列与目的基因一致,而且该重组载体能够在SW480细胞中表达.结论:成功构建了真核绿色荧光蛋白表达载体pEGFP-C1/PAK1,为研究PAK1在结直肠癌中的生物学功能奠定了基础.

AIM： To construct a recombinant eukaryotic fluorescent expression plasmid containing the coding region of p21-activated kinase-1 （PAK-1） gene and to detect its expression in SW480 cells. METHODS： Total RNA was extracted from human colorectal carcinoma cell line SW620 and used to amplify the PAK1 gene fragment by reverse transcription-polymerase chain reac- tion （RT-PCR）. The resulting PCR product was inserted into the plasmid pEGFP-C1 after re- striction endonuclease digestion and ligation. After verifying the correct insertion of the DNA fragment by endonuclease digestion and direct sequencing, the recombinant plasmid was trans- fected into SW480 cells to detect its expression in vitro. RESULTS： The sequence of the recombinant plasmid was verified by restriction digestion and DNA sequence analysis, and the target protein expression was detected in the cell cytoplasm of SW480 cells. CONCLUSION： A recombinant eukaryotic fluorescent expression vector carrying the PAK-1 gene （pEGFP-C1/PAK1） has been constructed successfully and provides a potent tool to investigate the role of PAK-1 in colorectal carcinoma.