pEGFP—C1-HSP27真核表达载体的构建及其稳定转染人胰腺癌SW1990细胞株的筛选

Construction of pEGFP-C1-HSP27 recombinant eukaryotic expression vector and screening of human pancreatic cancer SW1990 cell line stably expressing HSP27

目的构建表达热休克蛋白27（HSP27）的携带增强型绿色荧光蛋白（EGFP）的真核载体，建立稳定表达HSP72的人胰腺癌SW1990细胞株。方法采用RT—PCR法从SW1990细胞中扩增出带有BamHI、Hind Ⅲ酶切位点的HSP27基因片段，插入真核表达载体pEGFP—C1，鉴定正确后用重组质粒转染SW1990细胞，并筛选稳转细胞株。荧光显微镜观察HSP72的定位，RT—PCR、蛋白质印迹法检测转染细胞HSP27的表达。结果双酶切法鉴定和测序证实重组载体pEGFP—C1一HSP27的DNA序列完全正确，并成功转染SW1990细胞，筛选获得稳转细胞株。荧光显微镜见EGFP主要分布在胞质中，转染细胞的HSP27 mRNA表达明显增加（1．458±0．160比0．897±0．051，P〈0．05），且表达HSP27与EGFP的融合蛋白。结论成功构建真核表达载体pEGFP—C1-HSP27，并筛选获得稳转的细胞株。

Objective To construct pEGFP-C1-HSP27 recombinant eukaryotic expression vector and establish human pancreatic cancer SW1990 cell line stably expressing HSP27. Methods RT-PCR was applied to amplify human HSP27 cDNA from human pancreatic cancer SW1990 cells with a pair of specific primers carrying a restriction enzyme site BamH I or Hind 111 on each 5＇ end. HSP27 cDNA was inserted into pEGFP- C1 vector and then identified by restriction enzyme digestion and sequencing. Successful constructed pEGFP- C1-HSP27 or empty vector was transfected into SW1990 cells by lipofectamine 2000, respectively. The location of HSP72 was determined by fluoroscopy, RT-PCR and Western blot was used to detect the expression of HSP27 in transfected cell. Results The DNA sequence of pEGFP-C1-HSP27 recombinant plasmid was completely correct, and it was successfully transfected into SW1990 cell lines and stably transfected SW1990 cell lines were obtained, which were confirmed by restriction enzyme and sequencing. The expression of EGFP was distributed in cytoplasm, the HSP27mRNA expression was significantly increased （1.458 ± 0. 160 vs 0. 897±0.051, P 〈0.05）. In addition, it was showed that EGFP-HSP27 fusion protein was expressed. Conclusions The eukaryotic expression vector pEGFP-C1-HSP27 was constructed successfully and stably transfected SW1990 cell line expressing HSP27 was obtained.