真核表达载体pcDNA3．1-RASSF1的构建及其对肝癌细胞凋亡的影响

目的构建pcDNA3．1－RASSFl真核表达载体，并检测其对肝癌细胞系HepG2凋亡的影响。方法应用聚合酶链反应技术从人RASSFlcDNA中扩增出RASSF1基因后，以内切酶XhoI和EcoRI进行双酶切，将其克隆人用相同酶处理的载体pcDNA3．1；将重组质粒pcDNA3．1－RASSF1转染肝癌HepG2细胞，应用Westernblot法检测RASSF1的表达水平；用AnnexinV／PI法检测细胞的凋亡情况。结果酶切和测序结果表明，重组质粒pcDNA3．1．RASSF1构建成功；Westernblot法结果表明转染pcDNA3．1-RASSF1后HepG2细胞中RASSFl表达升高；AnnexinV／PI法用流式细胞术检测凋亡，空白组、pcDNA3．1组和pcDNA3．1-RASSFl组HepG2细胞的凋亡率分别为（5．8±0．42）％、（7．48±0．68）％和（35．1±3．15）％。结论真核表达载体pcDNA3．1-RASSF1构建成功；RASSF1蛋白在HepG2细胞中高表达，并促进细胞凋亡。

The construction of pcDNA 3.1 RASSF1 eukaryotic vector and the influence of RASSF1 on apoptosis of hepatocarcinoma cell line

Objective To construct pcDNA3.1 RASSF1 eukaryotic vector and observe the influence of RASSF1 on the apoptosis of hepatocarcinoma cell line HepG2. Methods RASSF1 gene was amplifled from human RASSF1 cDNA by polymerase chain reaction (PCR) and cloned into pcDNA3.1. The recombinant plasmid pcDNA3. 1 RASSF1 was transfected into hepatocarcinoma HepG2 cell line. The expression of RASSF1 was examined by Western blot. The influence of RASSF1 on the cell apoptosis was measured by Annexin V/PI assay. Results DNA enzyme digestion and sequencing results showed that recombinant plasmid pcDNA3. 1-RASSF1 was successfully constructed. RASSF1 protein was overexpressed in HepG2 cell line transfected with pcDNA3. 1-RASSF1 plasmid. The apoptosis rate of blank, pcDNA3. 1 and pcDNA3. 1-RASSF1 group was (5.8 ±0.42)%, (7.48 ±0.68)% and (35. 1 ±3. 15)%, respectively.Conclusion The pcDNA3. 1- RASSF1 eukaryotic vector was successfully constructed, RASSF1 protein overexpression could induce apoptosis in HepG2 cell line.