甲羟戊酸激酶mvk基因的真核表达及其对bxpc-3增殖凋亡的影响.docVIP

甲羟戊酸激酶基因真核及shRNA表达载体构建及其对BXPC-3细胞周期蛋白表达的影响 金 锐，王 芳，栾 康，罗 欣，张胜权（安徽医科大学，生物化学与分子生物学教研室，合肥 230032） 文章亮点： 1 重点探讨参与甲羟戊酸代谢途径的关键酶甲羟戊酸激酶对胰腺癌细胞部分生理功能的作用及影响。 2 采用基因重组及koockdown技术构建基因表达载体，研究对胰腺癌细胞的影响。 3 甲羟戊酸激酶基因过高表达及低表达都很大程度抑制了胰腺癌细胞的周期蛋白表达。 摘要 背景：胰腺癌是人类常见的消化系统恶性肿瘤，前人研究发现其与甲羟戊酸途径有密切关联。 目的 构建甲羟戊酸激酶（Mevalonate kinase，MVK）基因真核及shRNA表达载体并转染至BXPC-3细胞中，观察其对细胞周期蛋白的影响。 方法 从BXPC-3细胞中提取总RNA, RT-PCR获得目的基因MVK以及通过化学合成MVK（751）和 MVK（1089）位点寡核苷酸片段，构建真核表达及shRNA表达载体并转染至BXPC-3细胞，筛选稳定表达细胞系。 Western Blot分析转染后对BXPC-3细胞周期蛋白的影响。 结果与结论 成功获得甲羟戊酸激酶基因（MVK）真核表达（pcDNA3.1-mvk）及两个针对MVK shRNA表达载体（piLenti-RNAi-shRNA1/2）。通过抗生素筛选及Western-blotting分析获得了稳定的MVK过表达及低表达细胞株。Western Blot分析发现：与正常及对照细胞相比，过表达MVK的细胞株及MVK knockdown的细胞株中细胞周期蛋白表达均显著降低。 关键词 MVK；真核表达；基因重组；寡核苷酸；细胞周期蛋白；稳定表达；BXPC-3；过表达；低表达；shRNA The construction of eukaryotic and shRNA expression vector of Mevalonate kinase (MVK) gene and its effect on cyclins expression in BXPC-3 Jin Rui, Wang Fang, Luan Kang, Luo Xin, Zhang Shengquan （ Dept of Biochemistry and Molecular biology, Anhui Medicial University, Hefei,230032） ABSTRACT BACKGROND: Pancreatic cancer is a common digestive system of human malignancies, previous studies found that it was closely associated with the mevalonate pathway. OBJECTIVE: To construct the eukaryotic expression and shRNA expression vectors of mevalonate kinase (MVK) gene and study the effect on cyclins expression in BXPC-3 cells . METHODS: Total RNA was extracted from BXPC-3 cells, the desired gene MVK was obtained by RT-PCR and?acquiring chemical synthesis MVK (751) and MVK (1089) locus oligo fragments. After construction of eukaryotic and shRNA expression vectors using the genetic engineering, the recombinant was transfected into BXPC-3 and cell lines of stable expression were selected by antibiotic. Western Blot analyzed the expression of MVK and cyclins in BXPC-3. RESULTS AND CONCLUSION: MVK eukaryotic (pcDNA3.1-mvk)and two shRNA (piLenti-RNAi-shRNA1/2)expression vectors for MVK were successfully constructed. The cell lines