重组人凝血因子Ⅸ小基因及其无义突变体稳定细胞株的构建及意义

摘要

本研究构建重组人凝血因子Ⅸ小基因及其无义突变体稳定细胞株并探讨其意义.将人凝血因子Ⅸ(F9)小基因克隆到哺乳动物表达载体pCMV-Tag3B上；利用PCR定点突变技术得到在小基因121位氨基酸位置含有一个提前终止密码子(premature termination codon,PTC)的无义突变体；将构建的F9小基因及其无义突变体分别转染人肝癌细胞HepG2,经G418抗性筛选,单克隆化扩大培养,得到稳定细胞株,分别命名为HepG2-WT和HepG2-N.结果显示:通过酶切鉴定、PCR鉴定及DNA测序分析证明,小基因及其无义突变体表达载体构建成功；通过RT-PCR及基因组PCR均扩增到了大小正确的目的基因片段,证明了含有重组人凝血因子Ⅸ小基因及其无义突变体的稳定细胞株构建成功.结论:本研究成功构建了重组人凝血因子Ⅸ小基因及其无义突变体稳定细胞株,该细胞株可用于无义突变导致血友病的治疗药物的筛选和PTC通读药物的筛选等.%This study was purposed to construct the recombinant hF9 minigene and its stable nonsense mutant cell lines, and to investigate its significance. Minigene hF9 was cloned into the mammalian expression vector pCMV-Tag3B; a nonsense mutant containing a premature termination codon (PTC) in the 121st amino acid residue was obtained by PCR site-directed mutagenesis; minigene hF9 and nonsense mutant were respectively transfected into HepG2 cells with G418 treatment to get stable HepG2-WT and HepG2-N cell lines. The results confirmed that the minigene hF9 and nonsense mutant were constructed successfully. The gene of interest was amplified by RT-PCR from the stable cell lines, and the minigene hF9 was expressed in the stable cell lines. It is concluded that the recombinant hF9 minigene and its stable non-seuse mutant cell lines are constructed successfully. The cell lines can be used to screen the drugs treating the nonsense mutation-caused hemophilia according to PTC read-through approaches.