用pLNCX2携带人MCPH1基因shRNA重组逆转录病毒载体的构建及鉴定  

Construction and Identification of Recombinant Retrovirus Vector Ex- pressing shRNA Targeting Human MCPH1 Gene

作　　者：陈显兵[1] 刘锦红[1] 谭志鑫[1] 袁成福[1,2] 

机构地区：[1]湖北民族学院医学院病理学教研室,湖北省恩施市445000 [2]重庆医科大学生化与分子生物学教研室,重庆市430016

出　　处：《医学分子生物学杂志》2012年第1期16-20,共5页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.30800410）;湖北省教育厅（No.Q20092904）

摘　　要：目的建立逆转录病毒介导的MCPH1基因RNA干扰表达体系并观察其在宫颈癌Caski细胞中对MCPH1表达的影响。方法将人MCPH1基因RNA干扰双链DNA片段重组到逆转录病毒质粒pLNCX2中，构建携带人MCPHI基因RNA干扰的逆转录病毒载体pLNCX2-shRNA—MCPH1，经PT67细胞包装后，产生的重组逆转录病毒感染宫颈癌细胞株Caski细胞，并用G418筛选产生稳定的细胞克隆，用RT—PCR和Western印迹检测细胞中MCPH1mRNA和蛋白表达的变化。结果重组逆转录病毒质粒经测序鉴定正确，逆转录病毒感染Caski细胞后用G418筛选出稳定的细胞克隆，RT—PCR和Western印迹检测人MCPH1mRNA和蛋白表达水平明显低于阴性对照组和未干扰组。结论携带人MCPHI基因RNA干扰双链DNA片段的逆转录病毒感染Caski细胞后能明显抑制MCPHImRNA和蛋白表达，为进一步研究MCPH1在宫颈癌中的作用奠定了基础。Objective To construct a retrovirus-mediated expression system containing double strands DNA to interfere with the RNA of microcephalin （ MCPH1 ） gene and study its influence on the expression of MCPH1 in Caski cells. Methods A recombinant retrovirus vector pLNCX2-shR- NA-MCPH1 was generated by cloning a double strand DNA interfering with human MCPH1 RNA in- to the retrovirus vector pLNCX2. Caski cells were infected with virus supernatant produced from PT67 cells. The stable Caski cell clones were generated with G418 selection. MCPH! mRNA and protein levels in the stable infected Caski cells were examined by RT-PCR and Western Blot- ting. Results The pLNCX2-shRNA-MCPH1 recombinant retrovirus vector was constructed correctly as confirmed by sequencing. Stably infected Caski cell clones were generated after G418 selection, and displayed significant decrease in MCPH1 mRNA and protein expression. Conclusion The pLNCX2-shRNA-MCPH1 retrovirus vector shows effective interference on the expression of MCPH1 at mRNA and protein levels, enabling further study on MCPHI function in cervical cancer.

关 键 词：逆转录病毒载体 SHRNA MCPH1基因 宫颈癌 

分 类 号：R739.4[医药卫生—肿瘤]