人MCPH1基因重组慢病毒的制备及稳定感染HeLa细胞系的建立  

Construction of a recombinant lentivirus expressing MCPH1 and establishment of its stably infected HeLa cell line

作　　者：麦力[1] 张冀[1] 刘革力[1] 卜友泉[1] 李韵[1] 樊建军[1] 宋方洲[1] 

机构地区：[1]重庆医科大学分子医学与肿瘤研究中心、基础医学院生物化学与分子生物学教研室,重庆400016

出　　处：《重庆医科大学学报》2014年第3期290-294,共5页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:30800410);高等学校博士学科点专项科研基金资助项目(编号:20070631011)

摘　　要：目的 :构建人MCPH1(microcephalin 1)基因重组慢病毒载体并建立稳定过表达MCPH1基因的宫颈癌HeLa细胞株。方法:将MCPH1基因重组到pLenO-DCE质粒中,构建重组质粒pLenO-DCE-MCPH1,经酶切和测序鉴定后,与慢病毒包装质粒共转染293T细胞,包装重组慢病毒,滴度测定后,感染HeLa细胞,用有限稀释法筛选稳定的细胞克隆,real-time PCR和Western blot分别检测细胞中MCPH1 mRNA和蛋白水平。结果:经酶切和测序鉴定,成功构建MCPH1重组慢病毒载体,并成功包装慢病毒,滴度为1.24×109 TU/ml;慢病毒感染HeLa细胞后经有限稀释法成功筛选到MCPH1稳定表达细胞株pLenO-DCEMCPH1-HeLa;real-time PCR证实pLenO-DCE-MCPH1-HeLa细胞株较正常组HeLa(P=0.000)和pLenO-DCE-HeLa组(P=0.000)的MCPH1 mRNA水平显著地增高;Western blot进一步证实pLenO-DCE-MCPH1-HeLa细胞株过表达MCPH1。结论:成功构建MCPH1重组慢病毒,并建立了稳定表达MCPH1基因的细胞株pLenO-DCE-MCPH1-HeLa,为进一步研究MCPH1在宫颈癌中的作用奠定了基础。Objective ：To construct a recombinant lentiviral vector expressing microcephalin 1 （MCPH1） and to establish a human cervical cancer HeLa cell line stably overexpressing MCPH1. Methods：A recombinant lentiviral vector pLenO-DCE-MCPH1 was generated by cloning human MCPH1 gene into the lentiviral vector pLenO-DCE. Being indentified by restriction enzyme digestion and sequencing,the recombinant lentiviral vector pLenO-DCE-MCPH1 was packaged in 293T cells with the packaging plasmids including pRsv-REV, pMDlg-pRRE and pMD2G. AecoMing to the expression level of green fluorescent protein （GFP）, the viral titer was tested by flow cytometry. After being infected with the lentiviral suspension, HeLa cells stably overexpressing MCPH1 were selected by limiting dilution analysis. Real-time PCR and Western blot examined MCPH1 mRNA and protein levels of stably infected HeLa cells. Results：DNA restriction enzyme digestion and sequencing demonstrated that the pLenO-DCE-MCPHI recombinant lentiviral vector was constructed successfully. The recombinant lentivirus with a titer of 1.24 ×10^9 TU/ml was generated from 293T cells with the packaging system. HeLa cells were infected with pLenO-DCE-MCPH! recombinant lentivirus, and then a stable cell line,pLenO-DCE-MCPH1-HeLa,was established by limiting dilution method. Real-time PCR showed the expression of MCPHI mRNA in pLenO- DCE-MCPH1-HeLa was significantly higher than that of HeLa（P=0.000） and pLenO-DCE-HeLa（P=0.000）. Western blot also demonstrated MCPH1 protein expression was increased in pLen0-DCE-MCPH1-HeLa than in HeLa and pLenO-DCE-HeLa. Conclusions ：Recombinant lentivirus expressing MCPH1 and stable cell line pLen0-DCE-MCPH1-HeLa are successfully constructed,which is conducive to studying the function of MCPH1 in cervical cancer.

关 键 词：慢病毒 MCPH1基因 宫颈肿瘤 单克隆细胞 

分 类 号：R737-33[医药卫生—肿瘤] R-331[医药卫生—临床医学]