慢病毒介导稳定表达MCM7、CDC6细胞株的建立

Establishment of lentivirus-mediated MCM7 or CDC6-overexpressing stable cell lines

[目的]构建包装微小染色体维持蛋白7(Mcm7)、细胞分裂周期蛋白6(Cdc6)过表达慢病毒,并筛选Hep G2和LO2稳定过表达细胞株。[方法]通过PCR获得人的mcm7、cdc6基因,采用双酶切法构建重组慢病毒过表达载体。慢病毒过表达载体与ps PAX2、p MD2. G包装质粒共转染293T细胞后包装出慢病毒。慢病毒感染Hep G2和LO2后,通过有限稀释法筛选获得稳转细胞株,再经q PCR以及Western Blot鉴定过表达效果,FACS检测周期分布。[结果]成功构建并包装出了慢病毒,慢病毒滴度为1. 3×106TU/m L,感染Hep G2和LO2细胞后筛选获得稳转细胞株,并从mRNA和蛋白水平验证,Mcm7稳转株过表达效率超过0. 4倍,Cdc6稳转株过表达效率超12倍,稳转株周期分布与母代细胞没有显著差异(P> 0. 05)。[结论]Mcm7、Cdc6稳转株构建成功,稳转株过表达效果明显,Mcm7、Cdc6的稳定过表达没有扰乱细胞周期进程。

[ Objective ] To prepare Minichromosome maintenance protein 7 （Mcm7） and cell division cycle 6 （Cdc6） over - expressing lentivirus and establish stable cell lines of HepG2 and LO2. [ Methods ] The genes of MCM7 and CDC6 were ob- tained by PCR, and then the recombinant lentiviral overexpression vector was obtained by double enzyme digestion methods, the lentiviral overexpression vector was transfected with psPAX2 and pMD2. G plasmids into 293T cells to package lentivirus. HepG2 and LO2 cells were infected with the lentivirals, and stable cell line were selected by limited dilution methods. The MCM7 and CDC6 expression in the stable cell lines were examed by qPCR and Western Blot methods, and cell cycle distribu- tion were analyzed by FACS. [ Results ] The lentiviral overexpression vector was successfully constructed, and Lentivirus titer was 1.3 x 106 TU/mL. After infection,MCM7 or CDC6 were overexpressing in HepG2 and LO2 stable cell lines,The relative expression of stables cell lines were detected base on the levels of mRNA and protein, Mcm7＇ s overexpression efficiency was more than O. 4 times, Cdc6＇ s overexpression efficiency was more than 12 times. Cell cycle profiles showed no significan differ- ence between stable cell lines and parents cells （ P 〉 0.05 ）. [ Conclusion ] lentivirus - mediated MCM7 or CDC6 - overex- pressing HepG2 and LO2 stable cell lines were prepared, and the overerexpression efficiency of stable cell lines was very significant, cell cycle process was not disturbed by overexpression of MCM7 and CDC6.