利用慢病毒载体建立过表达Nfe2l2基因的L929细胞株实验研究  

Establishment of L929 Cell Line Overexpressing Nfe2l2 Gene Using Lentiviral Vectors:an Experimental Study

作　　者：刘成[1] 汤剑明[1] 李洋[1] 杨将[1] 李倩男[1] 刘耀丹[1] 洪莉[1] LIU Cheng;TANG Jianming;LI Yang;YANG Jiang;LI Qiannan;LIU Yaodan;HONG Li(No.2 Department of Gynecology,Renmin Hospital of Wuhan University,Wuhan 430060,China)

机构地区：[1]武汉大学人民医院妇二科,湖北省武汉市430060

出　　处：《中国全科医学》2018年第30期3723-3729,共7页Chinese General Practice

基　　金：国家自然科学基金青年科学基金资助项目(81701424);中央高校基本科研业务费专项资金青年教师资助项目(2042017kf0114)

摘　　要：目的利用慢病毒载体建立过表达Nfe2l2基因的L929细胞株,为探讨Nfe2l2基因在结缔组织成纤维细胞细胞外基质(ECM)重构中的作用奠定实验基础。方法 2014年12月—2015年5月,通过双酶切将Nfe2l2目的基因连接到GV341载体(Ubi-MCS-3FLAG-SV40-puromycin),经扩增、转化、验证、测序、质粒抽提,将携带目的基因的工具GV341载体质粒及病毒包装辅助质粒Helper1.0、Helper2.0转染293T细胞,完成慢病毒包装及质量检测后获取慢病毒工具载体LV-Nfe2l2,病毒感染L929细胞72 h后筛选稳定表达的细胞株L929/LV-Nfe2l2,采用Realtime qPCR测定Nfe2l2的表达。结果经比对,构建的LV-Nfe2l2阳性克隆序列与目的基因Nfe2l2相符;L929细胞达到80%感染效率的最佳感染条件为:在ENi.S培养基中进行感染,设定感染复数(MOI)为8～10,感染时间为72 h。Real-time qPCR结果显示,经筛选的L929/LV-Nfe2l2细胞中Nfe2l2基因表达丰度为高(ΔCt值≤12)。结论本研究成功构建了一种过表达Nfe2l2基因的慢病毒载体;LV-Nfe2l2感染L929细胞后可实现目的基因的高表达,经筛选的L929/LV-Nfe2l2细胞株可用于后续实验研究。Objective To establish a L929 cell line overexpressing Nfe2l2 gene using lentiviral vectors,laying a basis for investigating the role of Nfe2l2 gene in ECM remodeling.Methods From December 2014 to May 2015,Nfe2l2 gene sequence was ligated into the GV341 vector(Ubi-MCS-3FLAG-SV40-puromycin)via restriction enzyme digestion.After amplification,transformation,verification,sequencing and plasmid extraction,the GV341 vector plasmids carrying the target gene,together with lentiviral packaging plasmids Helper 1.0 and Helper 2.0 were used to co-transfect 293T cells,and the lentiviral vector LVNfe2l2 was harvested after packaging and virus concentration test.After being infected by LV-Nfe2l2 for 72 h,the L929 cells were screened for stable L929/LV-Nfe2l2 cell lines.The expression of Nfe2l2 was determined by real-time qPCR.Results Sequence analysis showed that the positive clone of LV-Nfe2l2 was consistent with the target gene Nfe2l2.The optimal conditions for L929 cells to achieve 80%infectious efficiency are as follows:ENi.S medium,MOI of 8-10,infection time of 72 h.Real-time qPCR showed that the abundance of Nfe2l2 gene in L929/LV-Nfe2l2 cells was high(ΔCt≤12).Conclusion In this study,we successfully established a lentiviral vector overexpressing Nfe2l2 gene;target genes with high expression levels could be obtained by LV-Nfe2l2 infected L929 cells;the screened L929/LV-Nfe2l2 cell line can be used in further experimental studies.

关 键 词：转染 慢病毒属 Nfe2l2 成纤维细胞 细胞外基质 

分 类 号：R394[医药卫生—医学遗传学]