慢病毒介导稳定表达FRT-LacZ基因的MDCK细胞株构建  

Establishment of MDCK cell lines which stably express FRT-LacZ gene by lentiviral system

作　　者：谢宝明[1] 于国伟[2] 令世鑫[1,3] 马忠仁[1,3] 柏家林[1,3] 

机构地区：[1]西北民族大学生命科学与工程学院,甘肃兰州730030 [2]西北民族大学医学院,甘肃兰州730030 [3]甘肃省动物细胞工程技术研究中心,甘肃兰州730030

出　　处：《生物技术》2016年第1期23-28,69,共7页Biotechnology

基　　金：教育部创新团队发展计划("动物医学生物工程创新团队";No.IRT13091);国家自然科学基金项目("脂联素介导绵羊肌内;内脏和皮下脂肪细胞生脂的基因表达模式与分子调控";No.31260533);甘肃省自然科学基金项目("绵羊脂联素及其受体基因表达模式研究";No.1208RJZA188);甘肃省科技支撑计划项目("CHO基因工程细胞株构建及其生物反应器悬浮培养研究";No.1204FKCA182);校企合作项目("应用基因工程技术构建MDCK悬浮细胞应用于疫苗生产";No.h2011-19);西北民族大学研究生科研(实践)创新项目("新型γ-PGA高效工程菌株构建";No.Yxm2014188)资助

摘　　要：[目的]通过慢病毒载体系统获得稳定表达FRT-LacZ基因的MDCK细胞株。[方法]PCR扩增FRT-LacZ基因,亚克隆至慢病毒载体p LVX-PGK-Puro。经四质粒包装系统共转染293FT细胞包装重组慢病毒并检测病毒滴度,获得的慢病毒感染MDCK细胞,经嘌呤霉素筛选和β-半乳糖苷酶原位染色检测筛选鉴定转基因细胞株。[结果]测序证实,成功构建重组慢病毒质粒p LVX-FRT-LacZ-PGK-Puro。包装产生的慢病毒滴度为4.04×107TU/m L,重组慢病毒感染MDCK细胞后筛选获得稳定表达FRT-LacZ基因的MDCK细胞株。[结论]构建了稳定表达FRTLacZ基因的MDCK细胞株。[ Objective] The purpose is to establish MDCK cell lines stably expressing FRT- LacZ gene mediated by lentiviral system. [ Methods] The full -length sequence of FRT- LacZ gene was amplified by PCR and then subcloned into the Lentivi- ral vector pLVX - PGK - Puro. The recombinant Lentivirus was generated by co - transfection of four - plasmids into 293FT cells. The titer of virus was tested according to the expression level of LacZ. MDCK was infected by recombinant virus and stable cell line expressed FRT - LacZ gene was isolated by selecting with puromycin and identifying by $ - galactosidase staining. [ Results] DNA sequencing demonstrated that the lentivirus vector pLVX - FRT - LacZ - PGK - Puro was constructed success- fully The titer of lentivirus rLV - FRT - LacZ was 4.04 x 107TU/mL. The MDCK cell line was infected by rLV - FRT - LacZ and stable transfected cell line stably expressing FRT - LacZ gene was selected. [ Conclusion] MDCK cell line stably express- ing FRT- LacZ gene was established.

关 键 词：FRT-LacZ基因 慢病毒载体 MDCK细胞 位点特异性重组 

分 类 号：Q812[生物学—生物工程]