靶向MA104细胞多聚嘧啶序列结合蛋白shRNA慢病毒干扰质粒的构建及鉴定  

Construction and identification of lentiviral shRNA interference plasmid targeting polypyrimidine tract-binding protein in MA104 cells

作　　者：周艳[1] 解裕萍 吴晋元[1] 闫姗姗[1] 张磊[1] 李鸿钧[1] 

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所云南省重大传染病疫苗研发重点实验室,云南昆明650118

出　　处：《中国生物制品学杂志》2016年第1期7-12,共6页Chinese Journal of Biologicals

基　　金：云南省科技计划项目(2014BC008);"协和青年基金资助";"中央高校基本科研业务费专项资金资助"(33320140082)

摘　　要：目的构建靶向MA104细胞多聚嘧啶序列结合蛋白(polypyrimidine tract-binding protein,PTB)的shRNA慢病毒干扰质粒,并对其进行鉴定。方法根据Gen Bank中登录的PTB的基因序列设计并合成2条靶向MA104细胞PTB基因的shRNA干扰序列(shPTB1、shPTB2)和1条阴性对照序列(shControl),分别插入载体p LVshRNA-EGFP(2A)Puro后,构建慢病毒重组质粒;利用HEK293Ta细胞包装针对PTB基因的shRNA的重组慢病毒颗粒;感染MA104细胞后,经Resl-time PCR、免疫荧光和Western blot法分别检测MA104细胞中PTB基因的转录和蛋白表达水平。结果 PCR及测序鉴定证明3种重组慢病毒质粒构建正确。3种重组慢病毒质粒转染HEK293Ta细胞48 h后,均可见绿色荧光表达,3组细胞的病毒滴度均为2×106 TU/ml。3种重组慢病毒感染MA104细胞48 h后均可见绿色荧光表达。经嘌呤霉素加压筛选后,p LVshPTB1-EGFP-2A和p LVshPTB2-EGFP-2A组MA104细胞仅有少量细胞于细胞核中表达,p LVshPTB1-EGFP-2A组MA104细胞中PTB基因转录及蛋白表达的水平均低于p LVshPTB2-EGFP-2A组。结论成功构建了靶向MA104细胞PTB蛋白的shRNA重组慢病毒,shRNA序列可成功下调PTB在MA104细胞中的表达,为PTB在RV增殖过程中调控作用的研究奠定了基础。Objective To construct and identify the lentiviral shRNA interference plasmid targeting polypyrimidine tractbinding protein（PTB） in MA104 cells. Methods Two shRNA interference sequences targeting PTB in MA104 cells（shPTB1 and shPTB2）and a negative control sequence（shControl）were designed and synthesized according to the PTB gene sequence in Gen Bank, and inserted into lentiviral vector p LVshRNA-EGFP（2A） Puro respectively, and the constructed recombinant lentiviral plasmids were packaged in HEK293 Ta cells. MA104 cells were infected with the obtained recombinant lentiviruses, and determined for PTB gene transcription and protein expression levels by real-time PCR, IFA and Western blot. Results PCR and sequencing proved that three recombinant lentiviral plasmids were constructed correctly. Green fluorescence was observed in HEK293 Ta cells 48 h after transfection with the three recombinant lentiviral plasmids, and all the virus titers in the transfected cells were 2 × 106 TU / ml. Green fluorescence was also observed in the MA104 cells 48 h after infection with three recombinant lentiviruses. After pressure screening with puromycin, PTB was only expressed in the nuclei of a small quantity of MA104 cells transfected with recombinant plasmids p LVshPTB1-EGFP-2A and p LVshPTB2-EGFP-2A. However, the PTB gene transcription and protein expression levels in MA104 cells transfected with recombinant plasmid p LVshPTB1-EGFP-2A were lower than those with p LVshPTB2-EGFP-2A. Conclusion The lentiviral shRNA interference plasmid targeting PTB in MA104 cells was successfully constructed, and shRNA down-regulated the expression of PTB in MA104 cells successfully, which laid a foundation of study on regulatory effect of PTB in proliferation of rotavirus.

关 键 词：MA104细胞 多聚嘧啶序列结合蛋白 慢病毒质粒 RNA干扰 

分 类 号：Q782[生物学—分子生物学]