MIG7-shRNA逆转录病毒表达载体的构建及包装细胞株的建立  

Construction of MIG7-shRNA retroviral vector and the establishment of its package cell lines

作　　者：余飞[1] 盛冠男 陈冠男[2] 王南南[2] 宁莉莉[3] 王宇鹏[4] 屈波[2] Yu Fei;Sheng Guannan;Chen Guannan;Wang Nannan;Ning Lili;Wang Yupeng;Qu Bo(The Seventh Affiliated Hospital,Sun Yat-sen University,Guangdong Shenzhen 518107,China;Department of Oncological Surgery,Affiliated Hospital of Logistics University of PAPF,Tianjin 300162,China;Department of Scientific Research,Affiliated Hospital of Logistics University of PAPF,Tianjin 300162,China;Department of Oncology,Tianjin Beichen Hospital,Tianjin 300400,China)

机构地区：[1]中山大学附属第七医院,广东深圳518107 [2]武警后勤学院附属医院肿瘤外科,天津300162 [3]武警后勤学院附属医院科研科,天津300162 [4]天津市北辰医院肿瘤科,天津300400

出　　处：《现代肿瘤医学》2019年第10期1669-1674,共6页Journal of Modern Oncology

基　　金：国家自然科学基金面上项目(编号:81272547);天津市科技计划项目(编号:15ZXLCSY00040)

摘　　要：目的:构建迁移诱导基因(migration inducing gene 7,MIG7)的逆转录病毒表达载体并测序,初步建立其包装细胞株。方法:根据MIG7已知序列设计合成所需脱氧寡核苷酸序列,并与线性RNAi-Ready pSIREN-RetroQ-ZsGreen载体连接。PCR扩增热转化后的连接产物,检测和挑取阳性克隆和阳性菌落,提取质粒后测序,与Gen-Bank中特异性MIG7-shRNA基因序列对比。包装并获取MIG7-shRNA重组逆转录病毒,qRT-PCR测定MIG7-shRNA重组逆转录病毒滴度并转染MHCC-97H细胞株。结果:构建的MIG7-shRNA表达载体与设计的完全一致,成功获得MIG7-shRNA逆转录病毒表达载体质粒CTC698-1-1和CTC698-2N-1转染的PT67细胞克隆株,pSIREN-M1病毒和pSIREN-MN病毒颗粒数约为1.0×10~8v.p/ml,能高效转染MHCC-97H细胞株。结论:成功构建了MIG7的逆转录病毒表达载体并建立其包装细胞株。Objective:To construct the retrovirus expression vector of migration inducing gene 7(MIG7),then it will be sequence-confirmed and its package cell lines will be established.Methods:The DNA sequence which was needed was designed and synthesized according to the known MIG7 sequence.And it jointed with the linear RNAi-Ready pSIREN-RetroQ-ZsGreen vector.The products were amplified by using polymerase chain reaction(PCR)after thermal conversion.The positive clones and colony of bacteria were tested and selected.They were sequenced after the extraction of plasmid,and compared with the MIG7-shRNA in Gen-Bank.The retrovirus expression vector of MIG7 was packed and obtained.The viral titer was detected by qRT-PCR.Results:We established the PT67 package cell lines which were infected by the MIG7-shRNA vector CTC698-1-1 and CTC698-2N-1 successfully.The pSIREN-M1 and pSIREN-MN average viral titer was 1.0×10 8v.p/ml.Conclusion:The MIG7 retrovirus expression vector has been successfully constructed and its package cell line PT57-MIG7 has been established.

关 键 词：迁移诱导基因 逆转录病毒 载体构建 

分 类 号：R73-3[医药卫生—肿瘤]