JSRV-env慢病毒过表达载体的构建及BEAS-2B细胞稳转株的建立  

Construction of JSRV-env lentiviral overexpression vector and establishment of stably transfected strain of BEAS-2B cell

作　　者：杨小林 杜晓悦 段续接 张培 陈思旭 王振玲[3] 刘淑英[1,2] YANG Xiaolin;DU Xiaoyue;DUAN Xujie;ZHANG Pei;CHEN Sixu;WANG Zhenling;LIU Shuying(Ministry of Agriculture Key Laboratory for Clinical Diagnosis and Treatment of Animal Diseases,College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot 010018,China;Key Laboratory for Basic Veterinary Medicine of Inner Mongolia Autonomous Region,College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot 010018,China;Beijing Vocational College of Agriculture,Beijing 102442,China)

机构地区：[1]内蒙古农业大学兽医学院农业部动物临床诊疗技术重点实验室,内蒙古呼和浩特010018 [2]内蒙古农业大学兽医学院内蒙古自治区基础兽医学重点实验室,内蒙古呼和浩特010018 [3]北京农业职业学院,北京102442

出　　处：《中国兽医学报》2023年第11期2228-2236,共9页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(32072819);内蒙古科技重大专项计划资助项目(2021ZD0010);牛羊疫病防控与发育工程创新团队资助项目(2200001130);内蒙古草原英才创新团队资助项目(20151031)。

摘　　要：通过构建过表达JSRV-env的慢病毒载体,筛选获得稳定表达Env的人支气管上皮细胞株(human bronchial epithelial cells-2b,BEAS-2B),检测Env对细胞增殖能力和迁移能力的影响。PCR扩增目的基因env,将其插入pMT194质粒中,构建pMT194-JSRV-env慢病毒过表达质粒,将重组质粒同包装辅助质粒混合后与转染试剂复合物共转染293T细胞。在收集、纯化浓缩病毒后,感染BEAS-2B细胞并优化感染条件。根据慢病毒载体携带的抗性基因puro,用嘌呤霉素(Puro)筛选稳定细胞株。通过qPCR和Western blot从转录和翻译水平检测目的基因表达,通过CCK-8检测细胞增殖能力和划痕试验检测细胞迁移能力。结果显示:目的基因env扩增成功,并将其重组插入pMT194载体,酶切鉴定与预期相符,pMT194-JSRV-env表达载体构建成功;转染293T细胞env表达量极显著高于空白对照组和阴性对照组(P<0.001),JSRV Env重组慢病毒平均滴度为1.03×10~9 IU/mL;慢病毒感染BEAS-2B细胞的最佳感染复数(multiplicity of infection,MOI)为40,筛选细胞株的最佳Puro质量浓度为0.3 mg/L;荧光显微镜显示BEAS-2B细胞表达红色荧光;qPCR和Western blot检测结果均表明env表达量在试验组中极显著高于空白对照组和阴性对照组(P<0.001),Env-FLAG融合蛋白在宿主细胞成功表达;相较于空白对照组和阴性对照组,试验组细胞的增殖能力及迁移能力显著增强(P<0.05)。结果表明,成功构建JSRV-env慢病毒过表达载体,筛选出稳定表达Env的BEAS-2B细胞株,Env能够显著增加BEAS-2B细胞的增殖和迁移能力。To construct a lentiviral vector overexpressing JSRV-env,screen human bronchial epithelial cells-2b(BEAS-2B) stably expressing Env,and detect the effect of Env on cell proliferation and migration.The target gene env was amplified by PCR and inserted into the pMT194 plasmid to construct the pMT194-JSRV-env lentiviral overexpression plasmid.The recombinant plasmid was mixed with the packaging auxiliary plasmid and co-transfected with the transfection reagent complex into 293T cells.After collection,purification and concentration of the recombinant lentivirus,the virus was used to infect BEAS-2B cells,the infection conditions were optimized.The stable cell strain was screened by puromycin(Puro)according to the resistance gene puro carried by the lentiviral vector.The expression of the target gene was detected by qPCR and Western blot at the transcriptional and translational levels.The cell proliferation ability was detected by CCK-8and the cell migration ability was detected by scratch test.The target gene env was successfully amplified and inserted into the PMT194vector.The enzyme digestion identification was consistent with the expectation,and the PMT194-JSRV-env expression vector was successfully constructed.The expression of envin 293Tcells was significantly higher than that in blank control group and negative control group(P<0.001).The average titer of JSRV Env recombinant lentivirus was 1.03×109IU/mL.The optimal multiplicity of infection(MOI)of the recombinant lentivirus on BEAS-2B cells was 40,the optimal Puro concentration for screening cell strain was 0.3mg/L,and fluorescence microscopy showed that BEAS-2Bcells expressed red fluorescence.The results of qPCR and Western blot showed that the expression of envin the experimental group was significantly higher than that in the blank control group and the negative control group(P<0.001).The Env-FLAG fusion protein was successfully expressed in the host cells.Compared with the blank control group and the negative control group,the proliferation and migration abi

关 键 词：慢病毒载体 稳转株 囊膜蛋白 增殖 迁移 

分 类 号：S852.65[农业科学—基础兽医学]