牛源TAK1基因慢病毒载体构建及其在MDBK细胞中的表达  

Construction of Lentivirus Vector of Bovine TAK1 Gene and Its Expression in MDBK cells

作　　者：张帆 姜坤生 王禹淳 马金柱[1] 于立权[1] 宋佰芬[1,2] ZHANG Fan;JIANG Kun-sheng;WANG Yu-chun;MA Jin-zhu;YU Li-quan;SONG Bai-fen(College of Life Science and Technology,Heilongjiang Bayi Agricultural University,Daqing,Heilongjiang,163319,China;College of Veterinary Medicine,China Agricultural University,Beijing,100093,China)

机构地区：[1]黑龙江八一农垦大学生命科学技术学院,中国大庆163319 [2]中国农业大学动物医学院,中国北京100093

出　　处：《动物医学进展》2023年第9期6-11,共6页Progress In Veterinary Medicine

基　　金：国家自然科学基金项目(32272991)。

摘　　要：为了探究转化生长因子-β激活蛋白1(TAK1)在牛疱疹病毒1型(BHV-1)感染中的作用,构建牛源TAK1基因慢病毒载体,筛选TAK1基因稳转过表达细胞株。根据GenBank中牛源TAK1基因序列(登录号:NM-001081595.1)设计引物,PCR扩增目的片段后插入载体中,构建pCDH-CMV-TAK1-MCS-EF1-turboRFP-T2A-Puro慢病毒质粒,将其导入293T细胞进行慢病毒包装及滴度测定。用包装好的慢病毒感染牛肾上皮细胞(MDBK),筛选过表达TAK1基因的稳转细胞株,通过荧光显微镜和免疫印迹试验(Western blot)进一步验证该基因的过表达。重组质粒双酶切与测序结果表明成功建立了pCDH-CMV-TAK1-MCS-EF1-turboRFP-T2A-Puro慢病毒质粒。慢病毒滴度测定结果显示,过表达TAK1基因组病毒滴度为1.0×108 TU/mL,空载体对照组病毒滴度为5.0×108 TU/mL。重组慢病毒以MOI 180转染MDBK细胞,荧光显微镜观察到明显的红色荧光,表明慢病毒质粒转染成功。Western blot结果显示,在相应分子量处检测到目的条带,进一步表明TAK1基因被表达。成功构建了pCDH-CMV-TAK1-MCS-EF1-turboRFP-T2A-Puro慢病毒质粒,并筛选出了过表达TAK1基因的MDBK稳转细胞株,为深入研究TAK1在BHV-1感染中机制的研究奠定基础。In order to investigate the role of transforming growth factor-beta activator protein 1(TAK1)in bovine herpesvirus type 1(BHV-1)infection,lentivirus vector of bovine TAK1 gene was constructed and stably over-expressed cell lines of TAK1 gene were screened.According to the sequence of bovine TAK1 gene in GenBank(accession number:NM-001081595.1),primers were designed,and the target fragment was amplified by PCR and inserted into vector to construct pCDH-CMV-TAK1-MCS-EF1-turboRFP-T2A-Puro lentivirus plasmid,and introduced into 293T cells for lentivirus packaging and titer determination.Bovine kidney epithelial cells(MDBK)were infected with packaged lentivirus,and stably transformed cell lines with overexpression of TAK1 gene were screened.The overexpression of TAK1 gene was further verified by fluorescence microscope and Western blot.The results of double enzyme digestion and sequencing showed that pCDH-CMV-TAK1-MCS-EF1-turboRFP-T2A-Puro lentivirus plasmid was successfully established.The results of lentivirus titer assay showed that the titer of overexpressed TAK1 genome was 1.0×10^(8)TU/mL,and that of empty vector control group was 5.0×10^(8)TU/mL.The recombinant lentivirus was transfected into MDBK cells with MOI 180,and the red fluorescence was observed by fluorescence microscope,which indicated that the lentivirus plasmid was successfully transfected.Western blot showed that the target band was detected at the corresponding molecular weight,which further indicated that TAK1 gene was overexpressed.In this study,pCDH-CMV-TAK1-MCS-EF1-turboRFP-T2A-Puro lentivirus plasmid was successfully constructed,and MDBK stable cell line overexpressing TAK1 gene was screened,which laid a foundation for further study on the mechanism of TAK1 in BHV-1 infection.

关 键 词：TAK1基因 过表达 慢病毒载体 MDBK细胞 

分 类 号：S855.3[农业科学—临床兽医学]