山羊痘病毒G14-STV44-55疫苗株表达载体的构建  

Construction of GTPV G14-STV44-55 strain expression vector

作　　者：李有文[1,2] 魏风[1] 陈创夫[1] 王远志[1] 张辉[1] 任艳[1] 郭志儒[1] 

机构地区：[1]新疆石河子大学动物科技学院,新疆石河子832003 [2]新疆塔里木大学动物科学学院,新疆阿拉尔843300

出　　处：《中国兽医学报》2011年第9期1270-1275,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30560109);校长基金重点项目(TDZKZD08001)

摘　　要：PCR扩增山羊痘病毒疫苗株G14-STV44-55的TK(Thymidine kinase)基因作为侧翼序列;用XhoⅠ/NotⅠ消化含有报告基因的质粒pLSEG得到痘苗病毒(Vaccinia virus,VV)p7.5启动子调控大肠杆菌黄嘌呤-鸟嘌呤磷酸核糖基转移酶(E.coli.gpt)作为报告基因;人工合成VV I1L启动子用于调控表达外源基因,用Overlap PCR的方法将VV I1L启动子和VV p7.5启动子调控的gpt连接但使两启动子转录方向相背,并在VV I1L启动子下游引入XhoⅠ位点作为外源基因的插入位点,构建成表达载体元件I1L-P7.5-GPT。将I1L-P7.5-GPT插入TK基因的Acc65Ⅰ位点,构建成表达载体。将构建的载体转染山羊痘病毒G14-STV44-55株感染的羔羊睾丸细胞,用含有霉酚酸(Mycophenolic acid,MPA)等筛选试剂的培养基筛选重组山羊痘病毒(rGTPV)。结果显示,构建成以TK基因为侧翼、以VV I1L启动子调控目的基因,以gpt为报告基因的表达载体pGEM-TK-I1L-GPT,成功筛选到表达gpt的rGTPV,rGTPV至少可稳定传代15代。结果表明,构建了我国山羊痘病毒G14-STV44-55疫苗株的表达载体;TK基因是GTPV G14-STV44-55疫苗株的复制非必需区,以TK基因为插入位点获得的重组病毒可稳定传代15代以上。The thymidine kinase（TK） gene of G14-STV-44-55 strain was amplified from viral genome by PCR,and used as flanking sequence for transfer vector construction after sequence confirmation.The vaccinia virus p7.5 promoter regulated xanthine-guanine phosphoribosyltransferase（gpt） was obtain by XhoⅠ/NotⅠ digestion of plasmid pLESD,and used as positive selection marker for recombinant virus screening.The VV I1L promoter and downstream Xho Ⅰ site was synthesized for regulation/insertion of target gene.Then the p7.5-gpt and VV I1L was put at opposite transcription orientation by overlap PCR,resulted in a transfer vector element I1L-P7.5-GPT.This constructed element was inserted in the TK gene of strain G14-STV-44-55 subsequently for construction of transfer vector.Then the vector was transfected into lamb testis（LT） cells pre-infected with GTPV G14-STV-44-55 for homologous recombination.Recombinant virus was selected with cultured media containing mycophenolic acid（MPA） and confirmed by PCR.A transfer vector,which contains the TK gene as flanking sequence,the VV I1L promoter for regulation target gene,and p7.5 regulated gpt as selection marker was constructed.A recombinant GTPV（rGTPV） strain which genome contained the constructed transfer element I1L-P7.5-GPT within the interrupted TK gene,was obtain after homologous recombination and MPA selection.The rGTPV still contain the inserted sequence after 15 passages.A transfer vector of GTPV strain G14-STV-44-55 was constructed and named as pGEM-TK-I1L-GPT.The TK gene of G14-STV-44-55 was confirmed as nonessential gene for G14-STV-44-55 replication,and rGTPV with inserted sequence within TK gene was stable for at least 15 passages when cultured in lamb testis（LT） cells.

关 键 词：山羊痘病毒 载体构建 启动子 报告基因 

分 类 号：S852.65[农业科学—基础兽医学]