猪繁殖与呼吸综合征病毒ORF7基因的克隆高效表达与鉴定.pdfVIP

30 1 大 连 工 业 大 学 学 报 Vol. 30 No. 1 2 0 1 1 1 Journal of Dalian Polytechnic University Jan. 2 0 1 1 : 2011) 猪繁殖与呼吸综合征病毒ORF7 基因的克隆高效表达与鉴定 1 2 2 2 2 3 张秀 云 , 吴 斌 , 肇 慧君 , 贾 赟 , 刘 霞 , 张 瑞 ( 1.大连工业大学 生物工程学院, 辽宁 大连 116034; 2.辽宁出入境检验检疫局, 辽宁 大连 116001; 3. 河南农业大学 牧医工程学院, 河南 郑州 450002 ) : (PRRSV) VR2332 ORF7 ,RTPCR ORF7 , PCR pM 18T pM 18TN pET28a( + ) , pET28a( + )NBL21 ,(IPTG ) , 37 0. 2 mmol/ L IPTG 5 h N N Ni NTA , Westernblotting , N PRRSV , N : ( PRRSV) ; ( ORF7) ;; : Q786 : A Cloning, expression and identification of porcine reproductive and respiratory syndrome virus ORF7 1 2 2 2 2 3 ZHANG Xiuyun , WU Bin , ZHAO Huijun , JIA Yun , LIU Xia , ZHANG Rui ( 1.School of Bioen ineerin , Dalian Polytechnic University, Dalian 116034, China; 2.Liaonin EntryExit Inspection and Quarantine Bureau, Dalian 116001, China; 3.En ineerin Colle e of Animal Husbandry and Veterinary Science, Henan A ricultural University, Zhen zhou 450002, China) A stract: A pair of specific primers was designed according to gene sequences ORF7 of PRRSV VR2332 strain to amplify a complete ORF7 gene by RTPCR. T hePCR products were cloned into the vector pM 18T and sequenced. T he doubledigested fragment of the sequenced recombinant plasmid pM 18TN was obtained and connected to prokaryotic expression vector pET28a(+ ) . Recombinant plasmid pET28a(+ )N was converted to the host bacteria BL21 and the conditions (concentrations of IPTG, induction time, induction temperature) were optim