摘要
根据已发表的犬瘟热病毒 OP株核酸序列设计 2对引物 ,以犬瘟热病毒 OP株感染 Vero细胞收获病毒提取的RNA为模板 ,应用 RT- PCR扩增出 F基因的 76 9、832 bp片段。将 PCR产物按正确的阅读框架定向克隆进 p GEX- 6 p-1载体中谷胱甘肽 - S-转移酶 (GST)基因的下游 ,再将重组质粒转化入宿主菌 BL2 1 中 ,在 37℃ 1 .0 mmol/ L IPTG诱导下 ,F基因 2个片段获得了良好的表达。表达产物经聚丙烯酰胺凝胶电泳鉴定 ,确定所表达的融合多肽大小分别为5 4 0 0 0、5 6 0 0 0。将表达产物回收后混合免疫小鼠 ,IFA显示 ,免疫小鼠血清能与病毒感染细胞呈现特异反应 ,并表现出 1∶ 1 6的中和抗体活性 ,表明体外表达的
Two pairs of primers had been designed and synthesized based on the fusion(F) protein gene of the onderstepoort strain of canine distemper virus(CDV).The templates were the RNA of OP CDV which derived from the Vero cell culture of OP CDV.The 769 bp and 832 bp fragments of F gene were amplified by polymerase chain reaction respectively.PCR products were cloned into the expressing plasmid vector pGEX 6p 1.The recombinant was verified by restriction endonuclease analysis and sequencing.Then two recombinants were transformed into the host strain BL 21 and expressed by induction of 1 0 mmol/L IPTG at 37℃。The expressed products were identified by SDS PAGE and found to be 54 000 and 56 000 in size as the fusion proteins form with glutathione transferase(GST) protein.The protein bands were excised from the gel,minced and injected into mice once a week for five times.The specificity of antibody was detected positively against CDV infected Vero cell by indirect immuno fluorescent assay(IFA) and virus serum neutralization assay(SN).The results indicated that in vitro expression of F gene maintained part of the antigenicity of CDV.
出处
《兽医大学学报》
CSCD
北大核心
2003年第3期231-233,共3页
