猪圆环病毒2型ORF2基因的原核表达及其免疫原性检测  

Prokaryotic Expression and Purification of PCV-2 ORF2 Gene and Its Immunogenicity Detection

作　　者：梁晓艳[1] 李玉林[2] 李兆学[3] 王继美[2] 贾丽锋[1] 周贺娟[1] 王云龙[1,2] 

机构地区：[1]郑州大学,河南郑州450002 [2]河南省生物工程技术研究中心,河南郑州450002 [3]河南工业大学,河南郑州450002

出　　处：《动物医学进展》2010年第9期63-67,共5页Progress In Veterinary Medicine

摘　　要：以病猪脾脏组织为材料,提取总RNA,通过RT-PCR得到579 bp的猪圆环病毒2型(PCV-2)/ORF2片段,与原核表达载体pET32a重组,通过菌落PCR鉴定、双酶切鉴定及测序鉴定后,证实重组质粒pET32a/ORF2构建成功,将其转化入表达菌Balgold,通过IPTG诱导表达,利用Ni-NTA亲和层析纯化目的产物,Western blot验证其免疫原性,重组蛋白免疫Balb/c小鼠,检测小鼠体内中和抗体滴度。获得的重组蛋白包涵体的形式出现,表达量占菌体总蛋白的30%,变性纯化后纯度达90%以上,Western blot证实重组蛋白能与PCV-2阳性血清发生反应,免疫Balb/c小鼠45 d后中和抗体滴度达到1∶22。Pigs spleen was used for the extraction of total RNA,and the 579 bp fragment of PCV-2/ORF2 cDNA was amplified through RT-PCR method.The fragment was cloned into prokaryotic expression vector pET32a and transformed into Balgold.After the recombinant plasmid pET32a/ORF2 was confirmed by PCR,restriction enzyme digestion analysis and DNA sequencing,it was transduced by IPTG,the recombinant protein was purified by Ni-NTA affinity chromatography and identified by Western blot,then it was used to immunize Balb/c mice.The results demonstrated that target protein could be expressed in the form of inclusion bodies,it was made up 30% of the total bacterial protein.The purity is above 90%.The protein can be recognized by monoclonal antibodie of PCV-2,the titer of neutralizing antibody is about 1∶22 after immunization 45 d in mice.

关 键 词：猪圆环病毒 ORF2 原核表达 免疫原性 

分 类 号：S852.659.2[农业科学—基础兽医学]