猪圆环病毒2型ORF2基因的表达及活性研究  

Study on Expression and Bioactivity of PCV-2 ORF2 Gene

作　　者：陈涛[1,2] 闫若潜[2] 谢彩华[2] 吴志明[2] 张志凌[2] 张健[2] 刘英[1] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]河南省动物疫病预防控制中心,河南郑州450008

出　　处：《动物医学进展》2009年第2期35-38,共4页Progress In Veterinary Medicine

基　　金：河南省科技攻关项目(0624410098)

摘　　要：根据猪圆环病毒2型(PCV-2)基因序列设计1对特异性引物,PCR扩增PCV-2去核定位信号(nuclear localization signal,NLS)的Cap蛋白基因(ORF2),经BamHⅠ/HindⅢ双酶切后将其克隆到表达载体pQE30中并转化大肠埃希菌JM109,采用IPTG进行诱导表达,采用自行创新的纯化方法对表达的融合蛋白进行纯化,Western blot鉴定纯化后的重组Cap蛋白(rCap)活性。结果表明,成功克隆了无核定位信号的ORF2基因,其分子质量大小为579 bp;表达的rCap蛋白大小为24.1 ku左右,与预期大小相一致;经纯化后的rCap蛋白纯度在95%以上;Western blot鉴定结果表明,Cap蛋白可以和抗PCV-2抗体反应,具有很好的抗原性。本研究为进一步建立以Cap蛋白为包被抗原的PCV-2间接ELISA诊断方法以及PCV-2疫苗的研究奠定了基础。The primers were designed to amplify the capsid protein gene （ORF2） of defected-nuclear localization signals（d-NLS）according to the sequence of PCV-2 ORF2 gene, and the PCR production was subsequently cloned, sequenced and sub-cloned into the prokaryotic expression vector pQE30. The expressed fusion recombinant PolL-2 （rPolL-2） protein was purified under the process of denaturing by urea, refolding by a self-innovative renaturation buffer and dialyzing by PBS buffer. The immunological activity of the purified recombinant capsid prmein （rCap） was estimated by Western-blot analysis using the anti-PCV-2 antibody as the first antibody. The result showed that the cloned d-NLS ORF2 gene was consisted of 579 bp nucleotides, which encoded a protein of t92 amino acids. The fusion rCap protein was successfully expressed and purified with the molecular mass of about 24.1 ku and more than 95% pure on SDS-PAGE, which indicated that the correct fusion rCap protein was obtained. The Western-blot analysis demonstrated the rCap protein has good immunological activity to recognize the anti-PCV-2 antibodies. Our study revealed that the rCap protein with good bioactivity was successful obtained, which layed a foundation for further establishing the indirect ELISA diagnosis method of PCV-2 and exploitation the nuclear vaccine by using the rCap protein.

关 键 词：猪圆环病毒2型(PCV-2) 克隆 表达 纯化 活性 

分 类 号：S852.659.2[农业科学—基础兽医学]