鸡传染性贫血病毒VP3基因的原核表达及抗血清制备  

Prokaryotic expression and antiserum preparation of Chicken anemia virus VP3 gene

作　　者：孙芬芬 高玉龙[2] 潘伟[3] 王笑梅 SUN Fenfen;GAO Yulong;PAN Wei;WANG Xiaomei(Experimental Teaching Center of Morphology,Xuzhou Medical University,Xuzhou 221004;Poultry Immunosuppressive Disease Team,State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences;Department of Pathogenic Biology and Immunology,Jiangsu Province Key Laboratory of Immunity and Metabolism,Xuzhou Medical University,Xuzhou 221004,China)

机构地区：[1]徐州医科大学形态学实验教学中心,江苏徐州221004 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/禽免疫抑制病科技创新团队,哈尔滨150001 [3]徐州医科大学病原生物学与免疫学教研室/江苏省免疫与代谢重点实验室,江苏徐州221004

出　　处：《黑龙江畜牧兽医》2019年第13期7-10,172,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家肉鸡产业技术体系项目(CARS-42-G07)

摘　　要：为了研究鸡传染性贫血病毒(Chicken anemia virus,CIAV)VP3蛋白与病毒自身蛋白之间的相互作用,需对该蛋白进行原核表达制备相应兔源抗血清,试验通过提取CIAV M9905株基因组DNA,PCR扩增出VP3基因,构建重组质粒pET30a(+)-VP3,然后转化入大肠杆菌BL21(DE3)感受态细胞中,进一步通过IPTG诱导表达后用Ni-NTA亲和柱纯化获得融合蛋白,经SDS-PAGE分析后对融合蛋白进行乳化,免疫新西兰兔制备抗血清,并对其进行ELISA效价测定、Western-blot及IFA检测。结果表明:SDS-PAGE分析证实获得高纯度的重组蛋白VP3,大小约为22 ku,且其在大肠杆菌中主要以包涵体的形式存在;ELISA法检测制备的兔源抗VP3血清效价在1∶6 400以上;经Western-blot及IFA检测证实,VP3兔源血清可特异性结合Vero细胞表达的VP3蛋白。说明试验成功表达出CIAV VP3蛋白,制备的兔抗血清具有较好的特异性。In order to research the interaction between Chicken anemia virus(CIAV) VP3 protein and virus proteins,the VP3 protein needed to be expressed prokaryoticly and its anti-rabbit sera were prepared. The genomic DNA of CIAV M9905 strain was extracted in this experiment,then the VP3 gene was amplified by PCR,and recombinant prokaryotic expression vector pET30 a(+)-VP3 was constructed,which was transformed into Escherichia coli BL21(DE3) competent cells. After IPTG induction and the purification by the immobilized Ni-NTA resin,the fusion protein VP3 was obtained. After the SDS-PAGE analysis,the fusion protein VP3 was emulsified and New Zealand rabbits were immunized for the preparation of anti-VP3 sera. Anti-VP3 sera were determined by indirect ELISA for titer detection,by Western-blot and IFA methods. The results showed that the SDS-PAGE analysis comfirmed that the recombinant VP3 with high purity was obtained,with a molecular weight of 22 ku,and existed mainly in the form of inclusion body in Escherichia coli. The prepared rabbit anti-VP3 sera showed a titer greater than 1∶6 400 using ELISA method. Western-blot and IFA results confirmed that the sera could specifically bind to the VP3 protein expressed in Vero cells. The results suggested that the CIAV VP3 protein was successfully expressed in this experiment,and the prepared rabbit antisera had good specificity.

关 键 词：鸡传染性贫血病毒(CIAV) VP3蛋白 蛋白相互作用 原核表达 纯化 抗血清 

分 类 号：S852.659.2[农业科学—基础兽医学]