猪圆环病毒3型抗体ELISA检测方法建立及其应用  

Development and Application of an ELISA for Detection of Porcine Circovirus 3 Antibodies

作　　者：谢怡灵 李润成[1] 赵墩[1] 杜丽飞[2,3] 熊梦霞 宁柯铭 李双寅 黎满香 葛猛[1] XIE Yiling;LI Runcheng;ZHAO Dun;DU Lifei;XIONG Mengxia;NING Kerning;LI Shuangyin;LI Manxiang;GE Meng(College of Veterinary Medicine,Hunan Agricultural University,Changsha 410128,China;Hunan Animal and Veterinary Science,Changsha 410131,China;Hunan Xinguangan Agriculture and Animal Husbandry Co.,Ltd.,Changsha 410100,China)

机构地区：[1]湖南农业大学动物医学院,长沙410128 [2]湖南省畜牧兽医研究所,长沙410131 [3]湖南鑫广安农牧股份有限公司,长沙410100

出　　处：《中国动物传染病学报》2021年第3期34-41,共8页Chinese Journal of Animal Infectious Diseases

基　　金：湖南省自然科学基金(2019JJ50258);湖南省重点领域研发计划(2019NK2171)。

摘　　要：本研究将猪圆环病毒3型(PCV3)Cap蛋白截去核定位序列(NLS)后对应的dCap基因克隆至pET28a(+)并转入大肠杆菌BL21(DE3),得到重组菌后进行蛋白表达。采用镍离子亲和层析纯化dCap蛋白。将纯化的dCap蛋白作为抗原进行包被,建立检测PCV3抗体的间接ELISA方法,并对湖南省1175份临床血清进行检测。结果显示:dCap蛋白主要以包涵体形式表达,当以pH为10.83的碳酸盐缓冲液将纯化的dCap蛋白稀释到0.2μg/mL并进行包被,且血清50倍稀释,酶标二抗1∶8000稀释时,ELISA的反应条件最佳,确定S/P临界值为0.270,批内及批间差异均小于10%,本方法与PCV2、CSFV、PRV和PRRSV阳性血清无交叉反应。对湖南省11个地级市的29个猪场的血清流行病学调查结果表明:PCV3抗体猪场阳性率为100%(29/29),样品总阳性率为58.64%(689/1175),但阳性样品主要集中在肥猪和母猪,且阳性率在肥猪阶段明显上升,说明猪群感染PCV3主要是在育肥阶段。In the study,the gene of Cap protein without nuclear localization sequence(NLS)of Porcine circovirus type 3(PCV3)was cloned into pET28a(+)and expressed in E.coli BL21(DE3).The recombinant dCap protein was purified by Ni-IDA affinity chromatography and used as a coating antigen to develop an indirect ELISA for detection of antibodies to PCV3.Total 1175 clinical serum samples were collected from 29 pig farms in 11 cities from Hunan province and tested for the PCV3 antibody status.The results showed that the optimal working conditions for ELISA were 0.2μg/mL of the recombinant dCap protein diluted in the carbonate buffer with pH10.83,1:8000 HRP-conjugated goat anti-swine IgG and 1:50 dilution of serum samples.The threshold of negative-positive cutoff S/P value for defining positive samples was set to be 0.270.The difference of inter-assay and intra-assay were less than 10%.The ELISA method had no cross-reaction with antibodies to PCV2,CSFV,PRV and PRRSV.The serological investigation of PCV3 in Hunan province showed that all sampled 29 farms and 58.64%(689/1175)serum samples were positive.The positive rates were high in fattening pigs and sows.

关 键 词：猪圆环病毒3 CAP蛋白 间接ELISA 

分 类 号：S852.659.2[农业科学—基础兽医学]