猪圆环病毒3型四川株Cap蛋白的截短表达及其抗体间接ELISA方法的建立  

Truncated Expression of Cap Protein of Porcine Circovirus Type 3 and Establishment of Indirect ELISA Method for Its Antibody

作　　者：欧云文 潘琴 汪洋 代军飞 任绍科 张洋 翟佳佳 张杰[2,3] OU Yunwen;PAN Qin;WANG Yang;DAI Junfei;REN Shaoke;ZHANG Yang;ZHAI Jiajia;ZHANG Jie(Animal Disease Prevention and Control Center of Kaijiang County,Dazhou 636250,China;State Key Laboratory for Animal Disease Control and Prevention,College of Veterinary Medicine,Lanzhou University,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;Hebei Key Laboratory of Preventive Veterinary Medicine,Hebei Normal University of Science&Technology,Qinhuangdao 066004,China)

机构地区：[1]开江县动物疫病预防控制中心,达州636250 [2]中国农业科学院兰州兽医研究所,兰州大学动物医学与生物安全学院,动物疫病防控全国重点实验室,兰州730046 [3]河北科技师范学院,河北省预防兽医学重点实验室,秦皇岛066004

出　　处：《中国畜牧兽医》2024年第7期3056-3066,共11页China Animal Husbandry & Veterinary Medicine

基　　金：四川省自然科学基金(24NSFSC3002);四川省科技计划资助项目(2023JDRC0126);达州市科技计划资助项目(22CYRC0016)。

摘　　要：【目的】原核截短表达猪圆环病毒3型(Porcine circovirus type 3,PCV3)四川株Cap蛋白(1-197位氨基酸),并建立其抗体间接ELISA检测方法,为PCV3血清学调查提供材料。【方法】以PCV3四川分离株基因组为模板,PCR扩增获得Cap蛋白编码基因片段,将其克隆至原核表达载体pET-30a(+)中构建重组质粒pET30a-PCV3-Cap。经酶切和测序鉴定后,将重组质粒转化大肠杆菌BL21(DE3)感受态细胞,IPTG诱导表达,Ni-NTA树脂亲和层析纯化,获得具有良好抗原性的重组Cap蛋白。基于纯化的重组Cap蛋白建立间接ELISA方法,确定最佳抗原包被浓度、待检血清最佳稀释比例、酶标抗体稀释比例和阴阳性临界值,对间接ELISA方法的特异性、敏感性、重复性、相关性和符合率进行分析,并对2018-2022年采自川东北地区猪场的351份临床猪血清进行检测,分析该区域PCV3流行情况。【结果】试验成功构建pET30a-PCV3-Cap重组表达载体,实现重组Cap蛋白(1-197位氨基酸)的原核截短表达,蛋白大小约为26 ku,可与PCV3阳性猪血清发生特异性反应;确定最佳抗原包被浓度为1 ng/μL,待检血清最佳稀释比例为1∶100,酶标抗体稀释比例为1∶60000,阴阳性临界值D_(450 nm)为0.684;建立的间接ELISA方法与猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪圆环病毒2型(PCV2)、猪伪狂犬病病毒(PRV)、日本乙型脑炎病毒(JEV)和猪细小病毒1型(PPV1)阳性猪血清均不发生交叉反应,敏感度达1∶1600,批内变异系数和批间变异系数均<10%,与病毒中和试验(VNT)结果呈正相关,与Western blotting方法的阳性符合率为95.8%;2018-2022年川东北地区的351份猪血清样品阳性率为7.12%。【结论】本研究成功截短表达了PCV3四川株Cap蛋白(1-197位氨基酸),建立了特异性强、灵敏度高、重复性好和符合率高的PCV3间接ELISA方法,为PCV3的血清学调查和检测试剂盒的开发提供了材料。【Objective】This study was aimed to express truncatly Cap protein(1-197 amino acids)of Porcine circovirus type 3(PCV3)Sichuan strain in prokaryotic expression system,and develop an indirect ELISA(i-ELISA)method for rapid detection of PCV3 antibody,and provide material for serological investigation of PCV3.【Method】The genome of PCV3 Sichuan strain was used as template.The Cap protein coding gene of PCV3 was obtained by PCR amplification and inserted into the prokaryotic expression vector pET-30a(+)to construct the recombinant expression plasmid pET30a-PCV3-Cap.The recombined plasmid pET30a-PCV3-Cap was expressed in E.coli BL21(DE3)competent cells and was induced by IPTG.The recombinant Cap protein with good antigenicity was purified by Ni-NTA,and analyzed by SDS-PAGE and Western blotting.The i-ELISA was developed using the recombinant Cap protein as coating antigen.The optimal concentration of antigen,the optimal dilution ratio of serum to be tested,the dilution ratio of enzyme-labeled antibody and the threshold of negative and positive were determined,and the specificity,sensitivity,repeatability,correlation and coincidence rate of the i-ELISA were analyzed.The established i-ELISA method was used to detect 351 pig serum samples collected from pig farms in Northeast Sichuan from 2018 to 2022 to analyze the prevalence of PCV3 in the region.【Result】The recombinant expression plasmid pET30a-PCV3-Cap was successfully obtained,and the recombinant Cap protein(1-197 amino acids)was successfully expressed in E.coli BL21(DE3)competent cells could react specifically with positive pig serum of PCV3 with a relative molecular weight of 26 ku.The coating concentration of Cap protein was 1 ng/μL,and the detection positive threshold was D_(450 nm)>0.684.The dilution concentration of serum and HRP-rabbit anti-pig IgG were 1∶100 and 1∶60000,respectively.The i-ELISA was not cross reaction with positive sera of CSFV,PRRSV,PCV2,PRV,JEV and PPV1.The sensitivity of i-ELISA was 1∶1600.The method was of high duplicabilit

关 键 词：猪圆环病毒3型(PCV3) CAP蛋白 截短表达 ELISA 

分 类 号：S852.651[农业科学—基础兽医学]