基于猪细小病毒6型VP1蛋白间接ELISA抗体检测方法的建立及应用

doi:10.3969/j.issn.1674-7968.2024.06.021

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摘要猪细小病毒6型(Porcine parvovirus type 6, PPV6)是一种新发现的猪细小病毒,可导致猪(Sus scrofa)出现繁殖障碍,严重威胁养猪业健康发展。为建立一种检测PPV6抗体的间接ELISA方法,本研究以PPV6流行毒株高度保守的病毒颗粒蛋白(virion protein, VP1)(348~675 aa)的编码基因为目标片段,构建原核表达载体pET30a-PPV6-VP1,转入大肠杆菌(Escherichia coli) BL21 (DE3)感受态细胞,IPTG诱导表达,进行Ni-NTA树脂亲和层析纯化,获得具有良好的反应原性的重组VP1蛋白,大小约为40 kD。基于纯化的重组VP1蛋白建立间接ELISA抗体检测方法,优化出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,HRP-兔抗猪IgG稀释比例为1∶60 000,阳性临界值OD₄₅₀>0.62。该间接ELISA检测方法与猪蓝耳病毒(Porcine reproductive and respiratory syndrome virus, PRRSV)、古典猪瘟病毒(Classical swine fever virus, CSFV)、猪伪狂犬病毒(Pseudorabies virus, PRV)、日本乙型脑炎病毒(Japanese encephalitis virus, JEV)、猪圆环病毒2型(Porcine circovirus type 2, PCV2)、PPV1阳性猪血清均不发生交叉反应,敏感度达1∶3 200,批内变异系数和批间变异系数均<10%,与病毒中和试验(virus neutralization test, VNT)结果呈正相关,与Western blot结果的阳性符合率为96.6%,表明该间接ELISA检测方法拥有良好的特异性、敏感性、重复性和符合率。采用建立的间接ELISA方法对2018~2022年中国西部地区部分省份收集的452份猪血清进行检测,结果显示样品阳性率为5.31%,表明该病在我国西部地区存在不同程度的感染。本研究成功截短表达了PPV6 VP1 (348~675 aa)蛋白,建立了特异性强、灵敏度高、重复性好和符合率高的PPV6间接ELISA方法,为PPV6的血清学调查和检测试剂盒的开发提供了材料。

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关键词 ：猪细小病毒6型(PPV6), VP1蛋白, 截短表达, ELISA

Abstract：Porcine parvovirus type 6 (PPV6) is a new swine (Sus scrofa) Porcine parvovirus, which can cause symptoms of reproductive dysfunction and seriously threaten the healthy development of pig industry. In order to develop an indirect ELISA (iELISA) method for rapid detection of PPV6 antibody, the highly conserved encoded gene virion protein (VP1)(348~675 aa) of the popular PPV6 strain was used as the target fragment, and the gene was amplified from the DNA of PPV6 positive sample by PCR. The recombined plasmid pET30a-PPV6-VP1 was expressed in Escherichia coli BL21 (DE3) and was induced by IPTG. The recombinant VP1 protein with a relative molecular weight of 40 kD was purified by Ni-NTA. The iELISA was developed by using the recombinant VP1 protein as coating antigen, and studied the specificity, sensitivity, repeatability and coincidence rate of the method. The coating concentration of VP1 protein was 1 ng/μL, and the detection positive threshold was OD₄₅₀>0.62. The dilution concentration of serum and HRP-rabbit anti-pig IgG was 1∶100 and 1∶60 000, respectively. The iELISA was not cross reaction with positive sera of Porcine reproductive and respiratory syndrome virus (PRRSV), Classical swine fever virus (CSFV), Pseudorabies virus (PRV), Japanese encephalitis virus (JEV), Porcine circovirus type 2 (PCV2), PPV1. The sensitivity of iELISA was 1∶3 200. The method was of high duplicability with less than 10% variation of intra-and inter-batch coefficients. The result of iELISA was positive correlation with virus neutralization test, and the positive coincidence rate with Western blot method was 96.6%. The above results showed that the established iELISA method had good specificity, sensitivity, and repeatability. The positive rate of PPV6 antibody was 5.31% in 452 pig serum samples collected in some provinces in the western of China from 2018 to 2022, which preliminarily proved that the disease had been infected in the western of China. This study successfully truncated expression VP1 (348~675 aa) protein and established iELISA of PPV6 with better specificity, sensitivity, repeatability and coincidence rate, which provides material for serological investigation and test kit development of PPV6.

Key words： Porcine parvovirus type 6 (PPV6) VP1 protein Truncated expression ELISA

收稿日期: 2023-04-19

中图分类号： S855.3

基金资助: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); 天府青城计划-农业领军人才项目

通讯作者: *zhangjie03@caas.cn

引用本文:

欧云文, 潘琴, 汪洋, 代军飞, 任绍科, 张洋, 张杰. 基于猪细小病毒6型VP1蛋白间接ELISA抗体检测方法的建立及应用[J]. 农业生物技术学报, 2024, 32(6): 1462-1470.
OU Yun-Wen, PAN Qin, WANG Yang, DAI Jun-Fei, REN Shao-Ke, ZHANG Yang, ZHANG Jie. Establishment and Application of Indirect ELISA Antibody Detection Method Based on VP1 Protein for Porcine parvovirus Type 6. 农业生物技术学报, 2024, 32(6): 1462-1470.

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https://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2024.06.021 或 https://journal05.magtech.org.cn/Jwk_ny/CN/Y2024/V32/I6/1462

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