农业生物技术学报2024，Vol.32Issue(6)：1462-1470,9.DOI:10.3969/j.issn.1674-7968.2024.06.021
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
Establishment and Application of Indirect ELISA Antibody Detection Method Based on VP1 Protein for Porcine parvovirus Type 6
摘要
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.关键词
猪细小病毒6型(PPV6)/VP1蛋白/截短表达/ELISAKey words
Porcine parvovirus type 6(PPV6)/VP1 protein/Truncated expression/ELISA分类
农业科技引用本文复制引用
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):1462-1470,9.基金项目
四川省自然科学基金(24NSFSC3002) （24NSFSC3002）
四川省科技计划资助项目(2023JDRC0126) （2023JDRC0126）
达州市科技计划资助项目(22CYRC0016) （22CYRC0016）
天府青城计划-农业领军人才项目 （）
