中国畜牧兽医2024，Vol.51Issue(7)：3056-3066,11.DOI:10.16431/j.cnki.1671-7236.2024.07.030
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
摘要
Abstract
[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 D450 nm＞0.684.The dilution concentration of serum and HRP-rabbit anti-pig IgG were 1∶100 and 1∶60 000,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 1:1 600.The method was of high duplicability with less than 10％variation of intra-and inter-assay coefficients.The result of i-ELISA was positive correlation with that of virus neutralization test,and the coincidence rate with Western blotting method was 95.8％.The positive rate of PCV3 antibody was 7.12％in 351 pig serum samples collected in Northeast Sichuan from 2018 to 2022.[Conclusion]This experiment successfully truncated expression Cap protein(1-197 amino acids)and established i-ELISA of PCV3 with better specificity,sensitivity,repeatability and coincidence rate,which provided material for serological investigation and test kit development of PCV3.关键词
猪圆环病毒3型(PCV3)/Cap蛋白/截短表达/ELISAKey words
Porcine circovirus type 3(PCV3)/Cap 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,翟佳佳,张杰..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[J].中国畜牧兽医,2024,51(7):3056-3066,11.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）
