南方农业学报2024，Vol.55Issue(4)：1238-1248,11.DOI:10.3969/j.issn.2095-1191.2024.04.031
猪丁型冠状病毒NSP14蛋白截短表达及其间接ELISA抗体检测方法的建立
Truncated expression of porcine delta coronavirus protein NSP14 and establishment of indirect ELISA antibody detection method
摘要
Abstract
[Objective]The truncated NSP14 protein of porcine delta coronavirus(PDCoV)was expressed in prokaryo-tic,and the polyclonal antibody of PDCoV NSP14 protein was prepared,and an indirect enzyme-linked immunosorbent as-say(ELISA)method for detecting PDCoV antibody was established.It provided a reference for exploring the biological function of PDCoV NSP14 protein and conducting PDCoV monitoring and diagnosis.[Method]In this study,the prokaryo-tic recombinant plasmid pET32a-NSP14 of NSP14 truncated gene was constructed using PDCoV 1468B strain as a tem-plate,and after identified by double digestion and sequencing,the recombinant protein pET32a-NSP14 was obtained by prokaryotic expression system.They were purified and then reimmunized to New Zealand white rabbits to obtain poly-clonal antibodies against NSP14 protein,the Western blotting was performed and and its titer was determined by indirect ELISA method.NSP14 recombinant protein was used as antigen coated enzyme-linked immunosorbent assay plate,and a series of reaction conditions were optimized by square matrix titration method to construct an indirect ELISA method for detecting PDCoV antibody.[Result]The recombinant plasmid pET32a-NSP14 was constructed successfully,and the NSP14 protein was expressed as an inclusion body in Escherichia coli BL21(DE3)competent cells with a size of approxi-mately 40.2 kD.The NSP14 protein polyclonal antibody was produced successfully,with an antibody titer above 1∶512000,and it could specifically react with the purified recombinant NSP14 protein.After screening,the optimal reaction conditions of the indirect ELISA antibody detection method were determined:the optimal coating concentration of NSP14 recombinant protein was 4.0 μg/mL,and the coating condition was 37℃for 2.0 h;the blocking condition was 5% skimmed milk powder for 1.0 h;the serum to be tested was diluted 1∶400 and incubated for 2.0 h;the enzyme labeled sec-ondary antibody was diluted 1∶2500 and incubated for 60.0 min;the TMB substrate reaction time was 30.0 min.The results of specificity test showed that the OD450 values of PRRSV,JEV,PCV2,PPV,PEDV and CSFV in the positive se-rum of porcine disease were less than 0.235,indicating that the optimized ELISA method did not react with other positive serum of porcine virus and had good specificity.The repeatability test results showed that the intraassay and inte-rassay co-efficients of variation of the optimized ELISA method for detecting pig positive serum were less than 10.000%,indicating that the optimized ELISA method had good repeatability.[Conclusion]In this study,the polyclonal antibody of PDCoV NSP14 protein with high titer and good immunoreactivity has been successfully prepared,and an indirect ELISA antibody detection method with good specificity,sensitivity and repeatability is established,which provides reference for further study of the biological function of PDCoV NSP14 protein and PDCoV monitoring and diagnosis.关键词
猪丁型冠状病毒/NSP14蛋白/原核表达/多克隆抗体制备/间接ELISAKey words
porcine delta coronavirus/NSP14 protein/prokaryotic expression/polyclonal antibody preparation/in-direct ELISA分类
农业科技引用本文复制引用
刘思雨,何颖,卢冰霞,赵武,赵硕,许心婷,全琛宇,秦毅斌,欧阳康..猪丁型冠状病毒NSP14蛋白截短表达及其间接ELISA抗体检测方法的建立[J].南方农业学报,2024,55(4):1238-1248,11.基金项目
广西创新驱动发展专项(桂科AA17204057-1) （桂科AA17204057-1）
国家现代农业产业技术体系广西创新团队建设专项(nycytxgxcxtd-2023-15-02) （nycytxgxcxtd-2023-15-02）
南宁市科学研究与技术开发计划项目(20232039) （20232039）
广西农业科技自筹经费项目(Z202221) Guangxi Innovation Driven Development Special Project(Guike AA17204057-1) （Z202221）
Guangxi Inno-vation Team Construction Project of China Agriculture Research System(nycytxgxcxtd-2023-15-02) （nycytxgxcxtd-2023-15-02）
Nanning Science Research and Technology Development Project(20232039) （20232039）
Guangxi Agricultural Science and Technology Self-financing Project(Z202221) （Z202221）
