中国兽医科学2024，Vol.54Issue(1)：48-55,8.DOI:10.16656/j.issn.1673-4696.2024.0009
猪丁型冠状病毒截短S蛋白的原核表达及间接ELISA检测方法的建立与应用
Prokaryotic expression of truncated S protein of porcine deltacoronavirus and establishment and application of indirect ELISA detection method
摘要
Abstract
In order to establish a rapid and efficient method for serological detection of porcine deltacoronavirus(PDCoV),the recombinant plasmid pET-32a-S-S1b was constructed,and the recombinant S1b protein was obtained by prokaryotic expression.An indirect ELISA method for detecting PDCoV IgA an-tibody in serum was established.After optimizing the reaction conditions of the method,its specifici-ty,sensitivity and repeatability were verified.The results showed that the recombinant S1b protein was expressed in inclusion bodies,the size was 34.7 ku and the reactivity was good,and the best reaction conditions of indirect ELISA were as follows:the antigen was coated at a concentration of 250 ng/well and incubated overnight at 4 ℃,10 g/L BSA was incubated at 37 ℃ for 2 h as a blocking step,the tested serum was diluted at a ratio of 1:200 and incubated at 37 ℃ for 45 min,and the secondary antibody was diluted at a ratio of 1∶10 000 and incubated at 37 ℃ for 1 h,with a coloration time of 10 min.The cutoff value was determined to be 0.388 after testing 24 negative sera.The sensitivity of detecting positive serum was 1∶1 600,and the coefficient of variation of 10 PDCoV-positive sera inter-and intra-batch assay of ELISA were both less than 10％.This method demonstrated high specificity and did not show cross reaction with standard sera against porcine epidemic diarrhea virus,pseudorabies virus,classi-cal swine fever virus,porcine reproductive and respiratory syndrome virus,African swine fever virus,porcine circovirus and porcine enterovirus G.Compared to the results obtained from 30 serum samples using Western-blot,the coincidence rate was 96.66％.A total of 486 clinical serum samples were detect-ed by this method,with a positive rate of 32.72％.The method established in this study provides techni-cal support for the prevention and control of PDCoV.关键词
猪丁型冠状病毒/S蛋白S1b/间接ELISA/临床检测Key words
porcine deltacoronavirus/S protein S1b/indirect ELISA/clinical testing分类
农业科技引用本文复制引用
秦秋英,张政,黄夏玲,洪大林,隆美金,陈樱,韦祖樟,黄伟坚,欧阳康..猪丁型冠状病毒截短S蛋白的原核表达及间接ELISA检测方法的建立与应用[J].中国兽医科学,2024,54(1):48-55,8.基金项目
广西重点研发计划项目(桂科AB23026082) （桂科AB23026082）
大学生创新创业项目(202210593121) （202210593121）
广西自然科学基金项目(2021GXNSFAA196067) （2021GXNSFAA196067）
广西大学巴马产教融合研究院专项(巴人科20220015) （巴人科20220015）
