基于二聚体核衣壳蛋白的猪繁殖与呼吸综合征病毒抗体iELISA检测方法建立  

Development of Indirect Enzyme‑Linked Immunosorbent Assay Based on the Dimeric Nucleocapsid Protein for Detecting Antibodies against Porcine Reproductive and Respiratory Syndrome Virus

作　　者：赵啸 乔松林[2] 孙亚宁[2] 钱祁昇 李睿[2] 张改平[1,2] ZHAO Xiao;QIAO Songlin;SUN Yaning;QIAN Qisheng;LI Rui;ZHANG Gaiping(College of Veterinary Medicine,Northwest A&F University,Yangling 712100,China;Institute for Animal Health,Henan Academy of Agricultural Sciences/Key Laboratory of Animal Immunology,Zhengzhou 450002,China)

机构地区：[1]西北农林科技大学动物医学院,陕西杨陵712100 [2]河南省农业科学院动物疫病防控研究所/动物免疫学重点实验室,河南郑州450002

出　　处：《河南农业科学》2024年第11期135-146,共12页Journal of Henan Agricultural Sciences

基　　金：河南省重大科技专项(221100110600-03);国家生猪产业技术体系项目(CARS-35);河南省现代农业(生猪)产业技术体系项目(HARS-22-12-S)。

摘　　要：为制备稳定性好、免疫反应性佳的核衣壳蛋白(Nucleocapsid protein,N),设计表达二聚体N蛋白(N-dimer),并基于该蛋白质建立猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)抗体间接酶联免疫吸附试验(Indirect enzyme-linked immunosorbent assay,iELISA)检测方法。首先选取当前PRRSV国内优势毒株NADC30-like N蛋白基因序列,通过(GGGGS)3linker将N蛋白基因序列串联,经密码子优化合成后构建N-dimer重组表达载体。利用原核表达系统进行诱导表达,并通过镍离子螯合亲和层析法进行纯化。Western-blot分析其与PRRSV阳性和阴性血清的反应性;冻融法比较N-dimer与单体N蛋白(N-monomer)的沉淀析出情况;ELISA法比较N-dimer与N-monomer的免疫反应性。以N-dimer作为包被抗原建立PRRSV抗体iELISA检测方法,对该方法的反应条件进行优化,验证其特异性、敏感性、重复性,并应用于临床血清样品检测。结果表明,在16℃、0.2 mmol/L异丙基硫代半乳糖苷条件下诱导16 h,N-dimer获得大量可溶性表达。Western-blot结果表明,N-dimer能够与PRRSV阳性血清发生特异性反应;相较于N-monomer,N-dimer能够稳定保存,并且免疫反应性更佳。成功建立了基于N-dimer的PRRSV抗体iELISA检测方法,且该方法具有良好的特异性、灵敏性、重复性;与商业试剂盒结果进行比较,其阳性符合率为96.88%,总符合率为96.86%,符合率高。综上,成功制备了稳定性好、免疫反应性佳的N-dimer,并基于N-dimer建立了PRRSV抗体iELISA检测方法。In order to prepare N protein with good stability and immunoreactivity,N‑dimer was designed and expressed,and an iELISA detection method for PRRSV antibody was established based on this protein.Firstly,the NADC30‑like N protein gene sequence of the current dominant PRRSV strain in China was selected,and the N protein gene sequence was concatenated through a(GGGGS)3 linker.After codon optimization and synthesis,an N‑dimer recombinant expression vector was constructed.The recombinant protein was expressed using prokaryotic expression system and purified by nickel ion chelation affinity chromatography.Its reactivity with PRRSV positive and negative sera was analyzed by Western‑blot.Its stability after freezing and thawing and immunoreactivity were further compared with those of N‑monomer.An iELISA was subsequently developed based on N‑dimer as coating antigen,and evaluated for the specificity,sensitivity,and repeatability.The developed iELISA was finally applied to clinical serum sample detection.The results showed that under the conditions of 16℃and 0.2 mmol/L isopropylthiogalactoside,N‑dimer was obtained with a large amount of soluble expression after 16 hours of induction.Western‑blot results showed that N‑dimer could specifically react with PRRSV positive serum;Compared to N‑monomer,N‑dimer coule be stably stored and had better immunoreactivity.We successfully established a PRRSV antibody iELISA detection method based on N‑dimer,which had good specificity,sensitivity,and repeatability;Compared with the results of commercial test kits,the positive agreement rate was 96.88%,and the overall agreement rate was 96.86%,indicating a high agreement rate.In summary,this study successfully prepared N‑dimer with good stability and excellent immunoreactivity;A PRRSV antibody iELISA detection method was established based on N‑dimer.

关 键 词：猪繁殖与呼吸综合征病毒 核衣壳蛋白 二聚体 间接ELISA 临床检测 

分 类 号：S858.28[农业科学—临床兽医学]