山羊副流感病毒3型N蛋白原核表达及间接ELISA抗体检测方法的建立

doi:10.11843/j.issn.0366-6964.2017.01.018

畜牧兽医学报 ›› 2017, Vol. 48 ›› Issue (1): 150-156.doi: 10.11843/j.issn.0366-6964.2017.01.018

山羊副流感病毒3型N蛋白原核表达及间接ELISA抗体检测方法的建立

王咪^1，2，李文良^1*，郝飞¹，毛立¹，杨蕾蕾¹，张纹纹¹，江杰元^1*

（1. 江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室，南京 210014；2. 南京农业大学动物医学院，南京 210095）

收稿日期:2016-07-04 出版日期:2017-01-23 发布日期:2017-01-23
通讯作者: 李文良，副研究员，E-mail：kfliwenliang@163.com；江杰元，研究员，E-mail：1776556843@qq.com
作者简介:王咪(1993-)，女，湖北黄冈人，本科生，主要从事动物疫病诊断技术研究，E-mail：2287057429@qq.com
基金资助:
江苏省农业科技自主创新资金项目［CX(14)2090］;“十三五”国家重点研发计划（2016YFD0500900）

Prokaryotic Expression of N Protein of Caprine Parainfluenza Virus Type 3 and Establishment of Indirect ELISA Antibody Detection Method

WANG Mi^1,2，LI Wen-liang^1* ，HAO Fei¹，MAO Li¹，YANG Lei-lei¹, ZHANG Wen-wen¹, JIANG Jie-yuan^1*

(1.Key Laboratory of Veterinary Biological Engineering and Technology of Ministry of Agriculture, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China; 2. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China)

Received:2016-07-04 Online:2017-01-23 Published:2017-01-23

摘要/Abstract

摘要：

山羊副流感病毒3型可引起山羊呼吸道疾病，与支原体、细菌等混合感染引起广泛的发病与死亡，笔者拟通过建立基于N蛋白的间接ELISA抗体检测方法用于该病的监测。设计引物扩增目的基因N，克隆到质粒pET32a(+)上，构建重组质粒pET32a-N，并转化至大肠杆菌BL21中，IPTG诱导表达重组N蛋白并鉴定。通过条件优化建立ELISA抗体检测方法，并检测临床样品与HI试验进行比较。本试验成功构建重组质粒，诱导表达的蛋白质（47 ku）经SDS-PAGE与Western blot鉴定，证明其正确表达且具有良好的抗原性与特异性。利用纯化的蛋白质作为包被抗原优化间接ELISA反应条件，确定其最佳抗原包被浓度为1 μg•mL^-1、血清稀释度为1∶200、血清作用时间60 min、酶标二抗工作浓度为1∶6 000、二抗反应时间30 min、底物显色时间10 min。通过对138份临床血清样品检测发现其与HI试验结果符合率达88.41%。本研究建立的间接ELISA检测方法敏感、特异、准确，适用于临床样品的检测与血清流行病学调查。

Abstract:

Caprine parainfluenza virus type 3 (CPIV) can cause respiratory disease in goats, which could cause extensive morbidity and mortality when co-infected with Mycoplasma, bacteria and other pathogens. In this study, an indirect ELISA detection method based on CPIV3 N protein was established to monitor the disease. Primers were designed to amplify the N gene of CPIV3 JS2013 strain. Target gene was cloned into prokaryotic expression plasmid pET32a(+), to construct the recombinant plasmid pET32a-N, and then pET32a-N was transformed into Escherichia coli BL21 competent cells. Large amount expression of recombinant N protein was induced by IPTG. By optimizing the reaction conditions, an indirect ELISA was established. Clinical sera were tested by indirect ELISA and the results were compared with HI test. The recombinant plasmid was constructed successfully, and the expression of protein was identified by SDS-PAGE and Western blot, which confirmed that the protein was expressed correctly and had good immunogenicity and specificity. The purified protein was used as antigen and the indirect ELISA reaction conditions were optimized as follows: the coating antigen concentration was 1 μg•mL^-1, serum dilution was 1:200, serum reaction time was 60 min, HRP-labeled secondary antibody dilution was 1:6 000, reaction time of secondary antibody was 30 min, and substrate chromogenic time was 10 min. By testing of 138 clinical serum samples with the indirect ELISA and HI test, the coincidence rate between these two methods was 88.41%. The indirect ELISA established in this study was sensitive, accurate, which will be suitable and useful for the detection of clinical samples and large scale serological surveys.

中图分类号:

S852.659.5

王咪，李文良，郝飞，毛立，杨蕾蕾，张纹纹，江杰元. 山羊副流感病毒3型N蛋白原核表达及间接ELISA抗体检测方法的建立[J]. 畜牧兽医学报, 2017, 48(1): 150-156.

WANG Mi，LI Wen-liang，HAO Fei，MAO Li，YANG Lei-lei, ZHANG Wen-wen, JIANG Jie-yuan. Prokaryotic Expression of N Protein of Caprine Parainfluenza Virus Type 3 and Establishment of Indirect ELISA Antibody Detection Method[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(1): 150-156.

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参考文献

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山羊副流感病毒3型N蛋白原核表达及间接ELISA抗体检测方法的建立

Prokaryotic Expression of N Protein of Caprine Parainfluenza Virus Type 3 and Establishment of Indirect ELISA Antibody Detection Method

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