[1]王莉莉,于艳超,周长平,等.基于鸡毒支原体热休克蛋白DnaK的间接ELISA方法的评价及初步应用[J].中国预防兽医学报,2018,(09):795-800.[doi:0.3969/j.issn.1008-0589.201711042]
 WANG Li-li,YU Yan-chao,ZHOU Chang-ping,et al.The evaluation and application of the heat shock protein DnaK of Mycoplasma gallisepticum as coating antigen in indirect ELISA[J].Chinese journal of preventive veterinary medicine,2018,(09):795-800.[doi:0.3969/j.issn.1008-0589.201711042]
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基于鸡毒支原体热休克蛋白DnaK的间接ELISA方法的评价及初步应用
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2018年09
页码:
795-800
栏目:
诊断和检测技术
出版日期:
2018-09-17

文章信息/Info

Title:
The evaluation and application of the heat shock protein DnaK of Mycoplasma gallisepticum as coating antigen in indirect ELISA
文章编号:
1008-0589(2018)9-0795-06
作者:
 

王莉莉1于艳超2周长平1赵雅芝2潘 巧1张 琳1郝文君1王秀梅1*辛九庆1*

 (1. 中国农业科学院哈尔滨兽医研究所 兽医生物技术国家重点实验室/动物支原体病创新团队,黑龙江 哈尔滨 150069;
2. 吉林农业大学 动物科技学院,吉林 长春 130118)
Author(s):
 

WANG Li-li1 YU Yan-chao2 ZHOU Chang-ping1 ZHAO Ya-zhi2 PAN Qiao1 ZHANG Lin1HAO Wen-jun1 WANG Xiu-mei1* XIN Jiu-qing1*

 (1. Innovation team of Animal Mycoplasma Disease, Division of Animal Diseases, State Key Laboratory of Veterinary Biotechnology,
Harbin Veterinary Research Institute, Chinese Academy of Agricultural Science, Harbin 150069, China;
2. College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, China)
关键词:
鸡毒支原体DnaK免疫原性间接ELISA
分类号:
S852.61
DOI:
0.3969/j.issn.1008-0589.201711042
文献标志码:
A
摘要:
为研究鸡毒支原体(MG)重组热休克蛋白DnaK (rDnaK)的免疫反应原性及其作为ELISA方法中诊断抗原的应用价值,本研究将已构建的含有MG DnaK基因的重组质粒pET-30a-DnaK进行原核表达纯化,以多份MG的标准阳性血清和阴性血清作为一抗,进行western blot分析,结果显示rDnaK与MG的阳性血清发生特异性反应,与MG的阴性血清无反应条带,表明rDnaK具有较好的免疫反应原性。将其作为包被抗原包被酶标板,用HRP标记的兔抗鸡IgG作为酶标二抗,分别对抗原包被浓度、血清稀释度、酶标二抗稀释度及工作时间等反应条件进行了优化,建立了一种稳定检测MG抗体的间接ELISA方法。对该方法进行评估,灵敏性试验结果显示该方法与进口试剂盒相当;特异性试验结果显示包被抗原rDnaK不与NDV、IBV、ILTV等几种常见的禽呼吸道疾病的阳性血清发生交叉反应;重复性试验结果显示,批内变异系数为1.6 %~5.15 %,批间变异系数为2.9 %~5.98 %;符合率试验结果显示该方法与进口试剂盒的总体符合率为90.4 %;抗体持续期试验结果显示该方法最早能在鸡群免疫MG疫苗后一周检测到MG抗体,表明该诊断方法适用于MG感染的早期检测。采用该方法检测了来自全国5个省份的568份临床样品,其结果显示MG的感染率在23 %~51 %,对我国MG的流行状况做了初步的调查。
 
Abstract:
The main aim of this research was to study the immunogenicity of rDnak and the application value as coating antigen in ELISA. The rDnak of Mycoplasma gallisepticum (MG) was expressed in E.coli with the recombinant plasmid pET-30a-DnaK. The product was analyzed by western blot with MG positive and negative serum and the result showed that it had a strong immunogenicity with positive serum and could not react with negative serum. Then the purified rDnak was used as coating antigen for ELISA and HRP-tagged rabbit anti-chicken IgG as the second antibody, the concentration of coating antigen,

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                                              (本文编辑:彭永刚)

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更新日期/Last Update: 2018-09-28