[1]毛立,刘霞,李文良,等.边界病病毒E2 蛋白质的原核表达及间接ELISA 检测方法的建立[J].江苏农业学报,2015,(01):93-99.[doi:10.3969/j.issn.1000-4440.2015.01.014]
　MAO Li,LIU Xia,LI Wen-liang,et al.Prokaryotic expression of border disease virus E2 and establishment of an indirect ELISA for serum antibody detection[J].,2015,(01):93-99.[doi:10.3969/j.issn.1000-4440.2015.01.014]
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边界病病毒E2 蛋白质的原核表达及间接ELISA 检测方法的建立()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2015年01期
页码:
93-99
栏目:
畜牧兽医·水产养殖
出版日期:
2015-02-28

文章信息/Info

Title:
Prokaryotic expression of border disease virus E2 and establishment of an indirect ELISA for serum antibody detection
作者:
毛立1; 刘霞12; 李文良1; 杨蕾蕾1; 张纹纹1; 魏建忠2; 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/国家兽用生物制品工程技术研究中心，江苏南京210014；2.安徽农业大学动物科技学院，安徽合肥230036）
Author(s):
MAO Li1; LIU Xia12; LI Wen-liang1; YANG Lei-lei1; ZHANG Wen-wen1; WEI Jian-zhong2; JIANG Jie-yuan1
（1.Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences/Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture/National Center for Engineering Research of Veterinary Bio-products, Nanjing210014, China；2.College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China ）
关键词:
边界病病毒; E2蛋白质; 间接ELISA
Keywords:
border disease virus; E2 protein; indirect ELISA
分类号:
Q939.4
DOI:
10.3969/j.issn.1000-4440.2015.01.014
文献标志码:
A
摘要:
边界病病毒（Border disease virus，BDV）是导致绵羊和山羊繁殖障碍的重要病原。为了建立检测BDV特异抗体的ELISA方法，本研究将BDV 基因克隆入原核表达载体pET-32a(+)，并构建重组表达菌，经SDS-PAGE和Western blot鉴定目的蛋白质。以纯化的重组表达蛋白质为抗原，建立间接ELISA（rE2-ELISA）检测方法。通过反应条件优化，确定抗原包被浓度4.0 μg/ml；封闭液为20 g/L BSA；血清最佳稀释度为1∶50，孵育1 h；二抗最佳稀释倍数为1∶30 000，作用45 min；以TMB为底物显色10 min。用该ELISA方法检测瘟病毒属的CSFV、BVDV阳性血清，结果均为阴性。对187份山羊血清样品进行临床检测，与Svanova试剂盒检测结果阳性符合率为76.25%，总符合率为74.87%；上述检测方法同时与Western blot鉴定结果进行比较，结果显示，Western blot结果与rE2-ELISA检测结果的符合率为79.55%，高于与Svanova试剂盒检测的符合率（72.73%）。表明建立的间接ELISA检测方法可适用于临床BDV血清样品的抗体检测。
Abstract:
Border disease virus(BDV) is main pathogen causing reproductive manifestations of sheep and goat. To develop an ELISA for detection of the specific antibody of BDV， the antigenic region of BDV E2 protein was amplified by RT-PCR, and cloned into pET-32a(+). The positive plasmid was transformed to BL21 and the recombinant bacteria were obtained. The recombinant protein was identified by SDS-PAGE and Western blot. The indirect ELISA was developed successfully through the optimization of reaction system which involved coated antigen of 4.0 μg/ml, sealing buffer of 20 g/L BSA, serum sample at 1∶50 dilution and incubation for 1 h at 37 ℃, HRP conjugated rabbit anti-pig IgG at 1∶30 000 dilution and the reaction time of 45 min at 37 ℃, and colour development at room temperature for 10 min. Coating antigen had no cross reaction with the antibodies against BVDV and CSFV. The rE2-ELISA showed a BDV positive coincidence rate of 76.25% and a total coincidence rate of 74.87% with Svanova ELISA kit. Western blot identification revealed a coincidicen rate of 79.55% in 44 serum sample, higher than that identified by Svanova ELISA kit. It was indicated that the rE2-ELISA was suitable for large-scale epidemiological investigation for BDV infection.

参考文献/References:

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备注/Memo

备注/Memo:
收稿日期：2014-06-18 基金项目：江苏省农业科技自主创新基金项目[CX(14)2090] 作者简介：毛立（1985-），男，湖北荆州人，助理研究员，主要从事动物疫病诊断技术研究。（E-mail）mao-li@live.cn 通讯作者：江杰元，（E-mail）jieyuanj57@gmail.com
更新日期/Last Update: 2015-02-28