[1]乔绪稳,张元鹏,陈瑾,等.猪圆环病毒2型荧光抗体的原核表达及初步应用[J].江苏农业学报,2017,(03):610-617.[doi:doi:10.3969/j.issn.1000-4440.2017.03.018]
　QIAO Xu-wen,ZHANG Yuan-peng,CHEN Jin,et al.Prokaryotic expression and preliminary application of fluorescent antibody against porcine circovirus type 2[J].,2017,(03):610-617.[doi:doi:10.3969/j.issn.1000-4440.2017.03.018]
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猪圆环病毒2型荧光抗体的原核表达及初步应用()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2017年03期
页码:
610-617
栏目:
畜牧兽医·水产养殖
出版日期:
2017-06-30

文章信息/Info

Title:
Prokaryotic expression and preliminary application of fluorescent antibody against porcine circovirus type 2
作者:
乔绪稳1; 张元鹏1; 陈瑾1; 张浩明1; 侯立婷1; 杨利1; 郑其升1; 侯继波1;2
（1. 江苏省农业科学院国家兽用生物制品工程技术研究中心，江苏南京210014；2.江苏省动物重要疫病与人兽共患病防控协同创新中心，江苏扬州225009）
Author(s):
QIAO Xu-wen1; ZHANG Yuan-peng1; CHEN Jin1; ZHANG Hao-ming1; HOU Li-ting1; YANG Li1; ZHENG Qi-sheng1; HOU Ji-bo1;2
（1.National Research Center of Veterinary Biological Engineering and Technology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;2.Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009，China）
关键词:
猪圆环病毒2型（PCV2）; 荧光抗体; 原核表达; 直接免疫荧光检测
Keywords:
porcine circovirus type 2(PCV2); fluorescent antibody; prokaryotic expression; direct immunofluorescence assay
分类号:
S852.65+9.2
DOI:
doi:10.3969/j.issn.1000-4440.2017.03.018
文献标志码:
A
摘要:
本研究旨在利用原核大肠杆菌系统表达1株猪圆环2型病毒（Porcine circovirus type 2，PCV2）小分子荧光抗体，以及该荧光抗体用于PCV2病毒直接免疫荧光（Direct immunofluorescence assay, DIF）检测。选取1株筛选到的PCV2特异性纳米抗体基因V22，按照E.coli原核表达系统进行密码子优化重新合成，与荧光蛋白基因融合后插入pET28（a）中，20 ℃条件下诱导表达16 h，利用SDS-PAGE、Western-blot技术鉴定蛋白是否表达及其可溶性，并对可溶荧光抗体蛋白进行镍柱亲和层析纯化,纯化蛋白用于建立PCV2特异性DIF检测方法，同时对其工作条件、工作浓度、特异性以及稳定性进行验证，并与PCV2经典间接免疫荧光检测方法进行比较，验证其灵敏度。结果表明，PCV2荧光抗体基因在E.coli原核表达系统中表达，可溶蛋白占目的蛋白的60%左右；纯化后荧光抗体蛋白呈现明显绿色，具有良好的荧光活性，不同批次表达及纯化的荧光抗体效果一致，-20 ℃保存6个月后抗体效价不变；PCV2 特异性DIF试验结果表明该荧光抗体只对PCV2病毒感染细胞具有良好的反应原性，利用该抗体建立的直接免疫荧光法能够用于PCV2病毒滴度的测定，滴度测定结果与间接免疫荧光法检测结果一致。说明，在原核系统中成功表达PCV2的小分子荧光抗体，该荧光抗体蛋白具有良好的特异性、灵敏度及稳定性，用该抗体建立的PCV2特异性DIF操作简单，且具有较好的特异性和敏感性，对PCV2的检测有潜在应用价值。
Abstract:
Our experiment was to express recombinant antibody fused with EGFP fluorescent in E.coli for detecting porcine circovirus type 2(PCV2) in direct immunofluorescence assay (DIF). The plasmid pET28a- V22-EGFP vector was transformed into Escherichia coli BL21(DE3) and V22-EGFP protein was induced by IPTG. Expression and solubility of recombinant V22-EGFP protein was analyzed by SDS-PAGE and Western blot and purified by Ni-NTA His·Bind Resin. The purified V22-EGFP protein was used to establish PCV2 direct immunofluorescence assay. SDS-PAGE and Western blot results revealed that recombinant V22-EGFP protein was successfully expressed with 60% soluble protein. With a obviously green appearances the purified V22-EGFP protein showed great and stable fluorescence activity, even stored at -20 ℃ for 6 months. The DIF assay based on V22-EGFP showed good specifity to PCV2-infected PK-15 cells only. Compared to the conventional PCV2 IFA method, the PCV2 DIF based on recombinant V22-EGFP was more stable, sensitive and specific, so it enjoys a great potential application in PCV2 research.

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备注/Memo

备注/Memo:
收稿日期：2016-10-25 基金项目：公益性行业(农业)科研专项(201303046); 江苏省农业科技自主创新基金项目[CX(15)1604] 作者简介：乔绪稳(1988-)，女，江苏徐州人，硕士，助理研究员，主要从事猪用新型疫苗与技术研究。 通讯作者：郑其升，（Tel）025-84392088；（E-mail）njcvc1302@163.com。侯继波,（Tel）025-84392008;（E-mail）houjibo@jaas.ac.cn
更新日期/Last Update: 2017-06-29