猪腺病毒3型Hexon蛋白多克隆抗体制备与免疫活性鉴定

doi:10.16431/j.cnki.1671-7236.2015.01.001

›› 2015, Vol. 42 ›› Issue (1): 1-8.doi: 10.16431/j.cnki.1671-7236.2015.01.001

• 生物技术 • 下一篇

猪腺病毒3型Hexon蛋白多克隆抗体制备与免疫活性鉴定

汪镇南^1,2, 唐青海^2,3, 颜爱^1,2, 刘云福^1,2

1. 中国农业大学动物医学院, 北京 100193;
2. 湖南九鼎集团兽药公司兽医中心, 岳阳 414000;
3. 南阳师范学院南阳市兽医生物工程技术研究中心, 南阳 473061

收稿日期:2014-08-02 出版日期:2015-01-20 发布日期:2015-02-06
通讯作者: 唐青海 E-mail:qinghaitang109@126.com
作者简介:汪镇南(1981-), 男, 湖南人, 博士生, 研究方向:动物传染病学与新兽药研发
基金资助:
国家自然基金青年基金(31101837);河南省重点科技攻关项目(142102110101);南阳师范学院引进人才专项项目(70640)

Preparation and Immuno-activity Identification of Polyclonal Antibodies against Porcine Adenovirus Serotype 3 Hexon Protein

WANG Zhen-nan^1,2, TANG Qing-hai^2,3, YAN Ai^1,2, LIU Yun-fu^1,2

1. College of Veterinary Medicine, China Agricultural University, Beijing 100193, China;
2. Center for Veterinary Medicine, Hunan Jiuding Group Veterinary Pharmaceutical Company, Yueyang 414000, China;
3. Center for Nanyang Veterinary Biological Engineering Technology, Nanyang Normal University, Nanyang 473061, China

Received:2014-08-02 Online:2015-01-20 Published:2015-02-06

摘要/Abstract

摘要： 本研究旨在制备猪腺病毒3型(PADV-3)Hexon蛋白多克隆抗体.试验构建重组原核表达载体pET28a-Hexon,IPTG诱导重组蛋白的表达并进行Western blotting鉴定,目的蛋白与佐剂混合、乳化制备免疫原,免疫家兔制备Hexon蛋白多克隆抗体.采用免疫过氧化物酶单层细胞染色法(IPMA)检测抗体的免疫活性与抗体滴度.结果显示,Hexon蛋白原核表达以包涵体形式存在,分子质量约为105 ku;真核细胞表达的Hexon蛋白均定位于HEK293细胞质内,细胞核内无分布;IPMA测定所制备的多克隆抗体滴度为1:1 600,该抗体与体外培养的PADV-3及稳定表达Hexon蛋白的细胞均呈特异性反应.结果表明本试验制备的PADV-3型Hexon蛋白多克隆抗体免疫活性和特异性良好.

关键词: 猪腺病毒3型; Hexon基因; 多克隆抗体

Abstract: This study was aimed to prepare polyclonal antibodies against porcine adenovirus serotype 3 (PADV-3) Hexon protein.The recombinant prokaryotic expression plasmid pET28a-Hexon was constructed and transformed into BL21 (DE3), 1 mmol/L concentration of IPTG was used to induce the target gene expression which was then analyzed by SDS-PAGE and Western blotting.The expressed proteins were emulsified with freund's adjuvant, the mixture was used to immunize the rabbit to prepare the polyclonal antibodies against PADV-3 Hexon protein.The immunocompetence and titers of the antibody were identified by immunoperoxidase monolayer assay (IPMA).The results showed that prokaryotic expressed Hexon protein existed as inclusion bodies, with a molecular weight of 105 ku, eukaryotic expressed Hexon protein was only observed in the cytoplasm of HEK293 cells.PADV-3 Hexon-specific polyclonal antibody showed perfect activity which recognized the PADV-3 propagated in PK-15 cells and the recombinant Hexon protein expressed in HEK293 cells specifically, it was IPMA antibody positive when diluted in 1:1 600 ratio.The prepared PADV-3 Hexon polyclonal antibodies showed wonderful activity and specificity.

Key words: porcine adenovirus serotype 3; Hexon gene; polyclonal antibody

中图分类号:

S852.4

汪镇南, 唐青海, 颜爱, 刘云福. 猪腺病毒3型Hexon蛋白多克隆抗体制备与免疫活性鉴定[J]. , 2015, 42(1): 1-8.

WANG Zhen-nan, TANG Qing-hai, YAN Ai, LIU Yun-fu. Preparation and Immuno-activity Identification of Polyclonal Antibodies against Porcine Adenovirus Serotype 3 Hexon Protein[J]. , 2015, 42(1): 1-8.

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猪腺病毒3型Hexon蛋白多克隆抗体制备与免疫活性鉴定

Preparation and Immuno-activity Identification of Polyclonal Antibodies against Porcine Adenovirus Serotype 3 Hexon Protein

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