猪乙型脑炎病毒E蛋白结构域Ⅲ原核表达和抗原性分析

doi:10.16431/j.cnki.1671-7236.2016.04.012

›› 2016, Vol. 43 ›› Issue (4): 934-939.doi: 10.16431/j.cnki.1671-7236.2016.04.012

猪乙型脑炎病毒E蛋白结构域Ⅲ原核表达和抗原性分析

厚华艳^1,2, 丁玲玲^1,2, 吕茂杰^1,2, 杨保收^1,2

1. 天津瑞普生物技术股份有限公司瑞普生物研究院, 天津 300308;
2. 农业部生物兽药创制重点实验室, 天津 300308

收稿日期:2015-09-21 出版日期:2016-04-20 发布日期:2016-04-27
通讯作者: 杨保收 E-mail:bsyang@ringpu.com
作者简介:厚华艳(1987-),女,内蒙古赤峰人,硕士,研究方向:家畜疫苗研发,E-mail:flloveyf@163.com
基金资助:
瑞普生物研究院猪乙型脑炎耐热保护剂活疫苗专项基金项目(RPBVI20130008)

Prokaryotic Expression and Antigenicity Analysis of Porcine Japenese Encephalitis Virus Envelope Protein Domain Ⅲ

HOU Hua-yan^1,2, DING Ling-ling^1,2, LV Mao-jie^1,2, YANG Bao-shou^1,2

1. Ringpu Biological Research Institute, Tianjin Ringpu Biotechnology Co., Ltd., Tianjin 300308, China;
2. Key Laboratory of Biogenic Veterinary Drugs Innovation, Ministry of Agriculture, Tianjin 300308, China

Received:2015-09-21 Online:2016-04-20 Published:2016-04-27

摘要/Abstract

摘要： 为分析猪乙型脑炎病毒(JEV)E蛋白Ⅲ结构域的抗原性,本研究克隆了JEV疫苗株SA14-14-2的E蛋白结构域Ⅲ,并通过pET-28a载体进行融合表达和纯化。用纯化后蛋白作为免疫原,免疫8周龄BALB/c小鼠,通过SDS-PAGE、Western blotting、间接ELISA及间接免疫荧光方法(IFA)检测小鼠及猪抗体滴度,验证E蛋白结构域Ⅲ的抗原性。SDS-PAGE结果表明融合蛋白以包涵体形式表达;Western blotting、间接ELISA检测结果表明表达产物具有良好的抗原性;纯化的蛋白免疫BALB/c小鼠ELISA方法检测特异性抗体滴度可达1×10⁵;猪JEV阳性血清ELISA抗体滴度可达5.1×10⁴;IFA结果表明JEV E Ⅲ蛋白产生的抗血清能很好的识别乙型脑炎病毒抗原。以上结果表明,表达、纯化的重组JEV E Ⅲ蛋白具有良好的抗原性。本试验结果为建立以E蛋白结构域为抗原的诊断方法提供了重要依据。

关键词: 猪乙型脑炎病毒; E蛋白; 结构域Ⅲ; 诊断抗原

Abstract: In order to analyze the antigenicity of porcine Japanese encephalitis virus (JEV) E protein domain Ⅲ, which was expressed by pET-28a vector with His-tag and purified through Ni-NTA, the BALB/c mice were immunized with the purified protein.We identified the antigenicity of domain Ⅲ of E protein and the anti-mice and anti-porcine JEV E Ⅲ protein specific antibody titers by SDS-PAGE, Western blotting, indirect ELISA and IFA.SDS-PAGE results showed the expressed target protein existed mainly in the form of inclusion body.Western blotting, ELISA test results showed that the protein had good reactivity with anti-serum.The mice immunized with the purified JEV E Ⅲ protein generated 1×10⁵ anti-JEV E Ⅲ protein specific antibody titers by ELISA, and the porcine immunized with the porcine JEV generated 5.1×10⁴ anti-JEV specific antibody titers.The IFA results showed that JEV E Ⅲ protein anti-serum could identify JEV antigen.The above results showed that the recombinant JEV E Ⅲ had good antigenicity.These results provided important basis for development of diagnostic antigen for JEV.

Key words: porcine Japanese encephalitis virus; E protein; domain Ⅲ; diagnostic antigen

中图分类号:

S852.65

厚华艳, 丁玲玲, 吕茂杰, 杨保收. 猪乙型脑炎病毒E蛋白结构域Ⅲ原核表达和抗原性分析[J]. , 2016, 43(4): 934-939.

HOU Hua-yan, DING Ling-ling, LV Mao-jie, YANG Bao-shou. Prokaryotic Expression and Antigenicity Analysis of Porcine Japenese Encephalitis Virus Envelope Protein Domain Ⅲ[J]. , 2016, 43(4): 934-939.

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猪乙型脑炎病毒E蛋白结构域Ⅲ原核表达和抗原性分析

Prokaryotic Expression and Antigenicity Analysis of Porcine Japenese Encephalitis Virus Envelope Protein Domain Ⅲ

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