牛流行热病毒糖蛋白基因的原核表达与抗原性鉴定

doi:10.16431/j.cnki.1671-7236.2015.09.005

《中国畜牧兽医》---唯一指定的官方网站 ›› 2015, Vol. 42 ›› Issue (9): 2246-2253.doi: 10.16431/j.cnki.1671-7236.2015.09.005

牛流行热病毒糖蛋白基因的原核表达与抗原性鉴定

李志^1,2, 郑福英², 高闪电², 王素艳², 岳城¹, 殷宏^2,3

1. 新疆农业大学动物医学学院, 乌鲁木齐 830052;
2. 中国农业科学院兰州兽医研究所, 家畜疫病病原生物学国家重点实验室, 甘肃省动物寄生虫重点实验室, 兰州 730046;
3. 江苏省动物重要疫病与人兽共患病防控协同创新中心, 扬州 225009

收稿日期:2015-04-15 出版日期:2015-09-20 发布日期:2015-09-25
通讯作者: 岳城, 殷宏 E-mail:yuechengxnd@yahoo.com.cn;yinhong@caas.cn
作者简介:李志(1988-),男,甘肃泾川人,硕士生,研究方向:动物病毒分子生物学,E-mail:lizhi19880717@163.com
基金资助:
农业科技创新工程(ASTIP);国家农业公益性行业科研专项"重要牛羊虫媒病毒病防控关键技术研究与应用"项目(201303035);国家肉牛牦牛产业技术体系(NBCIS-CARS-38)

Prokaryotic Expression and Antigenic Characterization Identification of G Gene for Bovine Ephemeral Fever Virus

LI Zhi^1,2, ZHENG Fu-ying², GAO Shan-dian², WANG Su-yan², YUE Cheng¹, YIN Hong^2,3

1. College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China;
2. Key Laboratory of Veterinary Parasitology of Gansu Province, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China;
3. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Disease, Yangzhou 225009, China

Received:2015-04-15 Online:2015-09-20 Published:2015-09-25

摘要/Abstract

摘要： 为探索可用来开发牛流行热病毒(bovine ephemeral fever virus,BEFV)疫苗和诊断试剂的候选基因,本研究针对BEFV糖蛋白(G)基因设计了2对特异性引物,用PCR方法扩增基因片段,PCR产物经Xho Ⅰ和Nde Ⅰ双酶切后亚克隆到表达载体pET-30上,将鉴定正确的重组质粒(480-pET-30、1107-pET-30)转化大肠杆菌BL21(DE3)感受态细胞,培养阳性菌株D_{600 nm}值为0.6~1.0,37 ℃下用1.0 mmol/L IPTG诱导表达目的蛋白,将其纯化之后进行SDS-PAGE和Western blotting免疫原性分析。同时,应用间接ELISA、动物免疫试验及交叉反应试验对目的蛋白进行分析。结果表明,首次发现的1 107 bp基因片段是分段表达的,具有很好的生物活性和特异性,更适合作为开发疫苗和诊断试剂的候选基因,为今后建立BEFV的血清学诊断方法及疫苗研发提供了理论基础。

关键词: 牛流行热病毒; 原核表达; 免疫原性分析

Abstract: In order to explore specific candidate gene of antigens and diagnostics development for the bovine ephemeral fever virus (BEFV), the target gene sequences were derived from G gene by PCR amplification using two specific primers.The PCR products were digested by Xho Ⅰ and Nde Ⅰ and cloned into pET-30 vector, and the recombinant plasmids (480-pET-30, 1107-pET-30) were transformed into E.coil BL21 (DE3) cells, the D_{600 nm} of positive strains were 0.6 to 1.0.The recombinant strains were induced by IPTG (1.0 mmol/L) at 37 ℃.The characteristics of the target proteins were analyzed using SDS-PAGE and the immunogenicity was analyzed through Western blotting.At the same time, the target proteins were used as coating antigens to do ELISA and all rabbits were inoculated with recombinant proteins.The results showed that the expression feature of 1 107 bp gene fragment of G gene for the first time was segmented in vitro as well as has nicer biological activity and specificity, and it more be suitable as a candidate gene for molecular vaccine and diagnostics development.This study provided the theoretical foundation of the establishment of diagnostics technique and vaccine for BEFV.

Key words: bovine ephemeral fever virus (BEFV); prokaryotic expression; immunogenicity analysis

中图分类号:

S852.65

李志, 郑福英, 高闪电, 王素艳, 岳城, 殷宏. 牛流行热病毒糖蛋白基因的原核表达与抗原性鉴定[J]. 《中国畜牧兽医》---唯一指定的官方网站, 2015, 42(9): 2246-2253.

LI Zhi, ZHENG Fu-ying, GAO Shan-dian, WANG Su-yan, YUE Cheng, YIN Hong. Prokaryotic Expression and Antigenic Characterization Identification of G Gene for Bovine Ephemeral Fever Virus[J]. , 2015, 42(9): 2246-2253.

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牛流行热病毒糖蛋白基因的原核表达与抗原性鉴定

Prokaryotic Expression and Antigenic Characterization Identification of G Gene for Bovine Ephemeral Fever Virus

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