牛分枝杆菌重组蛋白TB27.4的表达、纯化及活性鉴定

中国畜牧兽医

牛分枝杆菌重组蛋白TB27.4的表达、纯化及活性鉴定

高新桃¹，李平俊²，鑫婷²，贾红²，郭晓宇²，张改梅²，李明²，刘淑清²，朱鸿飞²，许雷¹

1.中国农业科学院研究生院，北京 100081；2.中国农业科学院北京畜牧兽医研究所，北京 100193

收稿日期:2014-03-10 出版日期:2014-08-20 发布日期:2014-08-22
通讯作者: 朱鸿飞（1965—），男，江苏人，研究员，从事重大人畜共患病研究。E-mail：bioclub@vip.sina.com；Tel：010-62819061 许雷（1964—），男，江苏人，研究员，从事微生物分子生物学与基因工程研究。E-mail:xuleimgd@soho.com；Tel: 010-82109695
作者简介:高新桃（1986—），男，江苏人，硕士生，研究方向：微生物分子生物学与基因工程。
基金资助:
中国农业科学院科技创新工程（ASTIP-IAS-11）；中国农业科学院北京畜牧兽医研究所中央级公益性科研院所基本科研业务费专项资金项目（2014ywf-zd-1）。

Expression, Purification and Immunological Activity Identification of Recombinant Proteins TB27.4 of Mycobacterium bovis

GAO Xin-tao¹, LI Ping-jun², XIN Ting², JIA Hong², GUO Xiao-yu², ZHANG Gai-mei², LI Ming², LIU Shu-qing², ZHU Hong-fei², XU Lei¹

1. Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China; 2. Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received:2014-03-10 Online:2014-08-20 Published:2014-08-22

摘要/Abstract

摘要： 为探索TB27.4蛋白在牛结核病鉴别诊断中的作用，本试验以牛分枝杆菌Vallee Ⅲ株基因组DNA为模板，PCR扩增tb27.4全长基因片段，将其定向克隆到原核表达载体pET-32a(+)中，构建重组质粒pET-TB27.4，优化原核表达条件，并用AKTA Purifier对蛋白的纯化条件进行优化。SDS-PAGE结果显示重组蛋白为可溶性表达，且大小与理论值相符，用牛分枝杆菌阳性血清进行Western blotting检测有特异性条带，且可特异性地刺激牛分枝杆菌感染牛外周血淋巴细胞释放大量IFN-γ。结果表明，重组蛋白TB27.4具有良好的B细胞活性和T细胞刺激活性，为进一步研究其在牛结核病诊断中的作用奠定了基础。

关键词: 牛分枝杆菌; TB27.4; 表达; 纯化; 活性鉴定

Abstract: In order to evaluate the diagnostic properties of protein TB27.4 against bovine tuberculosis, the gene coding for TB27.4 was amplified from Mycobacterium bovis (strain Vallee Ⅲ) genomic DNA by PCR, then cloned into pET-32a(+). The recombinant plasmid was transformed into E.coli BL21(DE3) and parameters for protein expression and purification were optimized. Results of SDS-PAGE and Western blotting showed that the recombinant protein was expressed in soluble form, and could be recognized by serum from Mycobacterium bovis-infected cattle. During ELISA, the recombinant TB27.4 could specifically stimulate the PBMCs of Mycobacterium bovis-infected cattle to secrete high amount of IFN-γ. These results indicated that the recombinant protein TB27.4 had good reactogenicity and T cell activity.This study laid the foundation for further study of the role which the recombinant protein TB27.4 played in the diagnosis of bovine tuberculosis.

Key words: Mycobacterium bovis; TB27.4; expression; purification; activity identification

高新桃，李平俊，鑫婷，贾红，郭晓宇，张改梅，李明，刘淑清，朱鸿飞，许雷. 牛分枝杆菌重组蛋白TB27.4的表达、纯化及活性鉴定[J]. 中国畜牧兽医.

GAO Xin-tao, LI Ping-jun, XIN Ting, JIA Hong, GUO Xiao-yu, ZHANG Gai-mei, LI Ming, LIU Shu-qing, ZHU Hong-fei, XU Lei. Expression, Purification and Immunological Activity Identification of Recombinant Proteins TB27.4 of Mycobacterium bovis[J]. China Animal Husbandry & Veterinary Medicine.

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