牛分枝杆菌eis基因在耻垢分枝杆菌中的表达及其生物学活性鉴定

doi:10.16431/j.cnki.1671-7236.2017.07.025

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (7): 2079-2085.doi: 10.16431/j.cnki.1671-7236.2017.07.025

牛分枝杆菌eis基因在耻垢分枝杆菌中的表达及其生物学活性鉴定

张通明, 宋天琪, 鑫婷, 朱鸿飞, 贾红

中国农业科学院北京畜牧兽医研究所, 北京 100193

收稿日期:2017-02-13 出版日期:2017-07-20 发布日期:2017-07-22
通讯作者: 贾红 E-mail:jiahong80@126.com
作者简介:张通明(1991-),男,北京人,硕士,研究方向:预防兽医学,E-mail:tongmingz@foxmail.com
基金资助:
国家自然科学基金（31302130）；中国农业科学院科技创新工程（ASTIP-IAS-11）

Expression and Bioactivity Identification of eis Gene of Mycobacterium bovis in Mycobacterium smegmatis

ZHANG Tong-ming, SONG Tian-qi, XIN Ting, ZHU Hong-fei, JIA Hong

Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received:2017-02-13 Online:2017-07-20 Published:2017-07-22

摘要/Abstract

摘要：

试验旨在构建牛分枝杆菌eis基因的穿梭表达载体，鉴定其在重组耻垢分枝杆菌中的生物学活性。采用PCR技术扩增并克隆牛分枝杆菌eis基因，构建大肠杆菌-分枝杆菌穿梭表达载体pMV261-Mbeis，经双酶切和测序鉴定其正确性，利用电穿孔法将重组质粒转化至耻垢分枝杆菌mc²155中，采用SDS-PAGE和Western blotting技术检测牛分枝杆菌eis基因在耻垢分枝杆菌中的表达，质谱鉴定目的蛋白氨基酸序列。研究结果表明，成功构建了牛分枝杆菌eis基因穿梭表达载体pMV261-Mbeis；生长曲线表明负载重组质粒不会影响耻垢分枝杆菌的体外生长；SDS-PAGE和Western blotting检测证实了牛分枝杆菌eis基因在耻垢分枝杆菌中可表达出分子质量约44 ku的eis蛋白；质谱检测证明了该蛋白即为牛分枝杆菌eis蛋白。本研究构建的牛分枝杆菌eis基因穿梭表达质粒pMV261-Mbeis在耻垢分枝杆菌中具有生物学活性，为下一步研究表达产物eis蛋白的功能奠定了基础。

关键词: eis基因; pMV261; 耻垢分枝杆菌

Abstract:

This study was aimed to construct a shuttle expression vector of Mycobacterium bovis(M.bovis) eis gene and identify its bioactivity in recombinant Mycobacterium smegmatis (M.smegmatis). M.bovis eis gene was cloned by PCR and the shuttle expression vector pMV261-Mbeis was constructed, then it was identified by double digestion and sequencing. The recombinant plasmid was transformed into M.smegmatis mc²155 by electroporation. The expression of M.bovis eis gene in M.smegmatis was detected by SDS-PAGE and Western blotting, and the amino acids sequence of the target protein was identified by mass spectrometry. The growth curve of recombinant M.smegmatis mc²155 containing pMV261-Mbeis was successfully constructed.The results showed that pMV261-Mbeis did not affect the growth of M. smegmatis in vitro. The results of SDS-PAGE and Western blotting confirmed that the M. bovis eis gene expressed the eis protein which was about 44 ku in M. smegmatis. Mass spectrometry proved that the protein was the eis protein of M. bovis.The expression vector pMV261-Mbeis was successfully constructed and the expressed recombinant protein was proved to be have biological activities in M. smegmatis, which laid a foundation for the further study of the function of eis protein in M. bovis.

Key words: eis gene; pMV261; Mycobacterium smegmatis

中图分类号:

S855.2

张通明, 宋天琪, 鑫婷, 朱鸿飞, 贾红. 牛分枝杆菌eis基因在耻垢分枝杆菌中的表达及其生物学活性鉴定[J]. 《中国畜牧兽医》, 2017, 44(7): 2079-2085.

ZHANG Tong-ming, SONG Tian-qi, XIN Ting, ZHU Hong-fei, JIA Hong. Expression and Bioactivity Identification of eis Gene of Mycobacterium bovis in Mycobacterium smegmatis[J]. , 2017, 44(7): 2079-2085.

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牛分枝杆菌eis基因在耻垢分枝杆菌中的表达及其生物学活性鉴定

Expression and Bioactivity Identification of eis Gene of Mycobacterium bovis in Mycobacterium smegmatis

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