|本期目录/Table of Contents|

[1]梁锦屏,张京燕,陈少红,等.结核分枝杆菌Rv2623蛋白的表达、纯化及诱导特异性免疫应答研究[J].宁夏医科大学学报,2018,(06):681-684.[doi:10.16050/j.cnki.issn1674-6309.2018.06.013]
 LIANG Jinping,ZHANG Jingyan,CHEN Shaohong,et al.Prokaryotic Expression, Purification,and Specific Immune Response of the Rv2623 Protein of Mycobacterium Tuberculosis[J].Ningxia Medical University,2018,(06):681-684.[doi:10.16050/j.cnki.issn1674-6309.2018.06.013]
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结核分枝杆菌Rv2623蛋白的表达、纯化及诱导特异性免疫应答研究(PDF)
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《宁夏医科大学学报》[ISSN:1005-8486/CN:64-1029/R]

卷:
期数:
2018年06期
页码:
681-684
栏目:
论著
出版日期:
2018-06-30

文章信息/Info

Title:
Prokaryotic Expression, Purification,and Specific Immune Response of the Rv2623 Protein of Mycobacterium Tuberculosis
作者:
梁锦屏12 张京燕3 陈少红1 汤业珍1 张 倩1 韩怀钦1
(1. 宁夏医科大学基础医学院,银川 750004; 2. 宁夏医科大学总医院临床病原生物重点实验室,银川 750004; 3. 山西长治医学院附属和平医院检验科,长治 046000)
Author(s):
LIANG Jinping12 ZHANG Jingyan3 CHEN Shaohong1 TANG Yezhen1 ZHANG Qian1 HAN Huaiqin1
(1. School of Basic Medicine, Ningxia Medical University,Yinchuan 750004; 2. Ningxia Key Laboratory of Clinical and Pathogenic Microbiology, the General Hospital of Ningxia Medical University, Yinchuan 750004; 3.The Affiliated Hospital of Changzhi Medical University, Changzhi 046000)
关键词:
结核分枝杆菌Rv2623表达免疫原性
Keywords:
mycobacterium tuberculosisRv2623expressionimmunogenicity
分类号:
R392
DOI:
10.16050/j.cnki.issn1674-6309.2018.06.013
文献标志码:
A
摘要:
目的 对结核分枝杆菌潜伏期Rv2623蛋白进行原核蛋白表达、纯化,并对其免疫学特性进行初步研究。方法 应用生物信息学方法分析Rv2623蛋白序列,PCR扩增基因,克隆到pET30b载体上,在大肠埃希菌BL21菌株中以IPTG诱导表达重组蛋白。经SDS-PAGE及Western blot鉴定后,亲和层析法纯化蛋白。将重组Rv2623免疫C57BL/6小鼠,制备免疫小鼠脾细胞,ELISA检测上清液特异性IFN-γ含量,流式细胞术联合胞内因子染色法,分析脾细胞中分泌TNF-α+、IFN-γ+的多功能CD4+T细胞水平。结果 成功构建、表达并纯化得到31.7kDa的重组Rv26232蛋白,SDS-PAGE 及 Western blot 分析表明表达产物正确。重组Rv26232蛋白免疫小鼠后,诱导其脾细胞分泌的特异性IFN-γ水平(121.9pg·mL-1)高于PBS组(37.8pg·mL-1),同时诱导高水平能分泌TNF-α+、IFN-γ单阳或双阳的特异性多功能CD4+T细胞(P<0.05)。结论 本研究成功表达并纯化结核分枝杆菌潜伏期蛋白Rv2623,明确其具有良好的免疫原性,并能诱导特异性免疫应答,为后续新型疫苗的研发奠定基础。
Abstract:
Objective To clone the gene of Rv2623, then express the fusion protein of Rv2623 in E.coli DH5α and purify the expressed protein,and evaluate the immunological properties of the recombinant protein. Methods The Rv2623 gene was amplified from the genome of M.tuberculosis H37Rv by PCR and subcloned into the expression vector pET30b and expressed in E.coli BL21. The expressed protein was identified by SDS-PAGE analysis and Western blot method,and subsequently purified it by Ni-NTA purification system. Immuned the mice, the level of IFN-γ secreted from splenocytes were detected by ELISA. The number of antigen-specific CD4+T cells which secreted TNF-α and IFN-γ were counted by flow cytometry(FCM) and intracellular cytokines staining(ICS). Results The Rv2623 gene of M. tuberculosis could be cloned into pET-30b(+) expression vector, and got a high expression. The level of IFN-γ in the recombinant protein group(121.9pg·mL-1)were higher than that of the control group(37.8pg·mL-1)(P<0.05). Induce mice can make the specfic CD4+Th1 type immune response increased obviously(P<0.05). Conclusions The recombinant proteins Rv2623 were built and expressed successfully and verified the proteins can induce good CD4+Th1 type immune response,can be used to conduct the new vaccine of tuberculosis.

参考文献/References:


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备注/Memo

备注/Memo:
收稿日期:2018-02-14
基金项目:国家自然科学基金(81660001);宁夏卫生计生委重点科研项目(2014-NW-006);大学生创新创业计划项目(NXCX2017112)
作者简介:梁锦屏(1977-),副教授,博士,研究方向:感染免疫。 E-mail:liangjp68@163.com
更新日期/Last Update: 2018-06-30