[1]石洁,朱岩昆,郑丹薇,等.结核分枝杆菌mpt59和esxA融合基因编码蛋白的原核表达[J].郑州大学学报(医学版),2015,(06):761-764.
 SHI Jie,ZHU Yankun,ZHENG Danwei,et al.Prokaryotic expression and immunogenicity analysis of mpt59 and esxA fusion gene from Mycobacterium tuberculosis[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2015,(06):761-764.
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结核分枝杆菌mpt59和esxA融合基因编码蛋白的原核表达()
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《郑州大学学报(医学版)》[ISSN:1671-6825/CN:41-1340/R]

卷:
期数:
2015年06期
页码:
761-764
栏目:
论著
出版日期:
2015-12-20

文章信息/Info

Title:
Prokaryotic expression and immunogenicity analysis of mpt59 and esxA fusion gene from Mycobacterium tuberculosis
作者:
石洁1朱岩昆1郑丹薇1马晓光1王少华1李辉1邢进1#吴彩霞2
1河南省疾病预防控制中心结核病防控所 郑州 4500162河南省疾病预防控制中心门诊 郑州 450016
Author(s):
SHI Jie1ZHU Yankun1ZHENG Danwei1MA Xiaoguang1WANG Shaohua1LI Hui1XING Jin1WU Caixia2
1Henan Research Institution for Tuberculosis Control,Henan Province Center for Disease Control and Prevention, Zhengzhou 4500162The Outpatient Department,Henan Province Center for Disease Control and Prevention, Zhengzhou 450016
关键词:
结核分枝杆菌mpt59基因esxA基因血清学诊断原核表达
Keywords:
Mycobacterium tuberculosis mpt59 gene esxA gene serological diagnosis prokaryotic expression
分类号:
R446.6
摘要:
目的:获取结核分枝杆菌mpt59和esxA基因编码的原核表达融合蛋白,并将该蛋白初步用于结核患者的快速诊断。方法:PCR扩增含有柔性链接肽段的esxA核苷酸序列,并将其连接至原核表达载体pET28a上,再将mpt59连接至pET28aesxA上。该融合蛋白在大肠杆菌BL21中原核表达后,用镍珠子进行亲和纯化。采用垂直电泳和免疫印迹分析重组蛋白,并用于结核患者的诊断。结果:成功构建了原核表达载体pET28aesxAmpt59,转化大肠杆菌BL21后经诱导产生了高水平的表达产物。用该纯化蛋白进行ELISA检测,结果表明其诊断结核的灵敏度为97.8%,特异度为100%。结论:构建了含mpt59和esxA融合抗原基因的原核表达载体,并诱导表达了融合蛋白,为进一步研究结核分枝杆菌疫苗或诊断试剂奠定了基础。
Abstract:
Aim: To obtain bacterially expressed prokaryotic protein encoded by mpt59 and esxA from Mycobacterium tuberculosis, and to apply this protein for rapid diagnosis of tuberculosis patients.Methods: The nucleotide sequence of esxA gene containing flexible peptide linker was obtained from Mycobacterium tuberculosis using PCR and was linked to pET28a expression vector.Then mpt59 gene was cloned into the pET28aesxA.The fusion protein was expressed in E.coil BL21 and purified by NiNTA. The purified fusion protein was confirmed by SDSPAGE and Western blot analysis, and was used for TB diagnosis.Results: The recombinant expression vector pET28aesxAmpt59 was successfully constructed. The E.coli BL21 strains with recombinant vector showed high level expressions of fusion protein after IPTG induction. The ELISA assay with purified protein for detecting Mycobacterium tuberculosis antibody showed a sensitivity of 97.8% and a specificity of 100%.Conclusion: The expression of recombinant fusion protein of Mycobacterium tuberculosis mpt59 and esxA lays a basis for further studies on researches such as vaccine or diagnostic reagent.

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更新日期/Last Update: 1900-01-01