Construction and identification of Mycobacterium shuttle expression plasmid pJHSP70

吕琳; 曹红丹; 王丕龙; 刘少宁; 王丽娟; 向廷秀

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Construction and identification of Mycobacterium shuttle expression plasmid pJHSP70

更新时间：2026-01-09

- Construction and identification of Mycobacterium shuttle expression plasmid pJHSP70
- Journal of Army Medical University Vol. 30, Issue 10, Pages: 890-893(2008)
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  CLC： Q75;Q781;Q939.131
- Published：2008
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吕琳,曹红丹,王丕龙,刘少宁,王丽娟,向廷秀.分枝杆菌穿梭表达质粒pJHSP70的构建及鉴定[J].陆军军医大学学报(原第三军医大学学报),2008,30(10):890-893.

　LU Lin,CAO Hong-dan,WANG Pi-long,LIU Shao-ning,WANG Li-juan,XIANG Ting-xiu.Construction and identification of Mycobacterium shuttle expression plasmid pJHSP70[J].J Amry Med Univ (J Third Mil Med Univ),2008,30(10):890-893.
吕琳,曹红丹,王丕龙,刘少宁,王丽娟,向廷秀.分枝杆菌穿梭表达质粒pJHSP70的构建及鉴定[J].陆军军医大学学报(原第三军医大学学报),2008,30(10):890-893. DOI：

　LU Lin,CAO Hong-dan,WANG Pi-long,LIU Shao-ning,WANG Li-juan,XIANG Ting-xiu.Construction and identification of Mycobacterium shuttle expression plasmid pJHSP70[J].J Amry Med Univ (J Third Mil Med Univ),2008,30(10):890-893. DOI：

摘要

目的用结核分枝杆菌热休克蛋白70(heat shock protein70

HSP70)启动子改建分枝杆菌穿梭质粒pJEM11为分枝杆菌穿梭表达质粒pJHSP70，并对其功能进行鉴定。方法以卡介苗(BCG)基因组DNA为模板，利用聚合酶链反应(polymerase chain reaction

PCR)扩增HSP70启动子，克隆至pMD18-T载体，经鉴定后，再定向克隆入pJEM11的ApaⅠ位点与SnaBⅠ位点之间

构建pJHSP70载体

并对载体进行PCR及酶切鉴定，然后再将幽门螺杆菌（Helicobacter pylori

HP）尿素酶B亚单位(urease B subunit

UreB)基因克隆入pJHSP70载体

评价其在耻垢分枝杆菌（Mycobacteria smegmatis，M.smegmatis）mc

155中的表达情况。结果从BCG基因组中扩增出160 bp的基因片段，所构建的穿梭质粒经酶切和PCR鉴定与预期结果一致。测序结果证实插入片段正确，UreB基因在M.smegmatis中成功表达。结论成功改建穿梭质粒pJEM11为穿梭表达质粒pJHSP70。

Abstract

Objective To reconstruct shuttle plasmid pJEM11 into Mycobacterium shuttle expression plasmid (pJHSP70) with Mycobacterium tuberculosis HSP70 promoter and identify the function of pJHSP70. Methods The HSP70 promoter was amplified from the BCG DNA by PCR and cloned into pMD18-T vector. After identified by enzyme digestion analysis and sequencing

HSP70 promoter was subcloned into pJEM11 that was digested with Apa Ⅰ and SnaB Ⅰ

to form the plasmid pJHSP70. After pJHSP70 was amplified by PCR and identified with enzyme digestion analysis

Helicobacte

r pylori UreB (Hp UreB) gene was cloned into pJHSP70. The expression of Hp UreB protein in the Mycobacteria smegmatis mc

155 was detected by SDS-PAGE. Results The fragment of 160 bp was amplified from the BCG DNA by PCR. Sequencing and homologous analysis showed that the homology between the cloned HSP70 gene and related genes in GenBank was 100%. SDS-PAGE analysis showed that Hp UreB protein expressed in Mycobacteria smegmatis mc

155. Conclusion The Mycobacterium shuttle expression plasmid pJHSP70

which was based on shuttle plasmid pJEM11

has been constructed successfully.

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