[1]吕琳,曹红丹,王丕龙,等.分枝杆菌穿梭表达质粒pJHSP70的构建及鉴定[J].第三军医大学学报,2008,30(10):890-893.
 LU Lin,CAO Hong-dan,WANG Pi-long,et al.Construction and identification of Mycobacterium shuttle expression plasmid pJHSP70[J].J Third Mil Med Univ,2008,30(10):890-893.
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分枝杆菌穿梭表达质粒pJHSP70的构建及鉴定(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
30卷
期数:
2008年第10期
页码:
890-893
栏目:
论著
出版日期:
2008-05-30

文章信息/Info

Title:
Construction and identification of Mycobacterium shuttle expression plasmid pJHSP70
作者:
吕琳曹红丹王丕龙刘少宁王丽娟向廷秀
重庆医科大学附属第一医院:消化内科,实验研究中心
Author(s):
LU Lin CAO Hong-dan WANG Pi-long LIU Shao-ning WANG Li-juan XIANG Ting-xiu
Department of Gastroenterology, Experiment Research Center, The First Affiliated Hospital of Chongqing Medical University
关键词:
结核分枝杆菌热休克蛋白70启动子基因表达
Keywords:
Mycobacterium tuberculosis heat shock protein 70 promoter gene expression
分类号:
Q75;Q781;Q939.131
文献标志码:
A
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 smegmatisM.smegmatis)mc2 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, Helicobacter pylori UreB (Hp UreB) gene was cloned into pJHSP70. The expression of Hp UreB protein in the Mycobacteria smegmatis mc2 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 mc2 155.     Conclusion    The Mycobacterium shuttle expression plasmid pJHSP70, which was based on shuttle plasmid pJEM11, has been constructed successfully.

参考文献/References:

吕琳,曹红丹,王丕龙,等. 分枝杆菌穿梭表达质粒pJHSP70的构建及鉴定[J]. 第三军医大学学报,2008,30(10):890-893.

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更新日期/Last Update: 2008-05-26