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《Acta Academiae Medicinae Militaris Tertiae》 2008-10
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Construction and identification of Mycobacterium shuttle expression plasmid pJHSP70

LU Lin1,CAO Hong-dan1,WANG Pi-long1,LIU Shao-ning1,WANG Li-juan1,XIANG Ting-xiu2(1Department of Gastroenterology,2Experiment Research Center,The First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China)  
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.
【Fund】: 国家自然科学基金(30371318);; 重庆市卫生局重点项目(07-1-007)~~
【CateGory Index】: Q78
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