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

2

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

2

155.     Conclusion    The Mycobacterium shuttle expression plasmid pJHSP70

which was based on shuttle plasmid pJEM11

has been constructed successfully.