Expression of foreign gene in mycobacterium regulated by human Mycobacterium tuberculosis heat shock protein 70 promoter.
The DNA fragments of 150bp length promoter of human Mycobacterium (M.) tuberculosis heat shock protein (hsp) 70 and 650bp length foreign gene, the Schistosoma japonicum glutathione S-transferase (Sj26GST) gene, were obtained by amplification with polymerase chain reaction. And the 150bp DNA sequence upstream initiation codon ATG of the human M. tuberculosis hsp 70 promoter that contains the sequence TTGAG and ATCATA which consensus with E. coli promoter's -35 and -10 region respectively, as well as ribosome binding site GGAGG at position -12--8 upstream the ATG were determined by SangerDideoxyribonucleotide-mediated chain-termination method. Then, the human M. tuberculosis hsp70 promoter and Sj26GST cDNA were cloned into E. coli-mycobacteria shuttle plasmid pBCG- 2000 to construct E. coli-Mycobacterium expression shuttle plasmid pBCG- Sj26 that can express Sj26GST gene. The M. smegmatis were electroporated and the positivecolonies were selected by kanamycin. The M. smegmatis containing the vector pBCG-Sj26 can be induced by heating and hydrogen peroxide (H2O2) to express GST. The molecular weight of the recombinant GST (rGST) was 26,000. The rGST contents that were about 10 percent of the total bacterial protein were analyzed by density scanning after running SDS-PAGE. This study would provide scientific evidences for application of hsp70 promoter in expressing foreign gene in mycobacterium and development of mycobacterium as multiple-valent vectoral vaccine.
Cheng
J
J
Department of Medical Molecular Biology, Research Center, Experimental Medicine, Tongji Medical University, Wuhan.
Huangfu
Y
Y
Feng
Z
Z
Liang
J
J
Xiao
H
H
D000818:Animals; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000096:biosynthesis*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D009163:Mycobacterium bovis; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D012549:Schistosoma japonicum / Q000235:genetics*
D018840:HSP70 Heat-Shock Proteins; D005982:Glutathione Transferase