Construction of Shuttle Expression Plasmid and Stable Expression of Foreign Gene in Mycobacteria and E．Coli

By employing the pUC19 as a backbone,the regulatory and signal sequences which encode kanamycin resistance, and mycobacterial plasmid origin of replication (oriM) were cloned into the pUC19. The recombinant E. Coli-mycobacteria shuttle expression plasmid PBCG-8000 was constructed. The PBCG-8000 was able to replicate in both E. Coli and mycobacteria (including BCG) systems, and to confer stable kanamycin resistance upon transformants. The study should facilitate the development of BCG and other mycobacteria into multivalent vaccine vectors.

Expression of Foreign Gene in Mycobacterium Regulated by Human Mycobacterium Tuberculosis Heat Shock Protein 70 Promoter

The DNA fragments of 150bp length promoter 0f human Mycobacterium(M.) tuberculosis heat shock protein (hsp)7O and 650bp length foreign gene, the Schistosoma japonicum glutathione S-transferase(Sj26GST)gene,were obtained by amplification with polymerase chain reaction. And the 150p DNA sequence upstream initiation codon ATG of the human M. tuberculosis hsp7O 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 26000. 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.

The Construction of Schistosoma Japonicum Vaccine BCG-Sj26GST and Its Identification

Summary: The expression of foreign gene, Schistosoma Japonicum 26 ku antigen (Sj26GST), in Bacillus Calmette Guerin (BCG), Mycobacterium ( M. smegmatis ) and Escherichia coli ( E. coli ) were studied. The cDNA fragment encoding Sj26GST was amplified by PCR using plasmid pGEX, which could express Sj26GST in E. coli as template. The Sj26GST cDNA was cloned into the downstream of human M. tuberculosis heat shock protein (hsp) 70 promoter with correct reading frame, and then the DNA fragment containing hsp70 promoter and Sj26GST gene were subcloned together into E. coli Mycobacteria shuttle plasmid pBCG 2000 to construct the expression shuttle plasmid pBCG Sj26. The recombinant BCG and M. smegmatis mc 2 155, which were electroplated with pBCG Sj26, could express Sj26GST and the recombinant Schistosoma Japonicum vaccine BCG Sj26GST was made. The recombinant Sj26GST (rSj26GST) were soluble and could be observed on SDS PAGE at molecular weight of 26 ku. The content of rSj26GST accounted for 15 % and 10 % of total bacterial protein in BCG and M. smegmatis respectively. The results of Western blot showed the combination of rSj26GST with antibody of GST.