The construction ofSchistosoma Japonicum vaccine BCG-Sj26GST and its identification

Abstract

The expression of foreign gene,Schistosoma Japonicum 26 ku antigen (Sj26GST),in Bacillus Calmette-Guerin (BCG),Mycobacterium CM. smegmatis) andEscherichia coli (E. coli) were studied. The cDNA fragment encoding Sj26GST was amplified by PCR using plasmid pGEX, which could express Sj26GST inE. coli as template. The Sj26GST cDNA was cloned into the downstream of humanM. 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 intoE. coli-Mycobacteria shuttle plasmid pBCG-2000 to construct the expression shuttle plasmid pBCG-Sj26. The recombinant BCG andM. smegmatis mc²155, which were electroplated with pBCG-Sj26, could express Sj26GST and the recombinantSchistosoma 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 andM. smegmatis respectively. The results of Western blot showed the combination of rSj26GST with antibody of GST.

Keywords

BCG / M. smegmatis / shuttle plasmid / Schistosoma Japonicum 26ku antigen gene / vaccine / gene expression

References

[1] HussonR N, JamesB E, YoungR A. Gene replacement and expression of foreign DNA in mycobacteria. J Bacteriol, 1990, 172(2): 519-519

[2] HuangfuY M, ZhangD J, ChengJ Z, et al.. Construction ofshuttle expression plasmid and stable expression of foreign gene inMycobacterium andE. coli. J Tongji Med Uni, 1995, 15(3): 138-138

[3] 26Ku 1996, 25(3): 173

[4] S cDNA, 1998, 6(l): 1

[5] SambrookJ E F, FritschE F, MantiatisT. Molecular cloning, A Laboratory Manual, 1987WuhanCold Spring Harbor Laboratory Press97-97

[6] BCG, 1996,16(4): 42

[7] AldoviniA, YoungR A. Humoral and cell-mediated immune response to live recombinant BCG-HIV vaccines. Nature, 1991, 351(6): 479-479

[8] YasutomiY, KoenigS, HaunS S, et al.. Immunization with recombinant BCG -SIV elicits SIV-specific cytotoxic T lymphocytes in rhesus monkeys. J Immunol, 1993, 150: 3101-3101

[9] BCG, 1998,10 (Suppl): 27

[10] MckenzieK R, AdamsE, BrittonW J, et al.. Sequence and immunogenicity of the 70-kDa heat shock protein ofMycobacterium leprae. J Immunol, 1991, 147: 312-312