[Construction of recombinant BCG bearing Schistosoma japonicum 26Ku antigen gene and study on its immunogenicity on mice].
OBJECTIVE
To construct recombinant BCG vaccine bearing Schistosoma japonicum 26Ku glutathione S-transferase (Sj26GST) gene and determine its immunogenicity on BALB/c mice.


METHODS
Using techniques of molecular biology, human mycobacterium tuberculosis HSP70 promoter and Sj26GST gene were linked to produce a fused gene. The fused gene was cloned into an E. coli-Mycobacterium shuttle plasmid pBCG-2000 to construct an E. coli-Mycobacterium expression shuttle plasmid pBCG-Sj26 that could express Sj26GST gene. Then, the pBCG-Sj26 was introduced by electroporation into mycobacterium bovis BCG to construct a recombinant BCG vaccine bearing Sj26GST gene (rBCG- Sj26GST). The expression of Sj26GST gene in BCG was induced by heating. The lymphocyte stimulating index (SI), macrophage activity and IL-2, IFN-gamma levels of the serum and culture supernatant of spleen lymphocytes were tested after immunization of BALB/c mice with rBCG-Sj26GST vaccine.


RESULTS
The fused gene of HSP70 promoter and Sj26GST cDNA was inserted into an E. coli-Mycobacterium shuttle expression plasmid by analysing electrophoresis results on PCR products using plasmid pBCG-Sj26 as a templet. The content of rSj26GST contained 15% of total bacterial protein of BCG. The SI of the experimental group was 2.26 +/- 0.43, which was significantly higher than those in the control group (1.61 +/- 0.28, P < 0.05), vector group (1.48 +/- 0.30, P < 0.05) and BCG group (1.42 +/- 0.26, P < 0.05). The macrophage NO level of the experimental group was (357.42 +/- 84.11) nmol/ml which was significantly higher than those in the control group (183 nmol/ml +/- 33 nmol/ml, P < 0.01) and vector group (203 nmol/ml +/- 56 nmol/ml, P < 0.01). The serum IL-2 level of the experimental group was (267 pg/ml +/- 130 pg/ml), which was significantly higher than those in the control group (45 pg/ml +/- 15 pg/ml, P < 0.01) and vector group (52 pg/ml +/- 29 pg/ml, P < 0.05. Compared with the control group, the serum IFN-gamma level increased by 20%, the IL-2 level of the culture supernatant of spleen lymphocytes increased by 44%.


CONCLUSIONS
The foreign gene encoding Sj26 GST can be expressed in BCG. rBCG Sj26GST vaccine may induce stronger immune response in BALB/c mice than in control, vector and BCG groups.
Dai
W
W
Department of Medical Molecular Biology, Research Center of Experimental Medicine, Tongji Medical University, Wuhan 430030, China.
Huangfu
Y
Y
Zheng
B
B
D000818:Animals; D000947:Antigens, Helminth / Q000235:genetics / Q000276:immunology*; D001500:BCG Vaccine / Q000276:immunology*; D002478:Cells, Cultured; D003470:Culture Media; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors / Q000276:immunology*; D005982:Glutathione Transferase / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D007376:Interleukin-2 / Q000096:biosynthesis; D008214:Lymphocytes / Q000276:immunology; D008264:Macrophages / Q000276:immunology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D012549:Schistosoma japonicum / Q000201:enzymology* / Q000235:genetics; D013154:Spleen / Q000166:cytology / Q000276:immunology; D014614:Vaccines, Synthetic / Q000235:genetics / Q000276:immunology*
D000947:Antigens, Helminth; D001500:BCG Vaccine; D003470:Culture Media; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; D007371:Interferon-gamma; D005982:Glutathione Transferase