Zhonghua yi xue za zhi · 2000
[Construction of recombinant BCG bearing Schistosoma japonicum 26Ku antigen gene and study on its immunogenicity on mice].
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Dai W, Huangfu Y, Zheng B
Аннотация
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.
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.