Novel constructs of tuberculosis gene vaccine and its immune effect on mice.
A novel tuberculosis (TB) gene vaccine containing mouse granulocyte macrophage-colony stimulating factor (mGM-CSF) and a TB antigen (Ag85A) was developed in this study. The genes encoding Ag85A and mGM-CSF were amplified by PCR respectively from the Ag85A-containing pBSby5 and pC-mGM-CSF. The genes were then cloned into two different polylinker sites of plasmid pIRES, forming a novel TB gene vaccine construct pI85AGM. Following transfection of pI85AGM plasmid into 7721 cell line by Lipofectamine(TM), the expression of Ag85A and GM-CSF proteins was identified by Western blotting or RT-PCR. Then Balb/c mice were inoculated with the recombinant pI85AGM, pI85A, pIGM or plasmid alone, respectively. The activities of CTL, NK cells and the Ag85A-stimulated proliferation of spleen cells were measured by MTT method. The serum antibody against Ag85A was detected by ELISA. The results showed that the Ag85A and GM-CSF proteins could be expressed in 7721 cell line and the activity of CTLs and the proliferation of spleen cells were significantly increased in the pI85AGM-immunized mice, indicating that the pI85AGM-immunized mice could generate specific immune responses to Ag85A. This study might provide possibility for developing novel anti-TB gene vaccine.
Dou
Jun
J
Department of Pathogenic Biology and Immunology, Southeast University School of Basic Medical Science, Nanjing, China. njdoujun@yahoo.com.cn
Chen
Jun Song
JS
Wang
Jing
J
Chen
Guo Bin
GB
Zhao
Feng Shu
FS
Tang
Quan
Q
Fang
Xue Song
XS
Chu
Li Li
LL
Pan
Meng
M
D000217:Acyltransferases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood / Q000276:immunology; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007694:Killer Cells, Natural / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013154:Spleen / Q000166:cytology / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology / Q000378:metabolism; D032581:Tuberculosis Vaccines / Q000235:genetics* / Q000276:immunology*
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D018076:DNA, Complementary; D011994:Recombinant Proteins; D032581:Tuberculosis Vaccines; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D000217:Acyltransferases; C112580:antigen 85A, Mycobacterium tuberculosis