[Construction of recombinant adenovirus expressing Ag85B of Mycobacterium bovis and its cellular immunoproperties in mice].
OBJECTIVE
We constructed recombinant adenovirus rAd-Ag85B expressing antigen 85B of Mycobacterium bovis and analyzed cellular immune responses after mice immunization.


METHODS
We amplified the fbpB gene from genomic DNA of BCG by PCR and cloned into pMD18-T vector for sequence analysis. Then we constructed pDC516-Ag85B and co-transfected with plasmid pBHGfrtdeltaE1, 3FLP into Ad293 cells by lipofectamine 2000. Then we identified the recombinant Ad-Ag85B virus by RT-PCR, Western blot assay and TEM observation after negative staining. BALB/c mice were subcutaneously immunized with the dose of 10(8) TCID50 rAd-Ag85B or rAd. Two weeks after immunization, CD69 expression, T lymphocyte proliferation and ELISPOT were detected in spleen cells of immunized mice.


RESULTS
We obtained the correct recombinant adenovirus rAd-Ag85B expressed Ag85B antigen. The level of both splenic CD69+ CD4+ T and CD69+ CD8+ T cells of rAd-Ag85B group were significantly higher than those of rAd control. The SI index of rAd-Ag85B was much higher than rAd. In ELISPOT assay more IFN-gamma specific secreting cell spots other than IL-4 were detected in rAd-Ag85B group.


CONCLUSIONS
The recombinant adenovirus rAd-Ag85B can induce specific Th1 cell immune responses in mice.
Zhou
Haixia
H
Jiangsu Key Lab of Zoonosis, Yangzhou University, Yangzhou 225009, China. hannah5658zhou@yahoo.com.cn
Chen
Xiang
X
Ji
Yan
Y
Zhou
Weidong
W
Hu
Maozhi
M
Huang
Jinlin
J
Pan
Zhiming
Z
Jiao
Xin'an
X
D000256:Adenoviridae / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology*; D002417:Cattle; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D009163:Mycobacterium bovis / Q000235:genetics / Q000276:immunology*; D011897:Random Allocation; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D014380:Tuberculosis, Bovine / Q000276:immunology* / Q000382:microbiology
D000942:Antigens, Bacterial; D011994:Recombinant Proteins; C065036:antigen 85, Mycobacterium bovis