| Objective: The clinical, social, and economic burden of Bovine tuberculosis(bTB) remains high worldwide. The Bacilli Calmette-Guerin(BCG) vaccine which is currently available can protect younger animals but is less effective in adults.Therefore, these data highlight the need for new and more effective bTB vaccines.The antigen 85B(Ag85B) is the most abundant protein secreted by Mycobacterium tuberculosis in the early, which is able to elicit antibody-mediated and cell-mediated immunity, so the ag85 b gene can be used to development genetic engineering vaccine as a target gene. The human adenovirus type 5 is very popular in the field of gene therapy as an adenoviral vector. The recombinant adenovirus vector is a replication-defective vector owing to E1 deletion region of adenovirus, which can produce progeny viruses in AD293 cells that have complete E1 region. The foreign gene is inserted into the adenovirus vector to enable production of the protein under the control of the cytomegalovirus(CMV) promoter. The recombinant vaccine has been found to be equally safe and effective.Methods: Using the plasmid of pGEM-85 bL as template, the gene of the ag85 b was amplified by PCR amplification and the PCR product was inserted into the adenovirus vector pacAd5CMVK-NpA. The recombinant plasmid pacAd5CMV-ag85 b and the pacAd 5 9.2-100 Ad backbone were linearized by digesting with the Pac?enzyme, and subsequently were transfected into adenoviruses packing cells lines AD293 by cotransfection with X- tremeGENE HP. The positive recombinant adenovirus, which we named rAd5-CMV-ag85 b, was identified by PCR.The rAd5-CMV-ag85 b was amplified, purified with the Adenovirus cation maxi kit,and then determinated the virus titer by the method of TCID50. The expression of ag85 b gene and the biological activities of Ag85 B protein were analyzed using RT –PCR and IFA. Whereafter, the 6-week-old male BALB/c mice were injected with the rAd5-CMV-ag85 b, rAd5-CMV-NpA, BCG and 0.9% Nacl by multipoint intramuscular. After two weeks, the mice were injected again as strong immunogenicity. Two weeks later, the CD3+, CD4+, CD8+lymphocytes and TNF,IFN-?, IL-2 cytokines of immune mice was detected. The expression of IgG in the serum of immune mice also was investigated by ELISA.Results: 1. The ag85 b gene was obtained, the recombinant adenovirus vector pacAd5CMV-ag85 b was constructed successfully. The recombinant adenovirus rAd5-CMV-ag85 b was producted by packing in AD293 cells. 2. The rAd5-CMV-ag85 b was amplified and purified. The virus titer was detected which is1×108.22/ ml. The Ag85 b protein was expressed in high levels in AD293 cells and remained correct biological activities. 3. Among the rAd5-CMV-ag85b-immunized mice, the level of IgG was lower than the BCG control no obvious difference(P>0.05), which was higher than the group of rAd5-CMV-NpA and 0.9%Nacl, the difference was significant(P < 0.05). The number of CD4+T cells in the rAd5-CMV-ag85b-immunized mouse was higher than the group of 0.9%Nacl(P <0.05), which was higher than the group of rAd5-CMV-NpA but lower the BCG control(P>0.05). The number of CD8+T lymphocyte in the BCG control was higher the rAd5-CMV-ag85b(P>0.05), higher both the rAd5-CMV-NpA and 0.9%Nacl(P<0.05). The number of CD3+T lymphocyte in the group of rAd5-CMV-ag85 b was less than the BCG control but more the rAd5-CMV-NpA and 0.9%Nacl(P>0.05).Thenumber of lymphocyte which secreted TNF-? in the rAd5-CMV-ag85 b was higher the BCG control(P>0.05), the rAd5-CMV-NpA and 0.9%Nacl(P<0.05). The number of lymphocyte which secreted IL-2 in the rAd5-CMV-ag85 b was higher the BCG control,the rAd5-CMV-NpA and 0.9%Nacl(P>0.05). The number of lymphocyte which secreted IFN-? in the rAd5-CMV-ag85 b was higher the BCG control(P>0.05),the rAd5-CMV-NpA and 0.9%Nacl(P < 0.05). These results showed that the rAd5-CMV-ag85 b can efficiently protect BALB/c mouse from infection of bTB. The results provide a basis for further studies of the Bovine tuberculosis live carrier vaccine. |
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