Construction of Recombinant Adeno-Associated Virus Type 2 Containing Ag85A Gene of Mycobacterium tuberculosis

Objective To construct a recombinant adeno-associated virus type 2 ( rAAV-2 ) containing Ag85A gene of Mycobacterium tuberculosis, and to investigate the immunogenicity of the recombinant protein. Methods Ag85A gene was amplified by polymerase chain reaction (PCR) from the genome of Mycobacterium tuberculosis H37Rv strain. The amplified PCR product was cloned into the adeno-associated virus expressing vector pSNAV. The recombinant pSNAV-Ag85A was transfected into BHK-21 cell by using LipofectamineTM 2000. After transfection, the cells were screened with G418. The cells expressing Ag85A (BHK-Ag85A) were then infected with the helper virus for the packaging of the rAAV-2. After purification, rAAV-2-Ag85A was obtained. Expression of rAAV-Ag85A in BHK-21 was detected by Western blotting. Immune sera from rAAV-2-Ag85A immunized Balb/c mice were tested for anti-Ag85A antibodies by the ELISA method. Cytotoxicity of the T-lymphocyte (CTL) was determined using the ~(51)Cr release assay. Results The sequence of the amplified gene segment was identical to the Ag85A sequence reported in GenBank. The titer of the recombinant virus was 1×1012 virus particles/ml. The expressed Ag85A protein had a molecular mass of 32 kD. After immunization of Balb/c mice with rAAV-2-Ag85A, the titer of anti-Ag85A antibody was 1∶1 024. Specific CTL activity was also detected. Conclusion Recombinant virus expressing Ag85A was successfully constructed. rAAV-2-Ag85A can induce both humoral and cellular immune response. The results indicated that the rAAV-2-Ag85A may be developed as a vaccine for the prevention infection by Mycobacterium tuberculosis.