Objective: To construct the recombinant eukaryotic plasmid DNA expression vector encoding Mycobacterium tuberculosis antigen 85B (Ag85B) and to investigate its immunogenicity.
Methods: The gene encoding secreted form of Ag85B was amplified by polymerase chain reaction (PCR) from genome of Mycobacterium tuberculosis H37Ra strain, and was inserted into sites cut with Hind III plus EcoR I of eukaryotic expression vector pcDNA3 after restriction endonuclease digestion. The gene fragment encoding secreted form of Ag85B was inserted into the vector of E. coli JM109 strain and was confirmed by restriction endonuclease digestion. After 4 weeks since BALB/c mice were vaccinated by recombinant eukaryotic expressing vector, dot blotting and ELISA were used to detect the serum antibody against Ag85B and its titer.
Results: Recombinant eukaryotic expressing vector, namely pTB30s, constructed successfully based on the gene encoding secreted form of Mycobacterium tuberculosis Ag85B; pTB30s induced high titer specific antibody against Ag85B in immunized mice.
Conclusion: pTB30s as DNA vaccine should be further studied to confirm its stimulating role in cell-mediated immune responses in TB prevention.