A new strategy: high level expression and immunogenicity analysis of triplicate repeated multigenes in Mycoplasma hyopneumoniae.

Highly immunogenic nucleotide fragments from 3 genes of Mycoplasma hyopneumoniae strain 232 were selected using information software technology. After repeating each fragment three times, a total of 9 nucleotide fragments were joined together to form a new nucleotide sequence called Mhp232_1092bp. Mhp232_1092bp was directly synthesized and cloned into a pET100 vector and expressed in Escherichia coli. After purification, the proteins were successfully validated by SDS-PAGE and Western blotting using mouse His-tag antibody and pig anti-Mhp serum. BALB/c mice were intraperitoneally injected with purified proteins in the high-dose (100 μg), medium-dose group (50 μg) and low-dose (10 μg) groups. Mice in each group were injected on day 1, day 8 and day 15 of feeding, respectively. Serum samples were collected from all mice on the day before immunization and on day 22 after immunization. The antibody level in the mouse serum was detected using western blotting using purified expressed proteins as antigens. IL-2, TNF-α and IFN-γ were simultaneously detected in the mouse serum by ELISA. The results showed that the 60 kDa protein was successfully expressed and reacted specifically with the specific serum Mhp His-Tag mouse monoclonal antibody and pig anti-Mhp serum. From day 0 to day 22 of immunization, IFN-γ increased from 269.52 to 467.74 pg/mL, IL-2 increased from 14.03 to 145.16 pg/mL, and TNF-α increased from 6.86 to 12.37 pg/mL. The IgG antibody in mice increased significantly from 0 day to day 22 after immunization. This study suggests that the expressed recombinant protein may serve as one of the novel vaccine candidates for Mhp.