Construction of the recombinant adenovirus expressing the C-Terminal of the Mycoplasma hyopneumoniae p97 gene and its immune response

Abstract:

Abstract: [Objective] To construct recombinant adenovirus carrying C-Terminal of the adhesion factor gene p97 of Mycoplasma hyopneumoniae (Mhp) so as to provide basis for further studying new type Mhp vaccine. [Methods] We amplified p97 gene from the genome of Mhp and cloned into pShuttle-CMV plasmid. The correctly identified recombinant plasmid was linearized with PmeⅠand transformed into E. coli BJ5183-AD-1 competent cells containing adenovirus backbone vector to produce recombinant adenovirus DNA by homologous recombination. Purified recombinant adenovirus plasmid was linearized with PacI, and transfected into AD293 cells to obtain recombinant adenovirus. The recombinant adenovirus was identified by RT-PCR, indirect immunofluorescence assay and Western Blot, and purified by cesium chloride density centrifugation kit, then its titer was determined. Balb/c mice were immunized with recombinant adenovirus via intramuscular and intranasal routes and analysis the immunity results through humoral immunity,mucosal immunity and cell-mediated immunity aspect. [Results] Digestion by PacI proved successful homologous recombination. RT-PCR, Indirect immunofluorescence assay and Western Blot showed that the recombinant adenovirus transcribed and expressed P97 C- terminal protein successfully, the titer could achieve to 5×1011TCID50/mL after purification. Inoculation with the recombinant adenovirus by each route elicited P97 C-terminal protein specific serum and lung homogenate IgG and SIgA was induced by intranasal route, but the special lymphocyte proliferation was not induced by each route. [Conclusion] The recombinant adenovirus expressing p97 C-Terminal gene was successfully constructed and it induced special humoral and mucosal immunity but no cell-mediated immunity.