Construction of recombinant adenovirus expressing Mycoplsma hyopneumoniae p52 gene and its immune response.

To construct recombinant adenovirus carrying p52 gene of Mycoplasma hyopneumoniae,a p52 gene was amplified from the strain J and cloned into pShuttle-CMV plasmid.The recombinant plasmid pShuttle-CMV-P52 was linearized with PmeⅠ and then transformed into Escherichia coli BJ5183-AD-1 competent cells containing backbone vector pAdEasy-1 to homologous recombination.Purified recombinant adenovirus DNA was transfected into AD-293 cells to obtain complete recombinant adenovirus.The recombinant adenovirus was identified by RT-PCR and indirect immunofluorescence assay,then its titer was determined.BALB/c mice were immunized and the immunity was analyzed through humoral,mucosal and cell-mediated immunity aspects.The results showed that the inserted gene was 1 202 bp in length.Digestion with PacⅠ showed that homologous recombination was successful.The recombinant adenovirus was successful and P52 protein was expressed from it,and its titer was 1×106TCID 50/mL.Inoculation with the recombinant adenovirus via intramuscular and intranasal routes induced IgG and SIgA specific to P52 protein in blood and lung,but did not induce specific lymphocyte proliferation.It was confirmed that the recombinant adenovirus expressing the p52 gene was successfully constructed and it induced specific humoral and mucosal immunities but not cell-mediated immunity.