Construction Of The Recombinant Adenovirus Expressing The C-Terminal Of The Mycoplasma Hyopneumoniae P97 Gene And Its Immune Effects

Mycoplasma hyopneumoniae(Mhp) is the causative agent of Mycoplasma Pneumoniae of swine(MPS),causing huge economic losses for pig-raising industry of the world which is characterized by high morbidity and low mortality rates. There are some questions such as low output, high cost, large response and the immune ways can not be recepted for Mhp vaccines currently used, so it is necessary to develop an efficient, safe and inexpensive new vaccines to control this epidemic disease.The surface adhesion molecules of Mhp play a key role in pathogenicity, for their adhesion to the epithelial cells of pig respiratory is the first step of pathogenicity. P97 protein is the most important adhesion in study currently. Cilium binding sites are mainly located in the C-terminal of the protein carrying specific antigenic determinants of Mhp. The 670bp fragment of C-terminal of P97(P97 C) was amplified from Mycoplasma hyopneumoniae Yin-1 by PCR technique, the primers was designed according to the gene sequences of strain 232 published in GenBank. The sequence of interest was cloned into pShuttle-CMV vector. After correctly tested by PCR technique and double digesting, it was sequenced. The recombinant plasmid which was identified correctly was linearized by PmeⅠand transformed into E.coli BJ5183-AD-1 competent cells containing adenovirus backbone vector to produce recombinant adenovirus DNA by homologous recombination and the positive recombinant plasmid could be digested by PacⅠinto 30kb and 4.5kb fragments. The positive recombinant plasmid was digested by PacⅠand purified by phenol and chloroform method, then transfected into AD293 cells. After 10-14 days, obvious cytopathic could be observed in the microscope, after subcultured and three rounds of plaque picked, the recombinant adenovirus was identified by RT-PCR, indirect immunofluorescence assay and Western Blot, the results showed that the recombinant adenovirus transcribed and expressed P97 C-terminal protein successfully. The recombinant adenovirus were amplified and purified by small cesium choride density centrifugation kit, then determined its titer and the titer could achieve to 5×1011TCID50/mL indicated that we got high purity and high titer recombinant adenovirus which could express P97 C-terminal protein of Mhp.Balb/c mice were immunized with recombinant adenovirus for 5×107 TCID50 of each mouse via intramuscular and intranasal routes and giving booster immunization for the same dose afer two weeks. ELISA and Lymphocyte transformation test showed that inoculation with the recombinant adenovirus by each route elicted 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.The recombinant adenovirus expressing P97 C-terminal gene was successfully constructed and it induced special humoral and mucosal immunity but no cell- mediated immunity, providing some inspiration for the study of new Mhp vaccine.