Preparation And Identification Of Monoclonal Antibodies Against Expression Of The R1 Region Of P97 Gene Of Mycoplasma Hyopneumoniae In Escherichia Coil

The P97 adhesin of Mycoplasma hyopneumoniae (Mhp) is a memberanous protein carrying species-specific antigenic determinants. R1 region encodes characterized immunodominant. The recombinant containing R1 region gene of P97 of Mhp was induced to express by IPTG. The recombinant protein containing His tag sequence was purified by the metal chelation chromatography. It was proved that the purified recombinant protein had immune activity through Western blot analysis.Then, 100μg the purified recombinant protein were injected intraperitoneally to the 8 week-old female BALB/c mice ; and three days after final immunization ,spleen cells got from immunized mice were fused with the SP2/0 myeloma cells .Hybrid cells were screened out with the purified recombinant protein and unrecombinant protein by indirect ELISA, and positive clones were subcloned three times with limiting dilution method..Two hybridoma cell lines secreting monoclonal antibodies (MAbs) against recombinant protein, designed as A9H7 and H11E7 were established. Both of them were IgG1 subtype ,and their affinity index were 52μg/ml and 46μg/ml. Their ascitic fluids titer of ELISA were 1x106 and 1x105 respectively. Through Western blot analysis, it showed two MAbs specifically recognized the antigen of 97KD of Mhp strain 168. In Dot-ELISA, two MAbs could react with M.hp, but neither could bind with M.hyorhinis(Mh), M.flocculare(Mf) and M.gallisepticum(Mg).This suggested that two MAbs are specific to antigen of Mhp. Furthermore, MAb was used in the monoclonal blocking ELISA for specific detection of antibodies against Mhp. With this method, 15/36 serum samples were positive ,the same as IDEXX M.hp antibody test kit. Our experiment provides the basis for the future researching Mhp test kit and for establishing M.PS earlier diagnosis methods.