| Mycoplasma hyopneumoniae (Mhp) is the causative agent of Swine Enzootic Pneumoniae (SEP) which is called Mycoplasma Pneumoniae of swine(MPS) too, It is a disease found all over the world and has caused great loss. The characters of Mph are hyperinfection, chronicity, high morbidity and low mortality rate. Since the early detection of Mhp infection is difficult and there is no effective drugs (including vaccines), there are a lot to do to control this disease.Chaperones protein DnaK is a adventitial protein and the C termination of which is the major species-specific antigenic determinant of Mhp. The C termination of DnaK gene was amplified from Mycoplasma Hyopneumoniae Yin-1 by PCR technique, thus a 1000bp fragment which was named Dnakc was obtained. Dnakc was then cloned into pMD18T vector, and tested by PCR and double-digesting, then the positive clone was sequenced by Sanger's sequencing technique, which showed that the concordance of this gene sequence with the relative gene of Yin-1 published in GenBank was above 99%. The positive Dnakc gene was inserted into the expression vector pGEX-6p-1 and pET-28a, then were transformed into Escherichia coli(DE3) and expressed. Two recombinant proteins were obtained and were named 6P-1-DnakC and 28a-DnakC differently, SDS-PAGE showed the two recombinant proteins were 65KD and 42KD differently, Western-blot showed that they all had good immunogenicities.The recombinant protein 28a-DnakC was isolated and purified by Histag affinity chromatograph and SDS-PAGE showed the effect of purification was good. 8-week-old female BALB/c mice were injected intraperitoneally with 500μg recombination proteins 6P-1-DnakC, emulsified in complete Freund's adjuvant for the first immunization and in incomplete Freund's adjuvant for the next three times of injections with 2-week intervals, followed by an intravenous dose of the antigen without adjuvant 5 days prior to the fusion experiment. Splenocytes from the immunized mice were fused with SP2/0 myeloma cells and then the positive hybridoma were screened by indirect ELISA. The positive clones were subcloned three rounds by serial-limited dilution method. Two hybridoma cell lines secreting monoclonal antibodies (McAbs) against Mhp proteins were established and were named 1AD10 and2AF11 differently. The subtypes of the two McAbs (1AD10 and 2AF11) are IgG2b and IgG2a isotype, the light chains are k ones, and the titers are about 1:105 and 1:104 in indirect ELISA, additive ELISA showed that the two McAbs recognize different antigen epitopes. The result of Western–blot indicated that they could react specifically with Mhp, at 65KD and 42KD, and couldn't react with PGI, Y-goat, Mccp and Mh. McAbs against Dnak obtained in this experiment provide us with useful tools to further analyze the structure and function of Mhp and biologically diagnose MPS. |
