Preparation Of Monoclonal Antibody Against P97 Of Mycoplasma Hyopneuniae

Mycoplasma hyopneumoniae(Mhp)is the causative agent of Swine Enzootic Pneumoniae (SEP),a disease found on pig farms worldwide which is characterized by high morbidity and low mortality rates.Since Mhp can compromise the rnucociliary clearance mechanism of respiratory, thus predispose the pigs to secondary pulmonary infections and increase the mortality.Now,the early detection of Mhp infection is difficult and there is also no effective drugs(including vaccines) to control.Chaperones protein DnaK is memberanous protein carrying species-specific antigenic determinants of Mhp.Protein P97 is the adhesin of Mhp.The 500bp fragment of C termination of DnaK gene(DnaKc)and The 670bp fragment of C termination of P97(P97c)was amplified from Mycoplasma Hyopneumoniae Yin-1 by PCR technique.The sequence of interest was cloned into pET-30a vector.After tested with PCR technique and double digesting,which was sequenced by Sanger's sequencing technique.The result showed the gene sequence identity of Dnakc and P97c was above 99%with the strain of GenBank.The Dnakc and P97c gene were transformed into Escherichia coli DE3 and expressed,the recombination proteins name 30a-DnakC-P97C,and 30a-P97C differently,which were 57KD and 36 KD through SDS-PAGE analysis.Western-blot analyses had showed that they all had immunogenicity of Mhp.The recombinant protein 30a-P97C containing His tag sequence was purified by the metal chelation chromatograpHy.It was proved that the purified recombinant protein had specificity strain through SDS-PAGE analysis.The 8-week-old female BALB/c mice were injected intraperitoneally with 50 ug recombination proteins 30a-DnakC-P97C,emuLsified in complete Freund's adjuvant for the first immunization and in incomplete Freund's adjuvant for next three injections with 2-week intervals,followed by an intravenous dose of the antigen without adjuvant 3 days prior to the fusion experiment.Splenocytes from immunized mice were fused with Sp2/0 myeloma cells and positive hybridoma were screened by indirect ELISA.The positive clones were subcloned three rounds by serial-limited dilution method,four hybridoma cell lines secreting monoclonal antibodies(MAbs)against Mhp proteins,designed as A3C9 and B4D5 for P97C,were established.The subtypes of all the two MAbs(A3C9 and B4DS)were IgG2b and IgG2a isotype, and their light chains are k chain,and the titers of their ascitic fluids were about 1:10~5 and1:10~6 in indirect ELISA.The reselt of additive ELISA showed that the McAbs could recognize different antigen epitopes.The Western-blot result indicated that the two MAbs of A3C9 and B4D5 could react with Mhp,at 97KD.They all couldn't react with PGI,Y-goat,Mccp and Mh.The McAbs against Dnak and P97 could be used for further analysis of the structure and function and biological diagnosis of Mhp...