| 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 mucociliary 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. In this experiments, Mhp strain ZCF23 was selected to prepare monoclonal antibodies, in order to develop new diagnostic reagents for detection of Mhp infection. 1. Development of monoclonal antibodies against Mycoplasma hyopneumoniae using the whole proteins of Mhp strain ZCF23Mhp strain ZCF23, grown in KM2 medium containing horse serum, were harvested by super-centrifugation and suspended in PBS. The 8-week-old female BALB/c mice were injected intraperitoneally with 400 ug whole proteins of freeze-thaw-killed Mhp, 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 5 days prior to the fusion experiment. Splenocytes from immunized mice were fused with Sp2/0 myeloma cells and positive hybridoma were screened by indirect EL1SA. The positive clones were subcloned three rounds by serial-limited dilution method. Five hybridoma cell lines secreting monoclonal antibodies (MAbs) against Mhp proteins, designed as 11C6, 11B6-1, 14C11-1, 10C 11-2 and 12E7-1, were established. The subtypes of all the five MAbs were IgG3 and the titers of their ascitic fluids were about 1:10000 in indirect ELISA. The immimoblotting result indicated that the five MAbs could react with 90kD, 70kD, 60kD and 40kD proteins, respectively, and couldn't bind with control horseserum samples. The results suggested that these MAbs were specific to the proteins of Mhp.2. Cloning and expression of P36 gene of Mycoplasma hyopneumoniae strain ZCF23It was reported that the P36 protein, which carrys species-specific antigenic determinants, is a immunodominant protein after infection of Mhp naturally. And the P36 gene is very conserved phylogenetically. Based on the gene sequence in Genbank, primers were designed for PCR amplification of the P36-encoding gene from the genomic DNA of Mhp strain ZCF23. A desired PCR product of 948bp was obtained after agarose gel electrophoresis analysis, and then the PCR product was cloned into pGEM T Easy vector for sequencing. It was shown that there was only one base pair verified, from A to G, and its sequence has 99.9% homogeneity to that in Genbank. The homogenity of amino acid sequence is 100%. After prediction of the antigenic epitopes of amino acid sequence using the Protein Sequence Analysis software, there were 15 antigenic epitopes in the 315 amino acids. Subsequently, the P36 gene of Mhp strain ZCF23 was subcloned into the prokaryotic expression vector pGEX 6P-1 through EcoR I and Xho I double digestion, then the recombinant expression vector pGEX 6P-1-P36 was constructed and transformed into E.coli BL21. When the recombinant bacteria BL21(6P-1-P36) were induced with IPTG, the fusion protein GST-P36 was expressed. After disrupted by sonication, the SDS-PAGE result indicated that some of the GST-P36 fusion protein was soluble and some formed inclusion bodies.3. Development of monoclonal antibody against P36 protein of Mycoplasma hyopneumoniaeThe recombinant bacteria BL21(6P-1-P36) were induced with IPTG and disrupted by sonication. After super-centrifugation, the soluble fusion protein GST-P36 was purified by affinity chromatography on Glutathione Sepharose 4B beads. The 8-week-old female BALB/c mice were injected intraperitoneally with 400 u g inclusion bodies containing GST-P36 fusion protein, 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...
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