Cloning And Expression Of P65Gene From Mycoplasma Hyopneumoniae、Preparation Of The Monoclonal Antibody Against P65Protein And Establishment Of Blocking Elisa Assay For Antibody To Mycoplasma Hyopneumoniae

Mycoplasma hyopneumoniae (Mhp) was the causative agent of Mycoplasma pneumoniae of swine (MPS), and causes huge economic losses to the pig industry. Due to the lack of depth study for immunogenic and specific proteins of Mhp, the development of the pathogenic mechanism, serological detection methods and new recombinant subunit vaccine research about Mhp were restricted to some extent, so it affected the control of MPS. Protein P65, the immunodominance protein of Mhp, with no cross reaction with other mycoplasmas, is used as the target protein or gene for Mhp detection. In this study, membrane protein P65fragments of Mhp were expressed using the prokaryotic expression system, We employed the recombinant P65protein to immunize BALB/c mice for the preparation of monoclonal antibody, and employed the P65protein and the whole Mhp protein to screen the monoclonal antibody against these two antigens, then use the recombinant protein as coating antigen of ELISA, the P65monoclonal antibody with HRP-labeled was used as secondary antibody, in order to establish successfully a monoclonal antibody blocking ELISA detection method.1Accroding to the gene sequence of Mycoplasma hyopneumoniae(Mhp) P65reported in GenBank, primers were designed by the use of Primer5.0software. Gene was amplified by PCR, the front922bp fragment containing three rare codons in P65gene was obtained by gene synthesis, and the orther fragment of P65gene was amplified by PCR from Mhp168strain, then the mutated P65gene was obtained through the Splicing by overhang extension PCR(SOE-PCR) with the surplus segment connection, finally the mutated P65gene was inserted into expression vector pET-32a(+), the recombinant plasmid pET-32a(+)/P65was constructed. After it was successfully identified, the fusion protein was induced and expressed in E. coli BL21(DE3). When induced with1%IPTG after3h, the fusion protein got the highest amount. The fusion protein was purified by Protino Ni-TED2000Packed Columns. Western-blot analysis proved that the fusion protein have satisfactory immunogenicity.2.In this study, We employed the recombinant P65protein to immunize BALB/c mice for the preparation of monoclonal antibody, and employed the P65protein and the whole Mhp protein to screen the monoclonal antibody against these two antigens, then one strain of hybridoma cells,3G12, was obtained through parallel screening with ELISA.The results of western blot showed that the McAb can reacted with the Mhp168F350whole cell protein and the P65recombinant protein,it has no reaction activity with vetor protein and the proteins of Mycoplasma hyorhinitis.The isotype of the3G12was Ig IgGl. The ELISA titer which reacted with P65of ascites of3G12was1:4000000, the ELISA titers which reacted with Mhpl68of ascites of3G12was1:25600. Ascites by the commercialization of Protein G was purified to get the high purity monoclonal antibody.3. In this study, we use recombinant protein P65as coating antigen, purified monoclonal antibody with labeling HRP as secondary antibody toestablish successfully the monoclonal antibody blocking ELISA method by optimizing the reaction conditions. According to the following formula blocking rate, blocking rate (percentage inhibition/PI)=(negative serum OD450nm value-detected serum OD450nm)/negative serum OD450nm value by100%. Through the detection of known serum, using standard ROC curve analysis, when OD450is0.563, the sensitivity and specificity was the largest, known as the critical value, at this time the blocking rate of36.456%. When serum sample OD450<0.563, the serum is judged to be positive; vice versa, OD450<0.563, it is negative. Trials show that coincidence rate of the blocking ELISA and IDEXX kit was92.92%, the repeatability was good, and it has no cross reaction with other pathogens. It may be used for Mhp epidemiological investigations and disease diagnosis.In this study, We had obtained successfully the recombinant P65protein of Mhp by expressing in the E.coli and preparaed one strain hybridoma cells of monoclonal antibody against P65,3G12. Then we established successfully the monoclonal antibody blocking ELISA. It provided the support for Mhp detection and subunit vaccine, and also give a hand for the further study of the pathogenic mechanism of this disease, and provided the support for establishing Mhp serological diagnosis method...