Establishment Of Mycoplasma Bovis Quick Detection Methods Based Lipoprotein P-48

Mycoplasma bovis is an important pathogen of bovine respiratory disease, bovine pneumonia, mastitis, arthritis and keratoconjunctivitis can be caused by it, so it has becomed a large important risk to obstruct the development of cattle industry. With the continuous development of the cattle industry the prevalence range of Mycoplasma bovis disease has becomed more widely and the frequency of occurrence also increasing. On the one hand for this research starts late in our country, and there is no recognized specific diagnostic method in the world. On the other hand for the current study to the pathogenesis of this disease has not yet reached a consensus, therefore it has brought a lot of inconvenience for disease control and prevention. Based on this, in this study Mycoplasma bovis P48 gene was amplified and through the prokaryotic expression vector for P48 protein prokaryotic expression. Polyclonal and monoclonal antibodies were prepared to research the related functions of P48 protein, it is laid the foundation for the research of prevention, immunology detection and pathogenic mechanism. At the same time this trial also established a method for the differential diagnosis of Mycoplasma bovis and Mycoplasma agalactiae and other pathogens, which has a high practical value for the rapid and accurate diagnosis of Mycoplasma bovis clinical1. Prokaryotic expression and purification of Mycoplasma bovis P48 protein and preparation of polyclonal antibody.The specific primers were designed refer to the Mycoplasma bovis P45 strains lipoprotein P48 sequence in Gen Bank, five termination codon TGA were samesense mutated to TGG In the coding region. The P48 gene was cloned into the expression vector p ET-28 a to build the prokaryotic expression vector p ET-P48, expression plasmids were transformed into E. coli BL21(DE3) and successful fusion protein product r Mb P48 about the size 51 ku through IPTG induction. Immunizing the New Zealand white rabbit with purified reco Mycoplasma bovis inant protein to polyclonal antibodies and verify their immunogenicity by western-blot, the results showed that P48 antiserum can occur specific reaction with the purified His-P48 antigen.2. Subcellular initial positioning of mycoplasma bovis P48 protein.Whole cell proteins, membrane proteins, cytosolic proteins of Mycoplasma bovis were prepared and detection with P48 antiserum by western-blot, the results show P48 protein has good immunogenicity and exist in membrane proteins proteins and cytosolic proteins of mycoplasma bovis. membrane proteins proteins and cytoplasmic protein as an antigen were coated to ELISA test with prepared P48 antiserum and the distribution of P48 protein were tested by fluorescent antibody. The results showed that P48 protein is mainly present in Mycoplasma bovis membrane separation phase. Whole cell immunofluorescence test results further confirm lipoprotein P48 is located in the membrane surface of Mycoplasma bovis.3. Preparation of the monoclonal antibodies of mycoplasma bovis P48 protein.Two hybirdomas were got after Immunized BALB / C mice with purified reco Mycoplasma bovis inant protein and the passaged test showed that the two hybirdomas can secrete monoclonal antibodies steady. The cell titers remained around 2 000 and ascites titers remained 640 000 stabled. It has no cross-reaction with pasteurella etc which can caused bovine respiratory disease, with high titer, stability and specificity.4. The establishment of Mycoplasma bovis PCR detection methodThe specific primers were designed refer to the Mycoplasma bovis P45 strains lipoprotein P48 sequence in Gen Bank, Mycoplasma bovis, Mycoplasma agalactiae, Mycoplasma mycoides, synovial mycoplasma, Mycoplasma gallisepticum gene were expanded by PCR and its sensitivity analysis. The Mycoplasma bovis disease material and suspected Mycoplasma infections material from Lintao, Wuwei and other place were expanded by PCR after processed, the results show that The primers have a high specificity and sensitivity, there is no purpose of the strip In addition to Mycoplasma bovis outside, there are clear purpose bands appear after the fresh clinical samples were expanded by PCR, this shows that the diagnostic method for differential diagnosis of Mycoplasma bovis has high practical value.