Prokaryotic Expression And Biological Function Of Mycoplasma Bovis P59 Protein

Mycoplasma bovis can lead to calf pneumonia,arthritis,adult cow mastitis and other diseases,causing huge economic losses to the cattle industry.Studies have shown that Mycoplasma bovis colonizes host cells mainly through the adhesion of membrane proteins and causes damage and death of host cells through metabolites.At present,some known biological functions of lipoproteins such as 24 Ku,pMB67,P26 have been confirmed,but there are still many undiscovered lipoproteins.There are no reports on the study of P59 protein and its function.In this study,the bioinformatics method was used to analyze the MBOVPG45₀018 gene sequence encoding the P59 protein,and the protein was subjected to prokaryotic expression to study its biological function.At the same time,the SYBR Green I fluorescent quantitative real-time PCR detection method of Mycoplasma bovis was established to provide experimental data for the study of the pathogenic mechanism of Mycoplasma bovis and pathogen prevention and control.1.Bioinformatics method to analyze the nucleic acid sequence and amino acid sequence of Mycoplasma bovis P59 and predict its function: The bioinformatics analysis of the P59 nucleic acid sequence and amino acid sequence showed that the p59 protein may be an extracellular component protein that exists in the ABC transport system of Mycoplasma bovis,and it may participates in the process of nutrient uptake or transport.It has important biological functions to the metabolism of Mycoplasma bovis.2.Using prokaryotic expression of Mycoplasma bovis P59 protein to preparation of polyclonal antibodies: The P59 gene was amplified and importd into the expression vector pET30 a,then it was transformed into E.coli BL21?DE3?.The P59 recombinant protein was successfully induced and purified which was immunized New Zealand white rabbits to make polyclonal antibodies.3.The biological function of P59 protein:?1?Western-blot results of Mycoplasma bovis membrane protein,P59 recombinant protein and cytoplasmic protein showed that the protein was recognized by P59 rabbit polyclonal antibody,which indicated that the recombinant protein has a certain reactogen.ELISA experiments were performed by using Mycoplasma bovis membrane protein and cytosolic protein as coat antigens and P59 rabbit polyclonal antibody as antibodies.The results showed that the numerical value of membraneprotein OD450 nm was significantly different from that of cytoplasmic protein OD450nm?P < 0.05?.According to Western-blot and ELISA experimental results,P59 protein mainly exists in Mycoplasma bovis with the form of membrane protein.?2?The prepared P59 rabbit polyclonal antibody was used to observe the effect of polyclonal antibody serum on the Mycoplasma bovis adhere to EBL cells.Under the confocal microscope,the green fluorescence on the membrane surface of rabbit polyclonal serum group was less than that on the membrane surface of rabbit negative sera group.It indicated that P59 rabbit polyclonal antibody can block pathogens and reduce the number of Mycoplasma bovis adhered to EBL cells.It Also indicated that P59 protein may be an adhesion-related protein of Mycoplasma bovis.?3?After counted the colonies in the solid medium by the growth inhibition test for three times to calculate the average number,the growth inhibition rate of the P59 rabbit polyclonal antibody serum group was 45.53%.It further showed that the P59 rabbit polyclonal antibody serum can significantly affect the growth of Mycoplasma bovis.?4?P59 protein was used to stimulate Cow peripheral blood lymphocytes.The experimental results showed that the OD450 nm of Cow No.1,3,4,5,6,and 7 were significantly different from that of the blank control?P < 0.05?.And the Cow No.8 is especially different?P < 0.01?.It indicated that P59 protein as a stimulant can promote Cow peripheral blood lymphocytes' s propagation to some extent.4.The SYBR Green I quantitative real-time PCR method for detection of Mycoplasma bovis is established: The MBOVPG45₀018 gene sequence was selected to establish quantitative real-time PCR detection method of Mycoplasma bovis.In this study,The sensitivity of Mycoplasma bovis SYBR Green I quantitative real-time PCR detection method was 100 times higher than that of the conventional PCR method.It does not cross-react with a variety of pathogens,and the coefficient of variation was less than 3%.The positive detection rate of milk samples and nose swab samples was higher than that of the ordinary PCR.Its provided a more sensitive and more specific method for the detection of Mycoplasma bovis.