Study On P48 Gene And Antigenicity Of Mycoplasma Bovis Isolated In Xinjiang Province

Mycoplasma bovis?M.bovis?,also known as bovine molds,is one of the important pathogenic pathogens causing a variety of infection in cattle.In 1961,Hale and his team first isolated Mycoplasma bovine from the mastitis milk.In 1976,it was reported that Mycoplasma bovine is closely related to respiratory diseases.After the first isolated of Mycoplasma in China in 2008,the disease was gradually spread throughout the country,causing great economic losses to the cattle industry in China.The structure of Mycoplasma bovine is simple,and there is no cell wall in the outer the cell membrane of where A large amount of lipoprotein aggregated on.The lipoproteins on the surface of cell membranes play a very important role in regulating the function of the host immune system by Mycoplasma.P48 protein is one of the many surface lipoproteins of Mycoplasma bovine.It is a specific antigen of Mycoplasma bovine.In this experiment,the P48 gene of Mycoplasma mycoplasma isolated from Xinjiang was cloned and compared,and one of them was selected for the prokaryotic expression of the purified P48 protein.Its immunogenicity was explored to provide a basis theoretical for the research and development of the new Mycoplasma vaccine.The main contents and results were as follows:1 PCR detection and sequence analysis of Mycoplasma P48 gene from different isolates of XinjiangIn order to study the genetic characteristics of Mycoplasma P48 gene in different isolates of Xinjiang,the study was conducted on Mycoplasma bovine isolated from different regions of Xinjiang.Specific primers were designed based on the P48 gene sequence on NCBI,P48 gene was detected by PCR amplification and analyze its sequence.The results showed that 10 strains of Mycoplasma from different regions of Xinjiang were amplified by P48 specific target bands of 1341bp without exception.Through sequence analysis and homology comparison,the homologous between P48 gene of Xinjiang isolates and PG45 international standard strain and other domestic isolates P48 was 96.8%⁹9.8%which illustrate that the P48 gene is a conserved gene in Mycoplasma bovine.2 Cloning and prokaryotic expression of P48 gene of Mycoplasma bovine isolated from XinjiangIn order to determine the immunogenicity of Mycoplasma bovine P48 gene,it lays the foundation for further screening of immuno protective genes of Mycoplasma bovine.In this study,Mycoplasma bovis isolates in Xinjiang as the research object,the amplified P48 gene point mutation of Mycoplasma bovis isolates in Xinjiang by using Overlap PCR method.Constructing the prokaryotic expression vector pET-32a?+?-P48 plasmid.Transforming the successfully constructed prokaryotic expression of pET-32a?+?-P48 plasmid into Escherichia coli BL21?DE3?after screening.The recombinant protein P48 was obtained under the induction of inducer ITPG,and the size of the recombinant protein was about 66KDa.3 Study on the antigenicity of P48 proteinImmunization of purified recombinant P48 protein to Kunming female mice to prepare polyclonal antibody and using Western-blotting and ELISA to verify its reactivity and immunogenicity.The results showed that the purified recombinant protein of Mycoplasma P48 could produce a good immune response and the titer of serum antibody reached a high level(OD_450nm50nm value 1.126).The Western-blotting results show that rat serum of anti-Mycoplasma P48 recombinant protein,recombinant protein of Mycoplasma bovine P48 and Mycoplasma bovine total bacterial protein antigen can produce an obvious antigen antibody reaction.it indicates that the recombinant protein of P48 has good immunogenicity and reactivity and can be used as a candidate gene for the new vaccine of Mycoplasma bovine.Finally,the Mycoplasma bovine P48 protein was immunized to calves,using P48 protein as an antibody Sandwich and the titer of antibody was 0.84(OD_450nm).But using Mycoplasma bovine total bacterial protein as an antibody Sandwich then there was no significant difference between the test group and the control group.