Isolation And Identification Of Mycoplasma Bovis And Expression Of GAPDH Gene Fragment

Mycoplasma bovis is a prokaryote lack of cell wall,which present in the respiratory and reproductive tract of bovine,is likely to be infected with long-distance transport or immunocompromised of cattle,induce severe pneumonia,mastitis,can also lead to arthritis, cornealconjunctivitis,ear infections,genital tract inflammation,miscarriage or infertility and other diseases.Mycoplasma bovis disease prevalence in the world wild,the economic interests of the cattle industry had been damaged seriously:Because of Mycoplasma bovis calf pneumonia,the economic losses of Europe is1.44～192million a year;in recent years,this disease prevalence in Hubei,Guizhou,Ningxia,Inner Mongolia,Guangxi endemic,Chongqing and other provinces, autonomous regions.Lack of safe and effective vaccine is one of the reasons which cause Mycoplasma bovis diffuse.In order to select an antigen protein for the production of mycoplasma bovis vaccine,in the study.one Mycoplasma bovis was identified by16S rRNA sequence analysis and PCR amplification of oppD/F gene.which was isolated from bovine.The immunology value of the GAPDH gene was analysesed depend on bioinformatics software, sieved the fragment with higher antigen index to cloning and expression.The biological activity of recombinant protein was proved by animal testing,Western-blot test and indirect ELISA analysis.The results provided a reference for the trituration of genetic engineering vaccine of Mycoplasma bovis.This test covers the following aspects:1Mycoplasma bovis was identified by PCR The bovine with clinical symptoms of pneumonia was culled,collected lung tissue as sterile,the (?)lation and purification of pathogeny completed in the laboratory.A"omelette-like" colony was isolated on the Mading Shi medium in5%CO2and37℃,named CQ1. Depend on the sequence of16S rRNA and oppD/F gene,designed the universal primers and specific primers for Mycoplasma bovis,identified the isolates by PCR.The results showed that the similarity of the isolates16S rRNA sequence is99.9%and99.3%with Mycoplasma bovis strain PG45(accession numbers: AF332756),Mycoplasma agalactiae (accession number:AY526879).The target fragment of specific primers is1900bp,by sequencing, the repetition rate is100%with the Mycoplasma bovis oppD/F gene.During the drug susceptibility testing and animal testing, explored the drug susceptibility and virulence of the isolates.The isolates were sensitive to ciprofloxacin,ofloxacin,tetracycline, spectinomycin.Attacked by chest and the intranasal don’t lethal in BABL/C mice and Kunming mice.New Zealand rabbits,Z:ZCLA gerbils.2Expression of GAPDH Gene FragmentThe GAPDH gene sequence of Mycoplasma bovis PG45strain was used as a template to analyze the immunological information,screening280to921bp sequence for the purpose fragment of cloning and expression.The expression ratio and modality of recombinant protein was detected by SDS-PAGE.The recombinant plasmid pET-32a (+)/GAPDH was highly expressed in the form of soluble protein in E. coli BL21(DE3)host strain.3Immunological activity of the recombinant proteinThe oil emulsion vaccine made by bacterial protein was used to immunize rabbits,the agar diffusion test wsa the way for the detection of polyclonal antibody preparation effects. A precipitation line formed between the polyclonal antibody and whole-cell protein as the highest dilution ratio was1:32of the polyclonal antibody, and a single precipitation line formed between the polyclonal antibody and the purified recombinant protein.The recombinant protein can bind to polyclonal antibody specifically and showed a stronger reactogenicity was verified by Western-blot test. The recombinant protein was used as coating antigen to build an indirect ELISA for differential diagnosis,the results showed that the method can not distinguish negative or positive serum accurately.In summary.this study isolated a Mycoplasma bovis,provided the experimental material for the follow-up test;the GAPDH gene (280～921bp) was successfully expressed in the E. coli expression system formed by soluble protein;The agar diffusion test of polyclonal antibody,Western-blot test and indirect ELISA test results showed that the recombinant proteins provide with immunogenicity and reactogenicity.allowed the experimental basis for the further development of the genetically engineered vaccine.but don’t suit to build the diagnostic method.