Antigenicity analysis of membrane proteins EF-Tu and pdhβ of Mycoplasmopsis synoviae Guizhou strain

In order to study the immune response effect of Mycoplasmopsis synoviae protein EF-Tu and pdhβ, the experiment designed specific primers for the target genes, which were amplified by PCR and constructed a prokaryotic expression vector; bioinformatics analysis of its nucleotide and amino acid sequences was carried out using relevant softwares, and the target protein was induced to express and animal immunization test was carried out. The results showed that PCR amplification of EF-Tu and pdhβ genes yielded specific bands with sizes of 1 185 bp and 993 bp; recombinant plasmids pET32 a-EF-Tu and pET32 a-pdhβ were successfully constructed; the homology of EF-Tu gene between GZ-ZJ strain and other popular strains of Mycoplasmopsis synoviae Guizhou strain（MS-GZ-ZJ） was between 99.7% and 100%, and the homology between pdhβ gene and other popular strains was between 99.6% and 100%, which indicated that the EF-Tu and pdhβ genes of MS-GZ-ZJ strain were highly conservative. The molecular masses of EF-Tu and pdhβ proteins were 43 ku and 35 ku, respectively, which were stable hydrophilic proteins; the secondary structure of EF-Tu protein was dominated by α-helix, random coil and extended chain, and pdhβ protein was dominated by α-helix and random coil. The size of the expressed protein was consistent with the prediction. The EF-Tu protein was mainly expressed in soluble form, and the pdhβ protein was mainly expressed in inclusion bodies. Both EF-Tu and pdhβ recombinant proteins could specifically bind to MS-positive serum. EF-Tu recombinant protein could not induce cellular immune response alone, while pdhβ recombinant protein could stimulate chickens to produce a small amount of interleukin-4（IL-4）; the combination of the two recombinant proteins could induce a small amount of IL-2 and IL-4, but much lower than the vaccine group; the effect of humoral immune response was unclear. The results suggested that the predicted protein with good immunogenicity might not be able to induce good reactogenicity in animal body when being used alone.