Establishment Of RT-qPCR Internal Reference For Mycoplasma Hyopneumoniae And Its Application In Subunit Vaccine Candidate Antigen Identification

Mycoplasma hyopneumoniae(Mhp)is an important cause of Mycoplasma Pneumonia of Swine(MPS),which destroys the cilia of the respiratory epithelial cells,thereby impairing the clearance of the respiratory tract,impairing the function of the respiratory immune system,leading to infection by other respiratory pathogens and causing εrmous economic losses worldwide.RT-q PCR(quantitative reverse transcription PCR)is a commonly used method to analyze the relative quantitative relationship among multiple samples,in which the role of internal reference genes is indispensable,however there is no research on Mhp internal reference genes.In this paper,13 genes which were constantly expressed in two different strains filtered by omics analysis and one commonly used reference gene in other species were selected as the candidate genes.The ge Norm,Normfinder,Bestkeeper and Ref Finder software were used to evaluate the stability of gene expression for samples of different virulence and in different growth phases to screen the optimal reference gene for RT-q PCR analysis.Next,the differently expressed genes were screened by the established RT-q PCR method and the function of their recombinant proteins in the pathogenesis of Mhp was investigated.The potential of the selected virulence-related proteins as candidate subunit vaccine antigens was further evaluated in BALB/c mice in this study.In conclusion,the main findings are as follows:Establishment of RT-q PCR of Mhp: 14 candidate genes were expressed and amplified in 25 Mhp samples from strains with different virulence and growth phases.The results of stability evaluation of ge Norm,Normfinder,and Bestkeeper showed that the top four stable genes were D,M,C,N in Mhp samples of different virulence,and the top four stable genes were C,D,K,B in Mhp strains in different growth stages.The results of optimal internal reference gene analysis by different softwares were consistent with each other with slightly difference.Ref Finder was further used to proceed synthesize analysis to filter one most suitable reference gene in strains with both different virulence and various growth phases.It found that among all selected genes,C was the most suitable internal reference gene.Virulence-Related Antigen Analysis: The gene ζ,θ,and β were screened according to the established RT-q PCR method.The results of flow cytometry and colony hybridization showed that ζ,θ and β proteins could be detected on the surface of Mhp by corresponding antibodies.Three recombinant proteins were further found to play important roles in the adhesion of Mhp to host cells.Screening of candidate subunit vaccine antigens: Mice were immunized with ζ,θ,β and reported virulence associated proteins ι,κ and γ respectively,combined with Freund’s adjuvant.The results of animal experiments showed that the vaccines prepared by the six antigens could stimulate the production of Ig G antibodies in sera.Recombinant ι,κ,θ and β could also stimulate s Ig A antibodies in the bronchoalveolar lavage fluid(BALF)of mice.The lymphocyte proliferation reaction in the blood,lung and spleen of the immunized mice showed that β and ι could significantly increase the lymphocyte proliferation.Recombinant ι,κ,θ and β could also stimulate the production of CD8+ T cells in blood.The six antigens had no significant effect on the secretion of IFNgamma and IL-4,while θ could significantly increase the secretion of IL-17 in serum,suggesting that the immune responses induced by most antigens were CD8 + T cells.Some antigen could induce Th17 associated and mucosal immune responses.