Construction Of A Genomic Expression Library Of Mycoplasma Synoviae And Screening And Identification Of In Vivo-inducible Genes

Mycoplasma synoviae(MS)is one of the most important infectious diseases affecting the development of the laying hens and broilers industry.The sick chicken often displays clinical symptoms such as joint swelling,effusion,lameness,loss of appetite,decreased egg production rate and abnormal egg shell top.The immunity of MS-infected chickens is reduced and it is common to see MS mixed with Mycoplasma gallisepticum(MG),Newcastle disease(ND)and infectious bronchitis virus,making the condition of infected chickens worse.In recent years,the incidence of MS disease in the world increased year by year,seriously affecting the healthy development of chicken industry.The prevention and control of MS need to study its pathogenic mechanism,explore and design new molecular targets for anti-MS drugs,establish rapid and specific detection methods,and develop efficient new vaccines.All these need to study the functional genes of MS,and screen and identify candidate antigens.Therefore,in this study,we construct that genomic expression library of MS,prepare the screening antibody against in vivo-induce antigen(IVIA)of MS,and studied the specific expression protein of MS in vitro and in vivo by using in vivo-induced antigen technique(IVIAT).(1)The genome of MS G strain was extracted and digested with Sau3A I.200～2 000 bp fragments were taken after digestion,and the pET-30a/b/c system BL21 expression library of MS G strain genome was constructed.The library was identified by PCR.The results showed that 200～2 000 bp fragments were inserted into the recombinant plasmid.The transformation rate of the genomic library was 100%,and the insertion rate was 82.5%.(2)Chickens were artificially infected with MS G strain.Clinical signs were observed,pathological tissue sections were prepared,and the serum inflammatory factors were detected by ELISA.The results showed that there were bleeding spots in larynx and trachea,effusion in articular cavity,and pathological changes in lung,liver,spleen and trachea.The levels of IL-6,IL-8,IFN-γ and TNF-α cytokines were significantly increased after infection with MS(P<0.01).After 30 days of primary infection,serum samples were collected at 45 days after secondary infection to prepare mixed serum for IVIAT test.(3)Adsorption of mixed sera from the whole MS infection process using MS G strain,BL21 whole bacteria and both bacteriophage lysates and heat-denatured lysates,consuming the specific antibody of the protein expressed by the MS bacterial both in vivo and in vitro through adsorption,and leaving the specific antibody of the antigen expressed by the MS only in vivo.ELISA and Western blot analyses before and after the serum adsorption treatment showed that the irrelevant antibodies were largely removed from the serum,and screening antibodies for the MS in vivo-inducible antigen(IVIA)were obtained.(4)The in vivo-induced antigen technique was applied to screen MS for in vivo inducible expression of antigen genes.The results showed that 17 in vivo-inducible antigens of MS were obtained,namely,tRNA(Met)acetate cytidine ligase,ABC transporter permease,kinase ABC subunit A,endonuclease,thymidine kinase,transcriptional regulatory protein,DNA polymerase Ⅳ,DHH subfamily 1 protein,DNA helicase,L-lactate dehydrogenase,RGS structural domain-containing protein,CRISPR-associated nucleic acid endonuclease Cas9,protein GrpE,ECF transporter S component and 3 phase-change haemagglutinins.Among them,L-lactate dehydrogenase,RGS structural domain-containing protein,haemagglutinin protein,CRISPR-associated nucleic acid endonuclease Cas9 and protein GrpE were upregulated by more than 1,000-fold in vivo relative to in vitro expression.According to gene enrichment analyses,these antigens are involved in cellular processes,single biological processes,metabolic processes,localisation,response to stimuli,organisation of cellular components or biogenesis,and biological regulation.Real-time fluorescence quantitative PCR showed that mRNA levels of all 17 genes were upregulated in vivo relative to in vitro.In summary,this study successfully constructed an MS genome expression library,performed MS infection experiments and prepared an in vivo-induction antigen screening serum.17 MS in vivo-induction genes were identified,and the results of the study provide a reference for screening MS virulence factors and candidate genes associated with infection.