Global Transcriptional Analysis And Preliminary Application Of Mycoplasma Gallisepticum Strain S6 Under The Pressure Of Specific Antibodies

Mycoplasma gallisepticum（MG）is the pathogen that can cause chronic respiratory diseases（CRD）in chickens and infectious sinusitis of turkeys.The respiratory tract and reproductive tract of chickens can be infected by the pathogen.MG can cause inflammation of the air sacs,lungs,trachea,reproductive tract and result in weight loss and egg production decrease.In chickens,MG can exist and spread for a long time with high infection rate,causing serious losses to the poultry industry.MG has the characteristics of immune escape.However,the mechanism of that after colonizing the host,MG can continue to i nfect the host under the condition that the host produces a specific immune response still unknow.In this study,MG positive-serum was used to stimulate the MG-S6 in vitro to simulate the antibody pressure environment in chickens.Based on the Illumina Hiseq sequencing platform,the genome-wide expression level of the MG-S6 in the pressure of specific antibodies was analyzed,and the sequencing results were analyzed by transcriptomics.Combined with the metabolic inhibition test,we hope to screen out the key regulatory genes required for the growth and metabolism of MG.The main results of this study are as follows:1.Transcriptomic results and analysis of MG-S6 under the pressure of specific antibodies.10%（v/v）MG-S6 standard positive serum was added to the MG FM-4 culture as the experimental group,and the control group was added with 10%（v/v）SPF chicken serum.Three replicates were set for each group.Cultured the two groups of MG-S6 in37℃,5%CO₂for 12 h,then harvested the bacterial.Extracted the RNA of the experimental group and the control group.The extracted RNA samples were sent to Shanghai Personalbio Technology Co.,Ltd.in the refrigeration condition for sample quality evaluation.After passing the quality evaluation,transcriptome sequencing was performed.Using the Illumina Hiseq sequencing platform,the transcriptome sequencing results of MG-S6 in the pressure of special antibody were obtained,and the differentially expressed genes were analyzed.By analyzing and screening the transcriptomic data,totally there were 411differentially expressed genes（DEGs）,of which 181 were up-regulated genes and 230were down-regulated genes.GO analysis showed that DEGs mainly distributed in metabolic process,membrane and catalytic activity.KEGG enrichment analysis revealed DEGs mainly covered Ribosomes,Aminoacyl-tRNA biosynthesis,Glycolysis fructose and mannose metabolism,Pyrimidine metabolism,Amino acid biosynthesis,Purine metabolism,Oxidative Phosphorylation,Carbon metabolism,ABC transporter,Amino sugar and nucleotide sugar metabolism,Methane metabolism,Protein export.Randomly selected ten differentially expressed genes for the validation of transcriptome datas by qRT-PCR.Results were consistent with the transcriptomic data,demonstrating the reliability of the transcriptomic data.2.A preliminary study on the biological characteristics of the three antigens encoded by GCW＿RS03690,GCW＿RS01725 and GCW＿RS00885According to the transcriptome data,we choosed three DEGs:GCW＿RS03690,GCW＿RS01725,GCW＿RS00885,which were significantly up-regulated and had low expression in the control group.Respectively constructed three recombinant plasmids which named pET-28a-03690,pET-28a-01725,pET-28a-00885 by using the prokaryotic expression system.Then we purified the three recombinant proteins which named r03690,r01725,r00885.Mouse polyclonal antibody serum was prepared,and the recombinant protein and its high-level anti-serum were detected by Western blot and metabolic inhibition test.The results showed that the three recombinant proteins had good antigenicity.MI test showed that the prepared polyclonal antibodies all had a certain metabolic inhibitory effect on MG-S6,the metabolic inhibitory valences were 1:40,1:80,and 1:80.And the three antiserums also have a certain metabolic inhibitory effect on F strain and HS strain of MG.The subcellular localization of the three proteins were performed by Western blot and suspension immunofluorescence.Western blot analysis showed that anti-r01725serum and anti-r00885 serum had obvious serological reactions with MG-S6 whole protein and cytoplasmic protein while the surface of MG cells treated with anti-r01725 and anti-r00885 serum could bind with the FITC fluorescence secondary antibody exhibits green fluorescence when observed by the microscope.These results indicated that the protein encoded by GCW＿RS03690 was located in the cytoplasm and belonged to cytoplasmic proteins,while the proteins encoded by GCW＿RS01725and GCW＿RS00885 belonged to membrane proteins.In this study,a transcriptomic study of MG-S6 in the pressure of specific antibodies was carried out to gain a comprehensive understanding of the genes regulation of MG exposure with the positive serum.Here we demonstrate that three regulatory genes are related with the metabolic inhibitory effects of MG.This study provides a certain diagnostic target for the differential diagnosis of MG infection and immunity,and lays a certain foundation for the further study of the immune escape mechanism of mycoplasma.