Microarray Analysis Of Actinobacillus Pleuropneumoniae Genes Expression Involved In Virulence Factors Under In Vitro Growth Conditions Mimicking The In Vivo Environment

Porcine contagious pleuropneumoniae is a respiratory system disease caused by actinobacillus pleuropneumoniae.It is a worldwide disease causing tremendous economic loss to the swine industry.The occurrence tendency of porcine contagious pleuropneumoniae in our country is rising now,and it has brought serious economic damages to pig industry.At present,the study of the pathogenic mechanism of APP is the hot spot and bottleneck. In this research,we carried out the in vitro transcription analysis of the important virulent genes of APP,The main contents were as follows:1.The establishment of the stress growth models of APPObserving the growth characteristics of APP serotypel 4074 strain in many plate medium and fluid nutrient medium,we screened the best plate medium and fluid nutrient medium for APP.The results provided many foundation for further research.According to the stress environment that APP could be faced with in the process of infection,we designed and practiced four conditions,including high temperature(40,41,42℃), oxidative stress(1 mmol/L H₂O₂),low iron ion concentration(200μmol/L,300μmol/L,500μmol/L,1mmol/L Na₃CaDTPA),acidity condition(pH7.0,6.6,6.2,5.8).We cultured APP in these conditions in vitro in 10,20,30,40,60 min and selected the best models to simulate the internal infection environment and the cell growth condition.They were high temperature(41℃),oxidative stress(1 mmol/L H₂O₂),low iron ion concentration(200μmol/L Na₃CaDTPA),acidity condition(pH5.8). 2.The target genes amplification and clone of APP virulence factors expression microarrayAccording to the important virulence genes of APP serotypel 4074 strain,31 primer pairs were designed and selected by using Array Designer 2.0 software.30 virulence genes and 1 housekeeping gene were amplificated.All target genes were cloned into vector pMD18-T.5 genes which code the toxin protein were apxIA(358 bp),apxIC(322 bp), apxID(465 bp),apxIIA(452 bp),apxIVA(346 bp),6 genes which code outer membrane protein were omlA(474 bp),palA(408 bp),cpsIC(603 bp),afuA(446 bp),apaA(493 bp),8 genes which code metabolism genes were guaA(499 bp),dsbA(493 bp),ureC(552 bp), ohr(405bp),sodC(506bp),sodA(459 bp),dsbE(374 bp),fumB(452 bp),oxygen-sensitive(434 bp),3 genes which code stress regulation gene were mopA(528 bp),mopB(400 bp), dnaK(325 bp),7 genes which code transport related gene were dmsA(528 bp),hgbA(562 bp),tbpB(511 bp),tbpA(592 bp),fhuD(561 bp),exbB2(318 bp),glyA(456 bp),recF gene (319 bp)was the housekeeping gene and acrA gene(443 bp) was unknown function. Alignment of all 31 target genes sequenced showed all target sequence had above 95% homology with the sequences of the reports in the GenBank,it made sure that the authenticity of the target genes which would be used in the microarray cross experiment.3.The construction of APP virulence genes expression microarray3.1 The preparation of the fluorescent probes of APP virulence genes expression microarrayIn the study,we extracted the total RNA of the experiment and the control groups in 4 different growth models(high temperature(41℃),oxidative stress(1 mmol/L H₂O₂),low iron ion concentration(200μmol/L Na₃CaDTPA),acidity condition(pH5.8)) with Master-Pure^TM RNA Purification Kit.The results showed that the total RNA OD₂₆₀/OD₂₈₀ were between 1.72 and 2.19,the electrophoresis strips of 18s and 28s were cleared,and the purity was consistent with the experiment demand.The total RNA were reverse transcripted and purified,and the cDNA were signed with fluorescein(Cy3 and Cy5) and purified.The purified cDNA were worked as the fluorescent probes of the expression microarray.To lower the statistical error in this study,the total RNA of the control groups were signed with Cy3-dCTP and Cy5-dCTP respectively.In the research,we produced 24 probes for the microarray.3.2 The preparation of APP virulence genes expression microarrayThe target genes(300 ng/μL) prepared by isopropanol precipitation method were spotted onto a 14 column×15 row array of the amination slide by SpotArray 24 under 45% ambient humidity and at 22℃.A target gene was spotted 5 times.Spot diameter was 120μm and the distance between spots center was 300μm.Spotted microarrays were stored after hydration,crosslink under UV and washing.Then the microarrays were prehybridized 5 h at 42℃and hybridized 16 h at 42℃,microarrays were scanned with ScanArray(?) after washing and drying.The hybridization and scanning results suggested the quality of the microarrays fabricated this time were good enough to be used in experiments.3.3 The expression study of APP virulence genes in the mimicking environmentIn the study,the hybridized microarrays were scanned and the images and data were analyzed by using Dchip2006,MIDAS and SAM software.Assessment standard of different expression of a target gene was determined according to q-value(%) and Ratio. When q-value(%)≤5%and Ratio＞2,the target gene was assessed as up-regulation,and q-value(%)≤5%and Ratio＜0.5,the target gene was assessed as down-regulation.The differential expression genes were selected through hybridization,scanning and statistics analysis according q-value and Ratio.There were 3(tbpA,tbpBå’ŒhgbA),1(acrA) and 1(apaA) up-regulated genes and 2(afuAå’ŒdmsA),2(afuAå’ŒdmsA),2(apxIIAå’ŒafuA) down-regulated genes in the high temperature stress at 10 min,30 min and 60 min, respectively.There were 1(exbB2),5(ohr,apxIA,dsbE,tbpAå’ŒtbpB),5(exbB2,hgbA,tbpA,tbpBå’ŒsodA)up-regulated genes and 7(glyA,omlA,afuA,apxIA,apxIC,dmsAå’ŒsodC),1(apaA),5(exbB2,hgbA,tbpA,tbpBå’ŒsodA) down-regulated genes in the oxidative stress at 10 min,30 min and 60 min,respectively.There were 13(apxID,apxIIA,mopA,mopB,hgbA,tbpA,exbB2 and so on),7(hgbA,tbpA,tbpB and so on),7(hgbA,tbpA,tbpB and so on)up-regulated genes and 3(dmsA,afuAå’Œoxygen),2,(dmsA,afuA) 5 (dnaK,fumB and so on)down-regulated genes in the low iron concentration stress at 10 min,30 min and 60 min,respectively and there were 0,3(dnaK,afuAå’ŒdmsA),3(dsbE,ureC,arcA) up-regulated genes and 3(dnaK,afuAå’ŒdmsA),1(afuA),3(omlA,oxygen, dnaK) down-regulated genes in the acid stress at 10 min,30 min and 60 min,respectively.In the study,we constructed 4 genetic transcriptional control network and selected the key genes,they were exbB2(high temperature stress),apxIVA,dsbE,mopB,sodC(oxidative stress),apxID,dsbE,exbB2(low iron concentration) and fhuD,ureC(acid stress).These key regulation genes would be good to the selection of medicine target and the design of drug, and the results provided good foundation to the subsequent research.We also analyzed APP virulence genes expression with the statistics and estimated the gene expression tendency in 4 stress models.Most virulence genes were down-regulated in the high temperature stress.Most virulence genes were up-regulated at the beginning of the oxidative stress,and the expression levels were higher at the middle of the oxidative stress, the level of expression were near the peak value,at the end of the oxidative many of virulence genes were down-regulated.The results showed that the expression of APP virulence genes were time- specificity in the high temperature and the oxidative stress.To validate the authenticity of the cDNA microarray,we adopted the technique of real time PCR to verify the genes of different expression.The results showed that the mRNA level of 4 genes was coincidence with the results of cDNA microarray.From the results,it showed that the results of genes expression profile by cDNA microarray method were true and reliable.