【作者】 李林溪；

【导师】 韩文瑜；

【作者基本信息】 吉林大学 ， 预防兽医学， 2013， 博士

【摘要】 胸膜肺炎放线杆菌是引起猪传染性胸膜肺炎放线杆菌疾病的主要病原菌，该病是世界范围内流行并严重危害养猪业的重要传染病。胸膜肺炎放线杆菌具有多个血清型，其致病力和免疫原性各不相同，主要血清型之间缺乏免疫交叉保护，影响了其疫苗的开发。我们先前的研究通过MALDI-ToF质谱鉴定出了六个共同抗原蛋白，单链DNA结合蛋白（PA-Ssb）、噬菌体休克蛋白A（PA-PspA）、甘油醛-3磷酸脱氢酶（PA-GAPDH）、丙酮酸激酶（PA-Pk）、丝氨酸羟甲基转移酶（PA-Shmt）以及天冬氨酸转氨酶（PA-Ast）。以痤疮丙酸杆菌参考菌株设计引物克隆并测序了以上六个蛋白，通过Genebank以及其他生物信息学数据库对其进行了分析，并上传至Genebank数据库。构建了六个抗原蛋白的原核表达载体pGEX-PA-Ssb、pGEX-PA-Pspa、pGEX-PA-GAPDH、pGEX-PA-Pk、pGEX-PA-Shmt和pGEX-PA-Ast，并表达纯化出了六个抗原蛋白的GST融合蛋白。通过小鼠模型免疫并以胸膜肺炎放线杆菌血清1型和5a型感染，分别得到免疫保护率达到（对血清1型）20%（PA-GAPDH）至80%（PA-Ssb）和（对血清5a型）10%（PA-Ast）至60%（PA-Ssb）。免疫保护作用最佳的抗原蛋白PA-Ssb与胸膜肺炎放线杆菌多个血清型均具有较高同源性的两段短肽，PH1（N`-AKNNSSGAGNSKDWGGN-C`）与ApxIV毒素同源，PH2（N`-VIESLSKG-C`）与ZnuA基因同源。人工合成的PH1和PH2短肽与胸膜肺炎放线杆菌多个血清型抗体均有较强的抗原抗体反应，其中PH1的反应较强。通过构建胸膜肺炎放线杆菌Nanoluc荧光素酶突变株，证明这一新型荧光素酶可以在胸膜肺炎放线杆菌内表达并催化Furimazine底物发光，是一种新型的报告基因。为了鉴定ApxIV毒素的调控子基因，通过基因工程方法构建了胸膜肺炎放线杆菌血清15型荧光突变库，采用IVIS系统对2万个以上的突变株进行了荧光筛选，没有筛选出有效的发光克隆。胸膜肺炎放线杆菌在体外培养条件下，其毒素ApxIV可以通过SDS-PAGE、免疫印迹、MALDI-ToF质谱、荧光定量PCR等方法检测出其基因的转录与表达。其表达受到培养条件不同以及钙离子浓度不同的影响。在含有5mM钙离子的增强型PPLO培养基中ApxIV的表达水平最高。本研究初步摸索了胸膜肺炎放线杆菌异源疫苗开发的一条途径，并首次以多种方法证明ApxIV毒素的体外表达。为今后胸膜肺炎放线杆菌的新型疫苗开发以及重要毒力因子的研究提供了一定基础。更多还原

【Abstract】 Actinobacillus pleuropneumoniae is the causative agent of acute and chronicpleuroneumonia that is responsible for substantial morbidity and mortality in the pigindustry. New improved vaccines that can protect against all serotypes and preventcolonization are required. In a previous study we showed that whole cells ofPropionibacterium acnes protected pigs from A. pleuropneumoniae serotype1and5and, there are common antigens between P.acnes and A.pleuropneumoniae, therefore,the basis for a promising heterologous vaccine. The aim of this study was to analysisthose protein antigens of P. acnes responsible for protection against A.pleuropneumoniae infection. Six P. acnes protein antigens that were recognized duringour priory study by sera raised against A. pleuropneumoniae were identified by2-DEand immunoblotting. Recombinant versions of all P. acnes proteins gave partialprotection (10-80%) against A. pleuropneumoniae serotype1and/or5infection in amouse challenge model. The best protection (80%serotype1;60%serotype5) wasobtained using recombinant P. acnes single-stranded DNA-binding protein. In part,protection against A. pleuropneumoniae infection may be mediated by small peptidesequences present in P. acnes single-stranded DNA-binding protein that arecross-reactive with those present in the A. pleuropneumoniae-specific RTX toxinApxIV and the zinc-binding protein ZnuA. The results suggest that P. acnes may be auseful vaccine to protect against different serotypes ofA. pleuropneumoniae.Nanoluc is a newly reported luciferase which catalysis furimazine substrate andglow luminescence100times stronger than commonly used firefly luciferase. It couldbe used as a reporter in mammalian cell. In this study, we are trying to integrate theNluc into of Actinobacillus pleuropneumoniae serotype15(HS143) genome under thecontrol of sodC promoter by construct a homologous recombination reporter plasmid and a mutant strain to evaluate if this new luciferase could expression and detect in A.pleuropneumoniae. Mutant strain APPS15::SodCNK cells PBS dilution andfurimazine substrate solution mixture could glow luminescence and detect by96wellsplate reader. Detect limitation is500CFU per well (1:50diluted substrate) or1:5,000diluted substrate (5*107CFU per well). Luminescence signal count will slowlyclimbed to the top in120minutes after sample loaded with NanoGlo buffer dilutedsubstrate. Another METK buffer diluted substrate reaction reach to signal top in60minutes and the signal variation is bigger than NanoGlo buffer. Single colonies placedon agar plate sprayed with substrate solution could be detect with IVIS imagingsystem, available for detect reporter gene expression. Our results indicate thatNanoluc luciferase could express in A. pleuropneumoniae and the luminescence ofthat could be detected by plate reader (suspension cells) or IVIS imaging system(colony) after catalysis furimazine substrate.ApxIV is one of the four A. pleuropneumoniae RTX toxin, generally consideredApxIV is unique only in the in vivo expression of A. pleuropneumoniae toxins. Theexistence of ApxIV has played an important role on A. pleuropneumoniae virulenceeffecting. Recent studies reported by gene chip and two-dimensional electrophoresisand mass spectrometry in the detection of a large number of genes or proteins isdetected in vitro transcription and protein ApxIV exist, but not specifically for theexpression of ApxIV further understanding. To confirm this conclusion, we used A.pleuropneumoniae serotype1,8and15were cultured in vitro, using reversetranscription PCR, quantitative PCR, SDS-PAGE, Western Blot and MALDI massspectrometry and other methods, from transcription, protein level and theantigen-antibody reaction proved that the ApxIV expression under in vitro cultureconditions. A. pleuropneumoniae in vitro expression under different conditions isquite different, with the culture medium and the calcium ion concentration. Under theconditions of PPLO broth contains L-cysteine hydrochloride, L-cysteinehydrochloride, glucose, Tween-80,5mM calcium, it can be detected the highestApxIV protein bands. Different concentrations of calcium ions on ApxIV protein leveldetection have an important influence, but weaker effect on the transcriptional level. Therefore, we speculate that calcium may be an important regulatory factor ApxIVposttranslational modification.In this study, cloning and expression of the six common antigen proteins betweenA. pleuropneumoniae and P. acnes and screen out two small peptides fragment of aprotein is a potential vaccine candidate, from the preliminary interpretation of theprotein levels of heterologous P, acnes immune protection Actinobacilluspleuropneumoniae infection mechanism. For the first time verified for the newluciferase Nanoluc expression and work effectively in A. pleuropneumoniae, prove itcan be a regulation studies tool and provides a new and convenient research method.From multiple angles proved A. pleuropneumoniae important virulence factor ApxIVexpression in vitro, initially discussed its expression regulation mechanism for thefurther study of this virulence factor and some help on A. pleuropneumoniaepathogenicity study and vaccine development.更多还原