【作者】 徐卓菲；

【导师】 陈焕春； 周锐； 熊远著；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 猪传染性胸膜肺炎(Porcine Contagious Pleuropneumonia,PCP)是由胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)引起的一种高度接触传染的呼吸道疾病。猪瘟(Swine fever,SF),欧洲人称为古典猪瘟(Classical swine fever,CSF),是由猪瘟病毒(Classical swine fever virus,CSFV)引起的一种高度接触性和致死性传染病。这两种重要的病原微生物所引起的疾病已经给全世界的养猪业造成了巨大的经济损失。随着高通量测序技术的发展,越来越多的基因组序列被测序完成,对基因组序列的比较分析为探索微生物的遗传变异提供了一个崭新的机会。我们利用生物信息学的分析方法和策略,对以上2种病原微生物的基因组序列进行了深入地比较与分析,描述了基因组序列结构与病原的致病能力/代谢机制之间的关系。主要的研究内容包括:1.APP的基因组测序与分析目前,APP根据表面脂多糖和荚膜多糖的不同划分成至少15个血清型,不同血清型菌株的毒力、抗原性和地域分布不尽相同。APP血清型的这些特点为发展安全有效的疫苗和简易的诊断方法带来了困难。APP代谢和致病的分子机制一直以来都是兽医界和诊断医学界研究的热点。但是APP基因组信息的缺乏已成为制约该细菌研究技术发展的瓶颈。因此,解析我国流行的优势血清3型APP的全基因组序列,不仅为国际APP基因组计划提供了“参考序列”,而且为该病防治技术的研究提供了理论依据和研究材料。在本研究中,我们对一株在中国流行的优势血清3型APP JL03株进行了基因组测序,并且首次对APP进行了全面的功能注释和分析比较。完成测序的JL03基因组由一个长度为2,242,062 bp的染色体组成,一共预测出2097个蛋白质的编码序列,6个核糖体rRNA操纵子和63个tRNA基因(GenBank登陆号:CP000687)。另外,加拿大测序完成的APP血清5b型L20株的长度约为2.27 Mbp,含有2012个CDS:而部分测序的APP血清1型4074株序列包含140个连接群(contig),长约2.07 Mbp,含有2132个CDS。在三个已测序的APP菌株中,JL03株为低毒力株,L20和4074株均为强毒株。通过APP三个菌株间以及APP与巴斯德菌科其他物种的比较基因组学的研究,详细描述了APP的代谢机制和致病性/毒力的特征。确认了APP代谢通路上的一系列基因,证实APP可以通过发酵和呼吸(有氧呼吸和无氧呼吸)两种方式产生能量(ATP),并且为阐明这一复杂的代谢网络提供了一个全面的遗传基础。在JL03基因组中,鉴定出一些特征性的基因组岛:同化硫还原基因串;Flp鞭毛合成基因串,参与细菌黏附作用;荚膜多糖合成基因串,参与细菌荚膜多糖的合成;脂多糖O抗原基因串,参与细菌O抗原的合成。同时,我们发现在L20株中存在一个37.7 kb的基因组岛,其上编码了8个噬菌体相关基因,该岛未在JL03株中发现。这些基因组岛的发现为进一步研究APP血清多样性的机制提供了基础。另外,我们描述了与细菌毒力相关的一整套基因,JL03株中的某些基因发生了遗传上的丢失或者不同类型的突变。以上分析证实了以往对该病原毒力决定簇的认识,为解释不同血清型菌株之间毒力的差异提供了理论依据。总之,APP基因组序列结构的解析和编码蛋白质功能的初步分析不仅证实了该菌部分生理生化的表型特征,并且为探索这个重要病原菌的毒力和代谢的特征提供了新的研究设想。2.CSFV的基因组测序与分析CSFV属于黄病毒科瘟病毒属,只有单一的血清型,是一种单正链的RNA病毒。在本研究中,我们对一株分离自中国河南的CSFV SWH/CA/2004株进行了基因组测序和分析比较。SWH/CA/2004基因组cDNA的序列长度为12296 nt(GenBank登陆号:DQ127910),5’NTR长度为373 nt,3’NTR的长度为226 nt以及一个开放读码框,编码一个3898个氨基酸的聚蛋白前体。对SWH/CA/2004株与其它已报道的CSFV株进行基因组的序列比对和进化分析后发现:不同株基因组的核苷酸的相似性在92.4%-97.9%之间,氨基酸的相似性在96.1%-98.4%之间。SWH/CA/2004与中国强毒株cF114/CA/2001株的遗传距离最接近,为0.013:而与中等毒力的GXWZ02/CA/2003株的遗传距离较远,为0.170。我们全面利用和分析了现有的不同地区来源的猪瘟病毒全长基因组的序列信息,揭示出CSFV的系统发生关系,为研究猪瘟病毒的遗传变异和分子流行病学提供了参考材料和研究基础。3.黄病毒科RNA聚合酶表达抑制的研究黄病毒科的依赖于RNA的RNA聚合酶(RNA-dependent RNA polymerase,RdRp)参与组成病毒的复制酶复合体,在病毒的复制循环中发挥重要的作用。在本研究中,针对三种病毒的RNA聚合酶基因高度保守的MotifV区域设计了相应的小干扰RNA分子(small interfering RNA,siRNA),用来研究siRNA分子在调控RNA聚合酶表达过程中的作用。我们通过荧光显微镜观察、流式细胞检测、Western blot检测和半定量荧光RT-PCR等方法验证了表达抑制的效果。由于筛选到的CSFV的siRNA能够十分有效地抑制RdRp-GFP融合蛋白在PK-15细胞中的表达,因此有望成为预防和免疫猪瘟病毒感染猪的候选疫苗。更多还原

【Abstract】 Porcine contagious pleuropneumonia(PCR),caused by Actinobacillus pleuropneumoniae(APP),is a severe contagious respiratory tract disease of pigs.Swine fever(SF,syn.Classical swine fever),caused by classical swine fever virus(CSFV),is also a highly contagious and often fatal disease of domestic pigs and wild boars.These diseases have caused considerable world wide economic losses in the swine industry.Huge amount of genome sequences have been obtained under the help of high-flux sequencing technology.The genomic comparisons and analysis provide an original insight to explore the genetic variation of microorganism.Based on our bioinformatics approaches,genome information of the above pathogens were comprehensively studied, which shows the relationship between the genomic structure and the mechanism of pathogenesis/metabolism.The main research was described as follows:1.Genome sequencing and analysis of APPAt present,APP has divided into at least 15 serotypes,differing in their surface lipopolysaccharide and capsule polysaccharide.As these serotypic characterizations,it is difficult for us to develop safe and effective vaccine and easy means of diagnosis.Moreover,a better understanding about the molecular mechanism of metabolism and pathogenesis of APP always has broad interesting in the field of veterinary as well as clinical medicine.Lack of comprehensive genomic information has become the major obstacle for tackling the scientific and technical problems in the study of this bacterium and related disease.With these considerations,we believe that complete genomic sequencing of serotype 3 of APP prevalent in China can provide reference sequence for international genome project.It also offers theoretical support for prevention and cure of this disease.In our study,we have sequenced the complete genome of A.pleuropneumoniae strain JL03,an isolate of serotype 3 prevalent in China.The detailed analysis and functional annotation for this organism was firstly performed.Its genome is a single circular chromosome of 2,242,062 base pairs containing 2,097 predicted protein-coding sequences,six ribosomal rRNA operons,and 63 tRNA genes(GenBank accession number CP000687).On the other hand,the genome of strain L20 of serotype 5b sequenced by Canada is approximately 2.27 Mbps,containing 2012 CDSs,and the incomplete genome of strain 4074 of serotype 1 is about 2.07 Mbps,consisting of 140 contigs encoding 2132 putative CDSs.Among the three serotypic strains,4074 and L20 were known as highly virulent strains but JL03 was a low virulent strain.Comparative genomics analysis using the available sequences of related bacteria detailedly described the characteristics of the mechanisms of pathogenesis and serotypic diversity of A.pleuropneumoniae.We identified a full spectrum of genes invovled in metabolic pathways and confirmed APP could generate energy via fermentation and respiration(aerobic and anaerobic).A complete metabolic network with various kinds of oxidation-reduction enzymes for catabolism and anabolism was comprehensively illustrated at the genomic level for this genus.Furthermore,several strain-specific genomic islands related to assimilatory sulfate reduction,biosynthesis of Flp fimbrial, capsular polysaccharide and lipopolysaccharide O-antigen were identified in JL03. Notably,strain L20 possesses a strain-specific genomic island of 37.7kb encoding eight phage-related proteins,which is absent in JL03.Discovery of such genomic islands provides a foundation for future research into the mechanisms of serotypic diversity of APP.We also described a complete set of genes related to the virulence of the bacterium, of which,some are either missing genetically or impaired by varies kinds of mutations in JL03.These data reconfirmed the knowledge of virulent determinants of APP,and also provided a foundation to explain the discrepancy of virulence between different serotypic strains.In short,preliminary analysis of the genomic sequence and the functions of the encoded proteins not only confirms the present physiological and pathological knowledge but also offers new insights into the metabolic and virulence characteristics of this important pathogen.2.Genome sequencing and analysis of CSFVCSFV containing only one serotype is a member of the genus Pestivirus within the family Flaviviridae.Here,we have sequenced the full genome of a novel CSFV strain, SWH/CA/2004,isolated from a hog pen in Henan Province,central China.It contains 12296 nucleotides(nt) in length.It is composed of a 373-nt 5’ terminal non-translated region(NTR),a 11697-nt open reading frame(ORF) encoding a polyprotein of 3898 amino acids,and a 226-nt 3’-NTR.Genome comparison of the SWH/CA/2004 isolate (GenBank Accession:DQ127910) with other known CSFV isolates was performed and analyzed.Corresponding segments from SWH/CA/2004 and other reported strains shared 80.4%-99.8%identity at the nucleotide level and 89.5%-99.8%identity at the amino acid level.From an evolutionary point of view,isolate SWH/CA/2004 is closely related to the highly virulent isolate cF114/CA/2001,with a pairwise distance of 0.013;and distantly related to the moderately virulent isolate GXWZ02/CA/2003,with pairwise distance 0.170.These analyses constitute a comprehensive study of the phylogenetics of CSF based on distinct regions of the genome and may provide the reference resource for future molecular epidemiology research and genetic variations of CSFV strains.3.Inhibition of expression of RNA polymerase in viruses of the the family FlaviviridaeThe RNA-dependent RNA polymerase(RdRp) in viruses of the family Flaviviridae plays an important role in the viral replication process and in the forming of a replicase complex.In this study,we used small interfering RNAs(siRNAs) corresponding to the highly conservative MotifV of RdRp gene of three viruses to examine their role in modulating the expression of RdRp. Evaluation of the expression of RdRps was performed by the fluorescence,flow cytometry,Western blotting,and real-time PCR.The plasmid-based CSFV siRNA is quite effective in silencing target-GFP fusion gene expression in PK-15 cells,which may provide favorable prevention and immunity of CSFV infection in swine.更多还原