猪链球菌2型不同毒力菌株的鉴别及Pan-genome分析

发布时间：2018-05-12 20:43

本文选题：猪链球菌2型 + 内标　；参考：《南京农业大学》2015年硕士论文

【摘要】：猪链球菌(Streptococcus suis,SS)是一种重要的人畜共患病病原,可引起人和猪的败血症、关节炎、脑膜炎、心内膜炎等多种疾病,严重威胁公共卫生安全。根据荚膜多糖抗原的不同,可将猪链球菌分为33个血清型(1/2,1-31,33),其中血清型2型(SS2)在世界范围内报道最多且致病性最强。我国曾爆发两次人感染SS2疫情,并造成严重的人员伤亡。目前,对SS2毒力因子的研究逐渐增加,但是其致病机理仍不明确。SS2不同菌株致病力存在差异,但是缺乏合适的毒力评价体系,严重阻碍对致病性SS2的鉴定。本研究将斑马鱼感染模型"内标"化,并成功筛选到若干SS2弱毒株。通过对SS2泛基因组(pan-genome)的分析,鉴定出强、弱毒株基因组的系列差异,为进一步研究SS2致病机理提供理论依据。1猪链球菌2型弱毒菌株的筛选综合考虑菌株分离时间和地域跨度,从本室菌种库挑选105株SS2菌株,通过PCR检测菌株中菌毛相关基因sbp2'和毒力相关基因mrp,epf,sly的分布,并鉴定出sbp2'-/mrp-/epf/sly-、sbp2'+/mrp+/epf+/sly+等6种基因型。利用斑马鱼感染模型测定不同基因型菌株的毒力,并以已知的弱毒株T15和强毒株ZY05719作为参考菌株,即"内标"。通过比较待检菌株和参考菌株的半数致死量(LD5O),评估待检菌株毒力水平。结果表明,我们成功筛选到16株SS2弱毒株,基因型为sbp2'-/mrp-/epf/sly-(13株)或sbp2'-/mrp+epf+/sly+(3株),这也说明菌毛相关基因sbp2'似乎比mrp,epf,sly更适合作为SS2毒力靶基因。2猪链球菌2型泛基因组分析选取7株SS2弱毒株完成基因组测序,并结合NCBI数据库,共获得10株强毒株和9株弱毒株全基因组信息,以此进行猪链球菌2型泛基因组(pan-genome)分析。结果显示,pan-genome由1,239个核心基因和2,436个可变基因组成,进化分析表明强、弱毒株进化差异明显,9株弱毒株又可分为Ⅰ、Ⅱ两组(两个分支)。噬菌体预测分析发现,弱毒株中含有更多前噬菌体序列。通过比较强毒株和弱毒株各自的核心基因组(core-genome),发现了强毒株共同特有的菌毛相关基因簇。通过比较强毒株和Ⅱ组弱毒株的core-genome,鉴定出强、弱毒株各自特有的53和58个基因。3 mrp基因型与猪链球菌2型菌株的毒力关系Pan-genome分析涉及的9株弱毒株的core-genome由1,459个基因组成,其中包括mrp基因,与我们筛选出的弱毒株PCR结果冲突(13株弱毒株呈mrp阴性),说明该基因序列可能存在多样性。本研究通过比对不同菌株中mrp序列,鉴定其保守区和高变区,并分别设计引物(mrp-1、mrp-2),检测不同SS2菌株的mrp基因型。分析发现,mrp基因在SS2菌株中广泛分布,但其序列存在差异。通过斑马鱼感染模型检测毒力,发现mrp-A型(mrp-1+//mrp-2+)菌株比B型(mrp-1+/mrp-2-)菌株毒力强;且实时荧光定量PCR结果表明A型菌株的mrp转录水平更高。4猪链球菌2型pnuC基因缺失株的构建及致病性分析比较10株强毒株和Ⅱ组弱毒株(7株菌)的core-genome,发现了强毒株特有的53个基因,包括位于同一操纵子上的nadR,pnu和mufT基因。我们以猪链球菌2型强毒株ZY05719为研究对象,采用融合PCR和同源重组方法,使用链球菌-大肠杆菌穿梭质粒pSET-4S成功构建了 pnuC基因缺失株△pnuC。通过斑马鱼模型,评价野生株ZY05719与缺失株△pnuC毒力水平,发现缺失株对斑马鱼的致病力相对于野生株明显降低。说明pnuC基因对SS2致病性的发挥具有重要意义。
[Abstract]:Streptococcus suis (Streptococcus suis, SS) is an important zoonosis pathogen, which can cause a variety of diseases such as septicemia, arthritis, meningitis and endocarditis, which seriously threaten public health. According to the different capsule polysaccharide antigen, the Streptococcus suis can be divided into 33 serotypes (1/2,1-31,33), of which the serotype 2 (SS2) is in the form of serum type (SS2). The most reported and pathogenicity in the world has been reported in the world. There have been two cases of human infection with SS2 and serious casualties. At present, the research on the virulence factor of SS2 is increasing gradually, but the pathogenesis is still not clear about the difference of the pathogenicity of different strains of.SS2, but the lack of a suitable virulence evaluation system has seriously hindered the pathogenicity. SS2 identification. This study made the "internal standard" of the zebrafish infection model and successfully screened several SS2 strains. Through the analysis of the SS2 pan genome (pan-genome), the series differences of the strong and weak strains were identified, which provided a theoretical basis for the further study of the pathogenesis mechanism of SS2,.1 of Streptococcus suis type 2. 105 strains of SS2 strain were selected from the strain storehouse of this room. The distribution of sbp2'and Virulence Related Genes MRP, EPF and sly were detected by PCR, and the genotype of sbp2'-/mrp-/epf/sly-, sbp2'+/mrp+/epf+/sly+ and other genotypes were identified by PCR. The known weak strain T15 and the strong strain ZY05719 were used as the reference strain, namely the "internal standard". By comparing the median lethal dose (LD5O) of the tested strains and the reference strains, the virulence level of the strains to be tested was evaluated. The results showed that we successfully screened 16 SS2 strains, the genotype of sbp2'-/mrp-/ epf/sly- (13 strains) or sbp2'-/mrp+epf+/sly+ (3 strains), which also indicated the bacteria. The hair related gene sbp2'seems to be more suitable than MRP, EPF, and sly as a SS2 virulence target gene for.2,.2, Streptococcus suis 2 type pan genome analysis, and selected 7 SS2 strains to complete genome sequencing. Combined with NCBI database, the whole genome information of 10 strains and 9 strains of weak strains was obtained, and the results of Streptococcus suis 2 genomes (pan-genome) analysis were performed. The results showed that pan-genome was composed of 1239 core genes and 2436 variable genes. Evolutionary analysis showed that strong and weak strains had obvious evolution differences. 9 strains of weak strains could be divided into group I, group II two (two branches). The phage prediction analysis found that the weak strains contained more pre phage sequences. By comparing the core genes of strong and weak strains, the core genes were compared. Group (core-genome), the common endemic fimbria genes of the strong strains were found. By comparing the core-genome of the strong and 2 groups, the virulence of the 53 and 58 genes of the strong and weak strains of.3 MRP and Streptococcus suis 2 strains were identified by Pan-genome analysis, and 1459 of the 9 strains of the weak strains were identified by 1459. The gene composition, including the MRP gene, was conflicting with the PCR result of the weak strain of the strain (13 strains of the weak strains were MRP negative), indicating that the gene sequence might have diversity. This study identified the conserved and hypervariable regions by comparing the MRP sequences of different strains, and set the primers (MRP-1, MRP-2) to detect the MRP genes of different SS2 strains. The analysis found that the MRP gene was widely distributed in the SS2 strain, but its sequence was different. The virulence of the mrp-A (mrp-1+//mrp-2+) strain was stronger than that of the B (mrp-1+/mrp-2-) strain by the zebrafish infection model; and the real-time fluorescence quantitative PCR results showed that the MRP transcriptional level of the A strain was higher than the pnuC gene deletion of the.4 Streptococcus suis type 2. The construction and pathogenicity analysis of 10 strains of strong virulent strains and 7 strains of strain (7 strains) found the 53 genes of the strong virulent strains, including the nadR, PNU and mufT genes on the same operon. We used the Streptococcus suis type 2 strain ZY05719 as the research object, using the fusion PCR and homologous recombination method, the use of Streptococcus - big core-genome The Enterobacteriaceae shuttle plasmid pSET-4S successfully constructed the pnuC gene deletion strain Delta pnuC. through zebrafish model and evaluated the virulence level of the wild strain ZY05719 and the deletion strain Delta pnuC. It was found that the pathogenicity of the missing strain on zebrafish was significantly lower than that of the wild strain. It indicated that the pnuC gene was of great significance to the pathogenicity of SS2.

【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.611

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