本文選題：豬鏈球菌2型 + 內(nèi)標�。� 參考：《南京農(nóng)業(yè)大學》2015年碩士論文

【摘要】：豬鏈球菌(Streptococcus suis,SS)是一種重要的人畜共患病病原,可引起人和豬的敗血癥、關(guān)節(jié)炎、腦膜炎、心內(nèi)膜炎等多種疾病,嚴重威脅公共衛(wèi)生安全。根據(jù)莢膜多糖抗原的不同,可將豬鏈球菌分為33個血清型(1/2,1-31,33),其中血清型2型(SS2)在世界范圍內(nèi)報道最多且致病性最強。我國曾爆發(fā)兩次人感染SS2疫情,并造成嚴重的人員傷亡。目前,對SS2毒力因子的研究逐漸增加,但是其致病機理仍不明確。SS2不同菌株致病力存在差異,但是缺乏合適的毒力評價體系,嚴重阻礙對致病性SS2的鑒定。本研究將斑馬魚感染模型"內(nèi)標"化,并成功篩選到若干SS2弱毒株。通過對SS2泛基因組(pan-genome)的分析,鑒定出強、弱毒株基因組的系列差異,為進一步研究SS2致病機理提供理論依據(jù)。1豬鏈球菌2型弱毒菌株的篩選綜合考慮菌株分離時間和地域跨度,從本室菌種庫挑選105株SS2菌株,通過PCR檢測菌株中菌毛相關(guān)基因sbp2'和毒力相關(guān)基因mrp,epf,sly的分布,并鑒定出sbp2'-/mrp-/epf/sly-、sbp2'+/mrp+/epf+/sly+等6種基因型。利用斑馬魚感染模型測定不同基因型菌株的毒力,并以已知的弱毒株T15和強毒株ZY05719作為參考菌株,即"內(nèi)標"。通過比較待檢菌株和參考菌株的半數(shù)致死量(LD5O),評估待檢菌株毒力水平。結(jié)果表明,我們成功篩選到16株SS2弱毒株,基因型為sbp2'-/mrp-/epf/sly-(13株)或sbp2'-/mrp+epf+/sly+(3株),這也說明菌毛相關(guān)基因sbp2'似乎比mrp,epf,sly更適合作為SS2毒力靶基因。2豬鏈球菌2型泛基因組分析選取7株SS2弱毒株完成基因組測序,并結(jié)合NCBI數(shù)據(jù)庫,共獲得10株強毒株和9株弱毒株全基因組信息,以此進行豬鏈球菌2型泛基因組(pan-genome)分析。結(jié)果顯示,pan-genome由1,239個核心基因和2,436個可變基因組成,進化分析表明強、弱毒株進化差異明顯,9株弱毒株又可分為Ⅰ、Ⅱ兩組(兩個分支)。噬菌體預(yù)測分析發(fā)現(xiàn),弱毒株中含有更多前噬菌體序列。通過比較強毒株和弱毒株各自的核心基因組(core-genome),發(fā)現(xiàn)了強毒株共同特有的菌毛相關(guān)基因簇。通過比較強毒株和Ⅱ組弱毒株的core-genome,鑒定出強、弱毒株各自特有的53和58個基因。3 mrp基因型與豬鏈球菌2型菌株的毒力關(guān)系Pan-genome分析涉及的9株弱毒株的core-genome由1,459個基因組成,其中包括mrp基因,與我們篩選出的弱毒株P(guān)CR結(jié)果沖突(13株弱毒株呈mrp陰性),說明該基因序列可能存在多樣性。本研究通過比對不同菌株中mrp序列,鑒定其保守區(qū)和高變區(qū),并分別設(shè)計引物(mrp-1、mrp-2),檢測不同SS2菌株的mrp基因型。分析發(fā)現(xiàn),mrp基因在SS2菌株中廣泛分布,但其序列存在差異。通過斑馬魚感染模型檢測毒力,發(fā)現(xiàn)mrp-A型(mrp-1+//mrp-2+)菌株比B型(mrp-1+/mrp-2-)菌株毒力強;且實時熒光定量PCR結(jié)果表明A型菌株的mrp轉(zhuǎn)錄水平更高。4豬鏈球菌2型pnuC基因缺失株的構(gòu)建及致病性分析比較10株強毒株和Ⅱ組弱毒株(7株菌)的core-genome,發(fā)現(xiàn)了強毒株特有的53個基因,包括位于同一操縱子上的nadR,pnu和mufT基因。我們以豬鏈球菌2型強毒株ZY05719為研究對象,采用融合PCR和同源重組方法,使用鏈球菌-大腸桿菌穿梭質(zhì)粒pSET-4S成功構(gòu)建了 pnuC基因缺失株△pnuC。通過斑馬魚模型,評價野生株ZY05719與缺失株△pnuC毒力水平,發(fā)現(xiàn)缺失株對斑馬魚的致病力相對于野生株明顯降低。說明pnuC基因?qū)S2致病性的發(fā)揮具有重要意義。
[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.

【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.611

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