豬鏈球菌2型抗吞噬相關(guān)基因的篩選及鑒定
發(fā)布時間：2018-11-02 10:09
【摘要】：豬鏈球菌(Streptococcus suis,S.suis)是一種重要的人畜共患病病原,可引起豬和人的廣泛性感染,表現(xiàn)為腦膜炎、敗血癥、關(guān)節(jié)炎及心內(nèi)膜炎等。在33個血清型中,豬鏈球菌2型(Streptococcus suis type2,SS2)流行最廣,致病性最強(qiáng),危害最重,給養(yǎng)殖業(yè)造成了巨大的經(jīng)濟(jì)損失,并危害公共衛(wèi)生安全。盡管已報道了該菌的多種毒力因子,但其致病機(jī)理仍不清晰。本研究通過轉(zhuǎn)座子突變技術(shù)篩選SS2抗吞噬相關(guān)基因,并對篩選所得hsdS基因的功能進(jìn)行驗證。研究結(jié)果有助于進(jìn)一步闡明豬鏈球菌2型抗吞噬機(jī)理。1、豬鏈球菌2型抗吞噬相關(guān)基因的篩選抗吞噬是SS2抵抗機(jī)體先天性免疫,逃避免疫殺傷,引發(fā)豬鏈球菌病的一項重要功能。為發(fā)掘SS2抗吞噬相關(guān)基因,進(jìn)一步研究SS2逃避宿主先天性免疫機(jī)制,本實驗利用含轉(zhuǎn)座子TnYLB-1的pMar4S質(zhì)粒構(gòu)建ZY05719菌株突變體文庫,通過BV-2細(xì)胞吞噬試驗獲取抗吞噬能力減弱突變株,從而分離、鑒定SS2抗吞噬相關(guān)基因。試驗成功構(gòu)建了含2000株突變株的突變體文庫,篩選出4株抗吞噬能力減弱的突變株(P0.05)。該4株突變株的吞噬率分別高于野生株:1.976、2.29、1.16和1.63倍,轉(zhuǎn)座子插入基因分別為:通透酶基因(ZY05719_RS02540),Ⅰ限制性修飾系統(tǒng)的S亞基基因(ZY05719_RS06850),1,4-二羥基-2-萘八異戊烯轉(zhuǎn)移酶基因(ZY05719_RS08285)和支鏈氨基酸轉(zhuǎn)運(yùn)蛋白相關(guān)基因(ZY05719_RS03750)。研究結(jié)果為進(jìn)一步研究SS2抗吞噬機(jī)制提供了基礎(chǔ)。2、豬鏈球菌2型hsdS基因抗吞噬功能的驗證hsdS是豬鏈球菌2型Ⅰ型R-M系統(tǒng)基因簇中的一個基因,編碼蛋白HsdS,形成限制性內(nèi)切酶和甲基轉(zhuǎn)移酶。本章通過構(gòu)建穿梭載體pSET2::hsdS導(dǎo)入至轉(zhuǎn)座子突變株MhsdS,從而構(gòu)建了突變株的互補(bǔ)株CMhsdS,驗證hsdS在SS2抵抗小鼠小膠質(zhì)細(xì)胞BV-2吞噬過程中的作用。試驗結(jié)果顯示互補(bǔ)株CMhsdS與突變株MhsdS相比,抗吞噬能力恢復(fù)了 45.4%,且差異顯著(P0.001)。此外,系列實驗顯示MhsdS在小膠質(zhì)細(xì)胞內(nèi)存活,全血存活,酸、氧耐受,刺激小膠質(zhì)細(xì)胞產(chǎn)生細(xì)胞因子及一氧化氮等方面的能力與野生株相比存在顯著差異(P0.05);而互補(bǔ)株CMhsdS與突變株相比,除全血存活和酸性耐受能力沒有得到恢復(fù),以上其他方面的能力均得到一定的恢復(fù),且與突變株存在顯著差異(P0.05)。轉(zhuǎn)錄組結(jié)果顯示突變株MhsdS與野生株ZY05719有56個差異表達(dá)基因,其中33個基因上調(diào)表達(dá),23個基因下調(diào)表達(dá),主要涉及過程包括能力代謝,細(xì)菌細(xì)胞壁和膜功能等。綜上所述,hsdS基因能夠促進(jìn)SS2抵抗小膠質(zhì)細(xì)胞的吞噬作用,同時在SS2致病過程中具有多種生物學(xué)功能。3、豬鏈球菌2型Ⅰ型R-M系統(tǒng)抗吞噬功能的分析本試驗所研究的SS2Ⅰ型R-M系統(tǒng)基因簇包含hsdR,hsdM,hsdS和hsdS'四個基因,其中hsdS是突變株MhsdS中轉(zhuǎn)座子TnYLB-1的插入基因,與SS2抵抗小膠質(zhì)細(xì)胞吞噬功能有關(guān)。為進(jìn)一步研究Ⅰ型R-M系統(tǒng)對SS2抵抗BV-2細(xì)胞吞噬功能的作用,本試驗利用溫敏型穿梭質(zhì)粒pSET4s分別成功構(gòu)建了hs R和hsdM的缺失株(△hsdM,△hsdR),hsdS的部分和全長缺失株(△hsdSp,△hsdSf),以及hsdS'的部分和全長缺失株(△hsdS'p,△hsdS'f)。與野生株相比,各缺失株生長速率無明顯差異,缺失株△hsdSf、△hs和△hsdR抵抗小膠質(zhì)細(xì)胞吞噬能力顯著降低(P0.05)。各缺失株誘導(dǎo)小膠質(zhì)細(xì)胞產(chǎn)生TNF-α的量均高于野生株,但僅缺失株△hsdSp和△hsdSf差異顯著(P0.05);而誘導(dǎo)產(chǎn)生MCP-1的量則基本低于野生株,除缺失株△hsdS'f和△hsdR外,其他差異均顯著(P0.05)。因此,hsdS的抗吞噬相關(guān)功能可能與整個Ⅰ型R-M系統(tǒng)的生物學(xué)功能相關(guān)。
[Abstract]:Streptinosuis, S. suis is an important pathogen of human animal, which can cause extensive infection of pig and human. Among the 33 serotypes, Streptinosuis type2 (SS2) is the most popular, most pathogenic and most harmful, causing enormous economic losses to the breeding industry and jeopardizing public health and safety. Although a variety of virulence factors have been reported, its pathogenesis is still unclear. In this study, SS2 anti-phagocyte-related genes were screened by transposable mutation technique, and the function of hsdS gene was verified by screening. The results of the study contribute to the further elucidation of the anti-phagocytizing mechanism of type 2. The screening of anti-phagocyte-related genes of Type 1 and GY2 is an important function of SS2 to resist the innate immunity of the organism, to avoid the immune killing, and to induce the anti-inflammatory disease. In order to find out the anti-phagocyte-related gene of SS2 and further study the mechanism of SS2 escape host, this experiment uses pMar4S plasmid containing TnYLB-1 to construct a mutant library of ZY05719 strain. Identification of SS2 anti-phagocyte-related genes. A mutant library containing 2000 mutants was successfully constructed, and 4 mutant strains (P0.05) were screened out. The four mutant strains were higher than wild strains: 1. 976, 2.29, 1. 16 and 1. 63 times, respectively. Transposon gene (ZY05719 _ RS02540), The S subunit gene (ZY05719 _ RS06850), 1, 4-dihydroxy-2-hepten-prenyltransferase gene (ZY05719 _ RS08285) and branched-chain amino acid transporter-related gene (ZY05719 _ RS03750) of restriction modification system. The results of this study provide the basis for further study of SS2 anti-eating mechanism. By constructing the shuttle vector pSET2:: hsdS, the mutant strain MhsdS was introduced to construct the complementary strain CMhsdS of the mutant strain, thus verifying the role of hsdS in the phagocytizing of small glial cells BV-2 in mice. The results showed that compared with MhsdS mutant strain MhsdS, the anti-eating ability of CMhsdS was 45.4%, and the difference was significant (P0.001). In addition, the series of experiments showed that the ability of MhsdS to survive, whole blood survival, acid, oxygen tolerance, stimulation of microglia to produce cytokines and nitric oxide were significantly different from those of wild strains (P0.05). In addition to the recovery of the whole blood survival and acid tolerance, the ability of the above-mentioned aspects was restored to a certain extent, and there was a significant difference with the mutant (P0.05). The transcription group showed 56 differentially expressed genes in mutant MhsdS and wild strain ZY05719, among which 33 genes upregulated and 23 genes were downregulated, mainly involved in the process including capacity metabolism, bacterial cell wall and membrane function. In conclusion, the hsdS gene can promote SS2 to resist the phagemogenesis of microglial cells, and has a variety of biological functions in the pathogenesis of SS2. The SS2 I-type R-M system of the type I R-M system of the type II type contains hsdR, hsdM, hsdS and hsdS "Four genes, in which hsdS is the inserted gene of transposons TnYLB-1 in mutant MhsdS, is associated with SS2 resistance to microglial cells." In order to further study the effect of type I R-M system on the devouring function of SS2 against BV-2 cells, we successfully constructed the deletion strains of hs R and hsdM, the partial and full length deletion strains of hsdS and hsdS 'using temperature sensitive shuttle plasmid pSET4s, respectively. Partial and full-length deletion strains (DbhsdS' p, uvhsdS 'f). Compared with wild strains, there was no significant difference between the growth rates of the deleted strains, and the loss of hsdSf, hshs and hshsdR were significantly lower than that of wild strains (P0.05). The results showed that there was no significant difference (P <0.05) between the deletion strains, hsdsp and HshsdSf (P0.05), but there was no significant difference (P0.05). Therefore, the anti-swallowing-related function of hsdS may be related to the biological function of the whole type I R-M system.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S852.611
本文編號：2305722
[Abstract]:Streptinosuis, S. suis is an important pathogen of human animal, which can cause extensive infection of pig and human. Among the 33 serotypes, Streptinosuis type2 (SS2) is the most popular, most pathogenic and most harmful, causing enormous economic losses to the breeding industry and jeopardizing public health and safety. Although a variety of virulence factors have been reported, its pathogenesis is still unclear. In this study, SS2 anti-phagocyte-related genes were screened by transposable mutation technique, and the function of hsdS gene was verified by screening. The results of the study contribute to the further elucidation of the anti-phagocytizing mechanism of type 2. The screening of anti-phagocyte-related genes of Type 1 and GY2 is an important function of SS2 to resist the innate immunity of the organism, to avoid the immune killing, and to induce the anti-inflammatory disease. In order to find out the anti-phagocyte-related gene of SS2 and further study the mechanism of SS2 escape host, this experiment uses pMar4S plasmid containing TnYLB-1 to construct a mutant library of ZY05719 strain. Identification of SS2 anti-phagocyte-related genes. A mutant library containing 2000 mutants was successfully constructed, and 4 mutant strains (P0.05) were screened out. The four mutant strains were higher than wild strains: 1. 976, 2.29, 1. 16 and 1. 63 times, respectively. Transposon gene (ZY05719 _ RS02540), The S subunit gene (ZY05719 _ RS06850), 1, 4-dihydroxy-2-hepten-prenyltransferase gene (ZY05719 _ RS08285) and branched-chain amino acid transporter-related gene (ZY05719 _ RS03750) of restriction modification system. The results of this study provide the basis for further study of SS2 anti-eating mechanism. By constructing the shuttle vector pSET2:: hsdS, the mutant strain MhsdS was introduced to construct the complementary strain CMhsdS of the mutant strain, thus verifying the role of hsdS in the phagocytizing of small glial cells BV-2 in mice. The results showed that compared with MhsdS mutant strain MhsdS, the anti-eating ability of CMhsdS was 45.4%, and the difference was significant (P0.001). In addition, the series of experiments showed that the ability of MhsdS to survive, whole blood survival, acid, oxygen tolerance, stimulation of microglia to produce cytokines and nitric oxide were significantly different from those of wild strains (P0.05). In addition to the recovery of the whole blood survival and acid tolerance, the ability of the above-mentioned aspects was restored to a certain extent, and there was a significant difference with the mutant (P0.05). The transcription group showed 56 differentially expressed genes in mutant MhsdS and wild strain ZY05719, among which 33 genes upregulated and 23 genes were downregulated, mainly involved in the process including capacity metabolism, bacterial cell wall and membrane function. In conclusion, the hsdS gene can promote SS2 to resist the phagemogenesis of microglial cells, and has a variety of biological functions in the pathogenesis of SS2. The SS2 I-type R-M system of the type I R-M system of the type II type contains hsdR, hsdM, hsdS and hsdS "Four genes, in which hsdS is the inserted gene of transposons TnYLB-1 in mutant MhsdS, is associated with SS2 resistance to microglial cells." In order to further study the effect of type I R-M system on the devouring function of SS2 against BV-2 cells, we successfully constructed the deletion strains of hs R and hsdM, the partial and full length deletion strains of hsdS and hsdS 'using temperature sensitive shuttle plasmid pSET4s, respectively. Partial and full-length deletion strains (DbhsdS' p, uvhsdS 'f). Compared with wild strains, there was no significant difference between the growth rates of the deleted strains, and the loss of hsdSf, hshs and hshsdR were significantly lower than that of wild strains (P0.05). The results showed that there was no significant difference (P <0.05) between the deletion strains, hsdsp and HshsdSf (P0.05), but there was no significant difference (P0.05). Therefore, the anti-swallowing-related function of hsdS may be related to the biological function of the whole type I R-M system.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S852.611
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 陸承平;姚火春;范紅結(jié);華修國;孫建和;顧宏偉;王楷;趙冉;濮俊義;張煒;;豬鏈球菌致病性研究及其公共衛(wèi)生意義[J];中國預(yù)防醫(yī)學(xué)雜志;2006年04期
，本文編號：2305722
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