猪链球菌2型抗吞噬相关基因的筛选及鉴定
发布时间：2018-11-02 10:09
【摘要】：猪链球菌(Streptococcus suis,S.suis)是一种重要的人畜共患病病原,可引起猪和人的广泛性感染,表现为脑膜炎、败血症、关节炎及心内膜炎等。在33个血清型中,猪链球菌2型(Streptococcus suis type2,SS2)流行最广,致病性最强,危害最重,给养殖业造成了巨大的经济损失,并危害公共卫生安全。尽管已报道了该菌的多种毒力因子,但其致病机理仍不清晰。本研究通过转座子突变技术筛选SS2抗吞噬相关基因,并对筛选所得hsdS基因的功能进行验证。研究结果有助于进一步阐明猪链球菌2型抗吞噬机理。1、猪链球菌2型抗吞噬相关基因的筛选抗吞噬是SS2抵抗机体先天性免疫,逃避免疫杀伤,引发猪链球菌病的一项重要功能。为发掘SS2抗吞噬相关基因,进一步研究SS2逃避宿主先天性免疫机制,本实验利用含转座子TnYLB-1的pMar4S质粒构建ZY05719菌株突变体文库,通过BV-2细胞吞噬试验获取抗吞噬能力减弱突变株,从而分离、鉴定SS2抗吞噬相关基因。试验成功构建了含2000株突变株的突变体文库,筛选出4株抗吞噬能力减弱的突变株(P0.05)。该4株突变株的吞噬率分别高于野生株:1.976、2.29、1.16和1.63倍,转座子插入基因分别为:通透酶基因(ZY05719_RS02540),Ⅰ限制性修饰系统的S亚基基因(ZY05719_RS06850),1,4-二羟基-2-萘八异戊烯转移酶基因(ZY05719_RS08285)和支链氨基酸转运蛋白相关基因(ZY05719_RS03750)。研究结果为进一步研究SS2抗吞噬机制提供了基础。2、猪链球菌2型hsdS基因抗吞噬功能的验证hsdS是猪链球菌2型Ⅰ型R-M系统基因簇中的一个基因,编码蛋白HsdS,形成限制性内切酶和甲基转移酶。本章通过构建穿梭载体pSET2::hsdS导入至转座子突变株MhsdS,从而构建了突变株的互补株CMhsdS,验证hsdS在SS2抵抗小鼠小胶质细胞BV-2吞噬过程中的作用。试验结果显示互补株CMhsdS与突变株MhsdS相比,抗吞噬能力恢复了 45.4%,且差异显著(P0.001)。此外,系列实验显示MhsdS在小胶质细胞内存活,全血存活,酸、氧耐受,刺激小胶质细胞产生细胞因子及一氧化氮等方面的能力与野生株相比存在显著差异(P0.05);而互补株CMhsdS与突变株相比,除全血存活和酸性耐受能力没有得到恢复,以上其他方面的能力均得到一定的恢复,且与突变株存在显著差异(P0.05)。转录组结果显示突变株MhsdS与野生株ZY05719有56个差异表达基因,其中33个基因上调表达,23个基因下调表达,主要涉及过程包括能力代谢,细菌细胞壁和膜功能等。综上所述,hsdS基因能够促进SS2抵抗小胶质细胞的吞噬作用,同时在SS2致病过程中具有多种生物学功能。3、猪链球菌2型Ⅰ型R-M系统抗吞噬功能的分析本试验所研究的SS2Ⅰ型R-M系统基因簇包含hsdR,hsdM,hsdS和hsdS'四个基因,其中hsdS是突变株MhsdS中转座子TnYLB-1的插入基因,与SS2抵抗小胶质细胞吞噬功能有关。为进一步研究Ⅰ型R-M系统对SS2抵抗BV-2细胞吞噬功能的作用,本试验利用温敏型穿梭质粒pSET4s分别成功构建了hs R和hsdM的缺失株(△hsdM,△hsdR),hsdS的部分和全长缺失株(△hsdSp,△hsdSf),以及hsdS'的部分和全长缺失株(△hsdS'p,△hsdS'f)。与野生株相比,各缺失株生长速率无明显差异,缺失株△hsdSf、△hs和△hsdR抵抗小胶质细胞吞噬能力显著降低(P0.05)。各缺失株诱导小胶质细胞产生TNF-α的量均高于野生株,但仅缺失株△hsdSp和△hsdSf差异显著(P0.05);而诱导产生MCP-1的量则基本低于野生株,除缺失株△hsdS'f和△hsdR外,其他差异均显著(P0.05)。因此,hsdS的抗吞噬相关功能可能与整个Ⅰ型R-M系统的生物学功能相关。
[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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.611
本文编号：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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.611
【参考文献】
相关期刊论文 前1条
1 陆承平;姚火春;范红结;华修国;孙建和;顾宏伟;王楷;赵冉;濮俊义;张炜;;猪链球菌致病性研究及其公共卫生意义[J];中国预防医学杂志;2006年04期
，本文编号：2305722
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