【作者】 马玉玲；

【导师】 陆承平；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2007， 博士

【摘要】 猪链球菌(Streptococcus suis,SS)是一种重要的人畜共患病痛原,根据菌体荚膜多糖抗原性的不同,分为35个血清型(1-34型,1/2型)。世界范围内以猪链球菌2型(Streptococcus suis type 2,SS2)流行最广,致病性最强。国内1991年在广东省首次分离鉴定到SS2,随后于1998年和2005年分别在江苏和四川部分地区暴发人感染SS2并致人死亡,危害严重。SS2感染已成为影响全世界养猪业的重要问题之一。分离和鉴定SS2的噬菌体,研究SS2的噬菌体对此类革兰氏阳性菌的遗传进化和环境生态学有重要的理论意义及实用价值。然而,目前对猪链球菌的噬菌体国内外迄今研究甚少,仅有一篇关于SS2溶源性噬菌体的报道。本试验对SS2同时进行了烈性和溶原性噬菌体的研究。从健康巴马香猪的鼻拭子样本中分离出一株SS2的烈性噬菌体,命名为SMP,并从形态学和分子水平对其特性进行研究。噬菌体SMP颗粒在电镜下观察,头部呈正六边形,直径约50nm,尾部约135nm,无非伸缩性,属于长尾噬菌体科。噬菌体SMP的宿主范围较窄,仅能裂解被测的24株SS2中的2株。SMP对热比较敏感,40℃以上的高温处理后活力明显下降,60℃处理后活力完全消失。SMP在pH6-10范围内比较稳定,在pH6.0以下失活较快。SMP的核酸属于双股线性DNA,可以被BglII、EcoRI、NcoI、BamHI和PstI消化,而不能被NotI消化。一步生长曲线表明潜伏期为20min,生长期为120min,平均裂解量为77PFU/Cell。本实验首次报道了从健康猪体内分离到SS2烈性噬菌体,这将为猪链球菌病的治疗开辟新的途径。用甘油梯度离心纯化噬菌体SMP颗粒,提取噬菌体基因组DNA,建立shotgun随机文库,从丈库中随机挑取克隆进行测序并完成序列组装,对SS2烈性噬菌体SMP进行基因组测序。通过限制性内切酶图谱分析,并结合酶切片段的变性、复性实验分析基因组末端。预测可能存在的开放读码框(ORF),对噬菌体的相关基因作进化树分析。测序结果表明噬菌体SMP基因组是36,216bp大小的线性dsDNA分子,其碱基组成为A=31.72%[11487];G=23.24%[8418];T=26.67%[9659];C=18.37%[6652];稀有碱基对为0[0];A+T=58.39%[21146]:C+G=41.61%[15070]。预测的169个大于150bp的ORFs中,48个被推定为编码的基因,大多数与已报道的噬菌体序列同源,包括头蛋白、末端酶大亚单位、复制蛋白、单股结合蛋白和主要的尾蛋白等。形态学特征和基因组序列的测定与分析表明,SMP在分类上属于长尾噬菌体科。噬菌体SMP的基因组序列的测定与分析,将为功能基因组学、溶原性机理、溶原性/裂性转换及有效诱导手段的研究、比较基因组学、噬菌体与宿主茵相互作用关系、噬菌体介导基因水平转移(尤其是致病基因、耐药基因)、生物多样性、噬菌体工程改造和噬菌体治疗等方面提供重要的线索。为了研究噬菌体整合酶基因在SS2中的分布情况,根据噬菌体整合酶基因设计引物,建立了PCR方法,并对扩增产物进行测序。结果显示,25株SS2致病菌株均扩增出目的片段,非毒力株T15、5株其它血清型猪链球菌及兰氏C群猪源链球菌未扩增出目的片段。经丝裂霉素C诱导后,25株SS2致病菌株均出现完全的细胞溶解,而非毒力株T15未出现溶解。电镜下观察SS2致病株HA9801和ZY05719诱导产生的瘩原性噬菌体,头部呈正六边形,无尾部。经DNaseⅠ等处理,鉴定其核酸类型为dsDNA。根据形态学特征和核酸性质,推测该噬菌体属于复层噬菌体科(Tectiviridae)。SS2致病株的噬菌体整合酶基因与SS2溶菌酶释放蛋白(mrp)等7种毒力相关基因有相关性,表明SS2的溶原性噬菌体可能与其致病性有关。对SS2溶原噬菌体的检测、是否能进行毒力基因的水平转移、基因水平转移的条件、溶原转换与细菌毒力的相关性等进行研究,这不仅可以从新的视角深入对猪链球菌的研究,而且通过对革兰氏阳性菌的噬菌体的深入研究,可将噬菌体研究提高到新的水平。更多还原

【Abstract】 Streptococcus suis(SS) is an important pathogen of human and animal. Thirty-five capsular serotypes or S.suis have been described(types 1/2 and 1 through 34).Streptococcus suis type 2(SS2) is considered to be the most-virulent serotype and prevalenced in wordwide.In China,SS2 was first isolated in 1991,later,human Streptococcus suis infection in Jiangsu province and Sichuan Province happened in 1998 and 2005,respectively.Study on the phages of SS2 would play a crucial role in the research on the ecology and evolution of such Gram-positive bacteria.However, very little is known of the phages of SS2 for that.only one paper about the temperate phage of SS2 has been published by far.A SS2 bacteriophage,named SMP,was isolated from nasal swabs of healthy Bama minipigs and was characterized at the microbiological and molecular level. Phage SMP had an isometric head of 50 nm,a noncontractile tail of approximately 135 nm.The host range of phage SMP was limited to 2 of 24 SS2 strains tested.The activity of SMP declined dramatically when heated above 40℃and stable at pH6-10. SMP nucleic acid belonged to double-stranded DNA and could be digested with BglⅡ, EcoRⅠ,NcoⅠ,BamHⅠand PstⅠbut not with NotⅠ.One-step growth curve for SMP showed a latent period of about 20min,a rise period of 120min,and an average burst size of about 77 PFU/cell.SS2 virulent phage SMP particles were purified with glycerol gradient centrifugation,and its genomic DNA was extracted in order to construct the shot-gun library.The randomly picked clones under went genome sequencing.The sequence was reconstructed,and then analyzed for the genome ends with restrictive endonuclease BamHⅠspectrum under denaturing Pannealing conditions.ORFs were predicted and phylogeny of phage-associated gene sequences was analyzed to determine the class of bacteriophage SMP.The genome of phage SMP is a 36216bp of linear double-stranded DNA.The base constituents of the genome were A= 31.72%[11487];G=23.24%[8418];T=26.67%[9659];C=18.37%[6652]; Ambiguous=[0];A+T=58.39%[21146];C+G=41.61%[15070]respectively.A total of 169 open reading frames(ORFs),of larger than 150 bp and 48 coding sequences, were detected in the phage SMP genome.A search using BlastP identified some sequences of high homology,including head protein,terminase large subunit,putative replication protein,single strand binding protein and major tail protein.The genome size of Siphophages range mostly from 22 to 121 kb and the study of molecular characteristics of phage SMP suggested that it should to be a member of the Siphoviridae family.To study the distributional characteristics of phage integrase gene in SS2,a PCR assay was developed and a 323bp distinct DNA target can be amplified in 25 strains of virulent SS2,while can not be amplified in avirulent strain T15,5 strains of other serotypes(SS1,SS7,SS9) and strains of group C streptococcus strains from pigs. The sequencing and restriction endonuclease analysis of the PCR products were also done.Complete cell lysis was observed with SS2 virulent strains but not with avirulent strain T15.Electron microscopy analysis of the lysate from SS2 virulent strains HA9801 and ZY05719 revealed the presence of phage particles.The induced phage has a small isometric nucleocapsid approximately 50 nm in diameter and has no tail and is therefore a member of the Tectiviridae family.The temperate phage and phage integrase gene can only detected from SS2 virulent strains but not from avirulent strain,and the detection of phage integrase gene was related to the virulence-associate factors of SS2,such as the muramidase-released protein gene (mrp),which suggested that the temperate phage of SS2 may be related to the pathogenicity of SS2.更多还原