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1.
大肠杆菌VT2噬菌体的分离与溶源转染   总被引:4,自引:1,他引:4  
本试验利用指示菌MC1061,经双层琼脂法纯化和PCR扩增vt2基因,分别从大肠杆菌O157菌株、牛粪、鸡粪和污水中分离获得5株含vt2基因的噬菌体.这些噬菌斑透明,直径为0.5-2 mm,对指示菌的感染效价均在109PFU/mL以上,抵抗氯仿和56℃C30min的作用.将噬菌体分离株SHφW1感染MC1061后,经PCR鉴定获得一株溶源菌株(MC1061/SHφW1).溶源株的LB培养滤液对Vero细胞产生了显著的病变效应,而MC1061在同等条件下培养的滤液无细胞病变,表明VT2噬菌体通过溶源将vt2毒力基因水平转移,证实了VT2噬菌体的转染与细菌毒力相关.  相似文献   

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根据GenBank中VT1、VT2毒素的基因序列设计合成2对引物,以大肠杆菌O157H7菌株DNA为模板,扩增vt1、vt2.诱导只扩增出vt2的菌株释放噬菌体,利用多种指示菌经双层琼脂平板法来分离纯化VT2噬菌体,观察噬菌斑的特征,提纯病毒粒子进行电镜观察,并对噬菌体中vt2基因检测、克隆和序列分析.结果显示VT2噬菌体感染MC1061在双层琼脂平板上形成的噬菌斑小而混浊,多呈磨玻璃样;而首次感染大肠杆菌CC118(λpir),此后用MC1061分离的噬菌体,再以MC1061为指示菌,在双层琼脂平板上形成小而清晰透明的噬菌斑.电镜下噬菌体头部呈六边形外廓,尾部细长无尾鞘结构.以噬菌体DNA为模板进行PCR扩增,检测到vt2特异性DNA带,克隆的vt2基因序列与GenBank中编码VT2毒素的核苷酸序列(X07865,NC_002655,BA000007,AF291819)的同源性分别达到99%,确定编码VT2毒素的基因位于噬菌体上,并获得VT2噬菌体()HY.  相似文献   

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根据GenBank中VT1、VT2毒素的基因序列设计合成2对引物,以大肠杆菌O157:H7菌株DNA为模板,扩 增vt1、vt2。诱导只扩增出vt2的菌株释放噬菌体,利用多种指示菌经双层琼脂平板法来分离纯化VT2噬菌体,观 察噬菌斑的特征,提纯病毒粒子进行电镜观察,并对噬菌体中vt2基因检测、克隆和序列分析。结果显示VT2噬菌 体感染MC1061在双层琼脂平板上形成的噬菌斑小而混浊,多呈磨玻璃样;而首次感染大肠杆菌CC118(λpir),此 后用MC1061分离的噬菌体,再以MC1061为指示菌,在双层琼脂平板上形成小而清晰透明的噬菌斑。电镜下噬 菌体头部呈六边形外廓,尾部细长无尾鞘结构。以噬菌体DNA为模板进行PCR扩增,检测到vt2特异性DNA 带,克隆的vt2基因序列与GenBank中编码VT2毒素的核苷酸序列(X07865,NC_002655,BA000007,AF291819) 的同源性分别达到99%,确定编码VT2毒素的基因位于噬菌体上,并获得VT2噬菌体(?)HY。  相似文献   

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根据GenBank中毒素基因vt1、vt2序列设计合成4对引物,以大肠杆菌O157菌株DNA为模板,扩增vt1、vt2,从只含有vt2的菌株中诱导释放噬菌体,以噬菌体DNA为模板进行PCR扩增,获得vt2、vt2-A、vt2-B3条特异性DNA带;将vt2-A、vt2-B扩增产物纯化后,分别插入pMD18-T载体,测序结果与相应序列比较,vt2-A和vt2-B亚单位的基因序列与GenBank中编码VT2毒素的A、B亚单位的核苷酸序列(X07865,NC_002655,:BA000007,AF291819)的同源性分别为98%~99%、96%~100%,确定vt2位于噬菌体,并为进一步研究大肠杆菌O157中VT噬菌体的毒力转导、VT2毒素的表达和应用奠定基础。  相似文献   

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根据GenBank中毒素基因 vt1、vt2序列设计合成 4 对引物,以大肠杆菌 O157 菌株 DNA为模板,扩增 vt1、vt2,从只含有vt2的菌株中诱导释放噬菌体,以噬菌体DNA为模板进行PCR扩增,获得vt2、vt2 A、vt2 B 3条特异性DNA带;将 vt2 A、vt2 B扩增产物纯化后,分别插入 pMD18 T载体,测序结果与相应序列比较,vt2 A和 vt2 B亚单位的基因序列与 GenBank中编码 VT2 毒素的 A、B亚单位的核苷酸序列(X07865,NC_002655, BA000007,AF291819)的同源性分别为98%~99%、96%~100%,确定 vt2 位于噬菌体,并为进一步研究大肠杆菌 O157 中VT噬菌体的毒力转导、VT2毒素的表达和应用奠定基础。  相似文献   

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根据GenBank中毒素基因vt1、vt2序列设计合成4对引物,以大肠杆菌O157菌株DNA为模板,扩增vt1、vt2,从只含有vt2的菌株中诱导释放噬菌体,以噬菌体DNA为模板进行PCR扩增,获得vt2、vt2-A、vt2-B 3条特异性DNA带;将vt2-A、vt2-B扩增产物纯化后,分别插入pMD18-T载体,测序结果与相应序列比较,vt2-A和vt2-B亚单位的基因序列与GenBank中编码VT2毒素的A、B亚单位的核苷酸序列(X07865,NC_002655,BA000007,AF291819)的同源性分别为98%~99%、96%~100%,确定vt2位于噬菌体,并为进一步研究大肠杆菌O157中VT噬菌体的毒力转导、VT2毒素的表达和应用奠定基础.  相似文献   

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从土壤中分离出一株卡那霉素链霉菌噬菌体SKJl,该噬菌体在卡那霉素链霉菌及林肯链霉菌菌苔上产生混浊的噬菌斑。从噬斑中长出的菌落后代对SKJl噬菌体的感染产生了抗性。单株传代未见自发释放游离噬菌体。紫外线照射亦未发现诱导现象。抗性菌株经10ug/ml丝裂霉素C处理后,其中一株能释放出约5.9×103pfu/ml游离噬菌体颗粒,推测这些抗性菌株可能是溶源性菌株。菌落原位杂交实验证实抗性菌株的DNA与SKJl噬菌体DNA有同源性,说明这些抗性菌株已被SKjL噬菌体溶源化,从而确证SKJl噬菌体为一温和性噬菌体。  相似文献   

8.
红霉素链霉菌2-62溶源性菌株的确证与特性研究   总被引:1,自引:1,他引:0  
我们从红霉紊产生菌Streptomyces erythreus的27株系谱菌株中分离到一株带确噬菌体的2—62菌株,并对2—62菌株作了多方面的考察。根据单菌落传代、孢子加热,柠檬酸钠洗涤消除噬菌体后仍继续释放噬菌体;2—62菌株释放的噬菌体经纯化后制备抗血清,用此抗血清处理2—62菌株的斜面孢子,以中和游离的噬菌体,这种孢子在培养过程中仍继续释放噬菌体;2—62菌株对其自身释放的噬菌体具有免疫力等特性,确证2—62菌株为溶源性菌株。用紫外线或丝裂霉素C进行诱导试验,噬菌体释放量增加不多。将2—62菌株释放的温和性噬菌体感染P32一102敏感菌,得到的溶源化菌林亦释放噬菌体,并且溶源化菌林的性状保持稳定。  相似文献   

9.
大肠杆菌VT噬菌体受体(vpr)基因突变株的鉴定   总被引:2,自引:0,他引:2  
近年来大肠杆菌O157引起的人和动物的发病率呈上升趋势^[1-3],O157的强致病性与很多因子有关,如:粘附因子、肠溶血素(Enterohaemolysin,Ehx)、Vero毒素(Vero toxin,VTs)等。VT可致人和动物的腹泻、出血性肠炎、溶血性尿毒综合证、血栓、血球减少性紫癜等疾病^[4],是O157主要的致病因子。大量的研究表明VT是由λ家族具有vT基因的噬菌体(vT—phage)编码的^[5],且在噬菌体感染宿主菌的裂解状态时由晚期调节基因调控毒素的产生。可见大肠杆菌O157所产的vT与vT噬菌体的感染有关。大肠杆菌表面是否存在噬菌体的受体为众多科学工作者所关注,1999年Saunders等在大肠杆菌染色体中克隆出一段与VT2噬菌体敏感性有关的基因^[6],将其定名为vpr(verotoxin phage receptor)。为了进一步确定vpr基因的功能,本试验利用同源重组的方法,将已经构建的含vpr基因片段的重组自杀性质粒pYYvpr,转化到含全长vpr基因的大肠杆菌MC1061,经PCR和southern blot鉴定,获得一株染色体同源基因突变株,并对其进行生物学特性研究,初步证实该基因与噬菌体的裂解性感染有关。  相似文献   

10.
目的:克隆并分析细菌性噬菌体φ97的整合酶基因(int)。方法:采用加接头的基因DNA片断为模板进行步移PCR,根据溶源性噬菌体φ297的染色体DNA上类似于噬菌体933W的整合酶基因的一个40个核苷酸设计引物,进行扩增、克隆、亚克隆、测序和序列分析。结果:得到了噬菌体φ297编码的整合酶基因(int)的完整序列,它的长度是1287bp,编码了428个氨基酸的Int蛋白质。将它们的序列与λ噬菌体的整合酶家族其它成员进行了比较,发现噬菌体φ297的整合酶基因(int)与噬菌体VT1-Sakai的整合酶基因有79%的同源性,噬菌体φ297的Int蛋白与噬菌体VT1-Sakai的Int蛋白在氨基酸序列上有82%的同源性。N-末端的氨基酸区域是完全保守的,而中心区和C-末端则显示出较大差异。结论:噬菌体φ297与λ噬菌体的int基因来源于同一基因库,噬菌体φ297可能属于λ噬菌体家族。  相似文献   

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It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.  相似文献   

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Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.  相似文献   

18.
Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.  相似文献   

19.
肝癌中HBV和HCV基因和抗原的分布及意义   总被引:1,自引:0,他引:1  
采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细  相似文献   

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For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.  相似文献   

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