志贺毒素2型噬菌体ΦMin27的stx2基因突变株构建及其感染特性

[目的]通过构建一株stx2基因突变的志贺毒素2型噬菌体,来观察它对不同血清型大肠杆菌的感染特性.[方法]利用九噬菌体的Red重组系统,将氯霉素抗性基因(cat)插入到大肠杆菌O157:H7 Min27株的stx2基因中,获得O157:H7 Min27的突变菌株Min27(Astx::cat).用丝裂霉素对该突变菌株进行诱导,结合抗性标记和PCR方法筛选出一株突变的志贺毒素2型噬菌体,命名为ΦMin27(△stx::cat).采用双层琼脂平板法和氯霉素抗性筛选的方法,观察ΦMin27(△stx::cat)感染21株不同血清型大肠杆菌后的裂解和溶原转换情况.[结果]2株血清型分别为O 60和O 138的大肠杆菌可以被ΦMin27(△stx::cat)溶原感染,表现出对氯霉素的抗性,但没有形成噬菌斑,而大肠杆菌MG1655株既可以被溶原又可以被裂解.溶原的菌株经丝裂霉素诱导后,能释放出感染性的ΦMin27(△stx::cat)颗粒,并对大肠杆菌MC1061进行裂解形成噬菌斑.[结论]ΦMin27(△stx::cat)可以感染和溶原特定的大肠杆菌,且溶原菌株能释放出原感染性的噬菌体,显示出ΦMin27噬菌体具有携带外源基因在若干不同血清型的大肠杆菌间水平转移的能力,为进一步研究Stx噬菌体的感染机理和志贺毒素表达调控奠定了基础.     

Stx2噬菌体溶原对大肠杆菌运动性及其相关基因表达的影响

【目的】通过基因缺失和噬菌体溶原转换研究大肠杆菌运动相关基因flhDC、fliA、fliD和fliE对Stx2噬菌体ΦMin27溶原菌的影响。【方法】本实验利用Red重组酶系统,构建了大肠杆菌MG1655的缺失株MG1655△flhDC、MG1655△fliA、MG1655△fliD及MG1655△fliE,并将flhDC片段、fliA片段、fliD片段和fliE片段连接pUC18后分别转化相应的突变株,得到相应的互补菌株。通过Stx2噬菌体ΦMin27的感染获得各缺失株的溶原株MG1655△flhDCФMin27、MG1655△fliAФMin27、MG1655△fliDФMin27及MG1655△fliEФMin27。随后测定了野生株、缺失株、互补株和溶原株的运动能力,并通过荧光定量PCR分析了flhDC缺失前后野生株和溶原株其他运动相关基因表达量的变化。【结果】Stx2噬菌体ФMin27溶原感染可促进MG1655的fliA和fliD基因的表达,增强宿主菌MG1655的运动特性;MG1655在flhDC基因缺失的状态下,fliA和fliD基因的表达同步出现上调,但运动性未发生变化,而MG1655△flhDC溶原菌丧失了运动特性,基因转录水平检测发现MG1655△flhDCФMin27与MG1655△flhDC相比,fliA和fliD基因的表达同步出现显著下调,而对fliE基因的表达几乎没有影响。fliA、fliD和fliE单个基因的缺失对大肠杆菌MG1655和Stx2噬菌体ΦMin27溶原菌的运动性几乎没有影响。【结论】提示fliA和fliD基因共同参与了鞭毛运动的调控,flhDC基因可影响噬菌体溶原菌株的运动性,为进一步研究噬菌体溶原与宿主基因之间的相互调节作用提供理论依据。     

江苏某地健康绵羊群产志贺毒素大肠杆菌体内分离株的分子流行病学及致病力

【目的】探讨江苏某羊场健康绵羊体内产志贺毒素大肠杆菌的带菌和流行情况,同时就分离株的致病力和对Vero细胞的毒性作用作了研究。【方法】基于本实验室已经建立的EHEC O157:H7 EDL933W株的stx1、stx2、eaeA、hlyA四个基因的多重PCR检测并配合选择性增菌、平板筛选等方法对STEC进行分离鉴定。【结果】在为期6个月的连续跟踪调查中,共分离到STEC菌株107株,分离率为19.4%(107/550)。分离株属于41种O血清型、62种O:H血清型,未定型(ONT)有22株,粗糙型(OR)1株。其中属于绵羊STEC的优势血清型有O5(2株)、O91(1株)、O103(1株)。本文检测到的优势血清型为O93,stx2阳性菌株的分离率较stx1阳性菌株的分离率高,LD50测定结果表明分离株对小鼠致病力不高,受试的3个分离株均不能致小鼠死亡。对107株stx阳性分离株噬菌斑试验表明,71株阳性菌株携带噬菌体(66.3%,71/109)。受试分离株进行Vero细胞毒性试验,其中有一个菌株stx基因阳性但不能使Vero细胞产生病变。【结论】绵羊是STEC的天然宿主,可健康带菌。虽然STEC分离株对小鼠的致病力较弱,但不能排除其对人类安全的威胁。STEC携带志贺毒素基因并不意味着一定表达志贺毒素,需对志贺毒素的表达及调控机理做进一步的研究。     

非致病性大肠埃希菌能增多大肠埃希菌O157:H7志贺毒素的产生

大肠埃希菌(简称大肠杆菌)O157：H7毒力因子为志贺毒素2(stx2),其基因由温和噬菌体编码,由晚期基因启动子调控表达。stx2的合成与释放需要诱导噬菌体溶菌周期,而且正常肠道大肠杆菌感染了毒素编码的噬菌体就能制造毒素和噬菌体,使毒素水平远远超过病原性菌株本身的产量,作者在体外以及鼠肠道验证了这一假设。     

一株强裂解性大肠杆菌T1样噬菌体新成员的分离与鉴定

【目的】自然界中噬菌体种类繁多,其裂菌功能在针对细菌耐药方面具有潜在应用价值。不同噬菌体也呈现出显著的基因多样性及宿主特异性。从上海某猪场仔猪肠内容物样品中分离、纯化大肠杆菌的裂解性噬菌体,分析其生物学特性和病毒学特征,为探索应用噬菌体治疗细菌性感染提供研究材料。【方法】采用双层琼脂平板法分离、纯化噬菌体,观察噬菌斑特征,通过电镜观察噬菌体形态特征,测定其裂菌谱、最佳感染复数、一步生长曲线和生物学特性,进行噬菌体全基因组测序和遗传进化分析。【结果】分离、纯化获得一株能高效裂解大肠杆菌K-12菌株的噬菌体,命名为v B_Eco S_SH2(SH2),噬菌斑呈圆形、大而透明、边缘整齐。电镜观察SH2的头部呈二十面体立体对称,尾部较长。噬菌体的潜伏期为10 min,暴发期为60 min,裂解量高达121 PFU/感染细胞,其最佳感染复数为0.1。基因组测序和比对结果表明,SH2的核酸类型为ds DNA,基因组全长为49 088 bp,G+C%含量为45%,Gen Bank登录号为KY985004,结合电镜观察及BLASTp分析,确定其属于有尾噬菌体目长尾噬菌体科成员。同源性及进化分析表明,该噬菌体为大肠杆菌T1样噬菌体的新成员。【结论】分离鉴定了一株裂解效率极高的大肠杆菌T1样噬菌体,并确认其为T1样噬菌体新成员,为研究大肠杆菌噬菌体及其抗菌应用提供了新的实验材料。     

猪链球菌2型Ⅳ型分泌系统组分VirD4与毒力相关性分析

摘要:【目的】构建猪链球菌2型强毒株05ZYH33毒力岛89 K上的Ⅳ型分泌系统组分VirD4敲除突变株,初步分析其活性和毒力,为进一步研究猪链球菌2 型在逃避宿主天然免疫杀伤中的作用提供基础。【方法】以05ZYH33基因组为模板,PCR扩增VirD4基因上下游同源臂,以穿梭质粒pSET1为模板,PCR扩增氯霉素抗性基因Cm,通过重叠PCR技术搭建上述3个片段并连接至温敏载体pSET4s,构建基因敲除载体pSET4s∷VirD4;通过同源重组构建基因敲除突变株ΔVirD4;通过体外全血杀伤实验、CD1小鼠竞争感染及攻毒实验 对突变株和野生株的毒力进行比较分析。【结果】获得了基因敲除突变株ΔVirD4,通过对比发现其毒力与野生株相比有所降低。【结论】猪链球菌2型Ⅳ型分泌系统组分VirD 与其毒力相关,并在早期抵抗天然免疫细胞杀伤中发挥一定作用。     

产生葡激酶的溶原性转换噬菌体的研究——Ⅱ.由双溶原性菌分离的转换和不能转换噬菌体的比较

我们曾报道了金葡菌66为双溶原菌,先后分离到两株噬菌体即α、β.α具有溶原性转换葡激酶能力,β则无。本文对这两株噬菌体特性作进一步比较,如溶血素的溶原性转换,噬菌斑的形态,溶原菌的免疫性,宿主特异性,血清型别,两株噬菌体DNA的酶切电泳图及溶原化菌株的噬菌体型等,表明菌株66是带有两个不同的亲和群前噬菌体的双溶原菌株。     

实时荧光定量PCR技术鉴别非O157∶H7大肠杆菌产志贺毒素菌株(STEC)的研究

本文运用实时荧光定量PCR的技术对菌株进行stx1基因、stx2基因、eaeA毒力基因检测;并对stx阳性、eaeA阳性的菌株进行O抗原基因rfbE(O157)、wzx(O26)、wbdI(O111)、ihp1(O145)、wzx(O103)检测。探究了实验室保存的94株非O157:H7大肠杆菌是否存在产志贺毒素菌株(STEC)存在;结果表明94株大肠杆菌中检出3株含有stx基因、12株含有eaeA基因;对stx和eaeA阳性菌株O抗原基因试验,检出2株含有wzx(O26)基;这2株大肠杆菌血清凝集试验结果为阳性。研究结果显示,实时荧光定量PCR技术具有特异性强,灵敏度高等特点,可用于产志贺毒素菌株(STEC)前期筛查。     

一株裂解性奶牛乳房炎源大肠杆菌噬菌体的分离鉴定及生物学特性分析

【目的】从新疆石河子地区奶牛粪样中分离裂解性大肠杆菌噬菌体(Escherichia coli phage),对其进行纯化及生物学特性分析。【方法】利用双层平板法从奶牛粪样中分离、纯化噬菌体,将纯化后的噬菌体浓缩液用醋酸双氧铀负染后通过透射电子显微镜观察其形态特征。对该噬菌体进行全基因组测序和遗传进化分析,同时测定噬菌体的宿主谱、最佳感染复数、一步生长曲线、热稳定性及酸碱稳定性。【结果】分离并纯化出一株裂解性噬菌体vB_EcoM_XJ2,噬菌斑圆形不透明,直径0.7 mm–1.2 mm;电镜显示其头部呈正多面体对称,有可伸缩性尾部;核酸类型为双链DNA,基因组大小为75.617 kb,G+C%含量为42.09%;其核酸序列与大肠杆菌噬菌体NJ01和vB_EcoP_SU10相似性高达94%。生物学特性研究显示该噬菌体能裂解多株临床分离的大肠杆菌;能耐受60°C左右高温,在pH 5.0–11.0范围内效价稳定;最佳感染复数为0.1,潜伏期为15 min,暴发期为95 min,裂解量约为10.6 PFU/cell。【结论】vB_EcoM_XJ2是一株在不同温度、不同酸碱性环境中有较强适应能力的裂解性肌尾科大肠杆菌噬菌体。     

高效广谱猪链球菌7型前噬菌体裂解酶的挖掘及活性研究

【目的】噬菌体具有防控耐药性病原菌的抗菌应用潜力,但是有些病原菌噬菌体的获得非常困难,研究发现大多数病原菌存在前噬菌体(prophage),且由前噬菌体编码的裂解酶(endolysin)具有良好的抗菌应用前景,本研究将挖掘猪链球菌前噬菌体及其编码的裂解酶。【方法】通过对GenBank中登录的数株猪链球菌前噬菌体裂解酶的基因信息分析,挖掘出一株猪链球菌7型菌株前噬菌体编码的裂解酶,研究其生物学活性。以猪链球菌7型菌株7917的基因组为模板,采用PCR技术扩增获得裂解酶基因ly7917,将其克隆至质粒pET28a(+)并转化大肠杆菌DH5α细胞,挑选基因序列正确的阳性克隆、抽提质粒、转化表达菌株大肠杆菌BL21,经IPTG诱导可高效表达裂解酶Ly7917。【结果】平板裂解试验结果显示Ly7917具有高效裂菌活性,能够裂解猪链球菌2型高致病力菌株HA9801;浊度递减试验结果显示该裂解酶能够高效裂解猪链球菌2型、7型、9型和马链球菌兽疫亚种参考株、金黄色葡萄球菌(包括耐甲氧西林金黄色葡萄球菌)等多种革兰氏阳性菌。【结论】基于前噬菌体挖掘的裂解酶Ly7917,具有高效广谱裂菌活性,为临床上利用裂解酶治疗耐药菌的混合感染提供了可能。     

Stereospecific 2'-amination and 2'-chlorination of adenosine by Actinomadura in the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin

2'-Amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin (2'-CldCF) are two nucleoside antibiotics produced by Actinomadura. The biosynthesis of these two nucleoside antibiotics has been studied by the addition of [U-14C]adenosine with or without unlabeled adenine to cultures of Actinomadura. By this experimental approach, it is possible to demonstrate that adenosine is the direct precursor for the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-CldCF. These conclusions are based on the observation that the percentage distribution of 14C in the aglyconic and pentofuranosyl moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were similar to the distribution of 14C in the adenine and ribosyl moieties of the [U-14C]adenosine (i.e., 48:52) added to cultures of Actinomadura. Experimentally, the percentage distribution of 14C in the (i) adenine:2-amino-2-deoxy-beta-D-ribofuranose of 2'-amino-2'-deoxyadenosine is 51:49; (ii) 8-(R)-3,6,7,8-tetrahydroimidazo[4,5-d]-[1,3-diazepin-8-o1]:2 -chloro-2- beta-D-ribofuranose of 2'-CldCF is 45:55; and (iii) adenine:ribose of the adenosine isolated from the RNA of Actinomadura is 42:58. Further proof that adenosine is the direct precursor for the biosynthesis 2'-amino-2'-deoxyadenosine and 2'-CldCF was demonstrated by the addition of 75 mumol of unlabeled adenine together with [U-14C]adenosine to nucleoside-producing cultures of Actinomadura. The percentage distribution of 14C in the aglycon and the sugar moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were 46:54 and 47:53, respectively; the percentage distribution of 14C in the adenine and ribose moieties of the adenosine isolated from the RNA of Actinomadura was 51:49. These data show that the hydroxyl on C-2' of the ribosyl moiety of adenosine undergoes a replacement by a 2'-amino or a 2'-chloro group to form 2'-amino-2'-deoxyadenosine or 2'-CldCF with retention of stereconfiguration at C-2'. Finally, Actinomadura can utilize inorganic chloride from the medium as demonstrated by the isolation of [36Cl]2'-CldCF following the addition of [36Cl]chloride to the culture medium. Mechanisms for the regioselective modification of the C-2' hydroxyl group and stereospecific insertion of the amino and chloro groups are discussed.     

Preparation of glycosides of 2-deoxy-2-methylamino-D-mannose, 2-deoxy-2-methylamino-D-glucose,and 2-deoxy-2-methylamino-D-galactose: observations on N-methylation of amides

Benzyl 2-[(benzyloxycarbonyl)methylamino]-2-deoxy-α-D-mannopyranoside (10) and its furanose isomer (9), the derived N-methyloxazolidinones 11 and 6, benzyl 2-[(benzyloxycarbonyl)methylamino]-2-deoxy-β-D-glucofuranoside (15) and methyl 2-deoxy-2-methylacetamido-β-D-galactofuranoside (20), were prepared from appropriate diethyl dithioacetals. They were considered the most suitable starting materials for synthesis of O-methyl-2-deoxy-2-methylamino-hexoses because of their ease of preparation and the presence of suitable blocking groups. Oxazolidinones were prepared from N-benzyloxycarbonyl derivatives of 2-amino-2-deoxy-D-mannose by using methanolic sodium methoxide. Their use in preparation of 2-deoxy-2-methyl-amino derivatives is discussed. The Kuhn reagent was used in these syntheses for N-methylating amides. However, certain amides containing comparatively bulky substituents in the vicinity of the NH group are resistant to methylation.     

Synthesis and physicochemical properties of 2'-deoxy-2',2'-difluoro-beta-D-ribofuranosyl and 2'-deoxy-2',2'-difluoro-alpha-D-ribofuranosyl oligonucleotides

We present procedures for nucleoside and oligonucleotide synthesis, binding affinity (Tm) and structural analysis (CD spectra) of 2'-deoxy-2',2'-difluoro-alpha-D-ribofuranosyl and 2'-deoxy-2',2'-difluoro-beta-D-ribofuranosyl oligothymidylates. Possible reasons for the thermal instability of duplexes formed between these compounds and RNA or DNA targets are discussed.     

Convenient synthesis of 2'-deoxy-2-fluoroadenosine from 2-fluoroadenine

A convenient synthesis of 2'-deoxy-2-fluoroadenosine from commercially available 2-fluoroadenine is described. The coupling reaction of silylated 2-fluoroadenine with phenyl 3,5-bis[O-(t-butyldimethylsilyl)]-2-deoxy-1-thio-D-erythro-pentofuranoside gave the corresponding 2-fluoro-2'-deoxyadenosine derivative (alpha/beta = 1:1) in good yield. The alpha- and beta-anomers were separated by chromatography, and then desilylated to give compounds 1a and 1b.     

Stereoselective Synthesis of 2-Amino-2-deoxy-d-arabinose and 2-Deoxy-d-ribose

An efficient method for the stereoselective synthesis of 2-amino-2-deoxy-d-arabinose and 2-deoxy-d-ribose is described.

The key step in this method was accomplished by the nucleophilic addition of methyl isocyanoacetate to 2,3-O-isopropylidene-d-glyceraldehyde with high erythro-selectivity (nearly 100%).

Subsequent intermolecular cyclization predominantly gave the desired oxazoline derivative (trans-form), in which two new chiral centers were formed. The oxazoline derivative was efficiently converted to both 2-amino-2-deoxy-d-arabinose and 2-deoxy-d-ribose.     

Stereospecific formation of alpha-hydroxycarboxylato oxo peroxo complexes of vanadium(V). Crystal structure of (NBu4)2[V2O2(O2)2(L-lact)2] x 2H2O and (NBu4)2[V2O2(O2)2(D-lact)(L-lact)] x 2H2O

An overview of structurally characterized alpha-hydroxycarboxylatodioxo- and alpha-hydroxycarboxylatooxoperoxovanadates(V) is presented and the geometric parameters of the V2O2 bridging core are discussed. The first case of a stereospecific formation of oxoperoxovanadates(V) is reported: The crystal structures of the isomeric compounds (NBu4)2[V2O2(O2)2(L-lact)2] x 2H2O and (NBu4)2[V2O2(O2)2(D-lact)(L-lact)] x 2H2O (lact = C3H4O3(2-), the anion of the lactic acid) differ mainly in the arrangement of the V2O2 core and in mutual orientation of the V=O bonds. The complexes with achiral ligands adopt the same structural type as the complexes formed from a racemic mixture of a chiral ligand, while the structure obtained using an enantiopure L,L-hydroxycarboxylate is different.     

Characterization of alpha 2 beta 2 and alpha 2 forms of kinesin

Bovine brain kinesin separates into two components on sucrose density gradient centrifugation. The predominant component is a heterotetramer of two 120 kDa alpha subunits and two 64 kDa beta subunits with an sedimentation coefficient of 9.6 S and a low Vm rate of microtubule-stimulated ATPase of 1.3 +/- 0.5 sec-1 at 25 degrees, pH 7.0. The minor element is a homodimer of two alpha subunits without beta subunits with a sedimentation coefficient of 6.9 S and a higher Vm rate of microtubule-stimulated ATPase of 7.0 +/- 1.9 sec-1. Microtubules stimulate the rate of release of ADP from the active site of the tetramer, but the rate of release is not fast enough to account for the rate of steady state ATP hydrolysis. Further complexity is indicated by biphasic release kinetics. In spite of the large difference in Vm ATPase rate for the two species, both drive the sliding of sea urchin axonemes over glass surfaces at the same velocity.