L-乳酸脱氢酶在大肠杆菌BL-21(DE3)中的表达

为了在大肠杆菌中构建利用葡萄糖生产L-乳酸的途径,以鼠李糖乳杆菌(Lactobacillus rhamnosus)LA - 04 -01基因组为模板,设计引物扩增L-乳酸脱氢酶基因L-ldh.将该基因连接到表达栽体pET-28a(+)上,并转化大肠杆菌Top10.通过卡那霉素抗性平板筛选,提取重组质粒pET28a-L-ldh并测序,结果正确.将pET28a-L-ldh转化大肠杆菌BL-21( DE3),通过卡那霉素抗性平板筛选,得到产乳酸的大肠杆菌基因工程菌.经IPTG诱导后,SDS-PAGE电泳,检测到目的蛋白条带,L-乳酸脱氢酶比酶活力达到9.44 U/mL.该基因工程菌通过摇瓶发酵,L-乳酸产量达到3 g/L,成功构建出一条在大肠杆菌中生产L-乳酸的新途径.     

肉类中大肠杆菌O157:H7多重PCR检测方法的建立

以编码大肠杆菌O157抗原的rfbE基因、 编码H7抗原的fliC基因以及编码毒力因子的eaeA基因为靶基因, 选择3对引物, 建立并优化了检测大肠杆菌O157:H7的多重PCR体系, 扩增产物分别为291 bp、625 bp、368 bp, 采用30株细菌验证了该多重PCR具有特异性。PCR检测的灵敏度在DNA水平上达到91.35 pg; 在存在干扰菌鼠伤寒沙门氏菌(Salmonella?typhimurium)的情况下, 当起始污染量为1.4 CFU/mL时, 37 ℃培养6 h 即可检出。在30份肉类样品中, 有3份检出了大肠杆菌O157:H7。本研究建立的多重PCR方法可特异、灵敏地实现对大肠杆菌O157:H7的检测。     

大肠杆菌O157:H7致病因子及其保护性免疫研究现状

大肠杆菌O157:H7是肠出血型大肠杆菌(EHEC)的主要病原血清型,它可产生特殊的粘附因子粘附靶细胞,产生Vero毒素和肠溶血素毒力因子.近几年对大肠杆菌O157:H7的毒力因子有了深入的了解,对致病机理作了一些探讨,用实验动物对保护性免疫进行了研究.本文对近几年来0157:H7大肠杆菌的致病因子及其主要的保护性免疫的研究作一简要综述.     

大肠杆菌O150 O-抗原基因簇的破译和dTDP-鼠李糖合成酶基因的鉴定

利用鸟枪法对大肠杆菌O150 O-抗原基因簇进行测序,序列全长13551bp,用生物信息学的方法进行序列分析,共发现11个基因,分别为鼠李糖合成酶基因(rmlB、rmlD、rmlA、rmlC)糖基转移酶基因(3个)、O-抗原转运酶基因(wzx)和O-抗原聚合酶基因(wzy),另外还有两个基因功能未知。用PCR的方法筛选出了针对大肠杆菌O150的特异基因,可以用于基因芯片或PCR方法对大肠杆菌O150的快速检测。另外,通过进化分析发现大肠杆菌O150的O-抗原基因簇中携带有典型的大肠杆菌鼠李糖合成酶基因,并且这些基因参与了O-抗原基因簇间的重组以形成新的基因簇的过程。     

大肠杆菌BL21(DE3)膜组分相关基因的敲除对重组蛋白胞外分泌的影响

摘要:【目的】探究Escherichia coli BL21(DE3)中膜组分相关的脂多糖合成基因waaF或msbB的敲除对重组蛋白胞外分泌的影响。【方法】运用Red重组技术将E.coli BL21 (DE3)染色体上的基因waaF或msbB敲除,构建敲除菌株E.coli BL21(ΔwaaF)、E.coli BL21(ΔmsbB)。将本实验室保存的带有β-呋喃果糖苷酶(β-fructofuranosidase,β-FFase)、青霉素G 酰化酶(penicillin G acylase,PGA)基因的重组质粒pET-ffase、pET-pga分别转入敲除菌株及出发菌株中,构建工程菌株E.coli BL21(ΔmsbB)/pET-ffase、E.coli BL21(ΔwaaF)/pET-ffase、E.coli BL21(DE3)/pET-ffase、E.coli BL21(ΔmsbB)/pET-pga、E.coli BL21(ΔwaaF)/pET-pga、E.coli BL21(DE3)/pET-pga。最后通过摇瓶发酵研究敲除菌株对β-FFase、PGA胞外分泌的影响。【结果】当诱导表达4 h,以出发菌株E.coli BL21(DE3)为宿主时,β-呋喃果糖苷酶β-FFase的胞外分泌量占总表达量的2.6%,以敲除菌株ΔmsbB为宿主时,胞外分泌量达到19.7%,而以敲除菌株ΔwaaF为宿主时,胞外分泌量达到50.9%。另外,当诱导表达24 h,以敲除菌株ΔwaaF为宿主时,青霉素G酰化酶PGA的胞外酶活是出发菌株中的4.1倍,达到1708 U/L。【结论】本研究成功构建了敲除菌株ΔmsbB和ΔwaaF,ΔmsbB能明显增强β-FFase的胞外分泌,而ΔwaaF对β-FFase和PGA的胞外分泌均有显著的强化作用。     

检测大肠杆菌(Escherichia coli) O157:H7免疫磁珠的制备、保存与应用

【背景】大肠杆菌(Escherichia coli) O157:H7是导致肠出血性大肠杆菌食源性疾病暴发的主要血清型,免疫磁珠(Immunomagnetic beads,IMBS)在E. coli O157的检测中发挥着重要作用,而免疫磁珠的稳定性、特异性、广谱性等性能指标关系着在实际应用中的使用效果。【目的】制备高效、稳定且具有广谱性的免疫磁珠,联合分子检测技术如环介导恒温扩增 (Loop-mediated isothermal amplification,LAMP)技术、PCR等,提高目标菌的检出率。【方法】采用新型的磁珠活化剂MIX&GO制备E. coli O157免疫磁珠,并进行广谱性以及特异性检测;针对6种试剂牛血清白蛋白(Bovine serum albumin,BSA)、酪蛋白(Casein)、海藻糖(Trehalose)、聚乙烯吡咯烷酮(Polyvinyl pyrrolidone,PVP)、抗坏血酸(Vitamin C)和防腐剂ProClin 300,利用正交试验L18(37)优化免疫磁珠保存液组分;采用IMBS-LAMP、IMBS-PCR、IMBS-生化、菌液-LAMP、菌液-PCR、显色平板-生化鉴定6种方式对20份生猪肉样品进行检测。【结果】利用MIX&GO活化剂制备的免疫磁珠捕获率最高达到81.5%±1.3%;免疫磁珠保存液最优配方为：牛血清白蛋白15.0 g/L,酪蛋白10.0 g/L,海藻糖10.0 g/L,PVP 2.0 g/L,抗坏血酸5.0 g/L,ProClin 300 2.5 g/L,保存6个月后免疫磁珠捕获率为75.5%;在20份生猪肉样品的检测中,自制磁珠和商品化磁珠与LAMP联用均检出9例阳性样品;IMBS-LAMP在6种检测方式中具有最高的检测灵敏度,但检出的样品会因磁珠抗体的差异而有所不同。【结论】与商品化磁珠相比,实验制备的免疫磁珠具有良好的特异性和广谱性,免疫磁珠-LAMP联用提高了目标菌的检出率,是一种高灵敏度、具有应用前景的检测方法。     

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。     

大肠杆菌BL21(pTrc-gsh)与酵母耦联合成谷胱甘肽的研究

谷胱甘肽 (ＧＳＨ)广泛存在于动、植物和微生物细胞内 ,有参与氨基酸的跨膜运输、维持细胞的还原状态等重要生理功能 ,在临床、保健品、食品等行业有广泛用途 ,如 :重金属解毒、抗氧化延缓衰老等 ,我国基本靠进口。开发高效、低成本的ＧＳＨ生产工艺势在必行。谷胱甘肽的制备有化学合成法[1 ] 、提取法[2 ] 、微生物发酵法[3] 、酶法[4] 等。由于酶法合成ＧＳＨ的产率高、后续的分离提取较简单而倍受关注。它是以ＡＴＰ为能量供体、由γ 谷氨酰半胱氨酸合成酶 (ＧＳＨＩ)和谷胱甘肽合成酶 (ＧＳＨＩＩ)连续催化合成的 :谷氨酸 半胱氨酸 Ａ…     

肠出血性大肠杆菌(O157：H7)的特异性荧光探针检测方法的建立

目的:建立一种real-time PCR,快速准确检测肠出血性大肠杆菌O157:H7。方法:以肠出血性大肠杆菌0157:H7 rfbE为待检靶基因,设计一对引物和一条Taqman探针,探针5’端用FAM基团标记,3’端用TAMRA标记。通过重组质粒的构建,建立并优化了大肠杆菌0157:H7的荧光定量PCR检测方法。结果:在人工污染样本无需富集的情况下,检测的最低DNA浓度是10拷贝/反应(3CFU/mL);特异性检测实验中,0157菌株检测结果均为rfbE阳性,而非0157:H7菌株检测结果均为阴性;重复性实验中,批内、批间变异系数均小于3%。结论:实验结果显示此荧光定量PCR方法特异性、灵敏度高,重复性好,可对分离的可疑大肠杆菌0157:H7菌株进行快速鉴定。     

糖丁基梭菌产丁醇途径在大肠杆菌中的构建及发酵

摘要:【目的】通过克隆来源于糖丁基梭菌(Clostridium saccharobutylicum DSM13864)丁醇合成途径的关键酶基因(thlA,bcs-operon和adhE),构建产丁醇大肠杆菌。【方法】以Clostridium saccharobutylicum DSM13864的基因组为模板,分别扩增丁醇途径关键酶基因thlA,bcs-operon(crt-bcd1-etfB2-fixB2-hbd)和adhE,构建了两个重组质粒pETDuet-bcs和pRSFDuet-thlA-adhE,并成功转入E.coli JM109(DE3)实现异源表达,使大肠杆菌具备产丁醇能力。在半厌氧条件下进行重组菌的发酵,并研究不同培养基对产丁醇的影响。【结果】该重组菌在半厌氧条件下经摇瓶发酵丁醇产量达到25.4 mg/L,通过优化培养基后,在TB发酵培养基中丁醇产量可达到34.1 mg/L。【结论】通过构建重组共表达质粒,将糖丁基梭菌来源的丁醇途径关键酶基因在大肠杆菌中表达,成功构建产丁醇大肠杆菌。该研究提供了一株易于操作的丁醇发酵重组大肠杆菌,避免了传统梭菌发酵丁醇生产中苛刻的厌氧条件、易产孢子等限制问题。     

Penicillin-binding proteins of pathogenic Escherichia coli strains

The penicillin-binding proteins of 11 pathogenic Escherichia coli strains, including enteropathogenic, enterotoxigenic, enteroinvasive, enteroaggregative, and enterohemorrhagic E. coli, were detected in gels following the labeling of isolated cell envelopes with [³H]benzylpenicillin. The electrophoretic profiles, sensitivities to and morphological changes induced by β-lactam antibiotics showed that the penicillin-binding proteins of most pathogenic E. coli possess structural and physiological functions similar to those of E. coli K12.     

Allelic subtyping of the intimin locus (eae) of pathogenic Escherichia coli by fluorescent RFLP

Intimin is a highly polymorphic protein encoded by the eae gene and plays a crucial role in the attaching-effacing phenotype of diarrheagenic Escherichia coli and related pathogens. We have developed a method to quickly and accurately uncover allelic variation at the eae locus through the use of fluorescent RFLP (fRFLP). Application of fRFLP to 151 eae -positive strains (including the newly described Escherichia albertii ) revealed 26 different fRFLP types that correspond to 20 of the 28 previously described eae alleles. Two sequence variants of the γ, ι, κ, and ζ alleles and three variants of ɛ were also observed. In addition to being reliable and accurate, the method can be easily adapted to accommodate new eae allelic sequences, as they become known.     

3种蒙药复方对致病性Escherichia coli O1的抑菌机理及对小鼠的保护率

【目的】研究3种蒙药复方对致病性Escherichia coli O1的抑菌机理及对感染E.coli O1小鼠的保护率。【方法】分别采用酶标比浊法、微生物粘附法、试剂盒法和邻硝基苯β-D-半乳吡喃糖苷(ONPG)法测定3种蒙药复方对E.coli O1生长曲线、细胞表面疏水性、细胞壁及细胞膜渗透性的影响。同时,采用人工感染小鼠法测定其对小鼠的保护率。【结果】3种蒙药复方均能不同程度地抑制致病性E.coli O1,且对人工感染E.coli O1的小鼠有保护作用,其中蒙药复方Ⅲ的保护率与环丙沙星相当,均达到50%;7味单药组成的蒙药复方Ⅲ水煎剂作用于E.coli O1细胞时,首先改变其表面疏水性,其次随作用时间的延长破坏其细胞壁的完整性,进而破坏细胞膜并改变其通透性,最终导致细胞结构的破坏,从而起到抑制作用。【结论】蒙药复方Ⅲ的体内外抑菌效果最佳,其效果与抗生素环丙沙星相当。     

Characterization of class 1 integrons-mediated antibiotic resistance among calf pathogenic Escherichia coli

Escherichia coli isolates from calf diarrhea cases (n=22) in the Beijing surrounding region in China were characterized for disease serotype, virulence factors, antimicrobial susceptibility pattern and class 1 integrons. 59% (n=13) of the isolates were positive for the int I1 gene. The presence and genetic content of class 1 integrons in 13 E. coli isolates were examined by PCR and sequencing. Sequencing analysis revealed six gene cassettes, which encoded resistance to trimethoprim (dfrA1, dfrA17), aminoglycosides (aadB, aadA1 and aadA5) and chloramphenicol (cmlA). The gene cassette arrays dfrA1-orf (45%) and aadB-orf-cmlA (32%) were most prevalent among these isolates. These data revealed the high prevalence of class 1 integrons among calf pathogenic E. coli isolates in the Beijing surrounding region in China, which may provide important and useful surveillance information reflecting specific antibiotic selective pressure.     

Ribosomal protein limitations in Escherichia coli under conditions of high translational activity

Details of the mechanism for ribosome synthesis have been incorporated in the single-cell Escherichia coli model, which enable us to predict the amount of protein synthesizing machinery under different environmental conditions. The predictions agree quite well with available experimental data. The model predicts that ribosomal protein limitations are important when the translational apparatus is in high demand. Ribosomal RNA synthesis is induced by an increase in translational activity, which, in turn, stimulates ribosomal protein synthesis. However, as the demand increases still more, the ribosomal protein mRNA must compete with the plasmid mRNA for ribosomes, and the efficiency of translation of ribosomal proteins is reduced. (c) 1994 John Wiley & Sons, Inc.     

Genetic and structural analyses of Escherichia coli O107 and O117 O-antigens

The O-antigen, consisting of many repeats of an oligosaccharide, is an essential component of the lipopolysaccharide on the surface of Gram-negative bacteria. The O-antigen is one of the most variable cell constituents, and different O-antigen forms are almost entirely due to genetic variations in O-antigen gene clusters. In this paper, we present structural and genetic evidence for a close relationship between Escherichia coli O107 and E. coli O117 O antigens. The O-antigen of E. coli O107 has a pentasaccharide repeating unit with the following structure: →4)-β- d -Gal p NAc-(1→3)-α- l -Rha p -(1→4)-α- d -Glc p NAc-(1→4)-β- d -Gal p -(1→3)-α- d -Gal p NAc-(1→, which differs from the known repeating unit of E. coli O117 only in the substitution of d -GlcNAc for d -Glc. The O-antigen gene clusters of E. coli O107 and O117 share 98.6% overall DNA identity and contain the same set of genes in the same organization. It is proposed that one cluster was evolved from another via mutations, and the substitution of a few amino acids residues in predicted glycosyltransferases resulted in the functional change of one such protein for transferring different sugars in O107 ( d -GlcNAc) and O117 ( d -Glc), leading to different O-antigen structures. This is an example of the O-antigen alteration caused by nucleotide mutations, which is less commonly reported for O-antigen variations.     

Lethality of hydrogen peroxide in wild type and superoxide dismutase mutants of Escherichia coli. (A hypothesis on the mechanism of H2O2-induced inactivation of Escherichia coli)

The toxicity of H₂O₂ in Escherichia coli wild type and superoxide dismutase mutants was investigated under different experimental conditions. Cells were either grown aerobically, and then treated in M9 salts or K medium, or grown anoxically, and then treated in K medium. Results have demonstrated that the wild type and superoxide dismutase mutants display a markedly different sensitivity to both modes of lethality produced by H₂O₂ (i.e. mode one killing, which is produced by concentrations of H₂O₂ lower than 5 mM, and mode two killing which results from the insult generated by concentrations of H₂O₂ higher than 10 mM). Although the data obtained do not clarify the molecular basis of H₂O₂ toxicity and/or do not explain the specific function of superoxide ions in H₂O₂-induced bacterial inactivation, they certainly demonstrate that the latter species plays a key role in both modes of H₂O₂ lethality. A mechanism of H₂O₂ toxicity in E. coli is proposed, involving the action of a hypothetical enzyme which should work as an O₂^-• generating system. This enzyme should be active at low concentrations of H₂O₂ (<5 mM) and high concentrations of the oxidant (>5 mM) should inactivate the same enzyme. Superoxide ions would then be produced and result in mode one lethality. The resistance at intermediate H₂O₂ concentrations may be dependent on the inactivation of such enzyme with no superoxide ions being produced at levels of H₂O₂ in the range 5–10 mM. Mode two killing could be produced by the hydroxyl radical in concert with superoxide ions, chemically produced via the reaction of high concentrations of H₂O₂ (>10 mM) with hydroxyl radicals. The rate of hydroxyl radical production may be increased by the higher availability of Fe²⁺ since superoxide ions may also reduce trivalent iron to the divalent form.     

Inducible stx2 phages are lysogenized in the enteroaggregative and other phenotypic Escherichia coli O86:HNM isolated from patients

We characterized two Shiga toxin-producing Escherichia coli (STEC) O86:HNM isolates from a patient with hemolytic uremic syndrome (HUS) or bloody diarrhea. Both of them did not possess the eaeA gene. However, the isolate from a HUS patient carried genetic markers of enteroaggregative E. coli (EAEC) and showed aggregative adherence pattern to HEp-2 cells. The other isolate from bloody diarrhea, which was negative with EAEC markers, was diffusely adhered to HEp-2 cells. The stx2 gene in both E. coli O86:HNM strains was encoded in each infectious phage, which was partially homologous to that of strain EDL933, a STEC O157:H7. These results will help to explain the genotypic divergences of STEC.