Regulation of expression of Escherichia coli pilus K99.

An immunoassay demonstrated that the assembled K99 pilus on the surface of Escherichia coli grown in minimal medium appeared during the logarithmic phase of growth, but the synthesis of K99 subunits, as measured by nonequilibrium two-dimensional gel electrophoresis, occurred throughout the life cycle of the cell. Contrary to other reports, the addition of glucose to the growth medium did not affect K99 pilus assembly or subunit synthesis, although in a K99+ adenyl cyclase (cya) mutant, subunit synthesis was reduced. There was no reduction in the amount of assembled K99 on the cell surface of the cya mutant compared with the wild-type parent. The addition of L-alanine to minimal medium repressed K99 synthesis. However, if L-threonine or L-isoleucine was also included in the growth medium, the effect of L-alanine was reduced. Chloramphenicol caused a complete inhibition of K99 subunit synthesis, but assembly proceeded normally. Growth at 18 degrees C inhibited both subunit synthesis and pilus assembly. Approximately 92% of all cellular K99 was associated with the outer membrane, and 4% was associated with the inner membrane. No K99 was detected in the cytoplasm.     

人mBAFF在原核系统的可溶性表达及功能鉴定

目的利用含重组质粒pQE80L-mBAFF的DH5α大肠杆菌，优化表达B细胞活化因子的突变蛋白（mBAFF）。方法研究mBAFF的体外诱导条件，包括诱导时间、IPTG浓度、诱导温度对表达量的影响。对表达条件进行优化后进行大量表达，经Ni^2＋ NTA亲和层析和SepharcryL S200凝胶过滤层析纯化。流式检测突变蛋白与细胞结合的能力以及MTT检测突变蛋白的生物学活性。结果最佳表达条件是诱导时间4h，诱导温度37℃，IPTG终浓度0．2mmol／L。mBAFF占菌体总蛋白的35％，蛋白纯度达98％以上，所获突变蛋白能与淋巴细胞表面受体结合，但不能刺激淋巴细胞增殖。结论优化可溶性表达后能提高mBAFF蛋白质在DH5α大肠杆菌表达，并经Ni^2＋ NTA亲和层析和Sephareryl S200凝胶过滤层析得到纯度高的表达产物，所获结果为进一步研究创造了条件。     

人CD20胞外区基因在原核系统中的表达

目的 将ＣＤ２０胞外区在大肠杆菌中表达,并研究表达产物作为抗原筛选抗体的可能性。方法 设计引物从ＰＵＣ１９／ＣＤ２０质粒上钓取取ＣＤ２０胞外区基因,克隆到ＰＥＸＳＥ１载体上,在大肠杆功各融合表达。结果 表达产物的相对分子建立了检测ＣＤ２０ｍＡｂ的ＥＬＩＳＡ方法。结论 为研制ＣＤ２０分子的ｍＲｂ奠定了基础。     

人GLIPR-2基因的克隆及在原核中可溶性表达

目的构建pQE80L-GLIPR-2载体,并检测融合蛋白RGS·His-GLIPR-2在原核中的表达水平。方法采用RTPCR方法,将克隆人GLIPR-2(glioma pathogenesis related-2,GLIPR-2)基因全长编码区的cDNA连接到原核表达载体pQE80L中,经IPTG诱导原核表达,通过Western blot检测融合蛋白RGS·His-GLIPR-2的表达水平。超声破壁后,通过SDS-PAGE电泳分析表达产物的表达形式。结果成功构建pQE80L-GLIPR-2载体;经IPTG的诱导,GLIPR-2可与RGS·His以融合蛋白的形式高效表达。经Western blot鉴定,pQE80L-GLIPR-2在原核内表达产物相对分子质量为18000,与预期融合蛋白大小一致。对表达产物可溶性分析表明,表达蛋白在上清液中和包涵体中均有表达。结论成功构建的重组质粒能够在大肠杆菌中高效表达可溶性目的蛋白,为进一步研究GLIPR-2基因的功能和意义奠定了基础。     

Regulation of L-carnitine metabolism in Escherichia coli

The metabolization of L-carnitine was studied using whole cells of Escherichia coli 044 K74. It showed features of an epigenetical control. L-carnitine and crotonobetaine were able to induce the carnitine-reducing system. Oxygen and nitrate as electron acceptors, gamma-butyrobetaine as final product of carnitine transformation in E. coli as well as glucose repress the carnitine metabolization. Other betaines and structurally related compounds did not show any effect neither as inductors nor as repressors.     

人PD-L2基因克隆及其在大肠杆菌中的表达

目的 克隆人PD-L2基因并构建PD-L2胞外区的原核表达载体，在大肠杆菌中进行表达。方法 以RT-PCR方法从活化的人外周血单个核细胞总RNA中克隆PD-L2基因的cDNA，构建PD-L2胞外区的原核表达载体，在大肠杆菌BL21(ED3)中进行表达并鉴定。结果 克隆到PD-L2基因cDNA编码区全长序列，经DNA测序证明其与已报道的序列一致。进而构建了PD-L2胞外区的原核表达载体，并在大肠杆菌表达，免疫印迹分析表明在IPTG诱导后表达PD-L2胞外区蛋白，相对分子质量Mr为22000，与理论值大小相符。结论 成功克隆PD-L2基因，其胞外区蛋白在大肠杆菌中获得表达，为进一步研究PD-L2功能提供了条件。     

人类神经型一氧化氮合酶原核表达载体的构建和在大肠杆菌中的表达（英文）

目的:构建在大肠杆菌中表达重组人神经型一氧化氮合酶的高表达系统。方法:用PCR方法从cDNA中扩增目的编码基因,然后将编码基因连接入表达载体pCWori+,将重组的质粒转染入大肠杆菌BL21进行蛋白高表达,经Western blot确认表达后,进行大量培养,通过层析方法精制此酶,并用吸收光谱法对酶的性质进行测定。结果:从该表达系统中可以获得3 mg/L培养基的高产量的一氧化氮合酶。结论:从该表达系统中可获得大量有活性的人一氧化氮合酶。     

Cloning, sequencing, and expression of the Escherichia coli cytolethal distending toxin genes.

A limited number of Escherichia coli isolates which produce an apparently novel toxin, termed cytolethal distending toxin (CDT), have been reported. The toxic activity produced by these strains causes certain cultured cell lines to become slowly distended and then disintegrate. DNA was isolated from the CDT-producing E. coli strain, 9142-88, and cloned into a cosmid vector. Plasmid DNA from a toxin-positive transductant was further subcloned until a plasmid with a 4-kb insert which still encoded the toxin activity was obtained. Nucleotide sequencing of a portion of this insert revealed the presence of three adjacent open reading frames. Further subcloning and deletion analysis suggested that the products of all three open reading frames may be required for toxin activity. Minicell experiments identified the products of all three open reading frames. The three proteins had predicted sizes of 27,753,29,531, and 19,938 Da, and all three appeared to have strong consensus leader sequences. None of the three predicted proteins had significant homology to known proteins.     

Expression of a human IgG4 antibody, BAB2, with specificity for the major Birch pollen allergen, Bet v 1 in Escherichia coli: recombinant BAB2 Fabs enhance the allergic reaction

BACKGROUND: Antigen recognition by antibodies of different isotypes can result in completely different effects as exemplified by Type I allergy. While the IgE-antibody-mediated release of biological mediators constitutes the immunopathological basis for the immediate symptoms observed in allergic patients, allergen-specific IgG antibodies are thought to have protective effects. METHODS: Cell lines secreting five human monoclonal IgG antibodies (BAB1-BAB5) with specificity for the major birch pollen allergen Bet v 1 were established from a birch-pollen-allergic patient who had received birch- pollen-specific immunotherapy. The influence of the Bet v 1-specific IgG antibodies on IgE binding to Bet v 1 was investigated. BAB2 was expressed in Escherichia coli as recombinant Fab, purified and tested for its ability to modulate Bet v 1-induced immediate-type skin reactions. RESULTS: The BAB antibodies belonged to different IgG subclasses (BAB1: IgG1; BAB2, BAB3, BAB5: IgG4; and BAB4: IgG2) reflecting a tendency towards Th2. BAB1 represented the only antibody which strongly blocked IgE binding to Bet v 1, whereas BAB 3-BAB5 had little effect on IgE binding. Surprisingly, natural BAB2 antibodies as well as recombinant BAB2 Fabs strongly enhanced IgE binding to Bet v 1 and Bet v 1-induced immediate-type skin reactions and thus represent 'enhancing antibodies'. CONCLUSION: The demonstration that anti-allergen IgG antibodies can also enhance IgE binding to a given allergen explains the unpredictability of specific immunotherapy as well as the controversy on the role of IgG in atopy.     

Regulation of the Shiga-like toxin II operon in Escherichia coli.

Investigations of the regulation of the bacteriophage-encoded Shiga-like toxin II (SLT-II) in Escherichia coli demonstrated that bacteriophages exhibit a regulatory impact on toxin production by two mechanisms. Firstly, replication of the toxin-converting bacteriophages brings about an increase in toxin production due to concomitant multiplication of toxin gene copies. Secondly, an influence of a phage-encoded regulatory molecule was demonstrated by using low-copy-number plasmid pADR-28, carrying a translational gene fusion between the promoter and proximal portion of slt-IIA and the structural gene for bacterial alkaline phosphatase (phoA). PhoA activity, reflecting the slt-II promoter activity, was significantly enhanced in E. coli strains which and been lysogenized with an SLT-I or SLT-II-converting bacteriophage (H-19B or 933W, respectively) or bacteriophage lambda. Both mechanisms are dependent on bacteriophage induction and hence are recA dependent. Moreover, the study revealed that the DNA-binding protein H-NS has a regulatory impact on both bacteriophage-mediated SLT-II synthesis and the activity of the slt-II promoter of plasmid pADR-28. While a slight impact of growth temperature on SLT-II expression was observed, no impact of either osmolarity, pH, oxygen tension, acetates, iron level, or utilized carbon source could be demonstrated.     

Regulation of human neutrophil type 3 complement receptor (iC3b receptor) expression during phagocytosis of Staphylococcus aureus and Escherichia coli.

Human neutrophils (PMN) express a receptor for iC3b, a cleavage product of C3b. CR3 is an important receptor for phagocytosis of opsonized bacteria and its expression is enhanced by cell activation. We examined PMN CR3 expression during phagocytosis using flow cytometry and a CR3-specific monoclonal antibody. After 30 min phagocytosis of opsonized S. aureus and E. coli, CR3 expression increased to 151% and 221% of controls, respectively. Unopsonized S. aureus had no effect on CR3; however, unopsonized E. coli enhanced CR3 expression despite not being phagocytosed. Time-kinetic studies indicated a rapid initial fall in CR3 after addition of bacteria to PMN, followed by enhanced expression within 5-10 min. The initial fall in CR3 probably represented CR3 internalization rather than receptor destruction, as superoxide dismutase, catalase and protease inhibitors had no effect on this. Correlation of CR3 expression with the PMN oxidative response, measured with the intracellular fluorescent probe DCF-DA, demonstrated a dichotomy. Opsonized S. aureus and E. coli caused an oxidative response from PMN but unopsonized E. coli, which caused significant CR3 up-regulation, did not. CR3 up-regulation with unopsonized and opsonized E. coli was markedly inhibited by Polymyxin B, suggesting a role for endotoxin. These experiments indicate that CR3 expression can be regulated during phagocytosis, and the mechanisms responsible are distinct from those involved in the oxidative burst. CR3 up-regulation following exposure to bacteria in vivo may enhance neutrophil function at sites of infection.     

Regulation of virulence and motility by acetate in enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli colonizes the human small intestine and causes severe diarrhea. Short-chain fatty acids are abundant in the intestine owing to the metabolic activity of the microflora and are important for intestinal health. Here, we found that acetate promotes the adherence of enteropathogenic E. coli O127:H6 to Caco-2 intestinal epithelial cells and its motility on semi-solid Luria–Bertani agar by activating the expression of locus of enterocyte effacement genes and flagellar genes, respectively. The effect of acetate on locus of enterocyte effacement gene expression is mediated by Ler, the master regulator of locus of enterocyte effacement genes, whereas the regulation of flagellar genes by acetate is dependent on the RNA polymerase sigma factor FliA. Conversely, formate, propionate, and butyrate had little or no effect on enteropathogenic E. coli O127:H6 adherence and motility. Finally, the acetate-mediated regulatory pathway was found to be a widespread mechanism used by a range of enteropathogenic E. coli to mediate bacterial virulence and motility. Therefore, upon entering the human small intestine, enteropathogenic E. coli may respond to the higher acetate level to enhance its virulence and motility, leading to efficient colonization of the target niche.     

尿道致病性大肠埃希菌感染对人膀胱上皮细胞基因表达谱的影响

目的 研究尿道致病性大肠埃希菌(UPEC)菌株132与人膀胱上皮EJ细胞的相互作用,分析该菌株感染对EJ细胞基因表达谱的改变.方法 UPEC132感染EJ细胞,用倒置显微镜观察细菌与细胞的黏附,计算黏附率,并通过激光共聚焦显微镜观察UPEC132对细胞的侵袭.感染UPEC132的EJ细胞与未经细菌感染的细胞提取总RNA,用人类全基因组寡核苷酸微阵列芯片分析差异表达基因,并采用RT-PCR对基因芯片数据进行验证.结果 UPEC132能够黏附于EJ细胞表面,黏附率为(73.20±5.26)%;激光共聚焦显微镜观察发现部分细菌位于细胞内部,证实该菌对EJ细胞具有侵袭性.UPEC132感染后的EJ细胞与未经感染的细胞相比,共有28个基因上调,1个基因下调,主要涉及细胞增殖、炎症反应、细胞凋亡等相关基因.结论 UPEC与尿路上皮细胞的相互作用激活宿主细胞内部多种应答反应与信号转导途径,本研究为深入探索UPEC致病机制奠定基础.     

Cloning and expression of the Pasteurella multocida toxin gene, toxA, in Escherichia coli.

A chromosomal DNA library of a toxigenic type D strain of Pasteurella multocida subsp. multocida was established in Escherichia coli. From this library two clones, SPE308 and SPE312, were identified by using a monoclonal antibody against the osteoclast-stimulating P. multocida toxin (PMT). Extracts of these clones showed cytopathic activity identical to that of extracts of toxigenic P. multocida. The recombinant plasmids, pSPE308 and pSPE312, directed the synthesis of a protein with an apparent molecular weight of 143,000 which could be specifically detected by anti-PMT antibody. The recombinant toxin, which was located in the cytoplasm of E. coli, was purified by affinity chromatography with immobilized monoclonal antibody and was shown to react in a manner identical to that of PMT in a quantitative structural test using a series of monoclonal antibodies as well as in all quantitative functional tests used, i.e., tests for dermonecrotic activity and mouse lethality and the embryonic bovine lung cell test for cytopathic activity. The gene encoding this toxic activity was named toxA and was found to be present in the chromosome of toxigenic strains only of P. multocida. A probe spanning the toxA gene therefore has potential in the diagnosis and surveillance of progressive atrophic rhinitis in pigs.     

Bactericidal, bacteriolytic and opsonic activity of human serum against Escherichia coli

The effect of human serum on Escherichia coli was studied with serum-sensitive and serum-resistant strains. The bactericidal effect of human serum on serum-sensitive strains of E. coli depended on the activation of the classical complement pathway. The role of activation of the alternative pathway was less important. After incubation in sub-bactericidal concentrations of serum these strains were also easily phagocytosed by polymorphonuclear leukocytes (PMNL). Strains of E. coli of certain O-types required not only an intact classical pathway but also the presence of specific antibodies for effective killing by serum and effective phagocytosis by PMNL, despite rapid activation of complement and rapid deposition of C3 on the bacterial surface in the absence of antibody. Capsulate strains O1K1 and O78K80 resisted the bactericidal effect of serum even in the presence of specific antibodies; phagocytosis by PMNL only occurred after opsonisation with specific antibodies.     

Viable but non-culturable state and toxin gene expression of enterohemorrhagic Escherichia coli O157 under cryopreservation

As major food-borne pathogens worldwide, Escherichia coli are capable of toxin production directly causing severe human disease. However, routine methods are incapable of detecting viable but non-culturable (VBNC) bacteria in food products and raw materials, leading to false-negative identification. In this study, VBNC E. coli O157 strains were acquired after cryopreservation at −20 °C, with and without freeze-thawing; morphology was observed to be of shorter rod-shape, and toxin expression remained at relatively high levels. PMA-PCR assay for VBNC detection was also validated. Therefore, these results suggest that VBNC E. coli O157 strains may represent a strong threat to public health and food safety.