α-氨基苄青霉素抗性转座子Tn2在E.coli K12乳糖操纵子Z基因中的插入区域特异性

转座子Tn2是大肠杆菌质粒RSF 1030上一段带有α-氨基苄青霉素抗性基因的DNA序列。这段序列具有转座能力,它能通过不同于一般的DNA重组机理从一个复制子转座到另一个复制子。已知,噬菌体Mu,插入序列IS1、IS2、IS3和抗药性转座子TnA、Tn5、Tn9、Tn10等均能使被插入的基因发生突变。已有报道,不同的转座子在E.coli K12乳糖操纵子Z基因中的插入模式不同。Mu噬菌体在Z基因     

大肠杆菌Nissle1917 L-天冬酰胺酶Ⅱ基因在大肠杆菌BL21中的表达与抗肿瘤活性

【目的】从大肠杆菌Nissle1917中获得L-天冬酰胺酶Ⅱ基因,并研究其抗肿瘤活性。【方法】以大肠杆菌Nissle1917基因组为模板PCR扩增L-天冬酰胺酶Ⅱ基因,克隆至可诱导表达载体pET28a上。将L-天冬酰胺酶Ⅱ表达载体pET28a-asp转化至大肠杆菌BL21(DE3)中并通过IPTG诱导表达,经聚丙烯酰胺凝胶电泳(SDS-PAGE)和液相色谱-质谱(LC-MS)对表达的L-天冬酰胺酶Ⅱ进行鉴定,并通过镍柱亲和层析纯化收集表达出的L-天冬酰胺酶Ⅱ。用纯化定量以后的L-天冬酰胺酶Ⅱ作用小鼠乳腺癌4T1细胞、人肝癌Hep-3B细胞和人脐静脉内皮细胞HUVEC。【结果】来自于大肠杆菌Nissle1917的L-天冬酰胺酶Ⅱ基因可在大肠杆菌BL21中高效表达并通过LC-MS得到鉴定,细胞毒性实验结果表明L-天冬酰胺酶Ⅱ对4T1细胞和Hep-3B细胞的生长具有较强的抑制作用,而对人脐静脉内皮细胞HUVEC的生长无明显抑制效果。【结论】来源于大肠杆菌Nissle1917的L-天冬酰胺酶Ⅱ能显著抑制4T1细胞和Hep-3肿瘤细胞的生长,而对人正常组织细胞的生长无明显抑制效果,为进一步研究L-天冬酰胺酶Ⅱ特异性抗肿瘤作用机制和对实体瘤的应用研究奠定了重要基础。     

零背景转座技术高效构建重组家蚕杆状病毒表达系统

摘要：【目的】获得零转座背景的基于家蚕核型多角体病毒(Bombyx mori Nucleopolyhedrovirus, BmNPV) Bac-to-Bac 系统,为高效经济构建重组BmNPV在家蚕体内表达目标蛋白提供新系统。【方法】利用R6Kγ作为复制子构建新的条件复制型杆状病毒转移载体pRADM,同时封闭BmNPV-Bacmid（BmBacmid）宿主菌（Escherichia coli BmDH10Bac）的Tn7转座受体位点attTn7,获得新的封闭型宿主菌E.coli BmDH10Bac△Tn7。【结果】由于pRADM无法在宿主菌E.coli BmDH10Bac中复制,封闭了attTn7位点的宿主菌也不能再和BmBacmid竞争与转移载体的重组,显著提高了转座效率。封闭宿主菌的attTn7位点,能使转座效率提高近4倍,使用条件复制型转座载体pRADM时,转座效率提高近10倍。而用pRADM转座E.coli BmDH10Bac△Tn7时,转座阳性率为100％。避免了获得重组病毒DNA的鉴定程序,缩短了获得重组蛋白所需时间。用携带红色荧光蛋白基因DsRed的重组质粒pRADM-Red转座E.coli BmDH10Bac△Tn7,获得重组BmBacmid转染BmN细胞,红色荧光蛋白在细胞中得到高效表达。【结论】结果表明pRADM和E.coli BmDH10Bac△Tn7是一种零背景高效构建重组BmNPV的新系统。     

鸡毒支原体通用型mini-Tn4001转座子的构建

为了实现以鸡毒支原体（MG）为载体携带和表达其他呼吸道病原保护性抗原,达到多种病原体共同保护的效果,本研究拟构建一套mini-Tn4001转座子（pMT4）.该微型转座子具备以下特征：含四环素抗性基因;两臂是转座酶识别的Inner和Outer插入重复区;转座酶位于插入区之外,在整合起始点处促进转座;多克隆位点便于插入外源基因.这一微型转座子载体的成功构建为MG非必需区缺失工作奠定了基础,对下一步MG功能基因的靶向缺失技术以及外源蛋白的表达建立了有效的基因操作工具.     

一种包含转座子Sleeping Beauty和PiggyBac的新型多功能载体的构建

DNA转座子作为一种遗传学工具对脊椎动物的转基因、突变体产生、癌基因发现和基因治疗方面都有巨大的贡献. 目前,哺乳动物中应用最为广泛、活性最高的DNA转座子为重构于鲑鱼的Sleeping Beauty (SB)转座子和来源于甘蓝蠖度尺蛾 (cabbage looper moth Trichoplusia ni)的PiggyBac (PB)转座子. 本研究中,我们成功构建了包含PB和SB两种转座子的杂合转座载体,命名为PBSBD. 在杂合转座载体中融入了基因捕获框及loxp/Frt元件,用以实现转座过程中的基因捕获和条件性敲除. 在HepG2细胞中通过检测报告基因的表达情况及阳性克隆的定位,对构建的杂合转座载体PBSBD进行了活性的初步验证. 结果表明,PBSBD能够有效被2种转座酶识别,并能检测到报告基因的表达. 本研究所构建的杂合转座载体PBSBD结合2种转座酶,可以应用于大规模筛选突变基因和研究基因功能. 并且该杂合转座载体还可以利用SB转座酶的邻近转座特性,结合载体内所包含的loxp/Frt元件用以邻近区域DNA片段的条件性敲除,研究大片段DNA在生物体中的作用.     

构建表达IL 2的大肠埃希菌对小鼠肠炎的影响

目的以Escherichia coli Nissle 1917为基础建立一种与肠道菌群相关的新型白介素2(IL-2)递送方式,研究其对葡聚糖硫酸钠(DSS)诱导的实验性结肠炎的治疗作用。方法将小鼠随机分为4组(每组10只),以正常小鼠作为空白对照组,实验组小鼠用3%的DSS水诱导小鼠结肠炎模型,分别灌胃表达IL-2的菌株(E.coli 1917/IL-2)、空质粒转化的菌株(E.coli 1917/0)或PBS进行治疗5 d,定期评估各组小鼠的临床体征、疾病活动指数(DAI)、病理和免疫组织学变化。结果构建的益生工程菌E.coli 1917/IL-2可有效缓解DSS诱导的小鼠肠炎,小鼠DAI评分较低,体质量及结肠长度均高于对照组,肠黏膜组织中炎症细胞浸润较少。结论使用工程化益生大肠埃希菌编码免疫调节细胞因子的治疗策略为溃疡性结肠炎提供一种潜在的治疗方法。     

铜绿假单胞菌水解弹性蛋白能力相关基因的筛选与鉴定

【目的】研究铜绿假单胞菌弹性蛋白水解能力相关基因。【方法】应用人工Mu转座技术构建铜绿假单胞菌野生型菌株PA68的转座突变文库,从2000多个突变子中筛选得到4株弹性蛋白水解能力改变的突变子,并通过克隆及测序获得转座子插入位点侧翼的序列。将铜绿假单胞菌弹性蛋白酶结构基因lasB的转录启始区序列整合入载体pDN19lacΩ并将该重组质粒电转化入野生型菌株PA68及4个突变株中,对报告基因在不同菌株中的表达水平进行测定。【结果】发现4个突变株中Mu转座子分别插入lasA、galU、xcpZ和ptsP 4个基因。ptsP基因失活的突变株中,lasB基因的转录水平是野生型菌株的7%,xcpZ和lasA基因的失活使lasB基因的转录水平分别降低为野生株的54%和75%,galU基因的插入失活使lasB基因的转录上升了1倍。【结论】推测ptsP和galU基因很可能直接或间接地调控着弹性蛋白酶的生物合成。     

肠道免疫的安全靶向载体:大肠杆菌Nissle1917

长期以来,如何激发高效的肠道黏膜免疫应答来预防肠道感染始终是较为棘手的问题.本文旨在对大肠杆菌(Escherichia coli)Nissle 1917作为肠道黏膜免疫的安全靶向载体,调理胃肠道菌群紊乱、缓解溃疡性结肠炎以及利用益生菌固有特性或优化特性进行治疗的可能性等相关研究进展作一综述.大肠杆菌Nissle 1917(EcN)是一株可口服的优良益生菌,也可作为生物载体活苗候选株,兼有较强的肠道局部定殖能力和无免疫原性的特性.该菌株还可以作为载体靶向递呈TAT-凋亡素融合蛋白治疗结肠直肠癌,并在研发靶向递呈防御素治疗溃疡性结肠炎和克罗恩病上具有重要的功能.其基因修饰株能够原位递呈特定的抗原分子,有效激发特异性的黏膜免疫应答.重组大肠杆菌Nissle-HA 110-120具有体外表达特异性抗原的能力,但EcN菌体本身不会引起黏膜免疫应答,也不影响对自身抗原的外周免疫耐受.同时,EcN具有很好的安全性,尤其是因炎症导致肠道防御屏障破坏的时候,重组大肠杆菌Nissle-HA110-120在健康或患有急性结肠炎的小鼠体内都没有迁移、克隆扩增和激活特异性CD4+T淋巴细胞的作用.     

重度肥胖患者肠道内机会性致病菌Escherichia coli微多样性分析

本研究利用志贺氏菌显色培养基和ERIC-PCR指纹图谱技术,从一名重度肥胖患者肠道内分离出19种不同ERIC类型的分离物。选取不同ERIC类型的代表菌株进行16S r RNA基因分子鉴定并构建系统发育树,发现19种代表菌株的16S r RNA基因和埃希氏菌属(Escherichia)、志贺氏菌属(Shigella)的相似性达到99%。经生理生化反应鉴定,19种代表菌株均为肠杆菌科的肠埃希氏菌(Escherichia coli)。体外生物学分析发现19种E.coli代表菌株在生化特性、耐药性、毒性方面有明显差异。本研究表明来自一名重度肥胖患者肠道内同一种群的E.coli在菌株水平上具有丰富的微多样性,不同ERIC类型的E.coli在生理生化特性上存在较大的差异,且部分菌株具有潜在致病性。     

产L-苹果酸重组大肠杆菌的构建

基于产琥珀酸重组大肠杆菌E.coli B0013-1050的琥珀酸合成途径,利用Red同源重组技术结合Xer/dif重组系统敲除富马酸酶基因fumB、fumC,苹果酸酶基因maeB,构建L-苹果酸合成途径,最终得到重组大肠杆菌E.coli2030,该菌株在15 L发酵罐中,产L-苹果酸12.5 g/L,葡萄糖-苹果酸转化率为52.1%,同时对发酵产物中主要杂酸丙酮酸和琥珀酸的生产原因进行了初步的探讨与分析。为进一步提高L-苹果酸的转化率,整合表达来源于黄曲霉的苹果酸脱氢酶基因,构建重组菌E.coli 2040,在15 L发酵罐中产L-苹果酸14 g/L,葡萄糖-苹果酸转化率提高到60.3%。     

Genomic peculiarity of coding sequences and metabolic potential of probiotic Escherichia coli strain Nissle 1917 inferred from raw genome data

Probiotic Escherichia coli strain Nissle 1917 (O6:K5:H1) is a commensal E. coli isolate that has a long tradition in medicine for the treatment of various intestinal disorders in humans. To elucidate the molecular basis of its probiotic nature, we started sequencing the genome of this organism with a whole-genome shotgun approach. A 7.8-fold coverage of the genomic sequence has been generated and is now in the finishing stage. To exploit the genome data as early as possible and to generate hypotheses for functional studies, the unfinished sequencing data were analyzed in this work using a new method [Sun, J., Zeng, A.P., 2004. IdentiCS--identification of coding sequence and in silico reconstruction of the metabolic network directly from unannotated low-coverage bacterial genome sequence. BMC Bioinformatics 5, 112] which is particularly suitable for the prediction of coding sequences (CDSs) from unannotated genome sequence. The CDSs predicted for E. coli Nissle 1917 were compared with those of all five other sequenced E. coli strains (E. coli K-12 MG1655, E. coli K-12 W3110, E. coli CFT073, EHEC O157:H7 EDL933 and EHEC O157:H7 Sakai) published to date. Five thousand one hundred and ninety-two CDSs were predicted for E. coli Nissle 1917, of which 1065 were assigned with enzyme EC numbers. The comparison of all predicted CDSs of E. coli Nissle 1917 to the other E. coli strains revealed 108 CDSs specific for this isolate. They are organized as four big genome islands and many other smaller gene clusters. Based on CDSs with EC numbers for enzymes, the potential metabolic network of Nissle 1917 was reconstructed and compared to those of the other five E. coli strains. Overall, the comparative genomic analysis sheds light on the genomic peculiarity of the probiotic E. coli strain Nissle 1917 and is helpful for designing further functional studies long before the sequencing project is completely finished.     

The probiotic Escherichia coli strain Nissle 1917 interferes with invasion of human intestinal epithelial cells by different enteroinvasive bacterial pathogens

The probiotic Escherichia coli strain Nissle 1917 (Mutaflor) of serotype O6:K5:H1 was reported to protect gnotobiotic piglets from infection with Salmonella enterica serovar Typhimurium. An important virulence property of Salmonella is invasion of host epithelial cells. Therefore, we tested for interference of E. coli strain Nissle 1917 with Salmonella invasion of INT407 cells. Simultaneous administration of E. coli strain Nissle 1917 and Salmonella resulted in up to 70% reduction of Salmonella invasion efficiency. Furthermore, invasion of Yersinia enterocolitica, Shigella flexneri, Legionella pneumophila and even of Listeria monocytogenes were inhibited by the probiotic E. coli strain Nissle 1917 without affecting the viability of the invasive bacteria. The observed inhibition of invasion was not due to the production of microcins by the Nissle 1917 strain because its isogenic microcin-negative mutant SK22D was as effective as the parent strain. Reduced invasion rates were also achieved if strain Nissle 1917 was separated from the invasive bacteria as well as from the INT407 monolayer by a membrane non-permeable for bacteria. We conclude E. coli Nissle 1917 to interfere with bacterial invasion of INT407 cells via a secreted component and not relying on direct physical contact with either the invasive bacteria or the epithelial cells.     

Analysis of the genome structure of the nonpathogenic probiotic Escherichia coli strain Nissle 1917

Nonpathogenic Escherichia coli strain Nissle 1917 (O6:K5:H1) is used as a probiotic agent in medicine, mainly for the treatment of various gastroenterological diseases. To gain insight on the genetic level into its properties of colonization and commensalism, this strain's genome structure has been analyzed by three approaches: (i) sequence context screening of tRNA genes as a potential indication of chromosomal integration of horizontally acquired DNA, (ii) sequence analysis of 280 kb of genomic islands (GEIs) coding for important fitness factors, and (iii) comparison of Nissle 1917 genome content with that of other E. coli strains by DNA-DNA hybridization. PCR-based screening of 324 nonpathogenic and pathogenic E. coli isolates of different origins revealed that some chromosomal regions are frequently detectable in nonpathogenic E. coli and also among extraintestinal and intestinal pathogenic strains. Many known fitness factor determinants of strain Nissle 1917 are localized on four GEIs which have been partially sequenced and analyzed. Comparison of these data with the available knowledge of the genome structure of E. coli K-12 strain MG1655 and of uropathogenic E. coli O6 strains CFT073 and 536 revealed structural similarities on the genomic level, especially between the E. coli O6 strains. The lack of defined virulence factors (i.e., alpha-hemolysin, P-fimbrial adhesins, and the semirough lipopolysaccharide phenotype) combined with the expression of fitness factors such as microcins, different iron uptake systems, adhesins, and proteases, which may support its survival and successful colonization of the human gut, most likely contributes to the probiotic character of E. coli strain Nissle 1917.     

A single nucleotide exchange in the wzy gene is responsible for the semirough O6 lipopolysaccharide phenotype and serum sensitivity of Escherichia coli strain Nissle 1917

Structural analysis of lipopolysaccharide (LPS) isolated from semirough, serum-sensitive Escherichia coli strain Nissle 1917 (DSM 6601, serotype O6:K5:H1) revealed that this strain's LPS contains a bisphosphorylated hexaacyl lipid A and a tetradecasaccharide consisting of one E. coli O6 antigen repeating unit attached to the R1-type core. Configuration of the GlcNAc glycosidic linkage between O-antigen oligosaccharide and core (beta) differs from that interlinking the repeating units in the E. coli O6 antigen polysaccharide (alpha). The wa(*) and wb(*) gene clusters of strain Nissle 1917, required for LPS core and O6 repeating unit biosyntheses, were subcloned and sequenced. The DNA sequence of the wa(*) determinant (11.8 kb) shows 97% identity to other R1 core type-specific wa(*) gene clusters. The DNA sequence of the wb(*) gene cluster (11 kb) exhibits no homology to known DNA sequences except manC and manB. Comparison of the genetic structures of the wb(*)(O6) (wb(*) from serotype O6) determinants of strain Nissle 1917 and of smooth and serum-resistant uropathogenic E. coli O6 strain 536 demonstrated that the putative open reading frame encoding the O-antigen polymerase Wzy of strain Nissle 1917 was truncated due to a point mutation. Complementation with a functional wzy copy of E. coli strain 536 confirmed that the semirough phenotype of strain Nissle 1917 is due to the nonfunctional wzy gene. Expression of a functional wzy gene in E. coli strain Nissle 1917 increased its ability to withstand antibacterial defense mechanisms of blood serum. These results underline the importance of LPS for serum resistance or sensitivity of E. coli.     

Escherichia coli Nissle 1917 Targets and Restrains Mouse B16 Melanoma and 4T1 Breast Tumors through Expression of Azurin Protein

Many studies have demonstrated that intravenously administered bacteria can target and proliferate in solid tumors and then quickly be released from other organs. Here, we employed the tumor-targeting property of Escherichia coli Nissle 1917 to inhibit mouse B16 melanoma and 4T1 breast tumors through the expression of azurin protein. For this purpose, recombinant azurin-expressing E. coli Nissle 1917 was developed. The levels of in vitro and in vivo azurin secretion in the engineered bacterium were determined by immunochemistry. Our results demonstrated that B16 melanoma and orthotopic 4T1 breast tumor growth were remarkably restrained and pulmonary metastasis was prevented in immunocompetent mice. It is worth noting that this therapeutic effect partially resulted from the antitumor activity of neutrophils and lymphocytes due to inflammatory responses caused by bacterial infections. No toxicity was observed in the animal during the experiments. This study indicates that E. coli Nissle 1917 could be a potential carrier to deliver antitumor drugs effectively for cancer therapy.     

Detection and distribution of probiotic Escherichia coli Nissle 1917 clones in swine herds in Germany

AIMS: To verify the presence of Escherichia coli Nissle 1917 as a natural isolate in swine and to characterize in vitro probiotic properties as well as in vivo persistence in a feeding experiment. METHODS AND RESULTS: During studies on the intestinal microflora of pigs, we isolated E. coli Nissle 1917 sporadically from a pig population over a period of 1 year. The identity of the isolates as E. coli Nissle 1917 was verified by serotyping, Nissle-specific PCR, macrorestriction analysis (pulsed field gel electrophoresis) and the determination of in vitro probiotic properties in invasion and adhesion assays using a porcine intestinal epithelial cell line. Both the E. coli isolates and the E. coli Nissle 1917 strain showed strong reductions in adhesion of porcine enteropathogenic E. coli and invasion of Salmonella typhimurium with epithelial cells in vitro, with a probiotic effect. Screening of five epidemiologically unlinked swine farms and two wild boar groups showed one farm positive for E. coli Nissle 1917. A feeding experiment with four piglets showed viable E. coli Nissle 1917 in the intestine of three animals. CONCLUSIONS: The results of this study suggest that the E. coli Nissle 1917 strain is already partially established in swine herds, but the colonization of individual animals is variable. SIGNIFICANCE AND IMPACT OF THE STUDY: We report natural, long-term colonization and transmission of the probiotic E. coli Nissle 1917 strain in a swine herd, characterized individual persistence and colonization properties in swine and established an in vitro porcine intestinal epithelial cell model of probiotic action. The results of this study would have implications in the use of this strain as a probiotic in swine and contribute to a better understanding of the individual nature of intestinal bacterial persistence and establishment.     

Effects of monocolonization with<Emphasis Type="Italic">Escherichia coli</Emphasis> strains O6K13 and nissle 1917 on the development of experimentally induced acute and chronic intestinal inflammation in germ-free immunocompetent and immunodeficient mice

Germ-free immunocompetent (BALB/c) and immunodeficient (SCID) mice were colonized either by E. coli O6K13 or by E. coli strain Nissle 1917 and intestinal inflammation was induced by administering 2.5% dextran sulfate sodium (DSS) in drinking water. Controls were germ-free mice which demonstrated only mild inflammatory changes after induction of an acute intestinal inflammation with DSS as compared with conventional mice in which acute colitis of the colon mucosa similar to human ulcerative colitis is elicited. In mice monocolonized with the nonpathogenic E. coli Nissle 1917 the inflammatory disease did not develop (damage grade 0) while animals monocolonized with uropathogenic E. coli O6K13 exhibited inflammatory changes similar to those elicited in conventionally reared mice (damage grade 3). In the chronic inflammation model, immunocompetent BALB/c mice monocolonized with E. coli Nissle 1917 showed no conspicuous inflammatory changes of the colon mucosa whereas those monocolonized with E. coli O6K13 developed colon inflammation associated with marked infiltration of inflammatory cells. In contrast to germ-free immunodeficient SCID mice that died after application of DSS, the colon mucosa of SCID mice monoassociated with E. coli Nissle 1917 exhibited only moderate inflammatory changes which were less pronounced than changes of colon mucosa of SCID mice monoassociated with E. coli O6K13.     

李斯特菌溶血素基因的原核表达及其生物学特性

李斯特菌溶血素(LLO)是产单核细胞李斯特菌的主要毒力因子,利用PCR技术从血清型4b的产单核细胞李斯特菌菌株中扩增出编码LLO的hly基因,经克隆筛选和测序鉴定后,构建成该基因的原核表达质粒pGEX6P1hly,SDSPAGE结果表明：LLO与谷胱甘肽在大肠杆菌中已融合表达,融合蛋白的分子量为82kD;溶血实验证明融合蛋白具有较强的裂解真核细胞膜的作用,表明表达产物LLO具有生物活性,其溶血效价达2.26×101.4 HU/mg,这为进一步研究其致病与免疫机理、单抗研制和疫苗设计提供了条件。