用单链噬菌体载体克隆DNA

丝状单链DNA大肠杆菌型噬菌体M 13、fd及f1,近来被作为一类新的DNA克隆载体而发展起来了,它的优点显著超过其它载体,包括其它两类大肠杆菌寄主的克隆载体:质粒和噬菌体λ。本综述描述单链噬菌体载体的生物学及应用方法以及测量插入DNA大小和排列方向的快速方法,特别是单链载体对DNA定序和离体位点定向诱变     

荧光标记噬菌体快速检测K1荚膜大肠杆菌方法的建立及应用

【背景】大肠杆菌(Escherichia coli,E.coli)是引发新生儿脑膜炎和禽类脑膜炎最常见的革兰氏阴性菌,其中含K1荚膜大肠杆菌是重要的病原菌。目前,K1荚膜大肠杆菌的检测方法存在一些弊端。【目的】利用PNJ1809-36噬菌体的宿主特异性建立快速检测K1荚膜大肠杆菌的方法。【方法】用荧光染料SYBR Gold标记PNJ1809-36噬菌体,侵染33株受试菌,在荧光显微镜下观察,测定该方法的特异性;倍比稀释宿主菌DE058,用荧光标记噬菌体侵染,测定该方法的灵敏度;用荧光标记噬菌体检测8份模拟粪样,测定该方法的临床应用效果;测定4℃避光保存4个月的荧光标记噬菌体的效价和检测效果。【结果】33株受试菌中的9株K1荚膜大肠杆菌有8株可见环状荧光,1株未能检出;20株非K1荚膜大肠杆菌以及4株非大肠杆菌属细菌均不能观察到荧光,检测灵敏度达100CFU/mL。8份模拟粪样的检测结果显示,3份含有K1荚膜大肠杆菌的粪样均可见环状荧光,5份不含K1荚膜大肠杆菌的粪样均无荧光。荧光标记噬菌体4℃避光保存4个月后效价无明显下降,检测效果无明显变化,表明该荧光标记噬菌体在4℃避光条件下较稳定...     

检测噬菌体DNA法鉴别细菌的溶原性

根据前噬菌体的可诱导性,将细菌培养物经丝裂霉素C诱导,诱导液滤过除菌,经核酸酶处理和聚乙二醇(PEG 6000)浓缩,再用苯酚进行抽提。通过检测抽提物中有无DNA,以确定菌株的溶原性。实验证明从溶原菌诱导液中可提取DNA,同时表明该DNA确为溶原菌诱导出的噬菌体DNA,而非溶原性菌以同样方法不能取得DNAo用此方法,可以作为鉴别细菌溶原性的一个手段。     

细胞培养用牛血清污染大肠杆菌噬菌体的检测

用大肠杆菌噬菌体增殖法检测２１６批细胞培养用牛血清,结果１４２批（６５．７％）显示阳性结果。由此提示牛血清在采集制备过程中的污染严重,应引起重视。     

布氏菌噬菌体SA对各种布氏菌的裂解活性

布氏菌噬菌体SA是从猪种布氏菌培养物中分离到的。其裂解活性当菌液浓度为10^-4时,对光滑型猪种菌1、3型,牛种菌1、3、6型和沙林鼠种菌可产生混合性裂解,产生噬菌斑直径3—4mm。噬菌体SA原液或稀释浓度为10^-1、10^-2时,对羊种菌1、3型不裂解。对羊种菌2型、粗糙型牛种菌45/20、犬种菌和绵羊附睾菌产生混合性裂解或噬菌斑,噬菌斑为1—2mm或更小。     

产肠毒素大肠杆菌DNA疫苗重组酵母菌体微胶囊开发

产毒素大肠杆菌(enterotoxigenic Escherichia coli, ETEC)感染是制约畜牧业发展的一大因素。然而,抗生素的滥用势必引发细菌耐药与药物残留问题,且现有疫苗难以有效激发肠道免疫,预防效果不佳。因此需要开发一种安全的、能激活肠道免疫、适宜作为饲料添加剂的新型疫苗。本研究旨在通过基因工程技术,构建携带ETEC DNA疫苗的酵母菌体微胶囊(yeast-cell microcapsules, YCM),并通过动物实验探究饲喂YCM对动物肠道免疫系统的调节作用及其对肠道菌群的潜在影响。选用酿酒酵母菌体作为DNA疫苗的口服递送载体—“微胶囊”,合成密码子哺乳动物源优化的ETEC主要抗原K88核酸序列,利用重组DNA技术构建含有相应DNA疫苗表达盒的酵母穿梭载体,转化酿酒酵母菌株JMY1制备相应的YCM重组菌株。进一步通过荧光报告实验、胃肠液耐受实验、肠上皮细胞黏附实验、肠道滞留性评估、抗血清检测和肠道菌群检测等一系列的实验检测全面评估了YCM菌株的特性及作为口服疫苗的可行性。实验结果显示,DNA疫苗表达盒能够在哺乳动物中有效表达,YCM重组菌能够耐受长达8 h的胃肠液消化,并对肠上皮细胞具有良好的黏附力。小鼠饲喂实验结果表明,YCM重组菌能够在肠道内滞留至少2周,且其所携带的DNA疫苗表达盒能够进入肠道免疫系统并引发免疫反应,诱导产生特异性抗体。此外,饲喂YCM重组菌还能改善小鼠肠道菌群丰度,在调节肠道菌群方面展现了积极作用。综上所述,本研究制备了携带ETEC DNA疫苗的YCM重组菌株,全面评估了其作为口服疫苗的特性及可行性,确认了饲喂YCM重组菌能够诱导特异性免疫反应并调节肠道菌群,为ETEC相关动物疾病的免疫预防提供了参考和借鉴。     

抗肿瘤DNA疫苗的研究进展

抗肿瘤DNA疫苗是一种全新的肿瘤疫苗,在肿瘤防治中发挥着越来越重要的作用。大量研究表明,多种类型的抗肿瘤DNA疫苗都具有一定程度的抗肿瘤效应,有着良好的应用前景。目前,构建新型高效的抗肿瘤DNA疫苗已经成为肿瘤研究的热点。     

噬菌体DNA的快速抽提

介绍一种噬菌体DNA的快速抽提方法.用聚乙二醇沉淀噬菌体颗粒,然后经DEAE纤维素纯化处理和酚抽提.与传统的噬菌体DNA纯化方法相比,改进后的方法方便、快速、经济,可获得高纯度的噬菌体DNA.     

重组大肠杆菌高密度培养研究进展

重组大肠杆菌细胞高密度培养(High cell-density cultivation,HCDC)是获得高外源蛋白产率的一种重要策略,影响重组大肠杆菌高密度培养的因素主要有以下几个方面:重组体的构建及其稳定性,培养基成分,培养方式,培养条件及培养过程中抑制性代谢产物的积累等。从以上几个方面对近期的研究进展进行了综述。     

E.coli DNA抗肿瘤免疫的动态实验研究

５５只Ｂａｌｂ／ｃ小鼠,５只作为正常对照组,其余５０只荷瘤后随机分为１０组, ５组为 ＴＥＳ对照组, ５组为Ｅ． ｃｏｌｉＤＮＡ治疗组。荷瘤第４ｄ起,分别于治疗组和对照组小鼠皮下注射 ＤＮＡ（１００ｍｇ／Ｌ）或 ＴＥＳ（０． １ｍｏｌ／Ｌ）,０．３ｍｌ／只,隔日１次,共５次。于荷瘤第５,９,１３,１７,２１ｄ处置对照组和治疗组各一组小鼠,进行Ｅ．ｃｏｌｉＤＮＡ抗肿瘤免疫的动态研究。结果表明, Ｅ． ｃｏｌｉ ＤＮＡ体内抑瘤作用出现早、强且持久,主要是通过诱导肿瘤组织坏死所致,它可迅速持久地激活免疫系统,防止出现免疫低下或紊乱;对照组小鼠荷瘤早期可出现暂时的免疫功能增强,随荷瘤时间延长,逐渐降低至较低水平,并出现免疫紊乱。     

The Molecular Genetics of Superoxide Dismutase in E. Coli

The biological role and the regulation of superoxide dismutase (SOD) in E. coli have been investigated using genetics. Cloning of both E. coli SOD genes permitted construction of mutants completely lacking SOD. The conditional oxygen sensitivity of those mutants, together with their increased mutation rate, demonstrated the essential biological role of SOD. SOD-deficient mutants constitute a powerful tool to assess a possible role of O^?2 or SOD in biological processes. Complementation of their deficiencies by the expression of SOD originating from a different organism is used for screening libraries for SOD genes of other species. Regulation of MnSOD has been studied using protein and operon fusions with the lactose operon, and isolating regulation mutants. These studies reveal multiregulation of MnSOD including response to the superoxide mediated oxidative stress and response to variations of the intracellular redox state induced by metabolic changes.     

Phage P22 helps phage MB78 to overcome blockage of DNA maturation in rifampicin-resistant host

Bacteriophage MB78, a virulent phage ofSalmonella typhimurium cannot grow in rifampicin-resistant mutant (rif-39) of the host having altered RNA polymerase. The temperate phage P22 which cannot multiply in presence of the virulent phage MB78 can, however, help MB78 to overcome replication inhibition in rif-39. The processing of concatemeric phage DNA to monomer is blocked in this nonpermissive host. Superinfection with P22 induces synthesis of at least five P22 specific polypeptides which help phage MB78 in the processing of the concatemeric DNA and maturation of phage particles.     

提高大肠杆菌分泌表达重组蛋白的研究进展

大肠杆菌是表达重组蛋白的常见宿主之一。重组蛋白分泌到周质空间或胞外培养基中较之在胞内以包含体形式表达有许多优势。主要讨论大肠杆菌Ⅰ、Ⅱ型分泌机制,并总结近年来在提高重组蛋白分泌表达的策略方面取得的进展。     

致病性大肠埃希菌和沙门菌毒力岛基因的双重PCR检测方法的建立

目的实现对致病性大肠埃希菌（E．coli）、沙门菌（Salmonella）的同时检测,建立快速灵敏的双重PCR检测方法。方法以致病性大肠埃希菌和沙门菌毒力岛基因为研究对象,根据GenBank发表的大肠埃希菌和沙门菌毒力岛基因序列,分别设计合成了大肠埃希菌毒力岛irpl、irl）2和fyuA,沙门菌毒力岛mgtC、sseL和sopB等6对引物,以禽致病性大肠埃希菌（CVCC1565）菌株和沙门菌（ATCC9150）菌株的核酸混合物为模板,经引物特异性试验,引物组合,成功建立了快速鉴别检测致病性大肠埃希菌和沙门菌的双重PCR方法。结果特异性试验结果显示,引物irpl、irp2和fyuA仅能扩增出大肠埃希菌（CVCC1565）的特异性片段,大小分别是799、414和948bp;引物mgtC、sseL和sopB仅能扩增出沙门菌（ATCC9150）的特异性片段,大小分别是500、269和1000bp。敏感性试验结果表明大肠埃希菌和沙门菌的最低检测限分别为2．2×101CFU／mL和2．0×101CFU／mL。结论本研究建立的双重PCR方法具有特异性强、敏感性高、快速简便等特点,可用于致病性大肠埃希菌和沙门菌的联合检测与鉴别诊断。     

The detection of non-O157 E. coli in food by immunomagnetic separation

AIMS: To compare immunomagnetic separation (IMS) protocols (enrichment media and temperature) for the isolation of Escherichia coli serotypes O26 and O111 from four different foods. METHODS AND RESULTS: Foods (minced beef, cheese, apple juice and pepperoni) spiked with low numbers (<100 g(-1)) of stressed nalidixic mutant E. coli serotypes O26 and O111 were enriched in media based on buffered peptone water (BPW), tryptone soya and EC broths incubated at temperatures of 37 and 42 degrees C to optimize the IMS technique. BPW enrichments gave increased recoveries of both serotypes compared with tryptone soya and EC broths. Elevated temperatures of incubation at 42 degrees C were superior to 37 degrees C. CONCLUSIONS: Positive detection of low numbers of stressed target pathogens in all replicate tests was only possible using BPW enrichments. The majority of tests from alternative enrichments resulted in zero or single colonies recovered post-IMS. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimum IMS protocol would improve isolation rates of E. coli O26 and O111 from foods and lead to increased safety for the consumer. Sub-optimal IMS protocols could lead to foods being incorrectly labelled free from these pathogens.     

E. coli chromosomal DNA in a transgene locus created by microprojectile bombardment in tobacco

Transgenic Research -     

Fuctional organization of the outer membrane of escherichia coli: Phage and colicin receptors as components of iron uptake systems

The functional interaction of outer memberane proteins of E. coli can be studied using phage and colicin receptors which are essential components of penetration systems. The uptake of ferric iron in the form of the ferrichrome complex requires the ton A and ton B functions in the outer membrane of E. coli. The ton A gene product is the receptor protein for phage T5 and is required together with the ton B function by the phages T1 anf ?80 to infect cells and by colicin M and the antibiotic albomycin, a structural analogue of ferrichrome, to kill cells. The ton B function is necessary for the uptake of ferric iron complexed by citrate. Iron complexed by enterochelin is only transported in the presence of the ton B and feu functions. Cells which have lost the feu function are resistant to the colicins B, I or V while ton B mutants are resistant to all colicins. The interaction of the ton A, Ton B, and feu functions apparently permits quite different “substrates” to overcome the permeablility barrier of the outer membrane. It was shown for ferrichrome dependent iron uptake that the complexing agent was not altered and could be used repeatedly. Only very low amounts of ³H-labeled ferrichrome were found in the cell. It is possible that the iron is mobilized in the membrane and that desferriferrichrome is released into the medium without having entered the cytoplasm. Growth on ferrichrome as the sole iron source waw used to select revertants of T5 resistant ton A mutants. All revertants exhibited wild-type properties with the exception of partial revertants. In these 4 strains, as in the ton A mutants, the ton A protein was not detectable by SDS polyacrylamide gel electrophoreses of outer membranes. Albomycin resistant mutants were selected and shown to fall into 5 categories: (1) ton A; (2) ton B mutants; (3) mutants with no iron transport defects and normal ton A/ton B functions, which might be target site mutants; (4) mutants which were deficient in ferrichrome-mediated iron uptake but had normal ton A/ton B functions. We tentatively consider that the defect might be located in the active transport system of the cytoplasmic membrane; (5) a variety of mutants with the following general properties: most of them were resistant to colicin M, transported iron poorly, and, like ton B mutants, contained additional proteins in the outer membrane. The outer membrane protein patterns of wild-type and ton B mutant strains were compared by slab gel electrophoresis in an attempt to identify a ton B protein. It was observed that under most growth conditions, ton B mutants overproduced 3 proteins of molecular weights 74,000–83,000. In extracted, iron-deficient medium, both the wild-type and ton B mutant strains had similar large amounts of these proteins in their outer membranes. The appearance of these proteins was suppressed by excess iron in both wild-type and mutant. From this evidence it is apparent that the proteins appear as a response to low intracellular iron rather than being controlled by the ton B gene. The nature of these proteins and their possible role in iron transport is disussed.     

Quantitative real-time PCR technique for the identification of E. coli residual DNA in streptokinase recombinant product

Recombinant streptokinase is a biopharmaceutical which is usually produced in E. coli. Residual DNA as a contamination and risk factor may remain in the product. It is necessary to control the production procedure to exclude any possible contamination. The aim of the present study was to develop a highly specific and sensitive quantitative real-time PCR-based method to determine the amount of E. coli DNA in recombinant streptokinase. A specific primers and a probe was designed to detect all strains of E. coli. To determine the specificity, in addition to using NCBI BLASTn, 28 samples including human, bacterial, and viral genomes were used. The results confirmed that the assay detects no genomic DNA but E. coli’s and the specificity was determined to be 100%. To determine the sensitivity and limit of detection of the assay, a 10-fold serial dilution (10¹ to 10⁷ copies/µL) was tested in triplicate. The sensitivity of the test was determined to be 101 copies/µL or 35 fg/µL. Inter-assay and intra-assay were determined to be 0.86 and 1.69%, respectively. Based on the results, this assay can be used as an accurate method to evaluate the contamination of recombinant streptokinase in E. coli.