大肠杆菌

Objective To screening sensitive test to detesting on ESBLs of Klebsiella pneumonia(KL.pneumonia )and E scherichia,study relation between ESBLs and resistance to antibiotics of KL.pneumonia and E.coli,provide suggestions empirical treatment against the bacteria.

摘 要］目的：筛选检测肺炎克雷伯氏菌及大肠杆菌的超广谱的β内酰胺酶的敏感方法，并探讨肺炎克雷伯氏菌及大肠杆菌产ESBLs的情况与耐药的关系，为临床合理使用抗生素提供实验依据。

Since the post-translational modification process doesn't exist in E. Coli expression system, in order to investigate if the natural nsLTP has antimicrobial activity, we purified nsLTP from rice etiolated seedlings. They were shown to have the similar antifungal activity compared with E. Coli expressed nsLTP.

由于大肠杆菌表达产物缺少翻译后加工，为了与表达产物的功能研究相对照，还摸索了从水稻黄化苗中抽提nsLTP的方法，结果表明水稻苗中提取的nsLTP亦具有抑菌活性，抗性水平接近大肠杆菌表达产物。

Here, we demonstrated in this work that CsrA performs a negative regulator of the cel expression. cel, encoding lysis protein (LysE7), is located downstream of the colicin E7 structural gene in the ColE7 operon. Lysis protein was essential for colicin release and causes a decline in culture turbidity as well as lethality of the host cell when overproduced. Western blotting analysis of the level of LysE7 in the wild-type and csrA mutant strains was examined.

在本论文中，我们进一步发现到CsrA对於cel 基因的表现具有抑制的现象。cel 基因位在质体ColE7-K317上的E7大肠杆菌操纵子，可以制造出溶菌蛋白质(lysis protein， LysE7)，协助大肠杆菌素及免疫蛋白质的复合体运送至菌体外；但当菌体产生过多的溶菌蛋白质，则会导致宿主细胞的死亡，所以溶菌蛋白质的表现必须受到严密的调控，避免菌体产生过量，而CsrA则是目前第一个被发现到能调控溶菌蛋白质表现的因子。

The PCR products were examined by agarose gel electrophoresis. The target gene fragments were purified by gel extraction kit and ligated to cloning vector pMD18-T. The recombinant vectors were transformed into host strain E. coli K802 by lithium chloride method, screened and identified with PCR and restrictive enzymatic digestion. Their sequences were confirmed by DNA sequencing.(2) sTWEAK1 gene was subcloned into expression vector pProEx HTb and transformed into E. coli BL21. sTWEAK2 gene was subcloned into expression vector pMAL-C2x and transformed into E. coli TB1. The recombinant vectors were screened and identified with PCR and restrictive enzymatic digestion. The recombinant fusion proteins were induced to express with IPTG, detected by coomassie brilliant blue-stained SDS-polyacrylamide gel electrophoresis , and confirmed by Western blot analysis.(3) The sTWEAK1 fusion protein was purified with Ni-NTA Spin Kit.(4) The biological activity was assayed on transformed and tumor cells by microplate photometer after crystal violet or sulfur rodamine B staining.(5) The contents of IL-8 in the supernatant of 1990 cell cultures were determined by ELISA.(6) The morphological changes of the sensitive cells were observed by light and transmission electron microscopies.(7) The cell cycle and apoptotic rate were assayed by flow cytometry in 1990 and M85 cells.(8) The effect of fusion proteins on induction of NF-κB in 1990 and LOVO cells was detected with Dual-Luciferase Reporter Assay system.(9) The TWEAK gene was subcloned into Adeno-X Viral DNA with pShuttle vector and transfected into HEK293 cells by lipofectamine method.

(1)本研究用RT-PCR方法，从人组织细胞总RNA中扩增可溶性TWEAK胞外区（sTWEAK1和sTWEAK2）的cDNA序列及全长编码序列，用琼脂糖凝胶电泳分析PCR产物，胶回收目的基因片段，连接到pMD18-T克隆载体中，转化大肠杆菌K802，PCR和酶切筛选阳性克隆，全自动DNA测序验证序列；(2)sTWEAK1和sTWEAK2分别亚克隆到pProEx HTb和pMAL-C2x表达载体中，分别转化大肠杆菌BL21和TB1，PCR筛选和酶切鉴定，阳性克隆用IPTG诱导表达，表达产物用SDS-PAGE分析和Western blot验证融合蛋白；(3)用NTA-Ni Spin试剂盒初步分离纯化sTWEAK1融合蛋白；(4)用体外培养的肿瘤细胞和正常对表达产物进行活性检测，贴壁细胞用结晶紫染色法，悬浮细胞用磺酰罗丹明B染色法，酶标仪检测OD值；(5)敏感细胞用ELISA法检测细胞培养上清中IL-8的含量；(6)用光镜和电镜观察敏感细胞死亡和细胞凋亡情况；(7)用流式细胞仪分析表达产物对敏感细胞凋亡率和细胞周期的影响；(8)用双荧光素酶报告基因检测法，测定表达产物对敏感细胞NF-κB的影响；(9)用pShuttle穿梭质粒将TWEAK重组到腺病毒载体上，用脂质体转染法转染HEK293细胞，PCR鉴定重组质粒。

Coli disease. And the use of allicin sulbactam or amoxicillin by a certain percentage for the Joint duck E.coli disease prevention observed experimental results, the results showed that: Allicin, amoxicillin, and sulbactam sodium single drug to duck the E. coli O0701 MIC were 1024ug/mL.Compound MIC its portfolio of 3～4.5ug/mL than single drug decreased by about 64 times.

利用大蒜素与阿莫西林或舒巴坦钠按一定的比例联合用于鸭大肠杆菌病的防治观察实验效果，结果显示：大蒜素、阿莫西林、与舒巴坦钠单药对鸭大肠杆菌 O0701的 MIC 均为1024ug/mL ，其复方组合 MIC 为3~4.5 ug/mL ，较单药降低了约64倍。

The objectives of this study are:(1) To amplify Tb wbbL gene encoding rhamnosyl transferase bypolymerase chain reaction from the genomic DNA ofM.tuberculosis H37RV strain;(2) To clone PCR product of Tb wbbLgene into a cloning vector pMD18-T for sequencing;(3) To subcloneTb wbbL gene to an expression vector pET16b to construct pET16b-Tb wbbL; and to overexpress Tb WbbL protein in E.coli BL21(DE3)under different induction conditions;(4) To establish theco-expression system for expressing chaperons of pKJE7 plasmidand soluble Tb WbbL protein in E.coli BL21(DE3) under differentinduction conditions;(5) To test expressed WbbL protein by SDS-PAGEand Western blot methods.

本论文的目的是：(1)利用 PCR 方法从结核分枝杆菌 H37Rv 菌株的基因组DNA 中扩增出 Tb wbbL 基因；(2)将 Tb wbbL 基因克隆到pMD18-T 克隆载体中，经 DNA 序列测定证实为正确的基因；(3)将 Tb wbbL 基因亚克隆到 pET16b 表达载体中并通过改变不同的诱导条件在大肠杆菌 BL21(DE3)中表达 Tb WbbL 蛋白质；(4)建立在 BL21(DE3)大肠杆菌中共表达 Tb WbbL 蛋白质与分子伴侣的体系，优化表达条件以高效表达出可溶性 Tb WbbL蛋白质；(5)用 Western blot 方法鉴定所表达的 Tb WbbL 蛋白质为 Tb wbbL 基因产物。

Second, the genes coding for TPI and ALD were constructed in tandem to form two cistron by altering the base composition and the length of intercistronic region and cloned into 〓 expression system, an active, high-level co-expression was completed successfully.

采用〓启动子成功地获得了TPI—ALD-FBPase三基因活性共表达，构建了三基因共表达的大肠杆菌-蓝藻穿梭表达质粒，并在大肠杆菌中成功地进行了活性表达。

Finally, wetested its antibacterial activities by E.coli and S.aureus.Methods In this research, the recombinded gene was cloned into the secretion vector pTYB12 and expressed in the E.coli BL21DE3. We did the preliminary research on secretion-expression of the gene. During the purification procedure, we used the chintin beads to wash off the sundry protein, then dialysed the collecting liquid to get rid of the salinity and the other small molecular peptides.

将上述重组基因抗菌肽的氨基酸片段克隆并拼接到分泌表达载体pTYB12上转染大肠杆菌(E.coli BL21DE3)后进行分泌表达，破碎离心收集的诱导表达大肠杆菌，首次应用几丁质珠柱分离纯化系统进行分离纯化，分离洗脱杂蛋白后透析去除盐类物质及其它小分子肽，得到目的重组基因抗菌肽。

To date, only the structures of prokaryotic succinate:ubiquinone oxidoreductases, which share a similar enzymatic function with mitochondrial SQR, have been reported, including QFR from E.coli, QFR from Wolinella succinogenes and SQR from E.coli.

在此之前，只有原核生物的琥珀酸泛醌氧化还原酶的结构得到了解析，这包括大肠杆菌的 QFR ， Wolinella succinogenes 细菌中的 QFR 和大肠杆菌的 SQR ，这些蛋白有着与线粒体复合物 II 相类似的酶功能。

Three standard and two isolated strains of E.coli were used for drug sensitivity test on Chloramphenicol, Ciprofloxacin Lactate, Rifampicin, sulfamonomethoxine, Florfenicol, Ceftiofur Sodium as well as the antimicrobial synergists sulbactam sodium and TMP. The bacteriostasis results of the antibiotics against E.coli In vitro were compared when they were used separately or combined with the synergist.

试验用3株大肠杆菌标准株和2株地方分离株，选择氯霉素、乳酸环丙沙星、利福平、磺胺-6-甲氧嘧啶钠、氟苯尼考、头孢噻呋和抗菌增效剂舒巴坦钠、TMP进行药敏试验，比较几种药物单一或与增效剂合用对大肠杆菌的体外抑菌效果。

As she looked at Warrington's manly face, and dark, melancholy eyes, she had settled in her mind that he must have been the victim of an unhappy attachment.

每逢看到沃林顿那刚毅的脸，那乌黑、忧郁的眼睛，她便会相信，他一定作过不幸的爱情的受害者。

Maybe they'll disappear into a pothole.

也许他们将在壶穴里消失