大肠杆菌表达重组人IL-8分离纯化工艺的建立

目的　建立重组人IL - 8(rhIL - 8)大肠杆菌表达后的高效分离纯化工艺 ,以便获得高纯度的重组蛋白。方法　将已构建好的表达载体转化大肠杆菌HB10 1,经IPTG诱导进行高效表达。将离心收集到的菌体用超声破壁分离 ,收集包涵体和胞浆 ,其中包涵体部分经变性和复性处理后 ,与胞浆部分合并 ,进行肝素亲和层析柱分离 ,再经离子交换柱和凝胶层析柱过滤 ,得到最终纯品。蛋白纯度用SDS -PAGE鉴定 ,ELISA法检测重组IL - 8含量。检测纯化后重组蛋白的生物学活性和热源质。结果　用大肠杆菌HB10 1表达重组人IL - 8,具有较高的表达量 (2 5mg/ml) ,其分泌的重组蛋白以包涵体和胞浆的形式存在。经多步柱层析纯化后 ,可获得高纯度的重组IL- 8蛋白 (纯度 >95 % )。该蛋白在体外具有趋化中性粒细胞游走的作用 ,兔温法检测热源质含量符合要求。结论　大肠杆菌可高效表达rhIL - 8,该工艺流程可从大肠杆菌载体中获得具有生物学活性的高纯度rhIL - 8。     

大肠杆菌临床分离株多重耐药过程中的主动外排机制

目的　探讨临床分离大肠杆菌多重耐药机制。方法　应用荧光法测定临床分离大肠杆菌对环丙沙星的主动外排功能 ;PCR及Southernblot测定主动外排系统AcrAB结构基因acrAB ;RT PCR测定acrAB表达水平 ;自动荧光测序法测定acrAB基因扩增产物DNA序列。结果　临床分离大肠杆菌多重耐药株细胞内环丙沙星稳态浓度显著低于敏感株 (0 .73mg/L± 0 .0 4mg/L比 2 .0 0mg/L± 0 .0 7mg/LA660 ,P <0 .0 0 1) ;临床分离大肠杆菌无acrAB缺失 ;多重耐药株acrAB表达水平显著高于其他菌株 ;多重耐药株以及敏感株acrAB基因扩增产物无点突变和缺失。结论　主动外排系统acrAB的高表达导致临床分离大肠杆菌产生多重耐药性 ;acrAB的表达可能受多重耐药操纵子调控。     

Department of Etiology and Department of Pharmacology

INTRODUCTION　　 Enteropathogenic E.coli (EPEC) is one of theimportant pathogens for infant diarrhoae and urinarytract infections in adults.The chemotherapy andchemoprophylaxis of EPEC infections by antibiotics arechallenged by the development of multi- resistant strainsand the colonization of pathogenic bacteria in vivo as aresult of gutflora dysfunction.Since late1980 s’,ithasbeen found that the infection of mammalian cells byEPEC occured in two steps:adhesion of bacteria to cells…     

Turcot's syndrome: Case report and review of the classification

Summary We report a case of association of a brain tumor with multiple colorectal polyposis and offer an analysis of the relevant literature with a view to revising the classification of the syndrome in relation to familial multiple polyposis and Gardner's syndrome. Differences emerged, depending on the brain tumor type, which suggests that this association may be classified as two distinct syndromes.     

不同抗生素杀菌过程导致细菌内毒素释放的异质性

目的初步了解各类抗生素杀菌过程导致细菌内毒素释放的不同特点。方法采用鲎基质偶氮法检测各类不同抗生素２倍最低抑菌浓度下对大肠埃希氏菌杀菌时细菌内毒素的释放量,计算杀菌开始至活菌数基本稳定阶段,平均每减少１个对数数量级（ｌｇ）菌落形成单位（ＣＦＵ）所导致的内毒素释放量（杀菌释放率）。结果各类抗生素致内毒素释放能力大致可以分为低、中、高三类,杀菌释放率的界限值分别为１０ｎｇ／ｉｇＣＦＵ和３０ｎｇ／ｉｇＣＦＵ。多粘菌素Ｂ具有低于所有品种的释放率。β－内酰胺类抗生素中的羟氨苄青霉素、棒酸和亚胺硫霉素具低释放率,氨苄青霉素、头孢氨苄、头孢呋辛、头孢哌酮、头孢他啶、氨曲南具高释放率。喹诺酮类的环丙沙星具高释放率,氧氟沙星具中等释放率。氨基甙类均具中等释放率,品种间无显著差异。此外,磷霉素也具有中等释放率。结论具有相似临床杀菌作用的抗生素可以具有差异明显的致内毒素释放能力,此认识有助于指导全身炎症反应综合征时抗生素的选择     

大肠杆菌HX88108膜孔蛋白初步研究

为了解大肠杆菌ＨＸ８８１０８耐药机理中有无膜孔蛋白缺失参与,用十二烷基磺酸钠－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）检测了大肠杆菌ＨＸ８８１０８的膜孔蛋白,并与膜孔蛋白标准株进行比较分析。结果发现：大肠杆菌ＨＸ８８１０８具有膜孔蛋白ＯｍｐＦ、ＯｍｐＣ,其耐药与膜孔蛋白缺失所致的抗生素进入减少无关     

Growth of Escherichia coli in propofol, lidocaine, and mixtures of propofol and lidocaine

BACKGROUND: Microorganisms grow rapidly in propofol. Extrinsic contamination of propofol is thought to be a source of postoperative sepsis and wound infection. We studied growth of a strain of Escherichia coli in thiopental, propofol, lidocaine, and mixtures of propofol and lidocaine. METHODS: The pathogen was exposed to 2.5% thiopental; 1.0% propofol; 1.0%, 2.0% and 4.0% preservative-free lidocaine; and propofol solutions containing 0.25%, 0.5%, 1.0%, 2.0%, or 4.0% lidocaine for 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h at room temperature, respectively. The inocula from these suspensions were cultured for 48 h at 37 degrees C after the antimicrobial activity of the local anesthetics in the inocula was inactivated by a 1:1000 dilution with distilled water. RESULTS: No organisms grew after exposure to 2.5% thiopental. The exposure of E. coli to propofol increased the colony count to approximately 90 times the control count. The colony counts of E. coli after exposure to 1.0%, 2.0% and 4.0% lidocaine and 0.25%, 0.5%, 1.0%, 2.0% and 4.0% lidocaine in 1.0% propofol were lower than the counts after exposure to 1.0% propofol (P = 0.0048, 0.0027, 0.0003, 0.0503, 0.0188, 0.0080, 0.0044, and 0.0001, respectively). The growth rate of the microorganism was significantly higher in cultures exposed to 1.0% propofol than that in cultures exposed to lidocaine alone or lidocaine-propofol mixtures (P < 0.0001, respectively). CONCLUSION: Lidocaine possesses bacteriostatic activity against E. coli. Addition of lidocaine to propofol confers its bacteriostatic activity to the mixture and may decrease the hazard of infection associated with the extrinsic contamination of propofol.     

采用定点基因缺失突变技术在大肠杆菌中分泌鲑鱼生长激素

目的：为获得与天然鲑鱼生长激素一致的基因工程产物,对已构建的鲑鱼生长激素基因分泌型表达质粒ｐＯｓＧＨ１５３进行改造,删除所编码表达产物氨基端多余的９个碱基,方法：采用ＰＡＬＴＥＲ系统进行寡聚核苷酸指导下的基因定点缺失突变。突变质粒ｐＯｓＧＨ１５８经限制性酶切图谱及Ｓｏｕｔｈｅｒｎ印迹杂交分析加以证,结果：含有表达质粒ｐＯｓＧＨ１５８的大肠杆菌株经ＩＰＴＧ诱导后提取周质帽白,经ＳＤＳ－ＰＡＧＥ及免疫     

(His)6-Arg-Arg-人胰岛素原在大肠杆菌中的高效表达

为了提高胰岛素原的表达量，用pQE-40质粒构建了(His)6-Arg-Arg-人胰岛素原[(His)6-Arg-Arg- human Proinsulin，RRhPI]的大肠杆菌表达系统。通过培养条件的优化，在摇瓶培养的条件下，目标产物：RRhPI以包涵体形式获得了高效表达，每升培养基收获湿菌体约27g，包涵体6g(干重1．8g)，RRhPI约540mg。该水平已超过现有文献报道的摇瓶培养的最高水平。