CONFORMATION STUDY OF THE LARGE FRAGMENT OF E. COLI DNA POLYMERASE I BY STM

The large fragment of E.coli DNA polymerase I is imaged by scanning tunneling microscope. The specimen is deposited on highly oriented pyrolytic graphite surface, and then covered with pure paraffin oil in order to maintain hydration of the molecules. Images of the enzyme reveal an ellipsoid shape of 5.5-6.0nm wide and 7.0 -7.5 nm long. The conformation of the enzyme is in agreement with the model derived from X- ray crystallography studies.

A simplified method for reconstituting active E.coli DNA polymerase Ⅲ

Genome duplication in E.coli is carried out by DNA polymeraseⅢ,an enzyme complex consisting of ten subunits.Investigations of the biochemical and structural properties of DNA polymeraseⅢrequire the expression and purification of subunits includingα,ε,θ,γ,δ′,δ,andβseparately followed by in vitro reconstitution of the polⅢcore and clamp loader.Here we propose a new method for expressing and purifying DNA polymeraseⅢcomponents by utilizing a protein coexpression strategy.Our results show that the subunits of the polⅢcore and those of the clamp loader can be coexpressed and purified based on inherent interactions between the subunits.The resulting polⅢcore,clamp loader and sliding clamp can be reconstituted effectively to perform DNA polymerization.Our strategy considerably simplifies the expression and purification of DNA polymeraseⅢand provides a feasible and convenient method for exploring other multi-subunit systems.

大肠杆菌表达重组Taq DNA聚合酶的分离和纯化

以插入Taq DNA聚合酶基因的pET-24a表达质粒转化E．coli菌株,表达Taq DNA聚合酶,用反复冻融的方法裂解菌体．表达产物为可溶性蛋白,采用Duo-Flow系统(Bio-Rad)及S柱,Tris缓冲液pH为7．5．以0-1 mol／L盐梯度洗脱方式,收集洗脱峰．透析去盐可得到高纯度的产品．经PCR反应与商品Taq酶的一个活性单位相比较,重组的Taq酶活性有1个单位．用该方法可以从1 L培养液中分离得到5．51 mg产品．

人γ心钠素在大肠杆菌中的高效表达

利用聚合酶链式反应（ＰＣＲ）技术扩增人γ心钠素（γ－ｈＡＮＰ）ｃＤＮＡ编码序列，在其５′和３′端分别引入ＥｃｏＲⅠ和ＢａｍＨⅠ限制性内切酶位点，定向克隆到表达质粒载体ｐＭＳ－３１ｂ，在大肠杆菌ｐｏｐ２１３６中高效表达出人γ心钠素融合蛋白，表达产物占菌体总蛋白的３０～４０％。双向免疫扩散法测定证明表达产物与人α心钠素（α－ｈＡＮＰ）抗血清呈阳性反应，纯化的表达产物经复性处理后能引起大鼠胸主动脉条舒张．

Pfu酶原核的高效表达及其活性分析

目的:在大肠杆菌中高效表达重组Pfu酶。方法:将pfu基因构建于载体pET28a上,重组质粒pET28a-pfu转化DH5α,获得了含pET28a-pfu重组表达菌株。在重组菌株OD600为0.2时,经终浓度为1 mmol/L的IPTG诱导表达10～12 h后,菌体超声波破壁后,80℃条件下热变性30 min去除部分杂蛋白;粗酶液再经Ni离子亲和层析进一步纯化。结果:获得了高纯度的重组Pfu酶,利用该酶成功扩增出目的基因片段。结论:纯化的Pfu酶具有较高的活性,比活性为62 500 U/mg,该研究表达的重组酶完全可以替代商用Pfu酶。

肠出血性大肠埃希菌(O_(157)∶H_7)的基因同源性的分析

目的 对江苏省徐州地区O157∶H7的病原学进行分析。方法 采用聚合酶链反应对O157∶H7菌株毒力基因谱进行检测 ,同时用脉冲凝胶电泳 (PFGE)和随机扩增多态性DNA(RAPD)方法对O157∶H7菌株的同源性分析比较。结果 流行地区分离的O157∶H7菌株 ,10 0 %携带Hly、eaeA基因 ,95 .35 %携带SLT2 基因 ,11.6 3%携带SLT1基因。脉冲凝胶电泳图谱表明流行地区分离的O157∶H7菌株与日本分离的O157∶H7菌株有明显差异 ,为不相关菌株 ;与国内标准菌株 882 36 4为近似型(相似 ,但不相同 )。流行地区患者分离菌株与外环境家畜家禽粪便及昆虫肠道分离菌株的脉冲凝胶电泳图谱完全相同。结论 携带O157∶H7菌株的家畜家禽可能是导致疫情发生的传染源。脉冲凝胶电泳方法用于O157∶H7病原学分析 ,对流行病学研究有重要意义。随机扩增多态性DNA方法用于O157∶H7病原学分析 ,技术简便、省时。