艰难梭茵毒素基因3''''末端重复区域基因片段的PCR克隆

目的探寻一种简单、快速、灵敏的检测艰难梭菌的方法.方法以艰难梭菌毒素A和毒素B基因3'末端高度保守重复区分别设计一对引物,进行PCR反应,同时在同一体系相同反应条件下使用相同引物分别以大肠杆菌和乳杆菌DNA为模板进行扩增,比较二者结果.结果从艰难梭菌成功克隆了毒素A基因3'端重复区域960 bp的基因片段及毒素B基因3'端重复区域1851 bp的基因片段,不同来源的菌株出现了相同的2条电泳带,并通过测序鉴定;而对照的大肠杆菌和乳杆菌株无特异电泳带出现.结论从艰难梭菌毒素A、毒素B基因3'末端重复区域克隆的基因片段具有保守性,可以作为基因探针在临床上进行艰难梭菌检测,该方法简便、灵敏,可直接用粪便标本进行检测.此外,这些基因编码的多肽具有较高的疏水性并存在着膜受体结合区域,可以此为基础进行基因工程蛋白质疫苗的研究.     

应用聚合酶链式反应快速检测嗜肺军团杆菌

嗜肺军团杆菌(LP)所引起的肺炎病死率高,易暴发流行,早期病原诊断困难。本研究应用聚合酶链式反应技术对嗜肺军团杆菌血清1、3、5型中巨噬细胞感染潜能基因(mip基因)的部分编码区域进行特异性扩增,经琼脂糖凝胶电泳、溴乙锭染色检测扩增产物。结果3型LP均出现650bp特异性阳性条带。检测灵敏度为80ng,本研究结果对嗜肺军团杆菌肺炎的早期诊断、及时治疗、降低病死率及控制暴发流行有重要意义。     

High level production of soluble single chain antibodies in small-scale Escherichia coli cultures

We have investigated the effect of growth and induction conditions on the production of soluble single-chain Fv antibody fragments in Escherichia coli under the control of wt lac promoter. The scFv was directed into the periplasmic space by a pelB leader sequence. Addition of sucrose to the medium gave a 15–25-fold increase in the yield of soluble scFv-phOx (3.0 mg/l) for bacterial shake-tube cultures and an increase of 80–150-fold (16.5 mg/l) for shake-flask cultures. Using flask culture in the presence of 0.4 M sucrose, a significant amount of scFv was released into the medium. We found that the scFv could be made to accumulate in the periplasm or be secreted into the medium by simply changing the incubation conditions and the concentration of the inducer. The ratio between soluble antibody fragments and insoluble scFv aggregates proved to be dependent on the strength of the promoter. Lowering the incubation temperature below 20°C had no effect on the yield of soluble antibody fragments in the periplasm, but they were no longer secreted into the medium. An example of high level production in shake-flask cultures and one-step purification by immobilized metal affinity chromatography (IMAC) is described for a soluble scFv specific for the T cell surface antigen CD3. The biological activity of the purified anti-CD3 scFv was demonstrated by flow cytometry. This method should be especially useful for the functional screening of a large number of clones in small-scale cultures.     

一种直接高效克隆PCR产物的载体制备

Ｂｌｕｅｓｃｒｉｐｔ经ＥｃｏＲ Ｖ酶切后，在Ｔａｑ酶作用下加入ｄＴＴＰ使其３’端加上碱基“Ｔ”而成为３’端突出的粘性末端载体。用此载体直接与ＰＣＲ产物连接进行克隆。克隆效率比采用平端载体克隆的效率提高约５０倍。     

解脲脲原体与自然流产关系的探讨

采用聚合酶链反应技术对３２例自然流产患者的宫颈粘液及其中２２例清宫组织进行解脲脲原体（ＵＵ）检测，并与３０例人工流产妇女比较。结果表明：自然流产与ＵＵ寄生于宫颈无关（Ｐ＞０．０５），与宫内感染ＵＵ明显相关（Ｐ＜０．０５）。     

IMMUNOLOGICAL DIAGNOSIS OF LIGHT CHAIN DISEASE ANALYSIS OF 11 CASES FOUND IN FUJIAN PROVINCE

The present paper reports 11 cases of light chain disease (LCD) sequently found in several citles over Fujian province, Immunological classification of this group of LCD ww as follows: six of me cases belonged to type λ, four of them were type κ, and another one was a double LCD. We found that LCD was common in Fujlan only next to multiple myeloma (MM) of IgG class and accounted for 20% of the total 55 MM cases found in recent yean.It to well known that In matt patients of LCD M protein or Bence Jones proteinemia (BJPemia) to not detectable by conventional electro-phoresis. Our studies show that by making serum protein along with urinary BJP electrophoresis on the same one gel plate the sltuation can be greatly Improved It not only favour* the recognition of smail and faint band or bands of free light chain in serum, but also provides a repid and sensitive way, i. e. , immunofixation, to directly detect urinary light chain on the gel plate Immediately after electrophresis has been run.     

听骨链多层螺旋CT曲面重建及临床应用

目的研究听骨链的CT曲面重建(CPR)方法,评估其临床应用价值。方法165耳(122例)行颞骨轴位高分辨率CT检查,对听骨链行CPR。50例(80耳)为正常听骨链,72例(85耳)为病变组。主要扫描参数:准直0.5mm,螺距0.875,重建间隔0.2~0.3mm,重建矩阵1024×1024。结果(1)正常听骨链组:1幅图像上清晰显示3个听骨及其关节。(2)临床应用:39例颞骨外伤中,听骨链异常的18例;锤砧关节半脱位及脱位各5例,砧镫关节半脱位及脱位分别为5例及6例,锤骨柄骨折1例。16例外耳发育不良中,骨性外耳道闭锁13例,膜性3例。骨性外耳道闭锁最常见的伴随听骨畸形是锤骨柄发育不全(10例),中耳腔变窄、听骨缺如3例,锤砧融合伴砧骨长突缺如1例。不伴随先天性外耳发育不良的听骨畸形2例,1例为双耳砧骨长突缺如,另1例为砧骨长突发育不全伴砧镫关节不连。慢性中耳乳突炎15例,6例合并胆脂瘤者听骨均有不同程度的破坏,鼓室硬化症1例。结论听骨链的多层螺旋CT曲面重建是诊断传导性耳聋的有效方法。     

异基因造血干细胞移植后巨细胞病毒的检测及临床意义

目的 为了降低异基因造血干细胞移植（allo-HSCT）后巨细胞病毒（CMV）感染的相关死亡率，寻求一种更快捷、更特异的诊断CMV感染的分子生物学方法。方法 2001年4月至2003年4月，从79例接受异基因造血干细胞移植的患者中采集了135份外周血标本。采用巢式聚合酶链反应（nested-PCR）方法检测患者外周血中CMVgB DNA，阳性标本做酶切分型，部分行序列测定。结果CMVgB DNA检测中有42例患者（53．9％）的66份标本（48．9％）阳性；对其中阳性患者的45份标本进行了酶切分型，结果 CMV gB1型21例（46．7％），CMV gB2型14例（31．1％），CMV gB3型7例（15．6％），CMV gB4型3例（6．7％）。先后有2种型别的CMV感染者有3例（3．8％）。经分析，CMV gB阳性和CMV gB阴性患者GVHD的发生率分别为81．0％和32．4％（P＜0.01）；CMV gB1、gB2、gB3以及gB4型Ⅱ～Ⅳ度急性GVHD及慢性GVHD的发生率分别为：81．0％、50．0％、42．9％和0。结论 Nested-PCR方法可快捷特异地检测CMV感染。CMVgB1、2型中，重度急性GVHD和慢性GVHD发生率较高。CMV gB基因分型检测可有效地指导临床抗病毒治疗，且对移植后患者的临床转归有一定的预测价值。     

成牙本质细胞中上游刺激因子1对骨桥素表达调控的研究

目的检测成牙本质细胞中上游刺激因子1（upstream stimulatory factor 1，USF1）对骨桥素表达的影响。方法培养成牙本质细胞MDPC-23，稳定转染PCMV-USF1和酸性-USF（A-USF）质粒，提取总RNA，半定量反转录聚合酶链反应检测骨桥素的表达水平，并对结果进行统计学分析。结果筛选出稳定转染USF1和A-USF的抗性克隆，USF1、A-USF转染组和对照组骨桥素相对灰度比值分别为：60．33％±4．51％、229．33％±7．09％、110．00％±15．62％，转染组与对照组差异有统计学意义（P〈0．01）。结论USF1可调控成牙本质细胞骨桥素mRNA转录，该作用被A-USF部分阻断。     

消毒器械供应链的信息化管理

[目的]对国内外消毒器械供应链的管理进行分析，为国内消毒器械供应链的跟踪管理提供依据。[方法]对国内外消毒器械供应链的信息化管理方式进行分析。[结果]消毒器械跟踪管理系统在国外已有成功应用的实例，效果良好；国内医疗机构至今还没有成功应用器械跟踪管理系统的相关报道。[结论]消毒器械跟踪管理系统是目前管理消毒器械供应链的有效途径。