幽门螺杆菌上皮接触诱导基因iceA1的克隆及序列特征

目的:克隆和分析镇江地区来源于不同疾病的(胃癌、溃疡和胃炎)幽门螺杆菌(Helicobacter pylori,H.pylori)的表皮接触诱导基因iceA1.方法:从胃十二指肠疾病患者胃黏膜组织中分离培养获得H.pylori,PCR扩增检测iceA1基因,并克隆至pMD18-T载体上,进行测序和序列分析.结果:克隆和测序了镇江地区来源于不同疾病的(胃癌、溃疡和胃炎)共12株H.pylori的i c e A1基因片段,并与标准菌株60190比对,结果显示镇江地区的H.pylori的iceA1基因中存在着3处框内缺失突变热点(780del6、809del5、914del7),这些缺失突变在溃疡和胃炎中均存在,但是胃癌株只存在809del5.对缺失片段周围的序列进行分析,这些缺失序列的两端基本都与同向重复序列相连,这可能与复制过程中滑动错配的小片段缺失模型有关.结论:iceA1序列的变异性有可能作为分析H.pylori群体遗传学的有用工具.     

西安地区幽门螺杆菌cagA, iceA基因与其所致疾病的相关性

目的: 研究幽门螺杆菌( H pylori) cagA、iceA基因及其不同组合对H pylori感染结局的影响, 探讨西安地区H pylori的优势致病基因型.方法: 用快速尿素酶试验(rapid urease test,RUT)筛选出H pylori阳性胃黏膜标本101例,细菌基因组DNA提取试剂盒提取DNA, 用聚合酶链反应(PCR)扩增尿素酶C( ureC)基因的方法筛选出H pylori阳性标本91例. 经PCR及琼脂糖凝胶电泳对cagA, iceA的基因亚型进行检测, 用χ2检验以及Fisher精确检验分析各基因及其不同组合与疾病的相关性.结果: cagA基因的阳性率为79.1%, iceA的总检出率为75.82%, 其中iceA1为50.5%, iceA2为38.5%, cagA+/ iceA1+的检出率高于其他组,单一基因及其不同组合在各疾病组中分布没有显著差异. iceA与cagA存在相关性. iceA2分别发现有229、334、439、549 bp以及229bp+334 bp的基因片段.结论: cagA+/ iceA1+是西安地区H pylori的优势致病基因型, cagA、iceA1、iceA2各单一基因以及其不同组合与感染的临床结局无关. iceA与cagA基因可能存在协同作用, 该地区iceA2基因有较大的变异性.     

肠聚集性大肠杆菌ybtE基因缺失突变株的构建

目的构建肠聚集性大肠杆菌ybtE缺失突变株,为下一步ybtE基因功能的研究奠定基础。方法应用PCR扩增肠聚集性大肠杆菌的ybtE基因,将其克隆至pUC18载体,酶切缺失988bp ybtE片段,并插入998bp的卡那霉素抗性基因,利用定向克隆技术将突变的ybtE片段克隆至自杀质粒pKTN701,形成重组自杀质粒。将携带重组自杀质粒的SM10λpir与EAEC17-2进行接合转移,利用同源重组,根据卡那霉素抗性筛选并鉴定接合子。结果经PCR、酶切和探针杂交鉴定,得到2株肠聚集性大肠杆菌ybtE缺失突变株。结论成功构建肠聚集性大肠杆菌ybtE基因缺失突变株。     

布鲁杆菌M16株pgm基因突变活菌苗株的构建和鉴定

目的 构建自杀性质粒载体,对布鲁杆菌M16株pgm基因进行精确突变,并对得到的pgm基因突变活菌苗株进行鉴定.方法 在puc19质粒载体上构建正向筛选标记--蔗糖敏感基因和反向筛选标记--卡那霉素抗性基因融合序列;用卡那霉素抗性基因融合序列对布鲁杆菌pgm基因进行修饰(插入突变),完成pucS1.6K自杀性质粒载体的构建;通过电转化获得布鲁杆菌M16株pgm基因突变菌株;应用PCR方法对pgm基因突变菌株进行鉴定.结果 鉴定结果显示,布鲁杆菌M16株pgm基因在卡那霉素抗性基因插入后失活,突变后的布鲁杆菌M16株pgm基因DNA片段长度约为3525 bp,与预期的相符,布鲁杆菌M16株pgm基因突变菌株构建成功.结论 构建的自杀性质粒载体能成功对布鲁杆菌进行精确毒力基因突变,为获得布鲁杆菌突变株提供了一个有效的技术平台,也为新型减毒活疫苗的研制奠定了基础.

Abstract：

Objective The construction of suicide plasmid vector could be used to make mutation of pgm gene which attenuates the virulent of Brucella melitensis strain 16, the research may lay a foundation for the development of novel live attenuated vaccines. Methods Sucrose sensitive gene as forward screening sign and fusion sequences of kanamycin resistance gene were constructed based on plasmid pucl9; pucS1.6K suicide plasmid vector was established by modifying pgm gene with fusion sequences of kanamycin resistance gene (insertion mutation); pgm gene mutation of Brucella melitensis strain 16 was obtained by electro transformation and mutation was confirmed by PCR amplification. Results The results showed that the identified Brucella melitensis strain 16 pgm gene was inactivated after insertion of kanamycin resistance gene, and the mutant pgm gene DNA fragment length was approximately 3525 bp, in line with expectations, Brucella pgm gene mutant melitensis strain 16 was successfully constructed. Conclusions The construction of suicide plasmid vector and precise mutation of Brucella melitensis strain 16 is successful, the study is not only provided an effective technology platform for constructing mutants of Brucella but also lays a foundation for the development of novel live attenuated vaccines.     

青岛地区幽门螺杆菌对左氧氟沙星耐药性及gyrA基因突变分析

目的分析青岛地区幽门螺杆菌(H.pylori)对左氧氟沙星的耐药现状,探讨H.pylori对左氧氟沙星耐药性与gyrA基因突变之间的关系。方法收集胃癌及胃炎患者的胃窦部黏膜标本,分离培养H.pylori,采用E-test法测定H.pylori对左氧氟沙星的最低抑菌浓度(MIC),提取H.pylori基因组DNA,PCR法扩增gyrA基因片段并对扩增产物测序、分析。结果从患者胃黏膜中成功分离出65例H.pylori菌株,23例对左氧氟沙星耐药(MIC>1μg/ml),占总菌株的35.4%,所有耐药菌株gyrA基因中的喹诺酮类耐药决定区(QRDR)都存在突变位点,且以第87位点的氨基酸发生突变为主,而敏感菌株中未发现基因突变位点。结论青岛地区H.pylori对左氧氟沙星的耐药率高于国内其他地区,表明gyrA基因突变是导致H.pylori对左氧氟沙星耐药的主要原因。     

利用融合PCR技术和T载体克隆技术构建结核分枝杆菌PhoPR双组份系统缺失突变载体

目的 基于融合PCR技术(SOE-PCR),T载体克隆技术,构建结核分枝杆菌(Mycobacterium tuberculosis,MTB)双组份系统缺失突变载体的方法.方法 利用SOE-PCR技术,将PhoP,PhoR和PhoPR基因上,下游的同源臂与卡那霉素抗性基因融合,构建PhoP,PhoR和PhoPR突变片段,然后将突变片段直接与T载体连接,将其转入E. coli DH5α感受态细胞并筛选抗性克隆,进而获得MTB PhoP,PhoR和PhoPR缺失突变载体.结果 成功构建MTB PhoPR双组份系统缺失突变载体,与传统方法相比,效率高,周期短.结论 基于融合PCR技术和T载体克隆技术成功构建MTB PhoPR双组份系统缺失突变载体,为下一步构建MTB突变株以及相关研究奠定基础.     

结核分枝杆菌H37Rv菌株Hsp16.3基因缺失突变株的构建与鉴定

目的 构建及鉴定结核分枝杆菌国际标准强毒株H37Rv菌株Hsp16.3基因缺失突变株。方法 体外培养结核分枝杆菌国际标准强毒株H37Rv菌株,并提取基因组DNA,扩增Hsp16.3基因两侧序列,分别插入到pKO质粒载体预定位点中,构建Hsp16.3基因置换型打靶载体,电转入结核分枝杆菌H37Rv菌株内,并与其基因组中的Hsp16.3基因同源交换,筛选出Hsp16.3基因缺失突变株。结果 通过卡那霉素筛选和蔗糖反筛选及PCR鉴定,并在含有潮霉素培养基上不能生长的菌株为Hsp16.3基因缺失突变株。结论 成功构建出结核分枝杆菌国际标准强毒株H37Rv菌株Hsp16.3基因缺失突变株。     

贵州省黔南州幽门螺杆菌克拉霉素的耐药性及23S rRNA基因突变的特征

目的:了解贵州省黔南州幽门螺杆菌(Helicobacter pylori,H.pylori)克拉霉素耐药相关基因突变情况.方法:采用改良琼脂稀释法检测分离自黔南州人民医院的74株H.pylori对克拉霉素的敏感性,选取H.pylori克拉霉素耐药菌株22株、敏感株10株,以及NCTC11637进行23S rRNA基因功能区V区片段的PCR扩增和测序,与GenBank中收录的H.pylori敏感菌株U27270相关序列进行比对和分析.结果:74株H.pylori克拉霉素耐药率为29.7%(22/74).共发现10种突变类型,其中在耐药菌株及敏感菌株23S rRNA V区均存在的基因突变包括:T2183C、T2245C和T2321C;仅在耐药株(22株)中检测到的突变包括:A2144G(4/22)、A2224G(4/22)、C2196T(1/22)、C2289 T(1/22)、A2435G(1/22)、C2695A(1/22)、T2712C(1/22).结论:黔南州医院分离的H.pylori克拉霉素耐药率较高;与耐药性相关的23S rRNA基因突变主要为A2144G和A2224G;本院菌株存在A2435G、C2695A、T2712C 3种尚未见报道的突变.     

山西省243株幽门螺杆菌药物敏感性及克拉霉素耐药基因突变特征分析

目的 了解山西幽门螺杆菌（Helicobacter pylori,H.pylori）对5种抗生素药物敏感性及其克拉霉素耐药相关基因突变特征.方法 收集临床分离的H.pylori243株,采用纸片扩散法检测H.pylori对5种抗菌药物的敏感性.选取所有耐克拉霉素及相当数量的敏感菌株,提取基因组DNA,PCR法扩增23SrRNA基因功能区并测序,测序结果采用DNAStar软件包分析.统计结果分析采用x2检验和Fisher精确概率法.结果 临床分离的243株H.pylori,药敏结果显示对甲硝唑,克拉霉素,阿莫西林,左氧氟沙星和呋喃唑酮5种药物的耐药率分别为：75.3％（183/243）,7.4％（18/243）,7.4％（18/243）,12.4％（30/243）,8.6％（21/243）,5种药物的耐药率有统计学意义（P＜0.05）.结论 H.pylori临床菌株对甲硝唑,左氧氟沙星,克拉霉素、阿莫西林及呋喃唑酮存在不同程度的耐药,以对甲硝唑耐药率最高.克拉霉素耐药菌株23SrRNA基因突变以A2143C为主,此突变可能与该地区H.pylori耐药性有关,此外,还发现了A2214G位点的突变.     

State of knowledge of helminth fauna of freshwater fishes of Poland

A total 89 fish and lamprey species has been recorded from Polish freshwater habitats. Twenty-seven of them (30.3%) have not been surveyed for parasitic helminthes. Some of the latter fishes are either rare or not easily accessible. Other live only in specific habitats in scattered localities. An important obstacle for studying parasite faunas of some fishes may be their status on an endangered species. Among the non-surveyed fishes, are those which have been relatively recently introduced to Poland or migrated there on their own. The present paper attempts to review all hitherto not studied helminthologically fish species, their habitats, localities and current protection status.     

高血压降压治疗目标的再认识

根据传统的高血压水平的定义,1993年WHO高血压治疗指南提出血压控制目标为<140/90mm Hg(1mm Hg=0.133kPa),但是并非所有患者都必须将血压降至同一水平,而应根据患者情况进行个体化治疗。Framingham进行的一项长达10～12年的心血管事件研究发现,第5年后,正常上限血压[收缩压(SBP     

Lack of correlation of degree of villous atrophy with severity of clinical presentation of coeliac disease

BACKGROUND AND AIM: Both the clinical presentation and the degree of mucosal damage in coeliac disease vary greatly. In view of conflicting information as to whether the mode of presentation correlates with the degree of villous atrophy, we reviewed a large cohort of patients with coeliac disease. PATIENTS AND METHODS: We correlated mode of presentation (classical, diarrhoea predominant or atypical/silent) with histology of duodenal biopsies and examined their trends over time. RESULTS: The cohort consisted of 499 adults, mean age 44.1 years, 68% females. The majority had silent coeliac disease (56%) and total villous atrophy (65%). There was no correlation of mode of presentation with the degree of villous atrophy (p=0.25). Sixty-eight percent of females and 58% of males had a severe villous atrophy (p=0.052). There was a significant trend over time for a greater proportion of patients presenting as atypical/silent coeliac disease and having partial villous atrophy, though the majority still had total villous atrophy. CONCLUSIONS: Among our patients the degree of villous atrophy in duodenal biopsies did not correlate with the mode of presentation, indicating that factors other than the degree of villous atrophy must account for diarrhoea in coeliac disease.