幽门螺杆菌cagA基因探针的制备与制备应用

目的 研究ｃａｇＡ＋Ｈｐ（幽门螺杆菌）与胃肠道疾病的关系，并探讨长臂光敏生物素标记的ｃａｇＡ基因探针的临床意义。方法 构建含ｃａｇＡ基因片段的重组质粒克隆株，以重组质粒ＤＮＡ为模板ＰＣＲ扩增ｃａｇＡ基因片段用长壁光敏生物素标记产物ＤＮＡ，对病人进行基因探针检测及ＰＣＲ、尿酶试纸检测。结果 浅表性胃炎、深度胃炎、胃溃疡、十旨肠 治疗慢性萎缩性胃炎及胃癌病人的检测结果是：尿酶检测阳性百分率为：７０．３     

cagA基因阳性幽门螺杆菌培养滤液对人胃粘膜上皮细胞系GES-1生长的影响

目的：研究cagA^ 幽门螺杆菌（Hp）培养滤液对人胃粘膜上皮细胞（GES－1）的作用及机制。方法：制备Hp培养滤液，PCR鉴定cagA基因。采用倒置显微镜、电镜、细胞生长曲线、克隆形成实验、单细胞微凝胶电泳及流式细胞仪等，观察Hp (cagA^ ）培养滤液对GES－1细胞的作用。结果：经Hp(cagA^ )培养滤液处理GES－1细胞，细胞核增大、畸形、核染色质变粗、核仁肥大、核分裂。生长曲线可见细胞增生活跃，增殖率195％。克隆形成试验显示细胞克隆形成能力增强，增殖率达到337．5％。流式细胞仪S期细胞比率显著高于对照组。Hp(cagA^ ）培养滤液可使GES－1细胞形成彗星现象。结论：Hp(cagA^ )培养滤液可以导致GES－1细胞的生长特性改变，呈现肿瘤细胞的形态学及生长特征。DNA损伤可能是cagA诱导GES－1细胞生长特性改变的机制之一。     

幽门螺杆菌中国株cagA全长基因克隆

世界范围内不同Hp菌株CagA分子变异较大，该变异可能导致不同菌株的cagA生物学作用乃至致病性不同。我国作为Hp感染较严重的国家之一，临床分离株中超过90％含有cagA基因，从克隆和分析全长cagA基因入手，开展cagA基因／CagA蛋白功能研究，对于揭示CagA在Hp致病中的分子机制具有现实意义。     

中国幽门螺杆菌cagA基因敲除突变株生物学特性分析

细胞毒素相关基因A蛋白（cytotoxin-associatedgeneA，CagA）是幽门螺杆菌（Hp）最重要的毒力因子之一。流行病学研究认为，CagA阳性肋感染与消化性溃疡和胃癌发生的关系比CagA阴性菌株更为密切，提示CagA可能是一种重要的疾病相关蛋白，在坳致病过程中发挥作用。由于却菌株自身的变异，CagA阳性株与CagA阴性株间除CagA基因外存在许多差异之处，因此，在自然状态下很难对CagA基因功能做出真实判断。     

浙江地区幽门螺杆菌cagA、vacA、iceA基因型别分析

据初步统计 ,全世界超过 5 0 %的人群有幽门螺杆菌 (Ｈｅｌｉｃｏｂａｃｔｅｒｐｙｌｏｒｉ,Ｈｐ)感染。其中西方国家的感染率为 2 5 %～ 5 0 % ,发展中国家有的高达 90 % 〔1〕。然而 ,只有少数感染者发展成消化性溃疡或胃癌 ,其中的原因尚不清楚。有研究表明 ,Ｈｐ的ｃａｇＡ、ｖａｃＡ、ｉｃｅＡ基因可能与疾病的发生有关。有关ｃａｇＡ、ｖａｃＡ与疾病的关系我国也有报道〔2〕。本文研究了浙江地区Ｈｐ菌株的ｃａｇＡ、ｖａｃＡ、ｉｃｅＡ基因型别并分析了与疾病的关系。材料和方法Ｈｐ菌株的来源 :Ｈｐ菌株分离自 60例胃、十二指肠疾…     

镇江地区幽门螺杆菌cagA、vacA基因及亚型与胃肠病患者的相关性

我们利用分子生物学技术，研究镇江胃肠疾病患者感染幽门螺杆菌（Hp）菌株的cagA基因型、vacA基因型，探讨基因型与胃肠疾病的关系。     

幽门螺杆菌ureB基因的克隆及序列分析

目的 对幽门螺杆菌（Hp)ureB核苷酸序列及推定氨基酸序列进行同源性比较分析,确定Hp菌株的ureB基因差异性,为疫苗的研究和诊断用品的评价提供依据。方法 自选设计引物,PCR扩增来自国内不同病人的4株Hp菌株的ureB基因,与pGEM-T载体克隆连接、测序;进一步同GenBank上发表的其他4株国外Hp菌株ureB的全基因序列进行比较分析。结果 8株Hp菌株的ureB基因序列出现了33种长度,CAPMF3和CPM630分别为1269bp和1680bp,其他为1710bp。6株1710bp的ureB基因序列的分析表明,国外3株Hp的ureB同源性较高,三者氨基酸的同源性达到100％。国内3株Hp的ureB变异较大,推定氨基酸同源性为98．7％－98．9％。结论 UreB作为保护性同时存在免疫保护覆盖率问题,研究疫苗和诊断用品时要注意选择使用在人群中流行占优势的Hp菌株的高度保守的UreB抗原肽,使其具有更高的覆盖率。     

幽门螺杆菌毒力基因cagA、cacA与胃十二指肠疾病的关系

国际上已有很多国家对本国幽门螺杆菌 (Ｈｅｌｉ ｃｏｂａｃｔｅｒｐｙｌｏｒｉ,Ｈｐ)分离株的ｖａｃＡ基因进行了研究和分析。Ａｔｈｅｒｔｏｎ等曾提出ＨｐｖａｃＡ基因镶嵌型模型 ,将信号序列分为三型 ,中间序列分为两型〔1〕 ,但来自欧洲和日本的报告显示不同地区Ｈｐ分离株具有较高多态性 ,具有地区、人群相关性特点。然而 ,目前国内尚未见这方面的研究报告。为了解我国Ｈｐ临床分离株ｖａｃＡ基因型特点以及不同ｖａｃＡ基因同胃、十二指肠疾病的关系以及ｃａｇＡ基因同Ｈｐ毒力的关系 ,本实验进行了以下研究。材料与方法菌株 :113株…     

幽门螺杆菌omp11基因的克隆及序列分析

目的　对幽门螺杆菌 (Helicobacterpylori,Hp)郑州分离株MEL Hp2 7和NCTC116 37株外膜蛋白基因 (omp11)进行克隆和测序 ;确定不同Hp菌株omp11基因序列的变异性 ,并对该基因编码多肽的化学及免疫学特性进行预测 ,为幽门螺杆菌疫苗抗原的筛选提供数据。方法　提取Hp染色体DNA ,用自行设计的PCR引物 ,从染色体DNA上扩增出omp11基因 ,将其克隆到载体pNEB193中 ,用重组质粒转化大肠杆菌 (E .coliJM10 9)。对重组质粒进行酶切鉴定 ,对插入的omp11基因片段进行测序 ,应用生物信息学软件Omiga2 .0、GeneDoc2 .3和GenBank、Swiss port数据库对 4个Hp菌株 (MEL Hp2 7、NCTC116 37、2 6 6 95、J99)的omp11基因序列进行同源性分析 ,并对该基因编码多肽的主要化学特征和抗原结构域进行预测。结果　MEL Hp2 7和NCTC116 37株omp11基因的核苷酸序列长度均为5 6 1bp ,不同菌株间核苷酸序列的同源性为 96 .6 %～ 98.0 % ,与国内的MEL Hp2 7株同源性最高的菌株是NCTC116 37,二者的同源性为 97.9%。 4株Hpomp11基因编码的氨基酸序列长度均为 186aa ,不同菌株间氨基酸序列的同源性为 98.9%～ 10 0 % ,与MEL Hp2 7株同源性最高的菌株是 2 6 6 95 ,二者的同源性为 99.5 %。预测HpMEL Hp2 7omp11基因编码多肽的相对分子质量 (Mr)     

幽门螺杆菌cagA蛋白的制备级复性和纯化

目的通过优化复性液的组成、pH值、稀释度等,建立幽门螺杆菌细胞毒素相关基因A(cytotoxin associated gene A,cagA)表达产物的制备级复性及纯化方法。方法以包涵体形式存在的cagA表达产物先用5mL/LTritonX-100进行初步纯化,使其纯度达到约90%;再用含8mol/L脲的变性剂溶解包涵体,用复性液[0.1mol/LTris-HCl、2mmol/L乙二胺四乙酸(EDTA)、0.1mmol/L氧化型谷胱甘肽(GSSG)、8mmol/L还原型谷胱甘肽(GSH)及50mmol/L精氨酸(Arg),pH8.0]稀释复性后,用DEAE-HP阴离子交换色谱进行纯化。结果用稀释法复性及液相色谱法纯化,一次可得到100~140mg的cagA。ELISA检测其抗原活性为1∶16000;SDS-PAGE检测纯度为98%。结论用制备级复性、纯化制备的cagA,可作为抗原用于制备检测抗幽门螺杆菌抗体的检测芯片,效果良好,并已获得国家新药批文。     

Detection of Helicobacter pylori cagA gene in gastric biopsies, clinical isolates and faeces

Purpose: Helicobacter pylori infection is common in the developing countries. The cagA gene is a marker of pathogenicity island (PAI) in H. pylori. The aim of this study was to determine the prevalence of cagA among dyspeptic patients in Bahrain directly from gastric biopsy and stool specimen. Methods: A total of 100 gastric biopsy samples, 16 clinical isolates and 44 faecal specimens were collected from Bahraini adult dyspeptic patients. cagA gene of H. pylori was assessed using polymerase chain reaction (PCR). Results: The cagA gene was detected in 59 (59%) from biopsy specimens, 10 (62%) clinical isolates and in 10 (22.7%) faecal specimens. The detection of cagA positive H. pylori was significantly higher in patients with duodenal ulcer (80%) compared to those with other endoscopic finding (42%) (P<0.05). Conclusions: Using PCR to detect cagA gene directly from biopsy is a rapid and reliable technique. However, using stool specimen for genotyping in our patients showed reduced sensitivity.     

Distribution of vacA alleles and cagA status of Helicobacter pylori in peptic ulcer disease and non-ulcer dyspepsia

The occurrence of cagA and vacA alleles among Helicobacter pylori isolates from Turkish patients and their relationship with ulcer disease outcome was investigated. Among isolates from 47 patients with peptic ulcer disease and 51 patients with non-ulcer dyspepsia, 72.3% and 44.4%, respectively, were cagA-positive (p 0.019). Most (88.8%) isolates were typed as vacA s1, and all of these were subtype s1a. The commonest (51.0%) vacA genotype was s1a m1. The results of multivariate analysis indicated that infection with cagA-positive H. pylori was the only variable associated with an increased risk of peptic ulcer disease (odds ratio, 3.01; 95% confidence interval, 1.27-7.10; p 0.012).     

The EPIYA-ABCC motif of Helicobacter pylori cagA gene and gastric carcinogenesis in Casablanca population

BackgroundH. pylori infection induce atrophic gastritis (AG) and intestinal metaplasia (IM) that can lead to gastric cancer (GC). The severity of gastric lesions is related to H. pylori genetic diversity. The oncogenic potential of H. pylori cagA virulence factor is linked to its high polymorphic EPIYA motifs.ObjectivesOur aim was to evaluate the association of EPIYA motifs with the risk of AG and IM in Casablanca population.MethodsA total of 210 patients suffering from gastric lesions (chronic gastritis, AG, and IM) was enrolled. H. pylori infection and the type of lesions were diagnosed by ureC PCR and histological examination, respectively. Detection of the cagA gene, and the type of EPIYA motifs, were carried out by PCRResultsThe prevalence of H. pylori and cagA gene was 95% and 37%, respectively. CagA-positive strains were associated with the risk of IM. The EPIYA motifs detected were: EPIYA-ABC (58%), EPIYA-ABCC (22%), and EPIYA-AB (20%). The EPIYA-ABCC motif was associated with the risk of IM (p-value = 0.007), compared to AG (p-value = 0.28).ConclusionThe EPIYA-ABCC motif might be a useful marker for the identification of patients at high risk of developing IM that can lead to GC.     

Relationship between bacterial load,morbidity and cagA gene in patients infected by Helicobacter pylori

One hundred and seventy-six biopsies of the gastric corpus and antrum from 97 patients were processed using classical and molecular methods in order to study the relationship between the factor cagA of Helicobacter pylori, bacterial load and morbidity. Bacterial load in patients with cagA was greater than in patients without it, both in the antrum and corpus (p < 0.01). There was a statistically significant association between cagA and consumption of proton pump inhibitors (adjusted odds ratio 3.11). Haemorrhage of the upper digestive tract was more associated with bacterial load than with the cagA gene (adjusted odds ratio 2.34 and 1.12, respectively), but none of these associations yielded statistical significance.     

标准菌株和临床菌株oipA基因的检测及其核苷酸序列比对

目的：检测幽门螺杆菌标准菌株NCTC11637及临床分离菌株Hp1和Hp2的oipA基因,分析其核苷酸序列,比对其与国际标准菌株Hp 26695的同源性.方法：常规方法培养幽门螺杆菌,提取DNA,PCR法扩增oipA基因,检测其核苷酸序列,并比较其与Hp 26695的同源性.结果：NCTC11637及Hp1、Hp2均表达oipA基因.其核苷酸序列与Hp 26695比对,NCTC11637有48个突变位点、Hp1有48个突变位点、Hp2有50个突变位点,同源性均为94%.NCTC11637与Hp1的同源性为100%、与Hp2的同源性为97%.结论：NCTC11637、Hp1、Hp2均表达oipA基因,但不同菌株oipA基因的核苷酸序列有所不同.     

Genotyping of the cagA gene of Helicobacter pylori on immunohistochemistry with East Asian CagA-specific antibody

The cytotoxin-associated antigen A (CagA) of Helicobacter pylori prevalent in East Asian countries, where the mortality rate due to gastric cancer is high, has been reported to be structurally different from that in Western countries, where the gastric cancer mortality rate is relatively low. Based on the structural features of the EPIYA motifs located at the carboxyl terminal of the protein, CagA was subdivided into two types: East Asian CagA and Western CagA. A recent study suggested that immunohistochemistry with anti-East Asian-specific antibody (α-EAS Ab), which was specifically immunoreactive with East Asian CagA but not with Western CagA, may be useful for diagnosis of the cagA genotype. To further evaluate the value of this diagnostic method in terms of sensitivity, specificity, and accuracy, 143 gastric biopsy specimens with α-EAS Ab were analyzed on immunohistochemistry and compared with the sequencing of the cagA gene. It was found that diagnosis of the cagA genotype of H. pylori on immunohistochemistry using the α-EAS Ab was highly sensitive (sensitivity 93.2%) and specific (specificity 72.7%), suggesting that immunohistochemical diagnosis of the cagA genotype is useful for diagnosis of the cagA genotype.     

长臂光敏生物素和光敏地高辛核酸探针检测医学真菌灵敏度的比较研究

建立应用长臂光敏生物素核酸探针和光敏地高辛核酸探针杂交。检测真菌的方法，并比较二者检测临床常见医学真菌的敏感性。设计并合成医学真菌通用引物，用PCR扩增白色念珠菌标准株的核糖体RNA基因，用长臂光敏生物素或光敏地高辛标记其纯化的扩增产物制备成相应的核酸探针，然后用上述两种探针对标本中的PCR扩增产物进行Southern杂交，检测医学真菌。通过用白色念珠菌感染大鼠，建立念珠菌病的实验动物模型，验证血培养法、PCR法和PCR扩增结合Southern杂交，检测真菌的敏感性。结果表明，这两种核酸探针可分别与白色念珠菌、热带念珠菌、新型隐球菌、黄曲霉菌、烟曲霉菌等9种真菌杂交呈阳性结果：与临床常见的细菌、病毒和哺乳动物组织细胞DNA杂交结果为阴性。利用实验动物模型比较了血培养法、PCR法和PCR扩增结合长臂光敏生物素核酸探针Southern杂交法检测真菌血症的灵敏度，实验证明后者的敏感性最高。总之，应用长臂光敏生物素核酸探针和光敏地高辛核酸探针与标本PCR扩增物杂交，检测上述真菌既特异和敏感，又不需放射性同位素。光敏地高辛核酸探针检测医学真菌的灵敏度略高于长臂光敏生物素核酸探针。