幽门螺杆菌尿素酶B亚单位编码基因的克隆与序列分析

目的 幽门螺杆菌(Helicobacter pylori)尿素酶B亚单位的编码基因(ureB)的克隆和序列分析，为H．pylori基因工程疫苗的研究奠定基础。方法应用PCR方法获得国内分离H．pylori菌株MEI，HP27和国际标准参考株H．pylori的ureB基因，通过定向克隆的方法分别插入克隆载体pNEB193中，用质粒酶切电泳和特异PCR方法鉴定重组质粒。克隆基因经测序后进行核苷酸和氨基酸的同源性比较。结果 重组质粒经双酶切后得到1．71kb的ureB基因片段，特异PCR可扩增出ureB基因片段，证实H．pylori ureB基因的重组克隆质粒构建成功。经测序，国内分离H．pylori MEL-HP27的ureB基因全长1710bp( Genbank收录号：AY295085)，编码由569个氨基酸残基组成的肽链，ureB基因序列与GenBank公布的H．pylori相应基因同源性高达96．08％～98．30％，氨基酸序列同源性在98．77％．99．82％之间。结论 成功克隆了MEL-HP27菌株的ureB基因，其核苷酸序列与国际参考株NCTC11637的同源性为97．67％。     

幽门螺杆菌热休克蛋白A亚单位编码基因的克隆与序列分析

目的 幽门螺杆菌(Helicobacter pylori)热休克蛋白A亚单位的编码基因(hspA)克隆和序列分析，为H.pyiori基因工程疫苗的研究奠定基础。方法 应用PCR方法获得国内分离H.pylori菌株MEI，HP27和国际标准参考株H.pylori的hspA基因，通过定向克隆的方法分别插入克隆载体pNEB193中，用质粒酶切电泳和特异PCR方法鉴定重组质粒。克隆基因经测序后进行核苷酸和氨基酸的同源性比较。结果 重组质粒经双酶切后得到0．35kb的hspA基因片段，特异PCR可扩增出hspA基因片段，证实H.pylori hspA基因的重组克隆质粒构建成功。经测序，国内分离HpMEL-HP27的hspA基因全长357bp(Genbank收录号：AY295084)，编码由118个氨基酸残基组成的肽链，hspA基因序列与GenBank公布的H.pylorl相应基因同源性高达95．20％～97．48％，氨基酸序列同源性在95．76％～97．46％之间。结论 克隆了H．pylori菌株MEL-HP27的hspA基因，其核酸序列与国际参考株NCTC11637同源性为97．48％。     

幽门螺杆菌47kDa微孔蛋白基因的克隆表达及特性鉴定

目的对人幽门螺杆菌(H.pylori)编码47kDa外膜微孔蛋白基因omp47克隆表达及特性鉴定,探索研制H.pylori疫苗的新途径。方法培养和收集H.pylori标准菌株NTTC11637及临床菌株,采用酚∶氯仿抽提、纯化基因组DNA。设计上下游引物,并以该基因组为模板,采用聚合酶链反应(PCR)扩增omp47基因片断。将目的基因和与克隆载体PGEM-T连接后,转化至E.coli DH5α及BL21,碱裂解提取质粒经BamHI和XhoI双酶切鉴定并进行序列分析。重组蛋白采用IPTG诱导表达后,经SDS-聚丙烯酰胺凝胶电泳(PAGE)鉴定、镍亲和层析纯化检测抗原活性。结果经酶切、测序分析表明,插入的基因片断为1284bp,编码427个氨基酸。与GenBank公布的H.pylori标准株26695、43504及J99序列比对,核苷酸同源性高达94%~96%,编码氨基酸同源性96%~99%。经SDS-PAGE检测,电泳图谱上显示一条相对分子量为47kda的新生蛋白带。Western blot检测表明重组蛋白有较好的抗原性。结论成功克隆了外膜微孔蛋白OMP47的编码基因,为H.pylori疫苗的研制和试剂盒的开发奠定了良好的基础。     

幽门螺杆菌NCTC 11637 cagT基因的克隆及序列分析

目的:克隆幽门螺杆菌(H pylori)NCTC 11637 cag T(HP0532)的编码基因,并分析其核苷酸序列.方法:应用PCR技术从H pylori基因组DNA中扩增cag T编码基因片段,克隆至pGEM-T载体后,再将其定向插入pQE30载体中,双酶切鉴定筛选阳性克隆,并进行序列分析.结果:NCTC 11637 cag T基因全长843 bp(GenBank登录号为EF114758),编码280个氨基酸,与GenBank公布的其他H pylori菌株基因序列的核苷酸同源性为97%-99%.结论:成功克隆了cag T基因,为进一步研究其生物学功能奠定了基础.     

镇江地区H pylori的cagA基因及其3′可变区结构的变化

目的:评估镇江地区H pylori临床株的cagA基因阳性率、了解菌株CagA蛋白的可能分型、其羧端可变区EPIYA的数目以及与临床结果之间有无关联.方法:培养分离来自临床胃十二指肠疾病患者的H pvlori,提取基因组DNA,通过PCR方法检测H pylori cagA基因的状况.随机选择不同病种的cagA基因阳性(cagA~ )的PCR产物.通过转化质粒,进行cagA~ PCR产物测序,通过ExPASY-Tranlation软件获得cagA基因相应的CagA蛋白的氨基酸序列.国际标准株NCTC11637的cagA基因以及CagA蛋白序列通过搜索NCBI(www.ncbi.nlm.nih.gov)数据库获得.结果:60株H pylori临床株中,56例为cagA~ ,阳性率达93.3%.20例测序的结果表明,CagA蛋白结构可分为2种类型,19株东亚型和1株西方型.所有19株东亚型均为Yamaoka分型的A型,所有20例菌株的EPIYA的数目均为3个,与临床结果无关联.结论:cagA基因阳性率在不同病种之间无差异.镇江地区H pylori临床株的CagA蛋白的一级结构,与日本、韩国菌株一样,主要为东亚型,其CagA蛋白羧端可变区EPIYA数目均为3个,与临床结果无关联.     

镇江地区H pylori的cagA基因及其3'可变区结构的变化

目的:评估镇江地区H pylori临床株的cagA基因阳性率、了解菌株CagA蛋白的可能分型、其羧端可变区EPIYA的数目以及与临床结果之间有无关联.方法:培养分离来自临床胃十二指肠疾病患者的H pylori,提取基因组DNA,通过PCR方法检测H pylori cagA基因的状况.随机选择不同病种的cagA基因阳性(cagA )的PCR产物,通过转化质粒,进行cagA PCR产物测序,通过ExPASY-Tranlation软件获得cagA基因相应的CagA蛋白的氨基酸序列.国际标准株NCTC11637的cagA基因以及CagA蛋白序列通过搜索NCBI(www.ncbi.nlm.nih.gov)数据库获得.结果:60株H pylori临床株中,56例为cagA ,阳性率达93.3%.20例测序的结果表明,CagA蛋白结构可分为2种类型,19株东亚型和1株西方型.所有19株东亚型均为Yamaoka分型的A型,所有20例菌株的EPIYA的数目均为3个,与临床结果无关联.结论:cagA基因阳性率在不同病种之间无差异.镇江地区H pylori临床株的CagA蛋白的一级结构,与日本、韩国菌株一样,主要为东亚型,其CagA蛋白羧端可变区EPIYA数目均为3个,与临床结果无关联.     

Cag A阳性幽门螺杆菌对胃癌细胞lncRNA DLEU2表达及上皮间质转化的影响

目的 探讨细胞毒素相关蛋白A(Cag A)阳性幽门螺杆菌（Hp）对胃癌细胞长链非编码RNA淋巴细胞白血病缺失基因2(DLEU2)表达及上皮间质转化的影响。方法 体外培养胃癌细胞系HGC-27、AGS、MGC-803、MKN-28以及正常胃黏膜上皮细胞系GES1，采用RT-qPCR法检测DLEU2表达，并选择DLEU2表达最高的胃癌细胞进行后续实验。将Hp NCTC标准菌株26695或11637分别与正常胃黏膜上皮细胞和胃癌细胞共培养0、3、9、12 h，采用RT-qPCR法检测DLEU2表达。采用Western blotting法检测与NCTC 26695或11637共培养3、9、12 h正常胃黏膜上皮细胞和胃癌细胞Cag A表达以及共培养3、9、12 h神经钙黏着蛋白（N-cadherin）、波形蛋白（Vimentin）表达。同时，分析与NCTC 26695或NCTC 11637共培养的胃癌细胞DLEU2、Cag A表达与N-cadherin、Vimentin表达的关系。结果 HGC-27、AGS、MGC-803、MKN-28细胞DLEU2相对表达量均高于GES1细胞（P均<0...     

幽门螺杆菌对甲硝唑的耐药性分析

目的探讨幽门螺杆菌（Helicobacter pylori,Hp）甲硝唑耐药菌株相关基因的突变与耐药性的关系。方法收集临床60例有胃部不适患者的胃活检组织,微需氧培养得到Hp菌株。E-Test法检测Hp对甲硝唑的最低抑菌浓度（MIC）,PCR扩增rdxA、frxA、fdxB,并对该基因进行测序,对耐药株的rdxA、frxA、fdxB的基因序列与标准株Hp26695相应序列进行同源性分析。结果 60例病人胃粘膜标本成功分离出11株Hp耐甲硝唑株（MIC均≥256μg/mL）。11株临床株和1株国际标准测序株（Hp26695）均扩增出rdxA、frxA、和fdxB;基因rdxA、fdxB和frxA突变有一定的规律性,除了存在共同突变外,还存在随机位点散在突变。结论 Hp对甲硝唑耐药性与rdxA、fdxB和frxA基因突变相关。     

外排泵ABC转运蛋白基因msbA和spab在幽门螺杆菌多重耐药中的重要作用

目的:探讨外排泵ABC转运蛋白基因msbA和spab在幽门螺杆菌(Helicobacter pylori,H.pylori)多重耐药中的作用.方法:从临床上胃炎和消化性溃疡患者的胃黏膜标本分离H.pylori.采用琼脂二倍稀释法测定氨苄西林、头孢曲松、四环素、克拉霉素、氧氟沙星和呋喃唑酮对H.pylori的最小抑菌浓度(MIC).应用氯霉素对敏感株和标准菌株进行体外诱导多重耐药.采用RT-PCR的方法分别测定敏感株和多重耐药株中msbA和spab基因的相对表达量.分别构建msbA和spab的基因敲除株,分别测定敲除前后菌株对9种抗生素的敏感性.结果:诱导出包括标准菌株NCTC11637在内的5株多重耐药株.多重耐药株中msbA和spab基因的相对表达量明显高于敏感株(1.8200±0.5310,1.8340±0.2726vs0.8420±0.0789,P=0.018,0.015).成功构建了msbA和spab基因敲除株(H.pyloriMZ1006△msbA和H.pylori MZ1006△spab),两株基因敲除株对4种抗生素的敏感性相对于野生株分别有明显的增加.在临床分离的20株H.pylori中均检测出...     

幽门螺杆菌尿素通道蛋白基因ureI的检测、克隆及序列分析

目的:检测镇江地区幽门螺杆菌(Helicobacterpylori,H pylori)尿素通道蛋白基因ureI,并进行克隆和序列分析.方法:从胃十二指肠疾病患者胃黏膜组织中分离培养获得60例H pylori,PCR扩增检测ureI基因,部分菌株的ureI基因克隆至pMD18-T载体上,进行测序和序列分析.结果:60例H pylori菌株的ureI检出率为100%,成功克隆了8株来源于慢性胃炎、消化性溃疡和胃癌的H pylori菌株ureI并进行了序列分析.结果表明不同来源H pylori菌株之间ureI核苷酸和氨基酸序列同源性均高达95.6%以上.结论:ureI基因在H pylori中高度保守,可以作为鉴定H pylori的分子诊断标志.     

Restriction fragment length polymorphism of adhesin gene hpaA from different Helicobacter pylori strains of Chongqing, China

AIM: To assess the variability of adhesin gene hpaA between different Helicobacter pylori (H pylori) strains with PCR-restriction fragment length polymorphism (RFLP). METHODS: Twelve different H pylori strains were chosen to amplify the 710-bp segments of gene hpaA. These strains were NCTC11637, SS1; Chongqing clinical isolates CCS9801, CCS9802, CCS9803, CCS9806, CCS9809, CCS9810, CCS9813, which were gained from patients of gastritis; Mongolia gerbil adapted H pylori strains (abbreviation MG), which were gained from the following steps: gastric mucosal specimens of Mongolia gerbils infected by clinical isolate CCS9803 were cultured and detected, the positive H pylori strains were named as the first generation of Mongolia gerbil adapted H pylori strains (abbreviation MG1) and then were subcultured with healthy Mongolia gerbil to generate MG2, in turn to gain the ninth generation (abbreviation MG9). All hpaA segments, obtained from 12 different H pylori strains, were digested by Hhal and HaeIll individually and analyzed by agarose gel electrophoresis. RESULTS: In all 12 strains, the 710-bp PCR products were successfully amplified and products were cloned to pMD18-T vector respectively, then the recombinant plasmids were digested simultaneously with Ncol and Xhol to recover the small fragments. The objective fragments from 12 different H pylori strains digested with Haelll could be seen as 4 types of bands and 5 types with Hha I. According to the hpaA RFLP patterns, the 12 H pylori strains could be divided into 5 groups: group I, NCTC11637 and SS1; group II, CCS9809, which RFLP type digested with HaeIll was the same as strains of group I, but Hhal RFLP showed difference compared with the other groups; group III, CCS9810; group IV, CCS9803; group V: CCS9801, CCS9802, CCS9806, CCS9813, MGl, MG3 and MG9. The sequence data of 12 hpaA segments were analyzed by DNAsis software and it was observed that: (1) The homologies of base pair and amino acid sequence between strains NCTC11637, SS1, CCS9809 were 99.6% and 98.9%, respectively; (2) The homology of base pair and amino acid sequence between CCS9803 and CCS9810 was 97.7% and 99.1%; (3) That of the rest strains, CCS9801, CCS9802, CCS9806, CCS9813, MGl, MG3, MG9 reached 99.4% and 98.4%; (4) The base pair homologies between all hpaA fragments of different sources were higher than 94.6%, therefore the correspondence of deduced amino acid sequence was higher than 96.8% between each other. CONCLUSION: The gene hpaA from different H pylori strains revealed variation, and this might provide an effective method for molecular epidemiological survey of H pylori.     

幽门螺杆菌基因组DNA芯片的研制

目的研制幽门螺杆菌(H.pylori)基因组DNA芯片。方法以H.pylori26695和J99作为模板,利用多聚酶链反应(PCR)扩增得到所需要的开放阅读框(open reading frame,ORF)片段。使用Genemachine点样仪进行点样,采用Cy3-dCTP或Cy5-dCTP以及Klenow片段标记H.pylori基因组DNA,并完成芯片杂交和数据读取。数据判断标准是标化后Cy3/Cy5比值(ratio)<0.5则认为不存在这一基因(记作0),若ratio值≥0.5则认为存在这一基因(记作1)。使用芯片重复性、信噪比以及假阳性率和假阴性率评估芯片质量。结果研制的H.pylori基因组DNA芯片共包括1 882个基因片段,相对应于1 636个ORF,其中H.pylori26695为1 549个,H.pyloriJ99为87个。信噪比(S/N)<2的基因点数约为10%。芯片的假阳性率分别为3.4%(H.pylori26695)和7.21%(H.pyloriJ99),假阴性率分别为0.27%(H.pylori26695)和0.24%(H.pyloriJ99)。芯片内点间重复率为98%,芯片间重复率为97%。结论成功制备了H.pylori基因组DNA芯片,所制备的芯片具有较高点一致性、信噪比和统计学重复性,为在全基因组范围内进行H.pylori的基础和应用研究提供了一工具。     

Disturbance of Apoptosis and DNA Synthesis by <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Infection of Hepatocytes

We evaluated the effects of infection of hepatocytes with the well-characterized Helicobacter species, H. pylori. Cell number doubled during each 24 h period in mock cultures or following infection with H. pylori 401C (CagA-, VacA-, BabA-, OipA-) (P < 0.05). In contrast, infection with the more virulent H. pylori NCTC11637 (CagA+, VacA+, BabA+, OipA+) resulted in cell arrest (P < 0.05). Furthermore, NCTC11637 activated caspase-3 and increased DNA fragmentation 6.1 +/- 1.2 fold (P < 0.01) and the number of apoptotic bodies 9.4 +/- 3.5 fold (P < 0.01) compared to controls. The effect was greater than with the less virulent strain 401C (3.8 +/- 0.6 fold and 3.9 +/- 1.7, respectively, P < 0.05). Strain NCTC11637 at low concentrations increased cellular DNA synthesis 139 +/- 6% (P < 0.05) but decreased it to 16 +/- 7% (P < 0.01) at high concentrations. In contrast, strain 401C increased DNA synthesis 155 +/- 14% of controls (P < 0.05) at high concentrations. The presence of intracellular NCTC11637 within hepatocytes increased DNA fragmentation 3.0 +/- 0.4 fold (P < 0.01) greater than in controls. H. pylori infection resulted in strain-species-dependent effects on hepatocytes, and virulent strain caused cell arrest and apoptosis of infected hepatocytes.     

幽门螺杆菌中国株cagA全长基因克隆及其分子特征分析

目的克隆幽门螺杆菌中国株MEL-Hp 27cagA全长基因,并进行分子特征分析。方法参考Hp26695基因组序列,分别针对cagA基因编码区保守序列和位于cagA基因上、下游的基因序列设计两对PCR引物,交叉分段扩增包括cagA基因编码区、5′、3′非编码区在内的全长基因序列,并对cagA基因调控序列、编码序列及源自中国的CagA分子EPIYA基序分布特点进行分析。结果Hp27cagA基因编码区长3510bp,编码1169个氨基酸。5′端非编码区长649bp,-10区、-35区分别位于起始密码子ATG上游89bp和154bp处,3′端非编码区长476bp。Hp27cagA基因编码区核苷酸序列与东亚菌株同源性为96%,与西方菌株的同源性仅为86%左右。Hp27CagA分子存在典型的EPIYA基序,属于ABD型,与国际参考株NCTC11637的AB-CCC型不同。比较源自中国的HpCagA序列的EPIYA基序分布特点,未发现中国Hp分离株之间存在CagAEPIYA基序与疾病结局(胃癌、慢性胃炎和十二指肠溃疡)的聚类关系。结论成功克隆幽门螺杆菌全长ca-gA基因;cagA基因编码区及非编码区序列存在东西方差异;未发现中国Hp分离株之间存在CagA EPIYA基序与疾病结局的聚类关系。     

Comparative genomics of the restriction-modification systems in Helicobacter pylori

Helicobacter pylori is a Gram-negative bacterial pathogen with a small genome of 1.64-1.67 Mb. More than 20 putative DNA restriction-modification (R-M) systems, comprising more than 4% of the total genome, have been identified in the two completely sequenced H. pylori strains, 26695 and J99, based on sequence similarities. In this study, we have investigated the biochemical activities of 14 Type II R-M systems in H. pylori 26695. Less than 30% of the Type II R-M systems in 26695 are fully functional, similar to the results obtained from strain J99. Although nearly 90% of the R-M genes are shared by the two H. pylori strains, different sets of these R-M genes are functionally active in each strain. Interestingly, all strain-specific R-M genes are active, whereas most shared genes are inactive. This agrees with the notion that strain-specific genes have been acquired more recently through horizontal transfer from other bacteria and selected for function. Thus, they are less likely to be impaired by random mutations. Our results also show that H. pylori has extremely diversified R-M systems in different strains, and that the diversity may be maintained by constantly acquiring new R-M systems and by inactivating and deleting the old ones.     

rdxA基因在幽门螺杆菌对甲硝唑耐药机制中的作用

目的 探讨rdxA基因变异与幽门螺杆菌(Hp)对甲硝唑耐药之间的关系。方法 随机收集快速尿素酶试验(RUT)阳性的病人(5l例)，取胃窦部黏膜l块，在体外微需氧培养。甲硝唑耐药率检测分别用E试验和临界点药敏法。PCR扩增HA的rdxA基因片断(886bp)并测序。对耐药株和敏感株的rdxA基因序列与标准菌株HA26695相应序列进行同源性分析。结果共分离出Hp临床分离菌47株。甲硝唑用E试验法检测耐药率为34％(16／47)，临界点药敏法为31．9％(15／47)，其中15株两种方法均耐药。测序15株Hp耐药株和8株敏感株的rdxA基因片断，与标准菌株HA26695相应序列对比，15株耐药株(MtzR)的rdxA基因核酸序列相似性范围在90％-95％之间，平均为92．5％，而8株敏感株(Mtzs)相似性范围在94％—95％之间，平均为94．5％。大多数耐药株rdxA基因的变异为点变异、单个碱基插入或缺失，未发现大片段序列的插入和缺失，且突变位点是不固定的。结论 在耐甲硝唑的大多数Hp临床分离株中，rdxA基因发生多处点变异，单个碱基插入或缺失，但未发现大片段序列的插入和缺失，且变异位点是不固定的。     

幽门螺杆菌5种候选疫苗抗原基因的克隆、表达及抗原性的鉴定

目的:构建含人Hpylori5种候选疫苗抗原Lpp20,HspA,UreaseA,CagA,UreaseB的编码基因的重组质粒并研究其抗原性.方法:应用PCR技术从Hpylori染色体中扩增编码Lpp20,HspA,UreaseA,CagA,UreaseB的基因片段,将其T-A克隆和测序,并与GenBank公布的其他Hpylori菌株基因序列比较,再将目的基因克隆至融合表达载体pGEX-4T-1上中进行表达,用GST亲和层析对其进行纯化,纯化产物用于对29株小鼠抗Hpylori-全菌单克隆抗体(mAb)的鉴定及与Hpylori感染患者血清进行Westernblot分析.结果:扩增的Lpp20,HspA,UreaseA,CagA,UreaseB基因全长分别为528bp,351bp,675bp,855bp,1704bp(GenBank登录号分别为DQ106902,DQ141574,DQ141577,DQ141575,DQ141576),与GenBank公布的其他菌株的核酸序列的同源性在95%-99%,表达Lpp20,HspA,UreaseA,CagA,UreaseB融合蛋白的相对分子质量分别约为48000,41000,52000,60000,91000Da,29株小鼠抗Hpylori全菌mAb中针对Lpp20,HspA,UreaseA,CagA,UreaseB抗原的分别为4,5,5,1,6株,5种抗原的纯化产物均可被Hpylori感染患者血清特异性识别.结论:重组表达的Lpp20,HspA,UreaseA,CagA,UreaseB均具有较好的抗原性.