幽门螺杆菌过氧化氢酶基因克隆及表达

目的 克隆幽门螺杆菌(Helicobacter pylori,HP)过氧化氢酶(katA)基因并在大肠杆菌中进行表达。方法 采用PCR扩增幽门螺杆菌katA全长基因,将其克隆入pET-11c载体中,经测序证实后,在大肠杆菌中进行表达,产物用Western blot检测其抗原性并进行N末端氨基酸的测序。结果 幽门螺杆菌katA基因全长1 518bp,编码氨基酸505个。在BL21(DE3)中的表达量约占细菌总蛋白的24.9％,表达产物经SDS-PAGE显示其相对分子质量与软件预测结果 58 000相符,N末端5个氨基酸测序结果与Hp中天然的katA完全一致,经Western blot检测可被 Hp全菌抗血清识别。结论 katA能在大肠杆菌中进行高效表达,具有良好的免疫反应性,可望为研究 Hp的致病机理、实验诊断及亚单位疫苗等提供充足的katA原材料。     

Epigenetics and Helicobacter pylori

Epigenetics regulates gene expression, cell type development during differentiation, and the cell response to environmental stimuli. To survive, bacteria need to evade the host immune response. Bacteria, including Helicobacter pylori (Hp), reach this target epigenetically, altering the chromatin of the host cells, in addition to several more approaches, such as DNA mutation and recombination. This review shows that Hp prevalently silences the genes of the human gastric mucosa by DNA methylation. Epigenetics includes different mechanisms. However, DNA methylation persists after DNA replication and therefore is frequently associated with the inheritance of repressed genes. Chromatin modification can be transmitted to daughter cells leading to heritable changes in gene expression. Aberrant epigenetic alteration of the gastric mucosa DNA remains the principal cause of gastric cancer. Numerous methylated genes have been found in cancer as well as in precancerous lesions of Hp-infected patients. These methylated genes inactivate tumor-suppressor genes. It is time for us to complain about our genetic and epigenetic makeups for our diseases.     

幽门螺杆菌hp1188基因的克隆及高效表达

目的克隆并表达幽门螺杆菌(Helicobacter pylori,H.pylori)hp1188基因,为研究幽门螺杆菌的黏附机制奠定基础。方法用PCR法从H.pylori标准株NCTC11637基因组DNA中扩增hp1188基因,克隆入原核表达载体pQE-30,构建重组原核表达质粒pQE30-hp1188,转化大肠杆菌DH5α,IPTG诱导表达。SDS-PAGE分析表达形式和表达量。表达蛋白经Ni2+-NTA树脂纯化,Western blot鉴定其反应原性。结果所构建的重组表达质粒pQE30-hp1188序列完整,插入的基因片段全长810bp,与GenBank中的hp1188基因同源性达98%。表达的重组蛋白相对分子质量约为30600,以1.0mmol/L IPTG诱导4h,表达量最高,破菌上清和沉淀中均有表达,可溶性蛋白表达量占全菌总蛋白的47%。重组蛋白纯化后纯度可达90%,并可被H.pylori患者血清识别。结论已成功克隆了H.pylori hp1188基因,并在大肠杆菌DH5α中获得高效表达。     

幽门螺杆菌尿素酶B亚单位基因的克隆及其高效表达

目的克隆幽门螺杆菌尿素酶B亚单位(UreB)基因,构建原核表达载体,并进行高效表达。方法以幽门螺杆菌基因组DNA为模板,PCR扩增UreB基因,双酶切后,与质粒pET-22b(+)连接,构建表达载体pET-22b(+)/UreB,分别转化E.coliBL21(DE3)、Origam(iDE3)和Rossetta(DE3),经IPTG诱导后,进行SDS-PAGE和Western blot分析。结果经酶切及测序,证明幽门螺杆菌UreB基因的原核表达载体构建正确。3种重组菌的诱导表达产物经SDS-PAGE分析,均可见相对分子质量为64000的目的蛋白条带,Rossetta(DE3)重组菌目的蛋白表达量最高,约占菌体蛋白的35%。Western blot结果表明,表达的目的蛋白具有良好的反应原性。结论已成功克隆了幽门螺杆菌UreB基因,并在大肠杆菌Rossetta(DE3)中获得了高效表达。     

幽门螺杆菌VacA-HpaA融合蛋白的表达及其免疫学特性

目的构建H.pylori细胞空泡毒素VacA与黏附素HpaA融合基因的原核表达载体,诱导其表达融合蛋白,并检测表达产物的抗原性与免疫原性。方法用PCR从pQE30-VacA质粒扩增出VacA基因,克隆至pTrc99A-HpaA载体中,与HpaA基因融合后,插入原核表达载体pQE30中,再将pQE30-VacA-HpaA转化入大肠杆菌DH5α,诱导表达并提纯融合蛋白,SDS-PAGE检测融合蛋白的表达,Bradford法检测融合蛋白含量,Western blot鉴定特异性。将融合蛋白免疫家兔,得到多克隆抗血清,用双向免疫扩散和ELISA检测免疫原性。结果SDS-PAGE显示融合蛋白相对分子质量约为65000,表达量在35%以上,主要以包涵体形式表达,蛋白含量为0·72mg/ml,具有良好的VacA和HpaA抗原性与免疫原性。结论VacA-HpaA融合蛋白已成功表达,且具有良好的免疫原性,为进一步研究制备H.pylori疫苗创造了条件。     

幽门螺杆菌粘附素基因hpaA的克隆、表达及鉴定

目的　克隆幽门螺杆菌粘附素基因hpaA ,构建其原核表达系统并鉴定融合蛋白免疫原性。方法　采用PCR技术从幽门螺杆菌总DNA中扩增hpaA基因 ,T -A克隆后测定核苷酸序列 ,构建pET30a的HpaA表达载体 ,在E .coliBL2 1DE3宿主菌中用IPTG诱导表达 ,Ni2 + 柱纯化后经Westernblot鉴定其免疫原性。结果　所克隆的hpaA基因与报道的相应核苷酸序列同源性为 94 . 8%～ 97. 3% ,氨基酸序列同源性为 94 . 6 %～ 97 .7% ,在 134～ 139位存在一段KRTIQK结构 ,HpaA融合蛋白在pET30a载体中可高效表达 ,经纯化后可获得高纯度的重组蛋白。结论　成功构建HpaA原核表达系统 ,所表达的融合蛋白具有较好的免疫原性 ,可作为Hp疫苗的候选抗原。     

幽门螺杆菌UreB与大肠杆菌LTB融合蛋白的表达及生物活性研究

目的 通过基因工程方法构建并表达幽门螺杆菌尿素酶B亚单位(UreB)和大肠杆菌不耐热肠毒素B亚单位(LTB)以基因形式的融合蛋白,并对其生物活性进行初步研究,为幽门螺杆菌疫苗的研究奠定基础。方法 用PCR方法从本室构建的融合基因克隆载体扩增出融合基因2 040bp的片段,将融合基因插入原核表达载体pET-11-c中。结果 经全自动测序仪测序,SDS-PAGE和免疫印迹以及N端氨基酸测序分析,证实融合蛋白已在BL21中表达,初步纯化后通过动物实验和酶联免疫吸附试验证实其相应的免疫原性和免疫反应性,以及其中LTB成分和GM1结合的特性。结论 融合蛋白表达方式为幽门螺杆菌分子内佐剂疫苗的研究奠定了基础。     

幽门螺杆菌双组分系统耐酸相关蛋白ArsS的原核表达及纯化

目的原核表达并纯化幽门螺杆菌双组分系统耐酸相关蛋白ArsS,为深入研究其功能及其在幽门螺杆菌耐酸机制中的作用奠定基础。方法以幽门螺杆菌菌株26695基因组为模板,采用PCR法扩增ArsS基因,插入pET-22b(+)载体,构建重组原核表达质粒pET-22b(+)-ArsS,转化E.coliBL21(DE3),0.5mmol/LIPTG25℃诱导表达,并采用亲和层析与分子筛层析对重组蛋白进行纯化。结果重组原核表达质粒pET-22b(+)-ArsS经双酶切及测序鉴定,证明构建正确;重组蛋白以可溶性形式表达,表达量占菌体总蛋白的30%以上;分子筛层析图谱显示,在150mmol/LNaCl条件下,目的蛋白层析效果较好,纯化后的重组蛋白纯度可达95%以上,浓度约为10mg/ml。结论已成功原核表达并纯化获得了高纯度的ArsS蛋白。     

不同预处理方法建立幽门螺杆菌感染动物模型的比较

目的 确定感染率高且稳定的幽门螺杆菌感染动物模型预处理方案。方法 取蒙古沙鼠200只,随机分为3个实验组及1个对照组。实验组沙鼠在断食、水12h后分别应用pH2盐酸、消炎痛+胃复安和50％乙醇3种不同方案进行预处理,对照组用生理盐水处理。此后继续断食、水12h,再灌喂Hp菌液（109cfu/ml）0.5ml/只。共3次,每次间隔12h。最后1次灌喂后2h给食、水。后每隔4周解剖一批动物,每组10只,进行分离培养、ELISA、PCR、快速尿素酶检测和病理切片检查。结果3组实验动物感染率不同,其中50％乙醇组沙鼠感染率相对较高,达到80％,其病理组织学变化与人体相应病变也极为相似。结论50％乙醇组沙鼠预处理方案感染效果最好,可作为Hp感染动物模型的预处理方法。     

幽门螺杆菌中性粒细胞激活蛋白的基因克隆与表达

目的　克隆、表达幽门螺杆菌中性粒细胞激活蛋白基因napA ,为研究幽门螺杆菌致病机理提供材料。方法　用PCR从幽门螺杆菌DNA中扩增出目的基因napA ,定向插入原核表达载体pQE30中 ,测序分析确认后 ,转化大肠杆菌DH5α ,IPTG诱导表达 ,表达蛋白以NI2 + NTA柱进行纯化。结果　PCR扩增出 4 35bp目的基因片段napA ,克隆入pQE30质粒。工程菌诱导后SDS -PAGE显示新生表达蛋白带 ,相对分子质量为 170 0 0 ,与预期一致 ,约占菌体总蛋白的 38% ,经Ni2 + NTA柱纯化后可获得纯度为 95 5 %重组蛋白。Westernblot显示重组蛋白具有良好的抗原性。结论　克隆napA基因成功 ,并在大肠杆菌DH5α中高效表达。     

Genetics of Host Protection against Helicobacter pylori Infections

This narrative review discusses the genetics of protection against Helicobacter pylori (Hp) infection. After a brief overview of the importance of studying infectious disease genes, we provide a detailed account of the properties of Hp, with a view to those relevant for our topic. Hp displays a very high level of genetic diversity, detectable even between single colonies from the same patient. The high genetic diversity of Hp can be evaded by stratifying patients according to the infecting Hp strain. This approach enhances the power and replication of the study. Scanning for single nucleotide polymorphisms is generally not successful since genes rarely work alone. We suggest selecting genes to study from among members of the same family, which are therefore inclined to cooperate. Further, extending the analysis to the metabolism would significantly enhance the power of the study. This combined approach displays the protective role of MyD88, TIRAP, and IL1RL1 against Hp infection. Finally, several studies in humans have demonstrated that the blood T cell levels are under the genetic control of the CD39⁺ T regulatory cells (T_REGS).     

幽门螺杆菌尿素通道蛋白基因(ureI)的克隆及其表达和纯化

目的　对幽门螺杆菌尿素通道蛋白基因ureI进行克隆、测序 ,并在昆虫细胞中表达及进行产物纯化。方法　克隆ureI基因 ,经测序正确后 ,酶切、连接到pFASTBACHb质粒上 ,与穿梭载体DH10BAC转座 ,获得Bacmid ureI质粒 ,转染Sf9细胞 ,采用Ni2 +螯合琼脂糖亲和层析纯化 ,经SDS PAGE和Westernblot鉴定。结果　克隆了ureI基因 ,并在昆虫细胞Sf9中表达纯化 ,蛋白纯度达 85 %以上 ,并与 6 His单抗特异结合。结论　表达及纯化的ureI蛋白为进一步研究打下了基础     

重组幽门螺杆菌粘附素的纯化工艺

目的纯化大肠杆菌表达的重组幽门螺杆菌粘附素。方法将表达HpaA蛋白的工程菌经高压均质机破菌获得包涵体,经洗涤、变性、复性,Q Sepharose High Performance阴离子交换层析和亲和层析分离纯化。采用SDS-PAGE和HPLC检测纯度,用Western blot检测其抗原性。结果纯化后HpaA蛋白纯度高达95％以上,具有良好的抗原性。结论建立了从包涵体中获得高纯度HpaA纯化工艺,为进一步的研究打下了基础。     

HpaA蛋白在幽门螺杆菌感染诊断中的应用

目的建立检测血清HpaA抗体的ELISA间接法,探讨以重组蛋白作为抗原在诊断H.pylori感染中的价值。方法将从临床分离菌株中获得的HpaA基因在大肠杆菌中表达,用Ni2+-NTA柱纯化表达的HpaA作为抗原,建立检测血清HpaA抗体的ELISA间接法,与诊断标准比较评价其应用的可行性。结果经超声破碎后,用SDS-PAGE分析显示,HpaA蛋白主要存在于上清中,纯化抗原检测临床标本中HpaA抗体的敏感性和特异性分别为100.0%和90.1%。结论以重组蛋白为抗原初步建立检测血清HpaA抗体的ELISA间接法敏感性、特异性高,为制备商品化的试剂盒奠定了基础。     

从包涵体中纯化重组人幽门螺杆菌尿素酶B亚单位

目的 建立一种有效的方法，从包涵体中纯化重组人幽门螺杆菌尿素酶B亚单位（rUreB）。方法重组大肠杆菌发酵后，表达的rUreB包涵体经洗涤、变性、复性，采用Q Sepharose Performance阴离子交换层析和Pheny1 Sepharose High Performance疏水层析分离纯化。使用 SDS－PAGE和HPLC检测纯度，选用 ELISA和 Western－bloning对纯化蛋白的生物学活性进行鉴定。结果 rUreB包涵体经洗涤和溶解后，rUreB的纯度＞70％。包涵体溶解液经阴离子交换层析和疏水层析后纯度超过95％。rUreB纯品具有良好的生物学活性。结论 建立了从包涵体中获得高纯度的rUreB的工艺，为进一步的研究打下了基础。     

幽门螺杆菌鞭毛蛋白B亚单位原核表达系统的构建及其产物的免疫性

目的　构建幽门螺杆菌 (Helicobacterpylori,Hp)鞭毛蛋白B亚单位 (flaB)原核表达系统 ,并鉴定其表达产物的免疫性。方法　采用PCR从幽门螺杆菌基因组DNA中扩增全长flaB基因 ,T A克隆后测定核苷酸序列 ,构建pET32a flaB表达载体 ,以E .coliBL2 1DE3为表达宿主菌 ,用SDS PAGE检测重组蛋白 (rFlaB)的表达水平。采用Hp全菌抗体的Westernblot和兔抗rFlaB血清的免疫扩散试验鉴定其免疫反应性和抗原性。结果　所克隆的flaB基因与文献报道的核苷酸序列同源性为 96 .31%～ 97.73% ,氨基酸序列同源性高达 99.4 1%～ 10 0 %。构建的原核表达系统pET32a flaB E .coliBL2 1DE3的rFlaB产量为细菌总蛋白的 4 0 %左右。Hp全菌抗体能识别rFlaB。rFlaB免疫家兔能获得高效价血清抗体。结论　已成功地构建了HpflaB高效原核表达系统 ,所表达的rFlaB有良好的免疫反应性和抗原性 ,可作为Hp疫苗的候选抗原     

Outer Membrane Vesicle Production by Helicobacter pylori Represents an Approach for the Delivery of Virulence Factors CagA,VacA and UreA into Human Gastric Adenocarcinoma (AGS) Cells

Helicobacter pylori infection is the etiology of several gastric-related diseases including gastric cancer. Cytotoxin associated gene A (CagA), vacuolating cytotoxin A (VacA) and α-subunit of urease (UreA) are three major virulence factors of H. pylori, and each of them has a distinct entry pathway and pathogenic mechanism during bacterial infection. H. pylori can shed outer membrane vesicles (OMVs). Therefore, it would be interesting to explore the production kinetics of H. pylori OMVs and its connection with the entry of key virulence factors into host cells. Here, we isolated OMVs from H. pylori 26,695 strain and characterized their properties and interaction kinetics with human gastric adenocarcinoma (AGS) cells. We found that the generation of OMVs and the presence of CagA, VacA and UreA in OMVs were a lasting event throughout different phases of bacterial growth. H. pylori OMVs entered AGS cells mainly through macropinocytosis/phagocytosis. Furthermore, CagA, VacA and UreA could enter AGS cells via OMVs and the treatment with H. pylori OMVs would cause cell death. Comparison of H. pylori 26,695 and clinical strains suggested that the production and characteristics of OMVs are not only limited to laboratory strains commonly in use, but a general phenomenon to most H. pylori strains.     

Characterization and Genomic Analysis of a New Phage Infecting Helicobacter pylori

Helicobacter pylori, a significant human gastric pathogen, has been demonstrating increased antibiotic resistance, causing difficulties in infection treatment. It is therefore important to develop alternatives or complementary approaches to antibiotics to tackle H. pylori infections, and (bacterio)phages have proven to be effective antibacterial agents. In this work, prophage isolation was attempted using H. pylori strains and UV radiation. One phage was isolated and further characterized to assess potential phage-inspired therapeutic alternatives to H. pylori infections. HPy1R is a new podovirus prophage with a genome length of 31,162 bp, 37.1% GC, encoding 36 predicted proteins, of which 17 were identified as structural. Phage particles remained stable at 37 °C, from pH 3 to 11, for 24 h in standard assays. Moreover, when submitted to an in vitro gastric digestion model, only a small decrease was observed in the gastric phase, suggesting that it is adapted to the gastric tract environment. Together with its other characteristics, its capability to suppress H. pylori population levels for up to 24 h post-infection at multiplicities of infection of 0.01, 0.1, and 1 suggests that this newly isolated phage is a potential candidate for phage therapy in the absence of strictly lytic phages.