幽门螺杆菌Ⅳ型分泌系统cagT基因的克隆表达及其重组蛋白对SGC-7901细胞增殖和分泌细胞因子功能的影响

[目的]初步研究幽门螺杆菌(Helicobacter pylori,H.pylori)NCTC 11637 cagT(HP0532)基因对SGC-7901细胞增殖和白细胞介素-8(IL-8)分泌的影响.[方法]应用PCR技术从H.pylori基因组DNA中扩增cagT编码基因片段,将其定向插入pQE30载体中,双酶切鉴定筛选阳性克隆.测序分析确认后,IPTG诱导表达,表达蛋白以Ni2 -NTA柱进行纯化,并经Western blot鉴定,纯化的蛋白作用于SGC-7901细胞,提取细胞总RNA,RT-PCR扩增IL-8,并用MTT法检测蛋白对细胞增殖的影响.[结果]成功克隆了cagT基因,全长843bp(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%.工程菌诱导后SDS-PAGE显示新生表达蛋白带,相对分子质量约为32kDa,经Ni2 -NTA柱纯化后可获得纯度为98%重组蛋白.经透析复性后的蛋白(终浓度10μg/mL)作用于SGC-7901细胞后,可诱导细胞因子IL-8在mRNA水平上的表达.不同浓度CagT蛋白与SGC-7901细胞作用,细胞增殖的程度随着蛋白浓度的增加逐渐降低,细胞的增殖受到抑制.[结论]CagT蛋白可刺激SGC-7901细胞IL-8的表达,并抑制细胞增殖,为进一步研究其生物学功能奠定了基础.     

幽门螺杆菌Cag-PAI hp0525基因的克隆表达及其重组蛋白HP0525对SGC-7901细胞增殖的影响

目的：由于文献报道幽门螺杆菌（Helicobacter pylori）是产生胃溃疡和胃癌的致病菌之一,一个重要的影响因素是由cag致病岛编码的四型分泌系统。Hp0525是Cag致病岛中重要成分,是一种内膜蛋白ATPase。而幽门螺杆菌中Cag蛋白的表达以及Cag PAI编码的各自蛋白功能研究得还很少,为进一步研究幽门螺杆菌的致病机制和研发幽门螺杆菌诊断试剂盒及疫苗,特克隆幽门螺杆菌NCTC 11637hp0525 (caga) 基因,并对其进行测序,构建原核重组质粒,表达HP0525蛋白,初步研究其对SGC-7901细胞增殖的影响。方法：应用PCR技术从H.pylori基因组DNA中扩增hp0525编码基因片段,克隆至pMD18-T载体后,再将其定向插入pET-30a载体中,双酶切鉴定筛选阳性克隆,以DNA自动分析仪进行序列测定。测序分析正确后,经IPTG诱导表达,表达蛋白以Ni2＋-NTA柱进行纯化,并经Western blot和MALDI-TOF鉴定,透析除盐后的蛋白,通过免疫新西兰大白兔和抗体效价的测定,纯化的蛋白作用于SGC-7901细胞,用MTT法检测蛋白对细胞增殖的影响。结果：成功克隆hp0525基因,全长993bp,编码330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%。工程菌诱导后SDS-PAGE显示新生表达蛋白带,相对分子质量为36 000,与预期一致,经Ni2＋-NTA柱纯化后可获得纯度为98％重组蛋白。蛋白作用于SGC-7901细胞后,结果呈现一定量的时间和剂量依赖性。它是一种ATPase,通过测定具有一定的活性。活性为4.40IU/ml结论：成功克隆hp0525基因,并在大肠杆菌BL21中表达,经过镍柱纯化后得到纯度较高的蛋白, MTT法来检测出重组蛋白抑制细胞增殖;同时具有一定的酶活力,为进一步研究其生物学功能奠定了基础。     

幽门螺杆菌热休克蛋白70基因的克隆与表达

从幽门螺杆菌染色体ＤＮＡ,用ＰＣＲ方法扩增得到了热休克蛋白７０基因。序列分析表明,我国Ｈｐ临床分离株Ｙ２的热休克蛋白７０基因与经全基因组序列测定的两株幽门螺杆菌２６６９５和ｊ９９有高度同源性。将该基因克性到融合分泌表达载体ｐＭＡＬ－ｐ２中,转化大肠杆菌,在ＩＰＴＧ诱导下表达出与预期大小相符的１１３ｋＤ的融合表达蛋白。该蛋白质３０℃诱导表达５ｈ后,可达到细菌周质总蛋白质的１９．４％。用免疫印迹分析表     

幽门螺杆菌cagA克隆表达

克隆表达4株幽门螺杆菌的cagA基因,以方便地获得大鼠CagA蛋白和重组表达质粒,为临床诊断CagA阳性幽门螺杆菌感染,以及进一步研究不同类型CagA功能及其与疾病关系提供材料。PCR扩增幽门螺杆菌的cagA基因,克隆至PinPoint^TMXa-1T载体,酶切鉴定连接方向,IPTG诱导正向连接克隆表达CagA融合蛋白并进行SDS－PAGE和Western blots鉴定。结果显示PCR扩增得到3.5-3.8kb的CagA基因,PCR及酶切鉴定得到正向连接的重组克隆,SDS－PAGE及Western blots证实正向连接的重组克隆表达CagA融合蛋白。构建了4种cagA的重组表达质粒,通过转化同一宿主菌可研究不同CagA的功能和致病性差异;通过亲和层析纯化融合蛋白可获大量CagA蛋白,用于血清学诊断CagA阳性幽门螺杆菌感染,及不同抗原性CagA与疾病之间的关系。     

幽门螺杆菌热休克蛋白A基因的克隆,表达及免疫原性 …

幽门螺杆菌的感染可以诱发人体产生胃炎和消化性溃疡,其组成成分热休克蛋白Ａ可刺激机体产生保护性的免疫反应,用ＰＣＲ方法从幽门螺杆菌的染色体ＤＮＡ上扩增出ＨｓｐＡ基因片段,将其插入原核表达载体ｐＥＴ－２２ｂ（＋）中,并在ＢＬ２１（ＤＥ３）大肠杆菌表达。经测序Ｈｓｐ基因片段有３５４ｂｐ组成,可编码１１８个氨基酸残基的多肽。     

幽门螺杆菌HP0762蛋白的原核表达纯化及多克隆抗体制备

目的:表达和纯化幽门螺杆菌HP0762蛋白,并制备该蛋白的多克隆抗体。方法:从幽门螺杆菌SS1中经PCR扩增得到了hp0762基因,将其克隆至含有6×His编码序列的原核表达载体pET-28a(+)中,再将重组质粒转化大肠杆菌BL21(DE3),在IPTG诱导下进行蛋白表达;用HiTrap Chelating HP亲和柱纯化重组蛋白,Western印迹进一步鉴定;以纯化后的蛋白为抗原免疫新西兰大耳白兔,制备该蛋白的多克隆抗体;用ELISA和Western印迹检测抗血清。结果:目的蛋白在大肠杆菌BL21(DE3)中获得了可溶性表达,纯化后纯度可达90%以上;制备了针对HP0762重组蛋白的抗血清,抗体ELISA效价为1:256000,Western印迹分析表明该抗体能特异性识别内源性HP0762。结论:完成了HP0762蛋白的原核高效表达与纯化,并制备了其高效价的多克隆抗体,为进一步对其进行疫苗研制与基因功能研究奠定了基础。     

幽门螺杆菌cag PAI编码的Ⅳ型分泌系统

幽门螺杆菌(Helicobacter pylori,H.pylori)是定植于人胃部特定的病原菌,感染呈全球分布,感染率高达50%以上。现已证实它是轻度胃炎,消化性溃疡及胃癌的主要病因。Ⅰ型H.pylori菌株含有一个约40kb的特殊基因片段,即cag致病岛(cytotoxin associated gene pathogenicity island,cag PAI),该片段只出现于致病相关菌株,基因呈高密度分布并编码一个分泌转运系统称为Ⅳ型分泌系统(type Ⅳ secretion system,TFSS),通过转运相关毒素而参与H.pylori诱导上皮细胞细胞内的酪氨酸磷酸化、细胞骨架重排、基垫结构形成、活化核转录因子NF-κB、诱导促炎细胞因子白细胞介素-8的表达等,故在H.pylori的致病中起着关键作用。近年来,研究者们致力于研究Ⅳ型分泌系统的功能,但是对于这个装置是如何转运蛋白进入宿主细胞的确切机制还是知之甚少,因此,对Ⅳ型分泌系统的研究将有助于进一步明确H.pylori致病机制,并为临床诊断和治疗提供新的靶点。     

幽门螺杆菌热休克蛋白A基因的克隆、表达及免疫原性研究

幽门螺杆菌的感染可诱发人体产生胃炎和消化性溃疡,其组成成分热休克蛋白Ａ（ＨｓｐＡ）可刺激机体产生保护性的免疫反应。用ＰＣＲ方法从幽门螺杆菌的染色体ＤＮＡ上扩增出ＨｓｐＡ基因片段,将其插入原核表达载体ｐＥＴ２２ｂ（＋）中,并在ＢＬ２１（ＤＥ３）大肠杆菌表达。经测序ＨｓｐＡ基因片段有３５４ｂｐ组成,可编码１１８个氨基酸残基的多肽。ＳＤＳＰＡＧＥ和免疫印迹分析检测发现,ＨｓｐＡ基因表达的蛋白质分子量约为１５ｋＤ,并证实该重组蛋白质可以被幽门螺杆菌感染阳性患者的血清所识别,同时将其免疫小鼠可刺激机体产生抗该重组蛋白质的抗体。ＨｓｐＡ有可能作为一种有效的蛋白质疫苗用于幽门螺杆菌感染的预防和治疗。     

幽门螺杆菌vacA基因的克隆和序列分析

空泡毒素是幽门螺杆菌产生的已知的唯一蛋白毒素,该毒素与感染者胃肠上皮务和溃疡形成密切相关,同时也是幽门螺杆菌免疫预防和免疫治疗的重要候选组分。从幽门螺杆菌NCTC11637染色体DNA中经PCR方法获得了2.9kb的该基因成熟肽全长序列,将该基因克隆至载体pET22b ,经PCR扩增和酶切鉴定后序列分析表明,该基因与已知序列完全一致。     

幽门螺杆菌细胞毒素相关基因致病岛及Ⅳ型分泌系统的研究进展

致病岛是指细菌染色体上一段具有典型结构特征的基因簇,主要编码与细菌毒力及代谢等功能相关的产物。Ⅳ型分泌系统指革兰阴性菌中由多种蛋白分子构成的、通过菌毛样结构向宿主细胞注入毒力因子的分泌系统。幽门螺杆菌细胞毒素相关基因致病岛及其编码的Ⅳ型分泌系统是幽门螺杆菌关键性致病因子,有可能成为药物作用的新靶标,是近年相关研究的热点。     

Role of type IV secretion in Helicobacter pylori pathogenesis

Helicobacter pylori is a highly successful human-specific gastric pathogen that colonizes more than half the world's population. Infection with this bacterium can induce gastric pathologies ranging from chronic gastritis to peptic ulcers and even cancer. Virulent H. pylori isolates harbour the cag (cytotoxin-associated genes) pathogenicity island, a 40 kb stretch of DNA that encodes components of a type IV secretion system (T4SS). This T4SS forms a pilus for the injection of virulence factors into host target cells such as the CagA oncoprotein. This is accomplished by a specialized adhesin of the pilus surface, the CagL protein, which binds to and activates host cell integrins for subsequent delivery of CagA across the host cell membrane. Injected CagA becomes tyrosine-phosphorylated by Src and Abl family kinases and mimics a host cell protein in binding and activation of multiple signalling factors. Here we review the recent advances in the characterization of phosphorylation-dependent and phosphorylation-independent signalling activities of CagA and the T4SS which include the induction of membrane dynamics, actin cytoskeletal rearrangements and the disruption of cell-to-cell junctions as well as proliferative, pro-inflammatory and anti-apoptotic nuclear responses. The contribution of these signalling cascades to H. pylori pathogenesis is discussed.     

Architecture of the Helicobacter pylori Cag-type IV secretion system

Type IV secretion systems (T4SS) are macromolecular assemblies used by bacteria to transport material across their membranes. T4SS are generally composed of a set of twelve proteins (VirB1-11 and VirD4). This represents a dynamic machine powered by three ATPases. T4SS are widespread in pathogenic bacteria where they are often used to deliver effectors into host cells. For example, the human pathogen Helicobacter pylori encodes a T4SS, the Cag-T4SS, which mediates the injection of the toxin CagA. We review the progress made in the past decade in our understanding of T4SS architecture. We translate this new knowledge to derive an understanding of the structure of the H. pylori Cag system, and use recent protein-protein interaction data to refine this model.     

A novel sheathed surface organelle of the Helicobacter pylori cag type IV secretion system

Type I strains of Helicobacter pylori (Hp) use a type IV secretion system (T4SS), encoded by the cag pathogenicity island (cag-PAI), to deliver the bacterial protein CagA into eukaryotic cells and to induce interleukin-8 secretion. Translocated CagA is activated by tyrosine phosphorylation involving Src-family kinases. The mechanism and structural basis for type IV protein secretion is not well understood. We describe here, by confocal laser scanning microscopy and field emission scanning electron microscopy, a novel filamentous surface organelle which is part of the Hp T4SS. The organelle is often located at one bacterial pole but can be induced by cell contact also along the lateral side of the bacteria. It consists of a rigid needle, covered focally or completely by HP0527 (Cag7 or CagY), a VirB10-homologous protein. HP0527 is also clustered in the outer membrane. The VirB7-homologous protein HP0532 is found at the base of this organelle. These observations demonstrate for the first time by microscopic techniques a complex T4SS-associated, sheathed surface organelle reminiscent to the needle structures of bacterial type III secretion systems.     

幽门螺杆菌cag PAI编码的Ⅳ型分泌系统

幽门螺杆菌(Helicobacter pylori,H.pylori)是定植于人胃部特定的病原菌,感染呈全球分布,感染率高达50%以上。现已证实它是轻度胃炎,消化性溃疡及胃癌的主要病因。Ⅰ型H.pylori菌株含有一个约40kb的特殊基因片段,即cag致病岛(cytotoxin associated gene pathogenicity island,cag PAI),该片段只出现于致病相关菌株,基因呈高密度分布并编码一个分泌转运系统称为Ⅳ型分泌系统(type Ⅳ secretion system,TFSS),通过转运相关毒素而参与H.pylori诱导上皮细胞细胞内的酪氨酸磷酸化、细胞骨架重排、基垫结构形成、活化核转录因子NF-κB、诱导促炎细胞因子白细胞介素-8的表达等,故在H.pylori的致病中起着关键作用。近年来,研究者们致力于研究Ⅳ型分泌系统的功能,但是对于这个装置是如何转运蛋白进入宿主细胞的确切机制还是知之甚少,因此,对Ⅳ型分泌系统的研究将有助于进一步明确H.pylori致病机制,并为临床诊断和治疗提供新的靶点。     

Crystal structure of the hexameric traffic ATPase of the Helicobacter pylori type IV secretion system

The type IV secretion system of Helicobacter pylori consists of 10--15 proteins responsible for transport of the transforming protein CagA into target epithelial cells. Secretion of CagA crucially depends on the hexameric ATPase, HP0525, a member of the VirB11-PulE family. We present the crystal structure of a binary complex of HP0525 bound to ADP. Each monomer consists of two domains formed by the N- and C-terminal halves of the sequence. ADP is bound at the interface between the two domains. In the hexamer, the N- and C-terminal domains form two rings, which together form a chamber open on one side and closed on the other. A model is proposed in which HP0525 functions as an inner membrane pore, the closure and opening of which is regulated by ATP binding and ADP release.     

幽门螺杆菌Ⅳ型分泌系统（T4SS）研究进展

Ⅳ型分泌系统（T4SS）广泛存在于革兰阴性菌中,细菌可通过该系统将生物大分子或毒力因子等运输至靶细胞中并发挥相应功能。目前在H. pylori中已发现了至少三种T4SS,其中研究较为透彻的是cag致病岛（cagPAI）编码的cagT4SS系统,此外可塑区编码的tfs3系统和comB系统也有相关的报道。H. pylori的T4SS作为其与致病相关的重要结构已受到很多学者关注,对该菌T4SS系统的研究有助于进一步明确H. pylori的致病机制,并为临床诊断和治疗相关胃十二指肠疾病提供新的靶点。本文将对H. pylori的T4SS相关研究进展作一简要综述。     

Helicobacter pylori cholesteryl glucosides interfere with host membrane phase and affect type IV secretion system function during infection in AGS cells

Helicobacter pylori infection is an aetiological cause of gastric disorders worldwide. H. pylori has been shown to assimilate and convert host cholesterol into cholesteryl glucosides (CGs) by cholesterol-α-glucosyltransferase encoded by capJ. Here, we show that CapJ-deficient (ΔcapJ) H. pylori resulted in greatly reduced type IV secretion system (TFSS)-associated activities, including the hummingbird phenotype of AGS cells, IL-8 production, CagA translocation/phosphorylation and CagA-mediated signalling events. Complementation of the ΔcapJ mutation with wild type cagJ or by adding CGs-containing lysates or exogenous fluorophore-tagged CGs reversed the mutant phenotypes. We also show that the wild-type but not ΔcapJ H. pylori recruited raft-associated components to sites of bacterial attachment. Fluorescence recovery after photobleaching (FRAP) analysis of AGS cells treated with fluorescence-tagged cholesterol/CGs revealed that there was a higher proportion of CGs associated with immobile fractions. CGs-associated membranes were also more resistant to a cold detergent extraction. Thus, we propose that CGs synthesized by H. pylori around host-pathogen contact sites partition in detergent-resistant membranes (DRMs), alters lateral-phase segregation in membrane and reorganizes membrane architecture. These processes together promote the formation of a functional TFSS and H. pylori infection.