幽门螺杆菌cag致病岛cagI基因功能的研究

发布时间：2018-04-09 16:16

  本文选题：幽门螺杆菌　切入点：cag致病岛　出处：《江苏大学》2011年博士论文

【摘要】：幽门螺杆菌(Helicobacter pylori, H. pylori)是人类常见的致病菌之一,是一种可长期定植于人类胃粘膜的革兰氏阴性螺旋形微需氧菌,在全世界范围内感染率超过50%。已证实,H. pylori感染是慢性胃炎、消化性溃疡发生的主要病因,并与胃癌、胃粘膜相关淋巴样组织(MALT)淋巴瘤的发生密切相关；其重要毒力因子细胞毒素相关基因A蛋白(Cytotoxin-associated gene A, CagA)是经cag致病岛编码的Ⅳ型分泌系统注入到宿主细胞内并发挥毒性作用。目前,cag致病岛编码基因的功能及其致病机理尚未完全明确。因此,本文以cag致病岛中的cagI基因为研究对象,通过分子生物学、免疫学及微生物学等技术探讨cagI基因的功能,旨为深入研究cag致病岛的致病机理奠定基础。 方法： 1.根据GenBank收录的H. pylori 26695全基因序列,利用生物信息学软件Primer 5.0自行设计cagI基因引物,获得cagI基因,T-A克隆后构建pMD18-T-cagI载体,测定序列；应用DNA Star、Clustal 1.8及Merga 4.0对其核苷酸及蛋白序列进行生物信息学分析研究,构建系统进化树。 2.构建pET-28a-cagI原核表达载体,转化表达宿主菌BL21(DE3)；经IPTG诱导后,SDS-PAGE电泳和Western Blot方法鉴定表达CagI蛋白,并以Ni2+-NTA柱分离纯化目的蛋白；纯化后的CagI融合蛋白免疫家兔,制备抗CagI多克隆抗体,应用酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELISA)检测血清抗体效价。 3.收集H. pylori,重悬并裂解菌体,经高速离心分离得到各菌体组分蛋白,包括周质间隙蛋白、胞质蛋白及膜蛋白(包括内膜和外膜),利用Western Blot法检测CagI蛋白的亚细胞定位。 4.收集H. pylori感染的GES-1细胞,重悬裂解,并分离细胞组分,Western Blot法检测CagI蛋白的转运。 5.将上述方法分离的H. pylori细菌膜组分与CagI多抗及Protein G琼脂糖珠子相孵育做免疫共沉淀并用Western Blot法检测互作蛋白及CagI蛋白的存在,将His-CagI大肠杆菌细胞总蛋白裂解产物与上述H. pylori亚细胞分离膜组分混合,加入到镍柱珠子中做Pull-down实验,分析与CagI互作的蛋白,MALDI-TOF质谱鉴定互作蛋白。 6.参考cagI基因测序结果,PCR扩增cagI基因编码区两侧翼序列,作为同源臂片段,构建出带卡那霉素抗性标志的打靶载体pBluescript/△cagI::KMr,采用电穿孔法将载体pBluescript/△cagI::KMr转化入受体菌株NCTC 11637中,并经PCR验证后获得了cagI基因缺失株,同理构建出cagL基因缺失株(阳性对照)。 7. cagI基因的缺失株与野生株分别与胃上皮细胞GES-1进行共培养后,检测其对CagA蛋白转运及其对细菌粘附作用的影响。 8.应用统计软件SPSS11.0分析数据,以均数±标准差表示,两组之间的比较采用t检验。 结果： 1.扩增获得的cagI基因全长1086bp,编码361个氨基酸,获得GenBank登录号为HM126476。其核苷酸序列与两株国际标准菌株(H. pylori 26695及J99)同源性分别为98.3%和98.1%,其氨基酸序列与H. pylori 26695同源性高达99.5%,基于核苷酸及氨基酸序列的系统进化树也说明了其同源性。DNA Star软件预测其编码蛋白相对分子量为39.37kDa； 2.构建获得了原核表达载体pET-28a-cagI, IPTG诱导后,经SDS-PAGE鉴定和Western Blot鉴定有目的蛋白表达；采用Ni2+-NTA柱梯度洗脱后,分离获得了目的蛋白CagI; CagI融合蛋白免疫家兔,获得了抗CagI多克隆抗体,其效价为1：1.6×105； 3. Western Blot结果显示CagI蛋白定位于菌体膜(包括内膜和外膜)上；CagI蛋白转运实验结果表明CagI蛋白没有转运到宿主细胞内,而阳性对照CagA蛋白转运到了宿主细胞内；免疫共沉淀、Pull-down及MALDI-TOF质谱实验结果表明CagI蛋白与CagA蛋白有相互作用； 4.PCR法扩增得到用于同源重组的cagI基因的同源臂片段F1(612 bp)和F2(796 bp),同时得到了cagL基因的同源臂片段F1’(651 bp)和F2’(666 bp),并分别与pBluescript SKⅡ(-)载体(带卡那霉素抗性基因的载体)相连接,构建出pBluescript/△cagI::KMr和pBluescript/△cagL::KMr载体；采用电穿孔技术将载体转化进入野生株NCTC11637菌体内,在含卡那霉素的哥伦比亚平板上筛出cagI基因及cagL基因的突变株,并用PCR方法及测序鉴定突变株； 5.缺失株及野生株分别与胃上皮细胞GES-1共培养后,发现cagI基因缺失株处理组,CagA蛋白转运明显降低,而cagL基因缺失株处理组,胃上皮细胞内未能检测到CagA的转运；菌落形成实验表明cagI基因缺失后细菌的粘附功能下降。 结论： 1.成功克隆cagI基因,明确该基因是H. pylori cag致病岛编码的Ⅳ型分泌系统结构基因之一,经生物信息学分析其核苷酸序列及氨基酸序列相对保守。 2.构建了cagI基因的原核表达载体pET-28a-cagI,获得并纯化了在大肠杆菌中异源表达的重组CagI蛋白,制备了抗CagI的多克隆抗体。 3.明确了CagI蛋白为一种膜相关蛋白质,位于菌细胞膜(包括内膜和外膜)上,不转运到宿主细胞内,与重要的毒力蛋白CagA蛋白有互作作用。 4.成功构建了pBluescript/△cagI::KMr和pBluescript/△cagL::KMr载体,获得了cagI基因及cagL基因缺失株。 5.表明了cagI基因缺失后不影响CagA蛋白转运至宿主细胞内,但CagA蛋白的转运量有所减少；并使H. pylori对宿主细胞的粘附能力下降。
[Abstract]:Helicobacter pylori ( H . pylori ) is one of the most common pathogens in human gastric mucosa , and is a Gram - negative spiral micro - aerobic bacteria which can be implanted in human gastric mucosa for a long time . The infection rate is over 50 % worldwide . It has been confirmed that H . pylori infection is the main cause of chronic gastritis and peptic ulcer , and is closely related to the occurrence of gastric cancer and gastric mucosa - related lymphoid tissue ( MALT ) lymphoma .
Cytotoxin - associated gene A ( A ) is a type IV secretion system encoded by cag pathogenicity island into the host cell and plays a toxic role . At present , the function of cag pathogenicity island coding gene and its pathogenic mechanism have not been completely defined . Therefore , this paper studies the function of cag pathogenicity island by molecular biology , immunology and microbiology , and aims to lay a foundation for further research on the pathogenesis of cag pathogenicity island .


Method :


1 . Based on the whole gene sequence of H . pylori 26695 in GenBank , a primer 5.0 self - designed with bioinformatics software Primer 5.0 was used to construct pMD18 - T - A 1 vector and sequenced after T - A cloning .
DNA Star , Clustal 1.8 and Merga 4.0 were used for bioinformatics analysis of their nucleotide and protein sequences , and the phylogenetic tree was constructed .


2 . constructing the prokaryotic expression vector of pET - 28a - gag I , and transforming and expressing the host bacterium BL21 ( DE3 ) ;
After IPTG induction , SDS - PAGE electrophoresis and Western Blot were used to identify the protein expressed by SDS - PAGE and SDS - PAGE and Western Blot .
The serum antibody titer was determined by enzyme - linked immunosorbent assay ( ELISA ) .


3 . H . pylori was collected and the cell components were isolated by high - speed centrifugation , including periplasmic space protein , cytoplasmic protein and membrane protein ( including inner membrane and outer membrane ) . Western Blot was used to detect the sub - cell localization of the CaI protein .


4 . GES - 1 cells infected with H . pylori were collected , the cells were resuspended and the cell fractions were isolated . Western Blot was used to detect the transport .


5 . The membrane components of H . pylori isolated by the above method were incubated with the two kinds of protein and protein G agarose beads for immune co - precipitation and Western Blot was used to detect the presence of each other . The protein was mixed with the above - mentioned H . pylori isolated membrane components . Then , a pull - down experiment was carried out in the nickel - column beads , and the proteins were identified by MALDI - TOF mass spectrometry and MALDI - TOF mass spectrometry .


6 . With reference to the results of gene sequencing , PCR was used to amplify the two flanking sequences of the gene coding region . As a homologous arm fragment , the pBluescrit / 鈻，

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