本文選題：幽門(mén)螺桿菌　切入點(diǎn)：cag致病島　出處：《江蘇大學(xué)》2011年博士論文

【摘要】：幽門(mén)螺桿菌(Helicobacter pylori, H. pylori)是人類(lèi)常見(jiàn)的致病菌之一,是一種可長(zhǎng)期定植于人類(lèi)胃粘膜的革蘭氏陰性螺旋形微需氧菌,在全世界范圍內(nèi)感染率超過(guò)50%。已證實(shí),H. pylori感染是慢性胃炎、消化性潰瘍發(fā)生的主要病因,并與胃癌、胃粘膜相關(guān)淋巴樣組織(MALT)淋巴瘤的發(fā)生密切相關(guān)；其重要毒力因子細(xì)胞毒素相關(guān)基因A蛋白(Cytotoxin-associated gene A, CagA)是經(jīng)cag致病島編碼的Ⅳ型分泌系統(tǒng)注入到宿主細(xì)胞內(nèi)并發(fā)揮毒性作用。目前,cag致病島編碼基因的功能及其致病機(jī)理尚未完全明確。因此,本文以cag致病島中的cagI基因?yàn)檠芯繉?duì)象,通過(guò)分子生物學(xué)、免疫學(xué)及微生物學(xué)等技術(shù)探討cagI基因的功能,旨為深入研究cag致病島的致病機(jī)理奠定基礎(chǔ)。 方法： 1.根據(jù)GenBank收錄的H. pylori 26695全基因序列,利用生物信息學(xué)軟件Primer 5.0自行設(shè)計(jì)cagI基因引物,獲得cagI基因,T-A克隆后構(gòu)建pMD18-T-cagI載體,測(cè)定序列；應(yīng)用DNA Star、Clustal 1.8及Merga 4.0對(duì)其核苷酸及蛋白序列進(jìn)行生物信息學(xué)分析研究,構(gòu)建系統(tǒng)進(jìn)化樹(shù)。 2.構(gòu)建pET-28a-cagI原核表達(dá)載體,轉(zhuǎn)化表達(dá)宿主菌BL21(DE3)；經(jīng)IPTG誘導(dǎo)后,SDS-PAGE電泳和Western Blot方法鑒定表達(dá)CagI蛋白,并以Ni2+-NTA柱分離純化目的蛋白；純化后的CagI融合蛋白免疫家兔,制備抗CagI多克隆抗體,應(yīng)用酶聯(lián)免疫吸附試驗(yàn)(enzyme-linked immunosorbent assay, ELISA)檢測(cè)血清抗體效價(jià)。 3.收集H. pylori,重懸并裂解菌體,經(jīng)高速離心分離得到各菌體組分蛋白,包括周質(zhì)間隙蛋白、胞質(zhì)蛋白及膜蛋白(包括內(nèi)膜和外膜),利用Western Blot法檢測(cè)CagI蛋白的亞細(xì)胞定位。 4.收集H. pylori感染的GES-1細(xì)胞,重懸裂解,并分離細(xì)胞組分,Western Blot法檢測(cè)CagI蛋白的轉(zhuǎn)運(yùn)。 5.將上述方法分離的H. pylori細(xì)菌膜組分與CagI多抗及Protein G瓊脂糖珠子相孵育做免疫共沉淀并用Western Blot法檢測(cè)互作蛋白及CagI蛋白的存在,將His-CagI大腸桿菌細(xì)胞總蛋白裂解產(chǎn)物與上述H. pylori亞細(xì)胞分離膜組分混合,加入到鎳柱珠子中做Pull-down實(shí)驗(yàn),分析與CagI互作的蛋白,MALDI-TOF質(zhì)譜鑒定互作蛋白。 6.參考cagI基因測(cè)序結(jié)果,PCR擴(kuò)增cagI基因編碼區(qū)兩側(cè)翼序列,作為同源臂片段,構(gòu)建出帶卡那霉素抗性標(biāo)志的打靶載體pBluescript/△cagI::KMr,采用電穿孔法將載體pBluescript/△cagI::KMr轉(zhuǎn)化入受體菌株NCTC 11637中,并經(jīng)PCR驗(yàn)證后獲得了cagI基因缺失株,同理構(gòu)建出cagL基因缺失株(陽(yáng)性對(duì)照)。 7. cagI基因的缺失株與野生株分別與胃上皮細(xì)胞GES-1進(jìn)行共培養(yǎng)后,檢測(cè)其對(duì)CagA蛋白轉(zhuǎn)運(yùn)及其對(duì)細(xì)菌粘附作用的影響。 8.應(yīng)用統(tǒng)計(jì)軟件SPSS11.0分析數(shù)據(jù),以均數(shù)±標(biāo)準(zhǔn)差表示,兩組之間的比較采用t檢驗(yàn)。 結(jié)果： 1.擴(kuò)增獲得的cagI基因全長(zhǎng)1086bp,編碼361個(gè)氨基酸,獲得GenBank登錄號(hào)為HM126476。其核苷酸序列與兩株國(guó)際標(biāo)準(zhǔn)菌株(H. pylori 26695及J99)同源性分別為98.3%和98.1%,其氨基酸序列與H. pylori 26695同源性高達(dá)99.5%,基于核苷酸及氨基酸序列的系統(tǒng)進(jìn)化樹(shù)也說(shuō)明了其同源性。DNA Star軟件預(yù)測(cè)其編碼蛋白相對(duì)分子量為39.37kDa； 2.構(gòu)建獲得了原核表達(dá)載體pET-28a-cagI, IPTG誘導(dǎo)后,經(jīng)SDS-PAGE鑒定和Western Blot鑒定有目的蛋白表達(dá)；采用Ni2+-NTA柱梯度洗脫后,分離獲得了目的蛋白CagI; CagI融合蛋白免疫家兔,獲得了抗CagI多克隆抗體,其效價(jià)為1：1.6×105； 3. Western Blot結(jié)果顯示CagI蛋白定位于菌體膜(包括內(nèi)膜和外膜)上；CagI蛋白轉(zhuǎn)運(yùn)實(shí)驗(yàn)結(jié)果表明CagI蛋白沒(méi)有轉(zhuǎn)運(yùn)到宿主細(xì)胞內(nèi),而陽(yáng)性對(duì)照CagA蛋白轉(zhuǎn)運(yùn)到了宿主細(xì)胞內(nèi)；免疫共沉淀、Pull-down及MALDI-TOF質(zhì)譜實(shí)驗(yàn)結(jié)果表明CagI蛋白與CagA蛋白有相互作用； 4.PCR法擴(kuò)增得到用于同源重組的cagI基因的同源臂片段F1(612 bp)和F2(796 bp),同時(shí)得到了cagL基因的同源臂片段F1’(651 bp)和F2’(666 bp),并分別與pBluescript SKⅡ(-)載體(帶卡那霉素抗性基因的載體)相連接,構(gòu)建出pBluescript/△cagI::KMr和pBluescript/△cagL::KMr載體；采用電穿孔技術(shù)將載體轉(zhuǎn)化進(jìn)入野生株NCTC11637菌體內(nèi),在含卡那霉素的哥倫比亞平板上篩出cagI基因及cagL基因的突變株,并用PCR方法及測(cè)序鑒定突變株； 5.缺失株及野生株分別與胃上皮細(xì)胞GES-1共培養(yǎng)后,發(fā)現(xiàn)cagI基因缺失株處理組,CagA蛋白轉(zhuǎn)運(yùn)明顯降低,而cagL基因缺失株處理組,胃上皮細(xì)胞內(nèi)未能檢測(cè)到CagA的轉(zhuǎn)運(yùn)；菌落形成實(shí)驗(yàn)表明cagI基因缺失后細(xì)菌的粘附功能下降。 結(jié)論： 1.成功克隆cagI基因,明確該基因是H. pylori cag致病島編碼的Ⅳ型分泌系統(tǒng)結(jié)構(gòu)基因之一,經(jīng)生物信息學(xué)分析其核苷酸序列及氨基酸序列相對(duì)保守。 2.構(gòu)建了cagI基因的原核表達(dá)載體pET-28a-cagI,獲得并純化了在大腸桿菌中異源表達(dá)的重組CagI蛋白,制備了抗CagI的多克隆抗體。 3.明確了CagI蛋白為一種膜相關(guān)蛋白質(zhì),位于菌細(xì)胞膜(包括內(nèi)膜和外膜)上,不轉(zhuǎn)運(yùn)到宿主細(xì)胞內(nèi),與重要的毒力蛋白CagA蛋白有互作作用。 4.成功構(gòu)建了pBluescript/△cagI::KMr和pBluescript/△cagL::KMr載體,獲得了cagI基因及cagL基因缺失株。 5.表明了cagI基因缺失后不影響CagA蛋白轉(zhuǎn)運(yùn)至宿主細(xì)胞內(nèi),但CagA蛋白的轉(zhuǎn)運(yùn)量有所減少；并使H. pylori對(duì)宿主細(xì)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，

本文編號(hào)：1727188