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)》2009年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 幽門(mén)螺桿菌(Helicobacter pylori,H.pylori)的細(xì)胞毒性蛋白A(CagA)是一個(gè)重要的毒力因子,與胃癌的發(fā)生密切相關(guān)。其是通過(guò)Cag致病島(cagpathogenicity island,Cag-PAI)編碼的Ⅳ型樣分泌裝置,轉(zhuǎn)運(yùn)進(jìn)入胃上皮細(xì)胞,發(fā)揮毒性作用。而Cag-PAI中編碼的基因功能及其致病機(jī)理均尚未十分明確。鑒此,本文初步研究了Cag-PAI中編碼的hp0523基因的功能,并初步探討其在CagA轉(zhuǎn)運(yùn)過(guò)程中的作用,旨在為深入研究Cag-PAI的致病機(jī)理奠定基礎(chǔ)。 方法: 1.利用PCR技術(shù)從H.pylori基因組DNA中擴(kuò)增獲取hp0523基因,T-A克隆后構(gòu)建pGEM-T-hp0523,進(jìn)行序列測(cè)定;并對(duì)其序列進(jìn)行生物信息學(xué)分析研究; 2.構(gòu)建pET-28a-hp0523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法和Western blot法鑒定表達(dá)后,并以Ni~(2+)-NTA柱分離純化目的蛋白;進(jìn)一步對(duì)純化蛋白進(jìn)行生物學(xué)活性研究; 3.擴(kuò)增獲得hp0523基因上下游同源臂片段F1、F2,構(gòu)建自殺質(zhì)粒pBlueKM40/△hp0523::Km~r,電擊轉(zhuǎn)化H.pylori后經(jīng)抗生素篩選hp0523基因缺失株,再進(jìn)行PCR鑒定; 4.采用hp0523基因缺失株和野生株與胃癌上皮細(xì)胞BGC-823進(jìn)行共培養(yǎng),而后采用Western blot法檢測(cè)CagA蛋白轉(zhuǎn)運(yùn)能力變化。 結(jié)果: 1.擴(kuò)增獲得的hp0523基因全長(zhǎng)為510bp,編碼169aa,與GenBank公布的26695、J99同源性為92～95%;蛋白相對(duì)分子量Mr預(yù)測(cè)為19.7kDa,等電點(diǎn)pI為9.53;蛋白二級(jí)結(jié)構(gòu)分析,在33～165位aa之間存在一個(gè)保守的SLT催化基序,第56位谷氨酸(GLU56)是催化活性的中心位點(diǎn),C端序列上含有一個(gè)肽聚糖結(jié)合域“AVGAY”,故預(yù)測(cè)hp0523基因可能是一個(gè)肽聚糖水解酶編碼基因; 2.構(gòu)建獲得了原核表達(dá)載體pET-28a-hp0523,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Ni~(2+)-NTA柱梯度洗脫后,分離獲得了目的蛋白HP0523; 3.HP0523經(jīng)復(fù)性處理后,采用溶菌斑點(diǎn)實(shí)驗(yàn)證實(shí)其具有溶菌活力;而后采用SDS煮沸法分離提取獲得細(xì)菌肽聚糖,進(jìn)行凝膠酶譜分析后,發(fā)現(xiàn)HP0523蛋白具有肽聚糖水解能力;理化特性研究表明,HP0523具有廣譜的溶菌能力,但酶活力較溶菌酶低;其最適酶活力pH值為6.0; 4.連接F1、F2后構(gòu)建自殺質(zhì)粒pBlueKM40/△hp0523::Km~r,電擊轉(zhuǎn)化后經(jīng)抗生素篩選,并經(jīng)PCR驗(yàn)證后獲得了hp0523基因缺失株;缺失株、野生株分別與胃癌上皮細(xì)胞BGC-823進(jìn)行共培養(yǎng)后,發(fā)現(xiàn)hp0523基因缺失株處理組,胃癌上皮細(xì)胞內(nèi)未能檢測(cè)到CagA的轉(zhuǎn)運(yùn)。 結(jié)論: 本研究認(rèn)為hp0523基因是一個(gè)肽聚糖水解酶的編碼基因,且其參與CagA的轉(zhuǎn)運(yùn),可能是Cag-PAIⅣ型樣分泌裝置的組成成分之一。
[Abstract]:Helicobacter pylori Helicobacter pylori (H.pylori), a cytotoxic protein, is an important virulence factor, which is closely related to the occurrence of gastric cancer. It is a type IV secretory device encoded by cagpathogenicity and Cag-PAI, which is encoded by Cag pathogenicity and Cag-PAI, and is transported into gastric epithelial cells. In this paper, the function of the hp0523 gene encoded in Cag-PAI and its role in the CagA transport process were preliminarily studied. The aim is to lay a foundation for further study on the pathogenesis of Cag-PAI. Methods:. 1. The hp0523 gene was cloned from H. pylori genomic DNA by PCR, then constructed pGEM-T-hp0523, sequenced and analyzed by bioinformatics. 2. PET-28a-hp0523 prokaryotic expression vector was constructed and transformed into the host strain BL21DDE3.The expression was identified by SDS-PAGE and Western blot after IPTG induction, and the target protein was isolated and purified by Ni~(2 column, and the biological activity of the purified protein was further studied. 3. The upstream and downstream homologous arm fragment F1 / F2 of hp0523 gene was amplified, and the suicide plasmid pBlueKM40/ hp0523: 1: Kmrwas constructed. After electroporation of H. pylori, the hp0523 gene deletion strain was screened by antibiotics and then identified by PCR. 4. Hp0523 gene deletion strain and wild strain were co-cultured with BGC-823 of gastric cancer epithelial cells, and then Western blot assay was used to detect the change of CagA protein transport ability. Results:. 1. The total length of hp0523 gene was 510bp, encoding 169aa, and the homology with 26695NJ99 published by GenBank was 92n9595. The relative molecular weight of the protein was predicted to be 19.7kDaand the isoelectric point Pi was 9.53.The secondary structure of the protein was analyzed, and there was a conservative SLT catalytic motif between 335AA and 165AA, and the relative molecular weight of the protein was 19.7 kDa and the isoelectric point Pi was 9.53A, respectively. Glutamate GLU56 is the central site of catalytic activity. The C-terminal sequence contains a peptidoglycan binding domain "AVGAY". It is predicted that the hp0523 gene may be a peptidoglycan hydrolase coding gene. 2. The prokaryotic expression vector pET-28a-hp0523 IPTG was constructed, the target protein was identified by SDS-PAGE and Western blot, and the target protein HP0523 was isolated by gradient elution with Ni~(2 column. 3. After renaturation, the bacteriolytic activity of HP0523 was confirmed by plaque test, and the bacterial peptidoglycan was extracted by SDS boiling method. The results of gel enzyme analysis showed that HP0523 protein had peptidoglycan hydrolysis ability. The physicochemical properties showed that HP0523 had broad spectrum bacteriolytic ability, but the enzyme activity was lower than that of lysozyme, and its optimum enzyme activity pH value was 6.0. 4. The suicide plasmid pBlueKM40/ hp0523: 1: Kmrwas constructed by ligation of F1 and F2. After electroporation, the deletion strain of hp0523 gene was screened by antibiotics and verified by PCR, and the deletion strain and wild strain were co-cultured with BGC-823 cells of gastric cancer epithelium, respectively. It was found that CagA transport could not be detected in gastric cancer epithelial cells treated with hp0523 gene deletion strain. Conclusion:. This study suggests that hp0523 gene is a gene encoding peptidoglycan hydrolase, and it is involved in the transport of CagA, which may be one of the components of Cag-PAI type 鈪，

本文編號(hào)：1611509