幽门螺杆菌Cag致病岛中hp0523基因功能的鉴定与分析

发布时间：2018-03-14 13:57

  本文选题：幽门螺杆菌　切入点：Cag致病岛　出处：《江苏大学》2009年硕士论文　论文类型：学位论文

【摘要】： 幽门螺杆菌(Helicobacter pylori,H.pylori)的细胞毒性蛋白A(CagA)是一个重要的毒力因子,与胃癌的发生密切相关。其是通过Cag致病岛(cagpathogenicity island,Cag-PAI)编码的Ⅳ型样分泌装置,转运进入胃上皮细胞,发挥毒性作用。而Cag-PAI中编码的基因功能及其致病机理均尚未十分明确。鉴此,本文初步研究了Cag-PAI中编码的hp0523基因的功能,并初步探讨其在CagA转运过程中的作用,旨在为深入研究Cag-PAI的致病机理奠定基础。 方法: 1.利用PCR技术从H.pylori基因组DNA中扩增获取hp0523基因,T-A克隆后构建pGEM-T-hp0523,进行序列测定;并对其序列进行生物信息学分析研究; 2.构建pET-28a-hp0523原核表达载体,转化表达宿主菌BL21(DE3);经IPTG诱导后,SDS-PAGE法和Western blot法鉴定表达后,并以Ni~(2+)-NTA柱分离纯化目的蛋白;进一步对纯化蛋白进行生物学活性研究; 3.扩增获得hp0523基因上下游同源臂片段F1、F2,构建自杀质粒pBlueKM40/△hp0523::Km~r,电击转化H.pylori后经抗生素筛选hp0523基因缺失株,再进行PCR鉴定; 4.采用hp0523基因缺失株和野生株与胃癌上皮细胞BGC-823进行共培养,而后采用Western blot法检测CagA蛋白转运能力变化。 结果: 1.扩增获得的hp0523基因全长为510bp,编码169aa,与GenBank公布的26695、J99同源性为92～95%;蛋白相对分子量Mr预测为19.7kDa,等电点pI为9.53;蛋白二级结构分析,在33～165位aa之间存在一个保守的SLT催化基序,第56位谷氨酸(GLU56)是催化活性的中心位点,C端序列上含有一个肽聚糖结合域“AVGAY”,故预测hp0523基因可能是一个肽聚糖水解酶编码基因; 2.构建获得了原核表达载体pET-28a-hp0523,IPTG诱导后,经SDS-PAGE鉴定和Western blot鉴定有目的蛋白表达;采用Ni~(2+)-NTA柱梯度洗脱后,分离获得了目的蛋白HP0523; 3.HP0523经复性处理后,采用溶菌斑点实验证实其具有溶菌活力;而后采用SDS煮沸法分离提取获得细菌肽聚糖,进行凝胶酶谱分析后,发现HP0523蛋白具有肽聚糖水解能力;理化特性研究表明,HP0523具有广谱的溶菌能力,但酶活力较溶菌酶低;其最适酶活力pH值为6.0; 4.连接F1、F2后构建自杀质粒pBlueKM40/△hp0523::Km~r,电击转化后经抗生素筛选,并经PCR验证后获得了hp0523基因缺失株;缺失株、野生株分别与胃癌上皮细胞BGC-823进行共培养后,发现hp0523基因缺失株处理组,胃癌上皮细胞内未能检测到CagA的转运。 结论: 本研究认为hp0523基因是一个肽聚糖水解酶的编码基因,且其参与CagA的转运,可能是Cag-PAIⅣ型样分泌装置的组成成分之一。
[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，

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