幽门螺杆菌抗原UreB的基因工程及免疫原性研究

发布时间：2018-05-20 15:35

本文选题：幽门螺杆菌 + 尿素酶B亚单位　；参考：《浙江大学》2005年博士论文

【摘要】：幽门螺杆菌(H.pylori)是定植于人的胃上皮的革兰氏阴性致病菌,它是慢性胃炎、胃溃疡的主要致病因子,并且与胃癌和胃淋巴瘤的发生密切相关。在 H.pylori各组分抗原中,被证明可诱导产生免疫保护作用的有尿素酶、热休克蛋白、空泡毒素、毒素相关蛋白等。尿素酶不仅是幽门螺杆菌重要的定植因子, 它还是一重要毒力因子。尿素酶包括A、B亚单位,分子量分别为29.5kDa、66.0 kDa,尿素酶B亚单位是无毒的、具有高免疫原性的保护性侯选抗原。本研究克隆了浙江省幽门螺杆菌的尿素酶B亚单位(UreB)基因,构建了表达载体分别转化烟草、水稻、大肠杆菌和乳酸乳球菌,并作相应的免疫研究。本研究对H.pylori浙江临床株的UreB基因的克隆和序列分析,证明了UreB 基因在不同菌株间的高度的同源性,这为UreB作为保护性抗原的H.pylori疫苗提供可很好的理论支持。本研究选用烟草作为材料来探索植物生产疫苗的可行性。通过构建植物表达载体pBIl21.ureB,以根癌农杆菌介导法转化烟草 (Nicotiana tabacum L.),获得50株转基因植株。经抗生素抗性筛选和PCR、 PCR-Southern鉴定,证实35株T_0代烟草植株中有ureB基因整合于基因组; RT-PCR和Western blot分别在转录水平和翻译水平显示该外源蛋白已在转基因基因植株中表达。本研究中获得的结果第一次报道H.pylori ureB基因在植物系统中表达,为通过转基因植物来生产UreB重组抗原用作抗H pylori感染的疫苗提供了较好的方法学。建立了农杆菌介导的利用潮霉素抗性作为筛选标记的水稻转化系统。以水稻成熟胚诱导的愈伤组织为材料,把H pylori的ureB基因连接到CaMV35S 启动子后构成pCAMBIA.13011.ureB表达载体,通过根癌农杆菌EHAl05的对水稻胚性愈伤组织进行感染,通过共培养和潮霉素抗性筛选,得到12株转基因再生植株。对所得12株再生苗进行PCR 检测,表明阳性苗比例为75%。 PCR—Southern结果进一步证明目的基因确已整合到水稻基因组中。Western-blot 结果同样显示UreB蛋白已经水稻中得到表达。本研究结果的获得,将为以后进行转基因后代遗传稳定性和基因表达水平的研究,以及以水稻作为载体进行动物口服免疫试验奠定基础。本研究为了阐明人胃幽门螺杆菌UreB及UreB部分片段的作为免疫原的特性,用穿梭质粒分别构建了分别构建pAMJ399-ureB、pAMJ399-f、pAMJ399-e、 pAMJ399-m,以此分别转化大肠杆菌JM109进行表达研究。经SDS-PAGE和 Western blot分析表明,含pAMJ399-ureB、pAMJ399-e的E coli均能表达外源蛋白或肽,其分子量大小分别为66kDa和28kDa,免疫印迹分析证实这些重组
[Abstract]:Helicobacter pylori (H.pylori) is a gram-negative pathogen that is colonized in the human stomach epithelium. It is slow.
The main causative factor of gastritis and gastric ulcer is closely related to the occurrence of gastric cancer and gastric lymphoma.
Among the H.pylori antigens, there are urease, heat shock eggs that have been proved to induce immune protection.
Vacuoles, toxin related proteins, etc. urease is not only an important colonizing factor for H. pylori.
It is also an important virulence factor. Urease includes A, B subunits, and the molecular weight is 29.5kDa, 66 respectively.
KDa, urease B subunit is a non-toxic and highly immunogenic candidate antigen.
The urease B subunit (UreB) gene of Helicobacter pylori in Zhejiang province was cloned and the expression vector was constructed.
Do not transform tobacco, rice, Escherichia coli and Lactococcus lactis, and make corresponding immunological studies.
In this study, cloning and sequence analysis of UreB gene from H.pylori Zhejiang clinical strain proved that UreB
The highly homologous gene among different strains is the H.pylori vaccine of UreB as a protective antigen.
It provides a very good theoretical support. This study uses tobacco as material to explore the feasibility of plant production vaccine.
PBIl21.ureB was transformed into tobacco by Agrobacterium tumefaciens mediated transformation.
(Nicotiana tabacum L.), 50 transgenic plants were obtained. After antibiotic resistance screening and PCR,
PCR-Southern identification confirmed that ureB gene was integrated into the genome of 35 T_0 generation tobacco plants.
RT-PCR and Western blot showed that the foreign protein was transgene at transcriptional level and translation level respectively.
The results obtained in this study are the first to report the H.pylori ureB gene in plants.
It is expressed in the system to produce UreB recombinant antigen through transgenic plants as an anti H pylori infection.
The vaccine provides a good methodology.
Agrobacterium tumefaciens mediated hygromycin resistance as a screening marker for rice transformation system was established.
Callus derived from mature embryo of rice was used as material to connect H pylori ureB gene to CaMV35S.
After the promoter, pCAMBIA.13011.ureB expression vector was formed through agrobacterium tumefaciens EHAl05.
Rice embryogenic callus was infected, and 12 transgenic plants were obtained through co culture and hygromycin resistance screening.
PCR regeneration of 12 regenerated plantlets was conducted. The results showed that the proportion of positive seedlings was 75%.
PCR - Southern results further proved that the target gene had been integrated into the rice genome.Western-blot.
The results also showed that UreB protein had been expressed in rice.
The genetic stability and gene expression level of transgenic offspring were studied, and rice was used as carrier.
The basis of oral immunity test of action objects is laid.
The purpose of this study is to elucidate the immunogenicity of Helicobacter pylori UreB and UreB fragments from human stomach.
PAMJ399-ureB, pAMJ399-f and pAMJ399-e were constructed by shuttle plasmid respectively.
PAMJ399-m was transformed into Escherichia coli JM109 for expression study respectively. SDS-PAGE and
Western blot analysis showed that E coli containing pAMJ399-ureB and pAMJ399-e could express exogenous protein.
The molecular weight of protein or peptide was 66kDa and 28kDa respectively. Western blot analysis confirmed these recombinant proteins.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392

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