本文選題：幽門(mén)螺桿菌 + 尿素酶B亞單位��； 參考：《浙江大學(xué)》2005年博士論文

【摘要】：幽門(mén)螺桿菌(H.pylori)是定植于人的胃上皮的革蘭氏陰性致病菌,它是慢 性胃炎、胃潰瘍的主要致病因子,并且與胃癌和胃淋巴瘤的發(fā)生密切相關(guān)。在 H.pylori各組分抗原中,被證明可誘導(dǎo)產(chǎn)生免疫保護(hù)作用的有尿素酶、熱休克蛋 白、空泡毒素、毒素相關(guān)蛋白等。尿素酶不僅是幽門(mén)螺桿菌重要的定植因子, 它還是一重要毒力因子。尿素酶包括A、B亞單位,分子量分別為29.5kDa、66.0 kDa,尿素酶B亞單位是無(wú)毒的、具有高免疫原性的保護(hù)性侯選抗原。本研究 克隆了浙江省幽門(mén)螺桿菌的尿素酶B亞單位(UreB)基因,構(gòu)建了表達(dá)載體分 別轉(zhuǎn)化煙草、水稻、大腸桿菌和乳酸乳球菌,并作相應(yīng)的免疫研究。 本研究對(duì)H.pylori浙江臨床株的UreB基因的克隆和序列分析,證明了UreB 基因在不同菌株間的高度的同源性,這為UreB作為保護(hù)性抗原的H.pylori疫苗 提供可很好的理論支持。本研究選用煙草作為材料來(lái)探索植物生產(chǎn)疫苗的可行 性。通過(guò)構(gòu)建植物表達(dá)載體pBIl21.ureB,以根癌農(nóng)桿菌介導(dǎo)法轉(zhuǎn)化煙草 (Nicotiana tabacum L.),獲得50株轉(zhuǎn)基因植株。經(jīng)抗生素抗性篩選和PCR、 PCR-Southern鑒定,證實(shí)35株T_0代煙草植株中有ureB基因整合于基因組; RT-PCR和Western blot分別在轉(zhuǎn)錄水平和翻譯水平顯示該外源蛋白已在轉(zhuǎn)基因 基因植株中表達(dá)。本研究中獲得的結(jié)果第一次報(bào)道H.pylori ureB基因在植物 系統(tǒng)中表達(dá),為通過(guò)轉(zhuǎn)基因植物來(lái)生產(chǎn)UreB重組抗原用作抗H pylori感染 的疫苗提供了較好的方法學(xué)。 建立了農(nóng)桿菌介導(dǎo)的利用潮霉素抗性作為篩選標(biāo)記的水稻轉(zhuǎn)化系統(tǒng)。以水 稻成熟胚誘導(dǎo)的愈傷組織為材料,把H pylori的ureB基因連接到CaMV35S 啟動(dòng)子后構(gòu)成pCAMBIA.13011.ureB表達(dá)載體,通過(guò)根癌農(nóng)桿菌EHAl05的對(duì) 水稻胚性愈傷組織進(jìn)行感染,通過(guò)共培養(yǎng)和潮霉素抗性篩選,得到12株轉(zhuǎn)基因 再生植株。對(duì)所得12株再生苗進(jìn)行PCR 檢測(cè),表明陽(yáng)性苗比例為75%。 PCR—Southern結(jié)果進(jìn)一步證明目的基因確已整合到水稻基因組中。Western-blot 結(jié)果同樣顯示UreB蛋白已經(jīng)水稻中得到表達(dá)。本研究結(jié)果的獲得,將為以后 進(jìn)行轉(zhuǎn)基因后代遺傳穩(wěn)定性和基因表達(dá)水平的研究,以及以水稻作為載體進(jìn) 行動(dòng)物口服免疫試驗(yàn)奠定基礎(chǔ)。 本研究為了闡明人胃幽門(mén)螺桿菌UreB及UreB部分片段的作為免疫原的特 性,用穿梭質(zhì)粒分別構(gòu)建了分別構(gòu)建pAMJ399-ureB、pAMJ399-f、pAMJ399-e、 pAMJ399-m,以此分別轉(zhuǎn)化大腸桿菌JM109進(jìn)行表達(dá)研究。經(jīng)SDS-PAGE和 Western blot分析表明,含pAMJ399-ureB、pAMJ399-e的E coli均能表達(dá)外源 蛋白或肽,其分子量大小分別為66kDa和28kDa,免疫印跡分析證實(shí)這些重組
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類(lèi)號(hào)】：R392

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