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  本文選題：幽門螺桿菌　切入點：尿素酶B　出處：《華中農(nóng)業(yè)大學(xué)》2010年碩士論文

【摘要】： 幽門螺桿菌(Helicobacter pylori, H.pylori)是一種螺旋狀、微需氧的革蘭氏陰性桿菌,被世界衛(wèi)生組織列為Ⅰ類致癌因子(TypeⅠcarcinogen)。該菌與人的慢性活動性胃炎、胃和十二指腸消化性潰瘍、胃黏膜相關(guān)性淋巴組織淋巴瘤(MALT)以及胃腺癌的發(fā)生密切相關(guān)。目前臨床上針對H.pylori使用的抗生素治療方法并不理想,有著諸多缺點,因此利用疫苗進(jìn)行預(yù)防接種和治療有望成為控制H.pylori感染行之有效方法。尿素酶B亞單位(UreB)是H.pylori的一個重要保護(hù)性抗原,動物模型中也已經(jīng)驗證其作為疫苗抗原的安全性和有效性,尋找鑒定其有效的抗原表位對于H.pylori的診斷、治療和疫苗開發(fā)具有重要的意義。本研究的目的是要篩選出單克隆抗體A1H10、B3D9、A3C10所對應(yīng)的B細(xì)胞抗原表位,并對其免疫學(xué)功能進(jìn)行初步研究。本課題共分為以下三部分： 1.抗幽門螺桿菌UreB單克隆抗體A1H10、B3D9、A3C10抗原識別表位的鑒定 通過多次截短法分段表達(dá)19段UreB片段,并在此基礎(chǔ)上通過免疫印跡方法逐步確定單克隆抗體A1H10、B3D9、A3C10所識別的抗原表位。我們成功構(gòu)建了19個表達(dá)載體且均實現(xiàn)了表達(dá)。免疫印跡方法確定了單克隆抗體A1H10、B3D9、A3C10所對應(yīng)的包含15個氨基酸殘基的抗原表位,名稱分別為UP32(GGGTGPADGTNATTI)、UP35 (WMLRAAEEYSMNLGF)、UP38 (TLHDMGIFSITSSDS),分別位于H.pylori尿素酶B亞單位(UreB)上第158-172、181-195、349-363位氨基酸殘基。所鑒定出來的3個表位通過ELISA得到進(jìn)一步地證實,純化的融合表位蛋白能與相對應(yīng)的單抗及臨床感染H.pylori的病人陽性血清發(fā)生特異性的結(jié)合反應(yīng)。以鑒定出來的抗原表位核苷酸序列為依據(jù),采用化學(xué)合成法合成表位多肽,分別命名為UP32、UP35、UP38,純度為90%以上。經(jīng)ELISA或Dot blot方法鑒定,ELISA結(jié)果顯示單克隆抗體A1H10、B3D9、A3C10分別與合成肽UP32、UP35、UP38發(fā)生了特異性抗原抗體反應(yīng),說明單抗A1H10、B3D9、A3C10所識別的抗原表位分別為UP32、UP35、UP38。Dot blot方法鑒定結(jié)果表明單抗B3D9所針對的抗原表位為UP35。ELISA結(jié)果顯示合成肽與H.pylori的病人陽性血清也發(fā)生了結(jié)合反應(yīng),說明病人血清中產(chǎn)生了針對這些表位的抗體。 2.幽門螺桿菌尿素酶B亞單位(UreB)B細(xì)胞抗原表位的免疫學(xué)功能研究 將純化后的融合表位蛋白免疫小鼠,獲得了較高效價的抗血清,其效價高達(dá)1：51200,說明我們構(gòu)建的融合表位蛋白能夠很好地激發(fā)機(jī)體的體液免疫應(yīng)答。免疫印跡試驗證實融合表位蛋白產(chǎn)生的抗血清能夠與重組的截短片段UreBM及天然的幽門螺桿菌UreB發(fā)生特異性抗原抗體反應(yīng)。ELISA結(jié)果發(fā)現(xiàn),免疫的鼠抗血清能夠很好地識別相應(yīng)的表位合成肽,同時也能與天然UreB發(fā)生反應(yīng)。尿素酶活性抑制試驗表明,單抗A1H10、B3D9、A3C10對尿素酶的活性具有較好抑制作用,抑制率可達(dá)70%,多抗血清對尿素酶的活性也有一定的抑制作用,抑制率可達(dá)50%。 3.呈現(xiàn)表達(dá)串聯(lián)表位及其免疫學(xué)評價 通過基因重組的方法,將串連的表位基因片段敲入到本實驗室構(gòu)建好的減毒沙門菌株S.ty2ΔrfaHΔssaV染色體上,與外膜蛋白A (OmpA)的C-端融合表達(dá)于載體菌表面,得到重組菌S. ty2ΔrfaHΔssaV-U258,通過免疫印跡分析證實目標(biāo)蛋白獲得了表達(dá)。經(jīng)口胃途徑將表達(dá)系統(tǒng)S.ty2ΔrfaHΔssaV-U258免疫小鼠,高、低劑量組都有效地激發(fā)了機(jī)體產(chǎn)生針對外源抗原UreB的免疫反應(yīng),抗外源抗原的IgG滴度高達(dá)1：3200,抗載體菌的IgG滴度為1：800-1：3200,而且也能夠檢測到抗原特異性的分泌型IgA的產(chǎn)生,表明口服免疫的兩組均能刺激機(jī)體產(chǎn)生有效的黏膜免疫反應(yīng)。 總之,本研究通過分子生物學(xué)與免疫學(xué)結(jié)合的方法鑒定出了單克隆抗體A1H10、B3D9、A3C10所對應(yīng)的抗原表位,經(jīng)動物實驗驗證具有良好的免疫原性,為新型多聯(lián)多聚表位疫苗的開發(fā)提供了重要的理論基礎(chǔ)和實驗依據(jù)。
[Abstract]:Helicobacter pylori (Helicobacter pylori H.pylori) is a spiral, microaerophilic gram negative bacilli, WHO was listed as a class I carcinogen (Type I carcinogen). The pathogen of chronic active gastritis and stomach and duodenum, peptic ulcer, gastric mucosa associated lymphoid tissue lymphoma (MALT) and gastric cancer is closely related to the current clinical use of H.pylori for antibiotic therapy is not ideal, there are many shortcomings, therefore the use of vaccine and treatment of H.pylori infection control is expected to become effective methods. Urease B subunit (UreB) is an important protective antigen of H.pylori, animal model also has been verified as a safe and effective vaccine antigens, to identify the effective epitopes for H.pylori diagnosis, treatment and vaccine development has important significance The purpose of this study is to screen out the B cell epitopes corresponding to monoclonal antibodies A1H10, B3D9 and A3C10, and to preliminarily study their immunological functions. The research is divided into three parts.
Identification of 1. anti Helicobacter pylori UreB monoclonal antibody A1H10, B3D9, A3C10 antigen recognition epitope
Through several truncated fractional expression of 19 UreB fragments, and on this basis by immunoblotting method identified by monoclonal antibody A1H10, B3D9 epitope recognized by A3C10. We successfully constructed 19 expression vector and have achieved expression. Western blot confirmed A1H10 monoclonal antibody, B3D9, corresponding to A3C10 contains 15 amino acid epitopes, the name was UP32 (GGGTGPADGTNATTI), UP35 (WMLRAAEEYSMNLGF), UP38 (TLHDMGIFSITSSDS), located at H.pylori urease B subunit (UreB) on the 158-172181-195349-363 amino acid residues. The 3 epitopes were further confirmed by ELISA identified, combined with patient reaction positive serum purified fusion protein with the corresponding epitope of monoclonal antibody and clinical H.pylori infection specific. To identify the anti epitope nucleotide sequence according to the According to the synthetic epitope peptides by chemical synthesis, which were named UP32, UP35, UP38, the purity was more than 90%. By ELISA or Dot blot identification methods, ELISA results showed that the monoclonal antibody A1H10, B3D9, A3C10 respectively with the synthetic peptide UP32, UP35, the specificity of antigen antibody reaction UP38 that mAb A1H10 B3D9 A3C10, the identified epitopes were UP32, UP35, blot method identification results of UP38.Dot revealed that B3D9 antibody against epitope UP35.ELISA results showed that patients with H.pylori positive serum peptide has the binding reaction to that in serum of patients with the antibodies against these epitopes.
Immunological function of 2. Helicobacter pylori urease B subunit (UreB) B cell antigen epitopes
The purified fusion epitope protein immunized mice obtained high titer antiserum. The titer of 1:51200, we construct the table shows the fusion of humoral immune response protein is able to stimulate the body. Western blot showed that antiserum epitope fusion protein can produce truncated fragments of UreBM and recombinant and the natural UreB of Helicobacter pylori specific antigen antibody reaction.ELISA results showed that mouse antiserum immune to identify the epitopes of the corresponding synthetic peptides, also can react with natural UreB. Urease activity inhibition test showed that the monoclonal antibody A1H10, B3D9, A3C10 on the activity of urease has good inhibition, inhibition the rate of up to 70%, the activity of urease antiserum also has certain inhibitory effect, the inhibition rate is 50%.
3. presentation of expression in series epitopes and their immunological evaluation
By the method of gene recombination, the tandem epitope gene knock in the laboratory to construct the attenuated Salmonella strain S.ty2 Delta rfaH Delta ssaV chromosome, and outer membrane protein A (OmpA) C- terminal fusion expression on the carrier surface to obtain the recombinant bacteria S. bacteria, Ty2 Delta rfaH Delta ssaV-U258, through Western blot analysis confirmed that the target protein was expressed. The oral route expression system S.ty2 Delta rfaH Delta ssaV-U258 immunized mice, high, low dose group can effectively stimulate the immune response to exogenous antigen UreB, IgG titers of anti exogenous antigen up to 1:3200, IgG titers of anti bacteria was 1:800-1:3200 carrier, but also can detection of antigen-specific secretory IgA, indicating that the two groups of oral immunization can elicit mucosal immune response effectively.
In conclusion, this study through the identification of molecular biology and immunology with a monoclonal antibody A1H10, B3D9 epitope corresponding to A3C10, the animal experiment has good immunogenicity, provides an important theoretical basis and experimental basis for the new multi poly epitope vaccine development.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 翟偉強(qiáng);減毒傷寒沙門氏菌作為表面展示疫苗載體的應(yīng)用研究[D];華中農(nóng)業(yè)大學(xué);2011年

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本文編號：1661962