【摘要】： 目的 幽門螺桿菌(Helicobacter pylori,Hp)是一種定植于胃內(nèi)的革蘭氏染色陰性、螺桿狀、微需氧菌,目前全球近50%的人口感染Hp,在發(fā)展中國(guó)家,其感染率可高達(dá)70%以上。Hp的抗感染治療存在較大問題,使用單劑藥物根除率低,而使用多聯(lián)藥物費(fèi)用昂貴,且不能有效阻止Hp的再感染和復(fù)發(fā)。此外,耐藥菌株的增加也使抗Hp治療面臨越來越復(fù)雜的難題。種種因素使Hp疫苗有望成為治療Hp感染最有效的途徑。Hp自然感染時(shí),機(jī)體存在免疫耐受狀態(tài),激發(fā)的免疫應(yīng)答不能起到保護(hù)作用,因而必須在抗原選擇以及表位水平對(duì)抗原進(jìn)行改造,激發(fā)更有效的免疫應(yīng)答。表位疫苗是近年來發(fā)展起來的一種獨(dú)特的疫苗設(shè)計(jì)思路,是目前研制某些感染性疾病、惡性腫瘤疫苗以及自身免疫性疾病等疫苗設(shè)計(jì)的新方向。基于表位的疫苗設(shè)計(jì)可以特異性的增強(qiáng)保護(hù)性免疫反應(yīng)的強(qiáng)度而排除非保護(hù)性甚至是抑制性免疫反應(yīng)的影響。因此,尋找鑒定Hp有效的抗原表位對(duì)發(fā)展Hp疫苗具有重要的理論指導(dǎo)意義。 幽門螺桿菌可以產(chǎn)生大量的尿素酶,該尿素酶對(duì)細(xì)菌在體內(nèi)的定植及致病發(fā)揮著重要作用。尿素酶主要由A (UreA)和B (UreB)兩個(gè)亞單位組成,分子量分別約為30kD和63kD。業(yè)已證實(shí)尿素酶B亞單位(UreB)是幽門螺桿菌的一個(gè)重要保護(hù)性抗原,近年已有很多學(xué)者對(duì)其展開了多方面研究。本實(shí)驗(yàn)室前期通過基因工程方法構(gòu)建了重組幽門螺桿菌尿素酶B亞單位,制備了針對(duì)此重組UreB抗原的單克隆抗體,并在研究中發(fā)現(xiàn)其中一株單克隆抗體6E6具有抗Hp定植的作用。本研究擬通過體外尿素酶活性中和試驗(yàn)進(jìn)一步鑒定Hp尿素酶B亞單位單克隆抗體6E6的中和作用,同時(shí)通過生物信息學(xué)方法和截短法分段表達(dá)UreB片段,并在此基礎(chǔ)上采用表位作圖的方法篩選出單克隆抗體所對(duì)應(yīng)的B細(xì)胞抗原表位,分析其免疫學(xué)功能,為Hp新型表位疫苗的研制提供理論和實(shí)驗(yàn)依據(jù)。 方法 1.通過體外尿素酶活性中和試驗(yàn)對(duì)Hp尿素酶B亞單位單克隆抗體6E6的功能進(jìn)行分析,根據(jù)不同劑量和同一劑量不同時(shí)間的依賴關(guān)系,鑒定單克隆抗體6E6的中和Hp尿素酶活性的作用。 2.通過生物信息學(xué)方法對(duì)尿素酶B亞單位蛋白的親水性、抗原性和表面可及性等進(jìn)行分析,結(jié)合尿素酶B亞單位蛋白的三級(jí)結(jié)構(gòu)分析圖,依據(jù)尿素酶B亞單位蛋白功能、結(jié)構(gòu)穩(wěn)定性,選擇性的將UreB蛋白分成5段:U12、U13、U47、U15和U16,進(jìn)行克隆表達(dá)。 3.采用PCR方法從Hp NCTC11637基因組中擴(kuò)增U12、U13、U47、U15和U16目的基因片段,并構(gòu)建到原核表達(dá)載體pET-11c,通過酶切、測(cè)序鑒定陽(yáng)性重組子。將陽(yáng)性重組子轉(zhuǎn)化宿主菌大腸桿菌BL21 (DE3)中,IPTG誘導(dǎo)表達(dá),Tris-Tricine PAGE電泳確定目的蛋白表達(dá),通過免疫印跡方法初步確定單克隆抗體6E6識(shí)別B細(xì)胞抗原表位的區(qū)域。 4.根據(jù)免疫印跡方法初步確定的B細(xì)胞抗原表位的區(qū)域,采用步移合成表位多肽的方法合成4條15肽,通過ELISA和Dot blot方法雙重鑒定,精確定位單克隆抗體6E6所對(duì)應(yīng)的B細(xì)胞抗原表位。 5.將B細(xì)胞抗原表位與載體蛋白BSA應(yīng)用戊二醛進(jìn)行交聯(lián),然后將交聯(lián)物皮下免疫BALB/c小鼠,同時(shí)做單一表位組和PBS組對(duì)照。初免加弗氏完全佐劑,次免加弗氏不完全佐劑,分別于0、7、14、28天免疫接種,劑量為100μg/只/次。末次免疫 7天后,ELISA檢測(cè)免疫后抗血清,測(cè)定抗體水平。并通過免疫印跡方法檢測(cè)抗血清能否識(shí)別不同天然Hp菌株的尿素酶B亞單位蛋白。 6.通過B細(xì)胞抗原表位與單克隆抗體6E6競(jìng)爭(zhēng)抑制尿素酶活性的試驗(yàn)以及B細(xì)胞表位對(duì)臨床感染Hp患者的血清學(xué)分析試驗(yàn)鑒定此表位的作用。 結(jié)果 1.體外尿素酶活性中和試驗(yàn)表明,單克隆抗體6E6能夠中和尿素酶的活性,其抑制率隨劑量的增加而增強(qiáng),并且隨時(shí)間的延長(zhǎng),其抑制率降低。 2.通過生物信息學(xué)方法分析,選擇了UreB蛋白中5’端的第200、230、250、260、300和390位氨基酸分別作為構(gòu)建UreB部分抗原的分割點(diǎn),克隆U12(1～300aa)、U13(1～200aa)、U47(250～390aa)、U15(1～230aa)和U16(1～260aa) 5個(gè)目的片段。 3.成功構(gòu)建了包含目的片段U12(1～300aa)、U13(1～200aa)、U47(250～390aa)、U15(1～230aa)和U16(1～260aa)的5個(gè)表達(dá)載體pET11c-U12、pET11c-U13、pET11c-U47、pET11c-U15和pET11c-U16,Tris-Tricine PAGE電泳確定目的蛋白表達(dá),目的片段分子量分別約為33.0kD、22.0kD、15.4kD、25.3kD和28.6kD。免疫印跡方法初步確定單克隆抗體6E6所對(duì)應(yīng)的抗原表位位于UreB蛋白5’端的第200～230位氨基酸內(nèi)。 4.步移合成表位多肽U201-215、U206-220、U211-225和U216-230,經(jīng)ELISA和Dot blot方法雙重鑒定,單克隆抗體6E6與合成肽U211-225發(fā)生了特異性抗原抗體反應(yīng),說明單抗6E6識(shí)別的B細(xì)胞抗原表位即為U211-225。 5. B細(xì)胞抗原表位U211-225與載體蛋白BSA交聯(lián)免疫動(dòng)物后,產(chǎn)生了較高效價(jià)的抗血清,其效價(jià)高達(dá)1:32000。免疫印跡試驗(yàn)證實(shí)載體蛋白BSA和B細(xì)胞表位U211-225交聯(lián)物產(chǎn)生的抗血清能夠與4株Hp天然菌株的尿素酶B亞單位發(fā)生特異性抗原抗體反應(yīng)。 6. B細(xì)胞表位U211-225與抗幽門螺桿菌尿素酶B亞單位單克隆抗體6E6的競(jìng)爭(zhēng)抑制尿素酶活性試驗(yàn)表明,此B細(xì)胞表位U211-225能夠拮抗單克隆抗體6E6中和尿素酶的活性。血清學(xué)分析試驗(yàn)顯示,57份幽門螺桿菌感染患者血清中,有37份能夠識(shí)別此B細(xì)胞表位U211-225。 結(jié)論 1.單克隆抗體6E6能夠抑制尿素酶的活性,是針對(duì)Hp的一種中和性抗體。 2.單克隆抗體6E6所針對(duì)的B細(xì)胞抗原表位位于UreB蛋白5′端第211～225位氨基酸內(nèi)。 3. B細(xì)胞抗原表位U211-225是Hp的一個(gè)優(yōu)勢(shì)(顯性)表位,可以作為幽門螺桿菌的表位疫苗成分和診斷靶分子。
[Abstract]:objective
Helicobacter pylori (Hp) is a gram-negative, screw-like, micro-aerobic bacteria colonized in the stomach. At present, nearly 50% of the world's population is infected with Hp. In developing countries, the infection rate can be as high as 70%. The anti-infective treatment of Hp has great problems, the eradication rate of single drug is low, and the cost of using multi-drug is high. In addition, the increase of drug-resistant strains also makes anti-Hp treatment more and more complex. Various factors make Hp vaccine hopefully the most effective way to treat Hp infection. Epitope vaccine is a unique vaccine design idea developed in recent years. It is a new direction of vaccine design for some infectious diseases, malignant tumor vaccines and autoimmune diseases. Epitope-based vaccine design It can specifically enhance the intensity of protective immune response and exclude the influence of non-protective or even inhibitory immune response. Therefore, it is of great theoretical significance to identify the effective epitopes of Hp for the development of Hp vaccine.
Helicobacter pylori can produce a large number of urease, urease plays an important role in the colonization and pathogenesis of bacteria in vivo. Urease mainly consists of two subunits A (UreA) and B (UreB), the molecular weight of which is about 30 kD and 63 kD respectively. It has been confirmed that urease B subunit (UreB) is an important protective antigen of Helicobacter pylori. Many scholars have studied it in many aspects in recent years. In our laboratory, we constructed a recombinant urease B subunit of Helicobacter pylori by genetic engineering and prepared monoclonal antibodies against the recombinant UreB antigen. In our study, we found that one of the monoclonal antibodies, 6E6, had the effect of anti-HP colonization. The neutralization effect of monoclonal antibody 6E6 against Hp urease B subunit was further identified by urease activity neutralization test. The UreB fragment was segmented and expressed by Bioinformatics Method and truncation method. On this basis, the B cell epitopes corresponding to the monoclonal antibody were screened by epitope mapping, and its immunological function was analyzed. The development of epitope vaccine provides theoretical and experimental basis.
Method
1. The function of monoclonal antibody 6E6 to Hp urease B subunit was analyzed by urease activity neutralization test in vitro. The neutralization effect of monoclonal antibody 6E6 on Hp urease activity was identified according to the dependence of different doses and different time at the same dose.
2. The hydrophilicity, antigenicity and surface accessibility of urease B subunit protein were analyzed by bioinformatics method. According to the function and structural stability of urease B subunit protein, UreB protein was selectively divided into five segments: U12, U13, U47, U15 and U16.
3. Purpose gene fragments of U12, U13, U47, U15 and U16 were amplified from Hp NCTC11637 genome by PCR and constructed into prokaryotic expression vector pET-11c. The positive recombinant was identified by enzyme digestion and sequencing. Immunoblotting method preliminarily identified the region of B cell epitopes recognized by monoclonal antibody 6E6.
4. Four 15 peptides were synthesized by step-by-step synthesis of epitope polypeptides according to the region of B cell antigen epitopes preliminarily determined by Western blot. The B cell epitopes corresponding to monoclonal antibody 6E6 were accurately located by ELISA and Dot blot.
5. The B cell antigen epitope was cross-linked with the carrier protein BSA by glutaraldehyde, and then subcutaneously immunized BALB/c mice with the cross-linking substance. At the same time, the BALB/c mice were immunized with single epitope group and PBS group as control group.
After 7 days, ELISA was used to detect the anti-serum and the antibody level, and the immunoblotting method was used to detect whether the anti-serum could recognize the urease B subunit protein of different natural Hp strains.
6. The effect of B-cell epitope on urease activity was identified by competitive inhibition of B-cell epitope with monoclonal antibody 6E6 and serological analysis of clinical infected patients with HP.
Result
1. In vitro neutralization test of urease activity showed that monoclonal antibody 6E6 could neutralize the activity of urease, and its inhibition rate increased with the increase of dosage, and decreased with the increase of time.
2. By bioinformatics analysis, the amino acids at positions 200, 230, 250, 260, 300 and 390 of the 5'end of UreB protein were selected as the splitting points for constructing partial antigens of UreB. Five target fragments of U12 (1-300aa), U13 (1-200aa), U47 (250-390aa), U15 (1-230aa) and U16 (1-260aa) were cloned.
3. Five expression vectors including target fragments U12 (1-300aa), U13 (1-200aa), U47 (250-390aa), U15 (1-230aa) and U16 (1-260aa) were successfully constructed. The molecular weights of the target fragments were 33.0kD, 22.0kD, 15.4kD, 25.3kD, respectively. The antigen epitope of monoclonal antibody 6E6 was initially located in the 200-230 amino acids of UreB protein 5'.
4. The epitope peptides U201-215, U206-220, U211-225 and U216-230 were synthesized by step-by-step method. The monoclonal antibody 6E6 reacted with the synthetic peptide U211-225 by ELISA and Dot blot, indicating that the B cell epitope recognized by the monoclonal antibody 6E6 was U211-225.
5. The antiserum produced by the cross-linking of the B cell epitope U211-225 with the carrier protein BSA produced a high titer of 1:32000. The immunoblotting test showed that the antiserum produced by the cross-linking of the carrier protein BSA and the B cell epitope U211-225 could react with the urease B subunit of four natural strains of Hp.
6. Competitive inhibition of urease activity by B cell epitope U211-225 and monoclonal antibody 6E6 against urease B subunit of Helicobacter pylori showed that the B cell epitope U211-225 could antagonize the activity of urease neutralized by monoclonal antibody 6E6. Serological analysis showed that 37 of 57 sera from Helicobacter pylori infected patients could recognize the B. Cell epitopes U211-225.
conclusion
1. monoclonal antibody 6E6 can inhibit the activity of urease and is a neutralizing antibody against Hp.
2. The B cell epitope of monoclonal antibody 6E6 is located in the 211-225 amino acids of UreB protein 5'.
3. B cell epitope U211-225 is a dominant (dominant) epitope of Hp and can be used as an epitope vaccine component and diagnostic target molecule of Helicobacter pylori.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

【參考文獻(xiàn)】

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本文編號(hào)：2214916