幽門螺桿菌基因hp0410的克隆表達(dá)及HP0410的免疫原性研究
發(fā)布時間：2018-09-04 17:21
【摘要】： 一、研究背景和目的 長期以來，醫(yī)學(xué)界普遍認(rèn)為，精神壓力和生活方式等因素是慢性胃炎和消化性潰瘍發(fā)病的主要原因。1983年，澳大利亞科學(xué)家J Robin Warren和Barry J Marshall首次分離培養(yǎng)出幽門螺桿菌(Helicobacter pylori，Hp)，并證實Hp感染可以引起胃炎和消化性潰瘍，為此榮膺2005年諾貝爾生理學(xué)與醫(yī)學(xué)獎。幽門螺桿菌是世界范圍內(nèi)感染率最高的致病菌，也是慢性胃炎和消化性潰瘍主要病原菌，并與胃腺癌和胃黏膜相關(guān)B細(xì)胞淋巴瘤的發(fā)生密切相關(guān)。怎樣有效控制Hp感染，一直是國內(nèi)外學(xué)者所關(guān)注的焦點(diǎn)。 目前，國內(nèi)外常用三聯(lián)藥物(如鉍劑+甲硝唑+抗生素)治療胃炎和消化性潰瘍。但是，Hp容易產(chǎn)生耐藥性，導(dǎo)致抗生素治療失效。如果Hp未能根除，多達(dá)75％-80％的患者很快又舊病復(fù)發(fā)。鑒于抗生素療法的弊端，疫苗接種被認(rèn)為是控制Hp感染最有效的方法。 目前，Hp疫苗研究的熱點(diǎn)主要集中在尿素酶(Ure)，空泡毒素(VacA)和細(xì)胞相關(guān)毒素(CagA)，熱休克蛋白60(HSP60)等亞單位保護(hù)性抗原上。尿素酶難以誘導(dǎo)出全面而穩(wěn)定的保護(hù)性免疫應(yīng)答；CagA和VacA變異較大，且CagA是否為Hp感染所必須還存在有一定爭議；HSP60和人體自身蛋白有比較高的同源性，用HSP60免疫小鼠只能誘導(dǎo)局部保護(hù)性免疫應(yīng)答，并會導(dǎo)致小鼠腸胃炎，其作為疫苗的安全性和有效性尚需進(jìn)一步驗證。在幽門螺桿菌的體內(nèi)清除過程中，黏膜免疫發(fā)揮主要作用。因此，篩選出合適的候選疫苗，誘導(dǎo)抗Hp的保護(hù)性黏膜免疫，仍是Hp疫苗研究的主要方向之一。 黏附素在Hp感染定植過程中不可缺少的，且定位于Hp表面，具有良好的抗原性。特異性的抗黏附素抗體能有效的抑制Hp定植并將其清除。因此，黏附素已成為Hp疫苗候選抗原研究的主要方向之一。幽門螺桿菌黏附素研究熱點(diǎn)主要集中在兩類主要黏附素上：①可與人細(xì)胞表面的Le~b抗原結(jié)合的血型抗原結(jié)合黏附素(blood group antigen-binding adhesin, BAb)；②可與人的紅細(xì)胞緊密結(jié)合的N-乙酰神經(jīng)氨酰乳糖結(jié)合原纖維血凝素(N-acetylneuraminyl-lactose-binding haemagglutinin，NLBH)。NLBH長約1.4kb，分為3個閱讀框(ORF)，其中ORF2亦即hpaA基因，長783bp，，編碼幽門螺桿菌黏附素A(Helicobacter pylori adhesin A，HpaA)為Hp黏附定植過程所必須，hpaA突變的Hp菌株無法在小鼠體內(nèi)定植。hp0410是hpaA基因家族同系成員，長為750bp，很可能為Hp黏附因子的一種，具體功能尚不明確。hp0231是對Hp基因組閱讀框分析所預(yù)測出的基因，長為789bp，表達(dá)產(chǎn)物可能為分泌型的蛋白。目前，有關(guān)HP0410和HP0231的免疫原性以及其作為候選疫苗的前景尚未闡明。 本研究的主要目的是：①通過生物信息學(xué)分析，預(yù)測hp0231和hp0410編碼蛋白的性質(zhì)特征和免疫原性；②在大腸桿菌原核表達(dá)系統(tǒng)中克隆表達(dá)hp0410，并純化獲得重組蛋白；③用重組蛋白篩查胃潰瘍患者和健康人血清，檢測HP0410能否在體內(nèi)誘導(dǎo)出特異性抗體，并進(jìn)行免疫組化實驗，探討該蛋白是否可作為一種新的檢測Hp感染的方法；④建立噬菌體肽展示文庫，對HP0410的抗原表位進(jìn)行研究；⑤建立細(xì)胞模型，初步探討HP0410與胃上皮細(xì)胞的相互作用。通過以上研究，為構(gòu)建多嵌合表位重組疫苗，或以嗜酸乳桿菌為載體的活疫苗的研制提供理論依據(jù)和工作基礎(chǔ)。 二、研究方法 1、提取幽門螺桿菌標(biāo)準(zhǔn)株NCTC11639的基因組DNA，按照GenBank中幽門螺桿菌標(biāo)準(zhǔn)株22695的序列設(shè)計引物PCR，擴(kuò)增hp0231和hp0410基因，克隆入pMD18-T載體中測序，進(jìn)行生物信息學(xué)分析。 2、hp0231和hp0410克隆入表達(dá)載體pGEX-4T-1中，誘導(dǎo)表達(dá)，SDS-PAGE電泳鑒定。大量表達(dá)HP0410，GST柱純化重組蛋白。 3、利用純化HP0410從29株小鼠抗幽門螺桿菌單抗中篩選出抗HP0410單抗，特異性—敏感性實驗及免疫組化鑒定，進(jìn)行酶聯(lián)免疫吸附(ELISA)實驗，篩查85份胃潰瘍患者血清和90份健康人血清，分析ELISA結(jié)果。 4、篩選出抗HP0410單抗E018，利用該單抗和M13噬菌體12肽隨機(jī)肽庫，構(gòu)建HP0410抗原表位的抗原展示庫，陽性噬菌體測序，分析HP0410的抗原表位。 5、建立HP0410和細(xì)胞相互作用的模型，通過測定不同濃度、不同時間HP0410作用下SGC7901細(xì)胞IL-8和GRO-a分泌量的變化，以及細(xì)胞ELISA探討HP0410和SGC7901細(xì)胞的相互作用。 三、研究結(jié)果 1、生物信息學(xué)分析表明，HP0231和HP0410均為外膜蛋白，有跨膜結(jié)構(gòu)域和信號肽序列，具有良好的抗原性。hp0231和hp0410在Hp中普遍存在，BLAST分析表明這2個蛋白有很高的種屬特異性，為Hp所特有。 2、構(gòu)建重組菌株，高水平表達(dá)可溶性重組蛋白HP0410。利用純化的重組HP0410蛋白篩查29株單抗，獲得高特異性單抗一株(E018)。 3、HP0410篩查85份胃潰瘍患者血清，陽性94.1％，結(jié)果表明抗HP0410抗體在患者血清中普遍存在；篩查90份正常人血清，陽性74.4％，陽性檢出率與我國Hp感染率接近，表明Hp攜帶者體內(nèi)也存在抗HP0410抗體；免疫組化實驗表明利用抗HP0410單抗檢測HP0410，可作為一個有效的檢測Hp感染的方法。 4、分析噬菌體測序結(jié)果獲得HP0410的模擬空間表位，其中1個為模擬HP0410與E018特異性結(jié)合的抗原決定簇的候選表位。展示的抗原表位在HP0410上的分布符合生物信息學(xué)預(yù)測結(jié)果，且多處于的β轉(zhuǎn)角結(jié)構(gòu)中，這些表位的HP0410同源區(qū)與預(yù)測的HLAⅡ類分子結(jié)合結(jié)構(gòu)域一致。 5、HP0410不能顯著性的上調(diào)胃腺癌細(xì)胞SGC7901表達(dá)IL-8，也不能刺激細(xì)胞產(chǎn)生GRO-a，提示HP0410并非HP的炎性因子，符合其主要功能為介導(dǎo)黏附的預(yù)測；細(xì)胞ELISA結(jié)果呈陰性，提示E018針對的抗原決定簇即為HP0410的黏附結(jié)構(gòu)域亦可能HP0410不能直接與SGC7901細(xì)胞結(jié)合。 四、結(jié)論 1、生物信息學(xué)分析表明HP0410有良好的抗原性，可以誘導(dǎo)出特異性的體液免疫應(yīng)答反應(yīng)，提示HP0410可作為Hp的候選疫苗。 2、免疫組化實驗表明檢測HP0410可成為檢測Hp感染的新手段。 3、噬菌體肽庫展示的抗原表位與HP0410同源性不高，而與生物信息學(xué)預(yù)測的位置相符，提示獲得的表位為HP0410高抗原性位點(diǎn)的模擬空間表位，并且可被HLAⅡ類分子識別并遞呈，從而激發(fā)保護(hù)性免疫應(yīng)答。競爭-抑制實驗表明候選表位為E018所識別的抗原決定簇的模擬表位。 4、細(xì)胞實驗提示HP0410沒有明顯的致炎作用，利用其構(gòu)建的嵌合疫苗安全性較高。
[Abstract]:I. background and purpose
Medical professionals have long believed that stress and lifestyle are the main causes of chronic gastritis and peptic ulcer. In 1983, Australian scientists J. Robin Warren and Barry J. Marshall first isolated and cultured Helicobacter pylori (Hp), and confirmed that Hp infection can cause gastritis and peptic ulcer. Helicobacter pylori is the most common pathogen of chronic gastritis and peptic ulcer in the world. It is closely related to the occurrence of gastric adenocarcinoma and gastric mucosa-associated B-cell lymphoma. The focus.
At present, triple drugs (such as bismuth + metronidazole + antibiotics) are commonly used in the treatment of gastritis and peptic ulcer at home and abroad. Effective methods.
At present, the research hotspots of HP vaccine mainly focus on the protective antigens of urease, vacuole toxin (VacA), cell-related toxin (CagA), heat shock protein 60 (HSP60), etc. Urease is difficult to induce a comprehensive and stable protective immune response; CagA and VacA have great variation, and whether CagA is necessary for HP infection still exists. It is controversial that HSP60 has a high homology with human autoproteins. Mice immunized with HSP60 can only induce local protective immune responses and cause gastroenteritis in mice. The safety and efficacy of HSP60 as a vaccine need to be further verified. Mucosal immunity plays a major role in the process of clearance of Helicobacter pylori in vivo. It is still one of the main directions in the study of Hp vaccine to find suitable candidate vaccines and induce protective mucosal immunity against Hp.
Adhesin is indispensable in the process of H.pylori infection and is localized on the surface of H.pylori and has good antigenicity.Specific anti-adhesin antibodies can effectively inhibit and eliminate H.pylori colonization.Adhesin has become one of the main research directions of candidate antigens of H.pylori vaccine. Major adhesins included: 1) blood group antigen-binding adhesin (BAb), 2) N-acetylneuraminyl-lactose-binding haemagglutinin (NLBH), which binds tightly to human erythrocytes. B, divided into three reading frames (ORFs), of which ORF2, also known as hpaA gene, is 783 B P long and encodes Helicobacter pylori adhesin A (HpaA), which is necessary for the process of Hp adhesion and colonization. HpaA mutant HP strains can not be colonized in mice. Hp0410 is a member of the hpaA gene family, 750 B P long, and may be one of the Hp adhesion factors. Hp0231 is a 789 BP long gene predicted by reading frame analysis of the Hp genome. The expressed product may be a secretory protein. At present, the immunogenicity of HP0410 and HP0231 and their prospects as candidate vaccines have not been clarified.
The main objectives of this study were: (1) to predict the properties and immunogenicity of the proteins encoded by hp0231 and hp0410 by bioinformatics analysis; (2) to clone and express hp0410 in E. coli prokaryotic expression system and purify the recombinant protein; and (3) to screen the serum of patients with gastric ulcer and healthy people with recombinant protein and detect whether HP0410 can be detected in vivo Specific antibodies were induced and immunohistochemical assays were carried out to explore whether the protein could be used as a new method for detection of Hp infection; 4. phage peptide display library was established to study the antigen epitope of HP0410; 6. cell model was established to explore the interaction between HP0410 and gastric epithelial cells. The recombinant vaccine with multiple chimeric epitopes or the live vaccine with Lactobacillus acidophilus as the carrier will provide theoretical basis and work basis.
Two, research methods.
1. The genomic DNA of standard strain NCTC11639 of Helicobacter pylori was extracted, and primers PCR was designed according to the sequence of standard strain 22695 of Helicobacter pylori in GenBank. The genes of hp0231 and hp0410 were amplified and cloned into pMD18-T vector for sequencing and bioinformatics analysis.
2, hp0231 and hp0410 were cloned into expression vector pGEX-4T-1, induced expression and identified by SDS-PAGE electrophoresis. HP0410 was expressed in large quantities and purified by GST column.
3. Anti-HP0410 monoclonal antibodies were screened from 29 strains of mouse anti-Helicobacter pylori monoclonal antibodies by purified HP0410. The specificity-sensitivity test and immunohistochemical identification were carried out. Enzyme-linked immunosorbent assay (ELISA) was used to screen 85 sera from patients with gastric ulcer and 90 sera from healthy volunteers.
4. The anti-HP0410 monoclonal antibody E018 was screened out. Using this monoclonal antibody and M13 phage 12 peptide random peptide library, the antigen display library of HP0410 epitope was constructed. The positive phage was sequenced and the antigen epitope of HP0410 was analyzed.
5. The interaction model between HP0410 and SGC7901 cells was established. The changes of IL-8 and GRO-a secretion in SGC7901 cells under different concentrations and different time of HP0410 were measured. The interaction between HP0410 and SGC7901 cells was investigated by cell ELISA.
Three, research findings
1. Bioinformatics analysis showed that HP0231 and HP0410 were both outer membrane proteins with transmembrane domain and signal peptide sequence, and had good antigenicity.
2. The recombinant strain was constructed and the soluble recombinant protein HP0410 was highly expressed. 29 monoclonal antibodies were screened by purified recombinant HP0410 protein, and a high specific monoclonal antibody (E018) was obtained.
3, HP0410 screened 85 sera of gastric ulcer patients, positive 94.1%, the results showed that anti-HP0410 antibody was prevalent in the sera of patients; screening 90 normal sera, positive 74.4%, positive detection rate was close to the infection rate of Hp in China, indicating that the carrier of Hp also had anti-HP0410 antibody in vivo; immunohistochemical test showed that the use of anti-HP0410 monoclonal antibody detection. HP0410 can be used as an effective method to detect Hp infection.
4. By analyzing the results of phage sequencing, the simulated spatial epitopes of HP0410 were obtained, one of which was a candidate epitope for the antigenic determinant that mimicked the specific binding of HP0410 to E018. The distribution of the displayed epitopes on HP0410 was in accordance with the bioinformatics prediction, and most of them were in the beta-corner structure. The homologous regions of these epitopes were in accordance with the predicted HLA. Class II molecules are consistent with domains.
5. HP0410 could not significantly up-regulate the expression of IL-8 and stimulate the production of GRO-a in gastric adenocarcinoma cell line SGC7901, suggesting that HP0410 was not an inflammatory factor of HP, which accorded with the prediction of HP-mediated adhesion; the results of cell ELISA were negative, suggesting that the epitope of HP0410 targeted by E018 might not be a direct binding domain of HP0410. And then combined with SGC7901 cells.
Four. Conclusion
1. Bioinformatics analysis showed that HP0410 had good antigenicity and could induce specific humoral immune response, suggesting that HP0410 could be used as a candidate vaccine for Hp.
2, immunohistochemistry showed that detection of HP0410 could be a new way to detect Hp infection.
3. The antigenic epitope displayed by phage peptide library was not homologous to HP0410, but was consistent with the predicted position of bioinformatics. The obtained epitope was a simulated spatial epitope of HP0410 high antigenic site and could be recognized and presented by HLA class II molecule, thus stimulating protective immune response. Competition-inhibition test showed that the candidate epitope was E018. The mimic epitopes of the identified epitopes are identified.
4, cell experiments suggest that HP0410 has no obvious inflammatory effect, and the chimeric vaccine constructed by using it has high safety.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R378
本文編號：2222837
[Abstract]:I. background and purpose
Medical professionals have long believed that stress and lifestyle are the main causes of chronic gastritis and peptic ulcer. In 1983, Australian scientists J. Robin Warren and Barry J. Marshall first isolated and cultured Helicobacter pylori (Hp), and confirmed that Hp infection can cause gastritis and peptic ulcer. Helicobacter pylori is the most common pathogen of chronic gastritis and peptic ulcer in the world. It is closely related to the occurrence of gastric adenocarcinoma and gastric mucosa-associated B-cell lymphoma. The focus.
At present, triple drugs (such as bismuth + metronidazole + antibiotics) are commonly used in the treatment of gastritis and peptic ulcer at home and abroad. Effective methods.
At present, the research hotspots of HP vaccine mainly focus on the protective antigens of urease, vacuole toxin (VacA), cell-related toxin (CagA), heat shock protein 60 (HSP60), etc. Urease is difficult to induce a comprehensive and stable protective immune response; CagA and VacA have great variation, and whether CagA is necessary for HP infection still exists. It is controversial that HSP60 has a high homology with human autoproteins. Mice immunized with HSP60 can only induce local protective immune responses and cause gastroenteritis in mice. The safety and efficacy of HSP60 as a vaccine need to be further verified. Mucosal immunity plays a major role in the process of clearance of Helicobacter pylori in vivo. It is still one of the main directions in the study of Hp vaccine to find suitable candidate vaccines and induce protective mucosal immunity against Hp.
Adhesin is indispensable in the process of H.pylori infection and is localized on the surface of H.pylori and has good antigenicity.Specific anti-adhesin antibodies can effectively inhibit and eliminate H.pylori colonization.Adhesin has become one of the main research directions of candidate antigens of H.pylori vaccine. Major adhesins included: 1) blood group antigen-binding adhesin (BAb), 2) N-acetylneuraminyl-lactose-binding haemagglutinin (NLBH), which binds tightly to human erythrocytes. B, divided into three reading frames (ORFs), of which ORF2, also known as hpaA gene, is 783 B P long and encodes Helicobacter pylori adhesin A (HpaA), which is necessary for the process of Hp adhesion and colonization. HpaA mutant HP strains can not be colonized in mice. Hp0410 is a member of the hpaA gene family, 750 B P long, and may be one of the Hp adhesion factors. Hp0231 is a 789 BP long gene predicted by reading frame analysis of the Hp genome. The expressed product may be a secretory protein. At present, the immunogenicity of HP0410 and HP0231 and their prospects as candidate vaccines have not been clarified.
The main objectives of this study were: (1) to predict the properties and immunogenicity of the proteins encoded by hp0231 and hp0410 by bioinformatics analysis; (2) to clone and express hp0410 in E. coli prokaryotic expression system and purify the recombinant protein; and (3) to screen the serum of patients with gastric ulcer and healthy people with recombinant protein and detect whether HP0410 can be detected in vivo Specific antibodies were induced and immunohistochemical assays were carried out to explore whether the protein could be used as a new method for detection of Hp infection; 4. phage peptide display library was established to study the antigen epitope of HP0410; 6. cell model was established to explore the interaction between HP0410 and gastric epithelial cells. The recombinant vaccine with multiple chimeric epitopes or the live vaccine with Lactobacillus acidophilus as the carrier will provide theoretical basis and work basis.
Two, research methods.
1. The genomic DNA of standard strain NCTC11639 of Helicobacter pylori was extracted, and primers PCR was designed according to the sequence of standard strain 22695 of Helicobacter pylori in GenBank. The genes of hp0231 and hp0410 were amplified and cloned into pMD18-T vector for sequencing and bioinformatics analysis.
2, hp0231 and hp0410 were cloned into expression vector pGEX-4T-1, induced expression and identified by SDS-PAGE electrophoresis. HP0410 was expressed in large quantities and purified by GST column.
3. Anti-HP0410 monoclonal antibodies were screened from 29 strains of mouse anti-Helicobacter pylori monoclonal antibodies by purified HP0410. The specificity-sensitivity test and immunohistochemical identification were carried out. Enzyme-linked immunosorbent assay (ELISA) was used to screen 85 sera from patients with gastric ulcer and 90 sera from healthy volunteers.
4. The anti-HP0410 monoclonal antibody E018 was screened out. Using this monoclonal antibody and M13 phage 12 peptide random peptide library, the antigen display library of HP0410 epitope was constructed. The positive phage was sequenced and the antigen epitope of HP0410 was analyzed.
5. The interaction model between HP0410 and SGC7901 cells was established. The changes of IL-8 and GRO-a secretion in SGC7901 cells under different concentrations and different time of HP0410 were measured. The interaction between HP0410 and SGC7901 cells was investigated by cell ELISA.
Three, research findings
1. Bioinformatics analysis showed that HP0231 and HP0410 were both outer membrane proteins with transmembrane domain and signal peptide sequence, and had good antigenicity.
2. The recombinant strain was constructed and the soluble recombinant protein HP0410 was highly expressed. 29 monoclonal antibodies were screened by purified recombinant HP0410 protein, and a high specific monoclonal antibody (E018) was obtained.
3, HP0410 screened 85 sera of gastric ulcer patients, positive 94.1%, the results showed that anti-HP0410 antibody was prevalent in the sera of patients; screening 90 normal sera, positive 74.4%, positive detection rate was close to the infection rate of Hp in China, indicating that the carrier of Hp also had anti-HP0410 antibody in vivo; immunohistochemical test showed that the use of anti-HP0410 monoclonal antibody detection. HP0410 can be used as an effective method to detect Hp infection.
4. By analyzing the results of phage sequencing, the simulated spatial epitopes of HP0410 were obtained, one of which was a candidate epitope for the antigenic determinant that mimicked the specific binding of HP0410 to E018. The distribution of the displayed epitopes on HP0410 was in accordance with the bioinformatics prediction, and most of them were in the beta-corner structure. The homologous regions of these epitopes were in accordance with the predicted HLA. Class II molecules are consistent with domains.
5. HP0410 could not significantly up-regulate the expression of IL-8 and stimulate the production of GRO-a in gastric adenocarcinoma cell line SGC7901, suggesting that HP0410 was not an inflammatory factor of HP, which accorded with the prediction of HP-mediated adhesion; the results of cell ELISA were negative, suggesting that the epitope of HP0410 targeted by E018 might not be a direct binding domain of HP0410. And then combined with SGC7901 cells.
Four. Conclusion
1. Bioinformatics analysis showed that HP0410 had good antigenicity and could induce specific humoral immune response, suggesting that HP0410 could be used as a candidate vaccine for Hp.
2, immunohistochemistry showed that detection of HP0410 could be a new way to detect Hp infection.
3. The antigenic epitope displayed by phage peptide library was not homologous to HP0410, but was consistent with the predicted position of bioinformatics. The obtained epitope was a simulated spatial epitope of HP0410 high antigenic site and could be recognized and presented by HLA class II molecule, thus stimulating protective immune response. Competition-inhibition test showed that the candidate epitope was E018. The mimic epitopes of the identified epitopes are identified.
4, cell experiments suggest that HP0410 has no obvious inflammatory effect, and the chimeric vaccine constructed by using it has high safety.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R378
【參考文獻(xiàn)】
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