本文選題：幽門螺桿菌 + NAP　； 參考：《第一軍醫(yī)大學(xué)》2007年碩士論文

【摘要】： 一、研究背景和目的 幽門螺桿菌(Helicobacter pylori，Hp)是一種微需氧革蘭陰性螺旋桿菌，可導(dǎo)致胃炎和消化道潰瘍，并與胃腺癌、胃黏膜相關(guān)性淋巴樣組織(gastric mucosal-associated lymphoid tissue lymphoma，MALT)淋巴瘤病的發(fā)生密切相關(guān)，全球人口感染率超過50％，1994年被國(guó)際癌癥研究中心定為Ⅰ類致癌物。 目前，臨床上多采用質(zhì)子泵抑制劑和抗生素聯(lián)合治療，但存在耐藥性、病人依從性差、易復(fù)發(fā)等副問題，效果不甚理想。因此，疫苗是防治Hp感染的有效手段，篩選免疫保護(hù)力強(qiáng)的無毒抗原是研制疫苗的關(guān)鍵。研究較多的抗原主要有尿素酶B(Urease B)、空泡毒素(Vacuolatint cytotoxin A，VacA)、細(xì)胞毒素相關(guān)蛋白(Cytotoxin associated protein A，CagA)等，在動(dòng)物實(shí)驗(yàn)中雖顯示一定的免疫保護(hù)作用，但都具有一定的局限性，迄今未有理想的疫苗問世。 中性粒細(xì)胞激活蛋白(Neutrophil-activating protein，NAP)是近期發(fā)現(xiàn)的與黏附和炎癥相關(guān)的重要毒力因子，由napA基因編碼，分子量為15kDa，4股螺旋結(jié)構(gòu)的單體構(gòu)成十二聚體鐵蛋白，NAP與細(xì)菌DNA保護(hù)性蛋白(Dps)及鐵蛋白(Flp)具有高度同源性，存在于所有菌株中。NAP能持續(xù)趨化和激活嗜中性粒細(xì)胞，促進(jìn)中性粒細(xì)胞對(duì)胃上皮細(xì)胞的黏附，刺激胃黏膜上皮細(xì)胞快速反應(yīng)，通過上調(diào)VCAM-1、ICAM-1、E選擇素和前炎癥細(xì)胞因子及趨化因子促進(jìn)白細(xì)胞的募集，并促使中性粒細(xì)胞釋放反應(yīng)性氧代謝物，引發(fā)胃黏膜炎癥病變。多數(shù)Hp感染者血清中存在抗NAP抗體，動(dòng)物實(shí)驗(yàn)證實(shí)NAP免疫小鼠可抵抗Hp的感染，保護(hù)率達(dá)80％。可見，NAP是Hp重要的毒力因子及候選抗原。 本研究在克隆表達(dá)NAP的基礎(chǔ)上，展開NAP的表位組學(xué)研究，利用噬菌體肽庫展示技術(shù)，尋找NAP精確的抗原表位，為篩選更多的保護(hù)性抗原表位及制備多價(jià)合成肽亞單位疫苗奠定基礎(chǔ)；展開胃癌、消化道潰瘍、胃炎病人與正常人群抗NAP抗體水平的調(diào)查，探討NAP與胃癌之間的相關(guān)性，并對(duì)獲得的3株抗NAP單克隆抗體(monoclonal antibodies，mAb)進(jìn)行鑒定，探討其臨床應(yīng)用價(jià)值。 二、研究方法 1、根據(jù)GenBank提供的Hp 26695株基因組序列設(shè)計(jì)引物，以NCTC 11639基因組為模板，擴(kuò)增napA基因，連接pMD18-T載體，構(gòu)建napA／pMD18-T重組克隆，PCR及雙酶切鑒定后測(cè)序，將重組質(zhì)粒napA／pMD18-T與表達(dá)載體pGEX-4T-1用EcoRⅠ和XholⅠ雙酶切后連接，轉(zhuǎn)化宿主菌Top10，構(gòu)建融合表達(dá)載體napA／pGEX-4T-1，雙酶切后測(cè)序。對(duì)測(cè)序結(jié)果進(jìn)行生物信息學(xué)分析：利用BLAST工具進(jìn)行同源性搜索；利用DNAMAN軟件分析napA的DNA序列和編碼蛋白的特性；利用harvard大學(xué)的在線軟件進(jìn)行抗原表位預(yù)測(cè)；應(yīng)用Signal P3.0 server分析氨基酸序列中是否存在信號(hào)肽。 2、利用IPTG誘導(dǎo)表達(dá)目的蛋白，優(yōu)化表達(dá)條件，實(shí)現(xiàn)融合蛋白的可溶性表達(dá)，通過GST親和層析柱純化NAP蛋白，SDS-PAGE電泳及Western Blot鑒定表達(dá)蛋白。 3、利用間接ELISA法，以純化蛋白NAP與Hp陽性胃癌、消化道潰瘍、胃炎患者血清及正常人群血清進(jìn)行反應(yīng)，檢測(cè)NAP的免疫反應(yīng)性，比較NAP在胃病人群及正常人群的抗體水平，探討NAP與胃癌的相關(guān)性。 4、以天然的Hp全菌蛋白為抗原免疫BALB／C小鼠，利用雜交瘤技術(shù)制備mAb。利用間接ELASA法，鑒定mAb與腸道致病菌交叉免疫反應(yīng)，以NAP蛋白為抗原，從不與腸道致病菌發(fā)生交叉免疫反應(yīng)的mAb中篩選抗NAP mAb。利用鼠mAb亞類檢測(cè)試劑盒，檢測(cè)陽性雜交瘤細(xì)胞培養(yǎng)上清，鑒定抗NAP mAb的亞類；采用間接ELISA法測(cè)定抗NAP mAb抗體效價(jià)；選用間接ELISA方法，測(cè)定抗NAP mAb濃度，按公式Kaff=(n-1)／2(n[Ab']t-[Ab]t)計(jì)算親和常數(shù)。將抗NAP mAb與10株常見腸道致病菌進(jìn)行免疫組化試驗(yàn)，鑒定抗體反應(yīng)的特異性；將NAP mAb與Hp涂片樣本及Hp感染患者胃黏膜標(biāo)本進(jìn)行免疫組化試驗(yàn)，鑒定mAb反應(yīng)的敏感性。 5、利用噬菌體隨機(jī)7肽庫，以抗NAP mAb(E019)為固化抗原，通過ELASA法篩選陽性噬菌體克隆，競(jìng)爭(zhēng)結(jié)合抑制試驗(yàn)鑒定NAP的抗原表位。 三、研究結(jié)果 1、成功擴(kuò)增及克隆NCTC11639株napA基因，基因全長(zhǎng)435bp。結(jié)果已登錄GenBank，登錄號(hào)為DQ341279。 2、BLAST同源檢索顯示該基因與GenBank中公布的其它20株菌株的napA基因的核酸序列相比有高度同源性(94％～98％)；napA基因編碼蛋白與細(xì)菌DNA保護(hù)性蛋白(Dps)及鐵蛋白具有高度同源性，與人及哺乳動(dòng)物基因組DNA同源性很低(＜10％)。 3、經(jīng)二級(jí)結(jié)構(gòu)、親水性、抗原性指數(shù)、信號(hào)肽、三維結(jié)構(gòu)分析顯示有4個(gè)高抗原性肽段，分別是第4～24，55～77，95～103，118～140氨基酸序列，平均抗原指數(shù)為1.0236，其中118～140長(zhǎng)22個(gè)氨基酸殘基的肽段抗原性指數(shù)1.15，親水性強(qiáng)、含有1個(gè)β轉(zhuǎn)角，表明該肽段可能含有良好的抗原表位。 4、PCR證實(shí)napA在Hp菌株中普遍存在，與國(guó)內(nèi)外文獻(xiàn)報(bào)道相同，說明該基因高度保守。 5、構(gòu)建napA-pGEX-4T-1-E. coli Top 10高效可溶性原核表達(dá)系統(tǒng)，通過優(yōu)化表達(dá)條件，確定最佳表達(dá)條件為1mmol／L IPTG誘導(dǎo)濃度，誘導(dǎo)溫度30℃，誘導(dǎo)時(shí)間4 h。 6、表達(dá)產(chǎn)物經(jīng)GST親和層析純化，獲得純度較高且有生物活性的目的蛋白，分子量是44Kda。重組NAP與患者血清及購買的商品化兔抗全菌Hp抗體之間可發(fā)生特異性免疫反應(yīng)，不與正常人血清反應(yīng)，說明重組NAP具有良好的免疫反應(yīng)性。 7、Hp IgG抗體試劑盒檢測(cè)結(jié)果表明：64％正常人群Hp感染陽性，均值為0.46±0.27；70％胃癌患者Hp感染陽性，均值為0.55±0.13；68％胃潰瘍患者Hp感染陽性均值為0.43±0.21；64％胃炎患者Hp感染陽性，均值為0.68±0.13。 8、NAP抗體水平檢測(cè)結(jié)果表明：97.5％胃癌患者血清中NAP抗體檢測(cè)結(jié)果陽性，均值為1.01±0.13；92.9％胃潰瘍患者血清中NAP抗體檢測(cè)結(jié)果陽性，均值為0.98±0.17；85.7％胃炎患者血清中NAP抗體檢測(cè)結(jié)果陽性，，均值為0.89±0.07；正常人群血清中60％NAP抗體檢測(cè)結(jié)果陽性，均值為0.61±0.14。20～29歲與40～49歲年齡段抗體水平有明顯差異(~*P＜0.05)，其它年齡段NAP抗體水平無明顯差異。 9、從29株小鼠抗Hp全菌mAb中篩選獲得3株抗NAP的mAb，mAb經(jīng)亞類鑒定顯示全部為IgG1，抗體效價(jià)為1：16至1：32，抗體親和力為1×10~(-10)至5.2×10~(-12)mol／L。Western blot鑒定表明，NAP蛋白可與NAP mAb之間發(fā)生免疫學(xué)反應(yīng)。免疫組化結(jié)果表明，3株mAb特異性較高，敏感性強(qiáng)，能與Hp純培養(yǎng)物及Hp感染胃黏膜標(biāo)本發(fā)生強(qiáng)陽性反應(yīng)，不與其他腸道菌發(fā)生反應(yīng)。 10、利用噬菌體肽庫技術(shù)，初步篩選到NAP的線性表位(FAHLATQ)。 四、結(jié)論 1、本研究首次從國(guó)際標(biāo)準(zhǔn)株NCTC 11639基因組DNA中克隆中性粒細(xì)胞激活蛋白基因napA，Genbank登錄號(hào)DQ341279。 2、成功構(gòu)建napA-pGEX-4T-1-E. coli Top 10高效可溶性原核表達(dá)系統(tǒng)。重組NAP經(jīng)鑒定具有良好的免疫原性和免疫反應(yīng)性，可作為Hp基因工程亞單位疫苗的候選組分，并為研究Hp致病機(jī)制、免疫保護(hù)機(jī)制及診斷試劑奠定基礎(chǔ)。 3、NAP是高度保守的基因，是良好的免疫原。初步預(yù)測(cè)NAP有4個(gè)高抗原性肽段，其中118～140肽段可能含有良好的抗原表位。 4、成功制備高親和力抗NAP全菌mAb，3株mAb(E006、E019、E023)特異性高，敏感性強(qiáng)，具有良好的應(yīng)用前景。 5、Hp在廣東地區(qū)的胃病及正常人群中感染率較高；NAP抗體表達(dá)水平高顯示NAP與胃癌有一定關(guān)聯(lián)，NAP是胃癌發(fā)生高風(fēng)險(xiǎn)因子；患者年齡與NAP抗體水平無明顯相關(guān)性，此項(xiàng)研究為NAP病原學(xué)研究提供了理論基礎(chǔ)，為Hp感染相關(guān)胃癌的診斷、治療和疫苗研究打下基石。 6、首次篩選到NAP的線性表位(FAHLATQ)，位于預(yù)測(cè)肽段(118～140)內(nèi)，為下一步開展多價(jià)合成肽疫苗及蛋白質(zhì)組學(xué)研究奠定良好基礎(chǔ)。
[Abstract]:First, research background and purpose
Helicobacter pylori (Hp) is a micro aerobic gram negative spiral bacilli, which can lead to gastritis and peptic ulcers, and is closely related to gastric adenocarcinoma and gastric mucosa associated lymphoid tissue (gastric mucosal-associated lymphoid tissue lymphoma, MALT) lymphoma. The global population infection rate is over 50%, 1994. It is designated as type I carcinogen by the International Center for cancer research.
At present, the clinical use of proton pump inhibitors and antibiotics combined treatment, but the existence of drug resistance, patient compliance, easy to relapse and other problems, the effect is not ideal. Therefore, the vaccine is an effective means to prevent and control Hp infection, the screening of immunoprotective non toxic antigen is the key to the development of vaccines. The major antigen mainly includes the urease B (U Rease B), vacuolated toxin (Vacuolatint cytotoxin A, VacA), cytotoxin related protein (Cytotoxin associated protein A, CagA) and so on. Although it shows certain immune protection in animal experiments, it has some limitations, so far no ideal vaccine has been published.
Neutrophil-activating protein (NAP) is an important virulence factor associated with adhesion and inflammation. It is encoded by the napA gene, the molecular weight is 15kDa, the monomer of the 4 Strand spiral structure constitutes twelve polybody ferritin, and NAP is highly homologous to the bacterial DNA protective protein (Dps) and the ferritin (Flp). In all strains,.NAP can continue to chemotaxis and activate neutrophils, promote the adhesion of neutrophils to gastric epithelial cells, stimulate the rapid reaction of gastric epithelial cells, promote the recruitment of leukocytes by up regulation of VCAM-1, ICAM-1, E selectin and pro-inflammatory cytokines and chemokines, and promote the release of reactive oxygen generation by neutrophils. Most Hp infected people have anti NAP antibodies in the serum of most Hp infected people. Animal experiments confirm that NAP immune mice can resist Hp infection, the protection rate is 80%., and NAP is an important virulence factor and candidate antigen of Hp.
On the basis of cloning and expression of NAP, this study carried out the study of NAP's epitopes, using phage peptide library display technology to find the exact epitopes of NAP, laying the foundation for screening more protective epitopes and preparing polyvalent peptide subunit vaccines, developing gastric cancer, alcic ulcer, and anti NAP antibody in gastritis patients and normal people. The correlation between NAP and gastric cancer was investigated by a level survey, and 3 anti NAP monoclonal antibodies (monoclonal antibodies, mAb) were identified and their clinical value was discussed.
Two, research methods
1, according to the Hp 26695 genome sequence provided by GenBank, the primers were designed, the napA gene was amplified by the NCTC 11639 genome, the pMD18-T vector was connected, the recombinant clone of napA / pMD18-T was constructed, PCR and double enzyme digestion were sequenced, and the recombinant plasmid napA / pMD18-T was connected with the EcoR I and the double enzyme. Top10, a fusion expression vector napA / pGEX-4T-1 was constructed and sequenced after double enzyme digestion. Bioinformatics analysis of the sequencing results: using BLAST tool for homologous search; using DNAMAN software to analyze the characteristics of DNA sequence and encoded protein of napA; use Harvard University's line software to predict antigen epitopes; use Signal. P3.0 server analyzed whether there was a signal peptide in the amino acid sequence.
2, the expression of the target protein was induced by IPTG, the expression conditions were optimized, the soluble expression of the fusion protein was realized, the NAP protein was purified by GST affinity chromatography column, and the expression protein was identified by SDS-PAGE electrophoresis and Western Blot.
3, the indirect ELISA method was used to purify protein NAP and Hp positive gastric cancer, alimentary tract ulcer, serum of gastritis patients and normal population serum, to detect the immunoreactivity of NAP, compare the antibody level of NAP in the patients with gastric disease and normal population, and to explore the correlation between NAP and gastric cancer.
4, the BALB / C mice were immunized with the natural Hp whole bacteria protein as antigen, and the indirect ELASA method was used to prepare mAb. by hybridoma technology. The cross immunoreaction of mAb and intestinal pathogenic bacteria was identified, NAP protein was used as antigen, and the anti NAP mAb. using mAb subclass detection kit was screened and tested positive for anti NAP mAb. using mAb in mAb. Hybridoma cell culture supernatant, identification of anti NAP mAb subclass, indirect ELISA assay to determine the titer of anti NAP mAb antibody, indirect ELISA method, determination of anti NAP mAb concentration, and formula Kaff= (n-1) / 2 (n[Ab']t-[Ab]t) to calculate affinity constant. Specificity, NAP mAb and Hp smear samples and Hp infected patients gastric mucosal specimens were immunohistochemical test to identify the sensitivity of mAb reaction.
5, using the phage random 7 peptide library and the anti NAP mAb (E019) as the curing antigen, the positive phage clones were screened by the ELASA method, and the competitive binding inhibition test was used to identify the epitopes of NAP.
Three, the results of the study
1, the napA gene of NCTC11639 strain was successfully amplified and cloned, and the full-length 435bp. gene was registered in GenBank. The accession number was DQ341279..
2, BLAST homologous retrieval showed that the gene was highly homologous compared to the nucleotide sequences of the other 20 strains of napA published in GenBank, and the napA gene encoded protein was highly homologous to the bacterial DNA protective protein (Dps) and ferritin, and was very homologous to the human and mammalian genome DNA (< 10%).
3, through the two stage structure, hydrophilicity, antigenicity index, signal peptide and three-dimensional structural analysis, there are 4 high antigenic peptide segments, which are fourth ~ 24,55 ~ 77,95 to 103118~140 amino acid sequences, and the average antigen index is 1.0236. The peptide antigenicity index of 118~140 long 22 amino acid residues is 1.15, and the hydrophilic property is strong and contains 1 beta angles. The peptide segment may contain a good epitope.
4, PCR confirmed that napA is ubiquitous in Hp strains, which is similar to that reported both at home and abroad, indicating that the gene is highly conserved.
5, the napA-pGEX-4T-1-E. coli Top 10 high efficiency soluble prokaryotic expression system was constructed. By optimizing the expression conditions, the optimum expression condition was 1mmol / L IPTG induced concentration, the induction temperature was 30, and the induction time was 4 h..
6, the expression product was purified by GST affinity chromatography to obtain a purer protein with higher purity and bioactivity. The molecular weight of the recombinant NAP was a specific immune response between the recombinant NAP and the serum of the patient and the commercialized Rabbit anti whole Hp antibody, which did not react with the normal human serum, indicating that the recombinant NAP has a good immune response.
7, the detection results of Hp IgG antibody kit showed that 64% normal population Hp infection was positive, the mean value was 0.46 + 0.27, 70% gastric cancer patients were positive for Hp infection, the mean value was 0.55 + 0.13, the mean value of Hp infection in 68% gastric ulcer patients was 0.43 + 0.21, and the Hp infection was positive in the patients with gastric cancer, the mean was 0.68 + 0.13..
8, NAP antibody level detection results showed that 97.5% gastric cancer patients' serum NAP antibody test results were positive, the mean value was 1.01 + 0.13, and the serum NAP antibody test results of 92.9% gastric ulcer patients were 0.98 + 0.17, and the NAP antibody test results in the serum of 85.7% gastritis patients were 0.89 + 0.07, and the normal population serum was 60. The results of%NAP antibody test were positive. There was a significant difference between 0.61 0.14.20 to 29 years and 40~49 years old age antibody levels (~*P < 0.05). There was no significant difference in the level of NAP antibody in other age groups.
9, 3 strains of anti NAP mAb were screened from 29 mice resistant to Hp whole bacteria. MAb was identified as IgG1, and the antibody titer was from 1:16 to 1:32. The antibody affinity was 1 x 10~ (-10) to 5.2 * 10~ (-12) mol / L.Western. The immunological results showed that 3 strains were specific. It is highly sensitive and sensitive. It can react strongly with Hp pure culture and Hp infected gastric mucosa specimens, and does not react with other intestinal bacteria.
10, phage peptide library technology was used to preliminarily screen the linear epitope (FAHLATQ) of NAP.
Four. Conclusion
1, this study was the first to clone neutrophil activating protein gene napA, Genbank accession number DQ341279. from genomic DNA of international standard strain NCTC 11639.
2, successful construction of napA-pGEX-4T-1-E. coli Top 10 high efficiency soluble prokaryotic expression system. The recombinant NAP has good immunogenicity and immune response, which can be used as a candidate component of the Hp gene engineering subunit vaccine, and lay the foundation for the study of the pathogenesis of Hp, the mechanism of immune protection and the diagnostic reagents.
3, NAP is a highly conserved gene and a good immunogen. It is preliminarily predicted that NAP has 4 highly antigenic peptides, of which 118~140 peptides may contain good epitopes.
4, successful preparation of high affinity NAP resistant whole strain mAb and 3 mAb (E006, E019, E023) are highly specific, sensitive and promising.
5, Hp has high infection rate in gastric disease and normal population in Guangdong area; high expression of NAP antibody shows a certain association between NAP and gastric cancer, NAP is a high risk factor for gastric cancer, and there is no significant correlation between the age of the patients and the level of NAP antibody. This study provides a theoretical basis for the study of NAP pathogeny and the diagnosis of Hp infection related gastric cancer. Treatment and vaccine research are the cornerstone.
6, the linear epitope (FAHLATQ) of NAP was screened for the first time, located in the predicted peptide segment (118~140), and laid a good foundation for the next step to carry out polyvalent peptide vaccine and proteomics.
【學(xué)位授予單位】：第一軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R378

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相關(guān)期刊論文 前3條

1 李妍;寧云山;洪燕華;劉宜楚;羅軍;龍敏;董文其;李明;;抗幽門螺桿菌單克隆抗體的制備與鑒定[J];南方醫(yī)科大學(xué)學(xué)報(bào);2006年04期

2 徐建華,朱家勇;生物信息學(xué)在蛋白質(zhì)結(jié)構(gòu)與功能預(yù)測(cè)中的應(yīng)用[J];醫(yī)學(xué)分子生物學(xué)雜志;2005年03期

3 涂小萍,秦敏,楊國(guó)友,楊勇,周富昌,趙彬,劉瑛;幽門螺桿菌尿素酶單克隆抗體的研制及鑒定[J];微生物學(xué)免疫學(xué)進(jìn)展;2004年03期

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