本文關(guān)鍵詞： 腸出血性大腸桿菌O157:H7 單克隆抗體 表位　出處：《第三軍醫(yī)大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 腸出血性大腸桿菌(Enterohemorrhagic Escherichia coli,EHEC)O157:H7(本文簡(jiǎn)稱O157)是一種重要的人畜共患傳染病病原菌。自1982年被確認(rèn)為致病菌以來的20多年中,世界各地包括中國(guó)都有不同規(guī)模的暴發(fā)流行。EHEC O157:H7感染可使人患腹瀉、出血性結(jié)腸炎(hemorrhagic colitis,HC),還可在5～10%的病例中引發(fā)溶血性尿毒綜合征(hemolytic uremic syndrome,HUS)及血栓性血小板減少紫癜(thromboticthrombocytopenic purpura,TTP)等嚴(yán)重并發(fā)癥,嚴(yán)重者可導(dǎo)致死亡。O157的感染因具有暴發(fā)流行趨勢(shì)、強(qiáng)烈的致病性與致死性和抗生素治療可加劇病情的危險(xiǎn)性等特點(diǎn),已經(jīng)成為全球性的公共衛(wèi)生問題。 EHEC與宿主腸道粘膜上皮細(xì)胞的粘附是疾病發(fā)展的第一步。介導(dǎo)O157細(xì)菌粘附定植的主要結(jié)構(gòu)單元是Ⅲ型分泌系統(tǒng)(type-Ⅲsecretion system,TTSS)。EspA(Esp為E.coli secreted protein縮寫)是一個(gè)中空的三維立體管道結(jié)構(gòu),是組成腸出血性大腸桿菌O157:H7Ⅲ型分泌系統(tǒng)的重要成分�？缒さ鞍譚ir以及其他Ⅲ型分泌系統(tǒng)都是通過此通道進(jìn)入宿主的。EspA基因全長(zhǎng)579bp,表達(dá)的蛋白分子量約為25kDa,EspA在O157表面表達(dá),并以多聚體形式存在,具有很強(qiáng)的免疫原性,在O157致病過程中發(fā)揮重要作用。 本研究擬通過制備抗EspA的單克隆抗體,并以該單抗作為研究工具鑒定EspA B細(xì)胞表位及其免疫學(xué)性質(zhì),為研究EspA的生物學(xué)功能及其在粘附定植中作用提供依據(jù),并以之來闡明O157:H7的致病機(jī)理,尋找預(yù)防、控制和治療O157:H7感染的手段。 方法 1.抗EspA單克隆抗體制備和純化應(yīng)用傳統(tǒng)雜交瘤技術(shù)制備抗EspA的單克隆抗體,用飽和硫酸銨(saturated ammonium sulfate,SAS)沉淀法和HiTrap rProtein A柱純化單抗。 2.單抗生物學(xué)活性鑒定以非競(jìng)爭(zhēng)ELISA法測(cè)定單抗的親和常數(shù);以結(jié)合相加實(shí)驗(yàn)對(duì)單抗識(shí)別的表位進(jìn)行初步鑒定;以Western blot鑒定單抗與重組蛋白結(jié)合的特異性;以間接免疫熒光檢測(cè)單抗與O157 Sakai菌EspA蛋白結(jié)合的特性。 3.EspA表位的初步預(yù)測(cè)通過生物信息學(xué)對(duì)EspA蛋白的親水性、抗原性和表面可及性等進(jìn)行分析并結(jié)合EspA蛋白功能、結(jié)構(gòu)穩(wěn)定性,選擇性的將EspA蛋白分成6段:E1,E2,E3,E4,E5和E6。 4.EspA表位的構(gòu)建表達(dá)采用PCR方法從EHEC O157:H7 Sakai基因組中擴(kuò)增E1,E2,E3,E4,E5和E6目的基因片段,分別構(gòu)建到原核表達(dá)載體pET-28a和pET-22b中,通過酶切鑒定及送檢測(cè)序陽性重組子。將陽性重組子轉(zhuǎn)化宿主菌大腸桿菌BL21,IPTG誘導(dǎo)表達(dá),Tris-Tricine PAGE電泳確定目的蛋白表達(dá)。 5.EspA B細(xì)胞表位的篩選以Western blot鑒定方法將單抗與EspA的6個(gè)片段分別反應(yīng),初步確定針對(duì)EspA的表位區(qū)域,將這個(gè)區(qū)域采用步移合成法合成7條多肽,通過Dot blot和ELISA雙重鑒定,精確定位單克隆抗體所針對(duì)的B細(xì)胞抗原表位。 6.表位的免疫學(xué)性質(zhì)鑒定將EspA B細(xì)胞表位與載體蛋白BSA應(yīng)用戊二醛偶聯(lián),將偶聯(lián)物免疫BALB/c小鼠,同時(shí)做單一表位組和BSA對(duì)照組。分別于0、14、28,31天免疫接種,劑量約為100μg/只/次。末次免疫4天后,ELISA檢測(cè)血清效價(jià),測(cè)定抗體水平。并通過免疫印跡及免疫熒光方法檢測(cè)抗血清能否識(shí)別重組及天然EspA,以此評(píng)價(jià)由表位制備的抗血清的免疫功能。 7.FAS實(shí)驗(yàn)評(píng)價(jià)單抗及表位多抗的生物學(xué)功能通過免疫熒光實(shí)驗(yàn)分別檢測(cè)單抗及表位多抗阻斷宿主細(xì)胞激動(dòng)蛋白聚集的能力,并將兩者作比較。 結(jié)果 1.通過傳統(tǒng)的雜交瘤技術(shù)制備得到3株抗EspA單克隆抗體,并用HiTrap rProteinA柱對(duì)單克隆抗體進(jìn)行純化,純度達(dá)90%以上 2.通過免疫印跡、ELISA、免疫熒光、結(jié)合相加等實(shí)驗(yàn)對(duì)抗體的特異性、親和力、與天然蛋白的免疫反應(yīng)性、識(shí)別抗原表位等都得到了證實(shí)。 3.采用生物信息學(xué)和表位作圖相結(jié)合的方法應(yīng)用制備的單抗鑒定EspA抗原B細(xì)胞表位。PCR方法從O157基因組中分別擴(kuò)增出E1(198bp)、E2(240bp)、E3(306bp)、E4(375bp)、E5(345bp)和E6(270bp)片段的堿基序列,并分別成功構(gòu)建到原核表達(dá)載體pET28a和pET22b上,分別經(jīng)IPTG誘導(dǎo),Tris-Tricine PAGE證實(shí)目的蛋白的表達(dá),并經(jīng)免疫印跡證實(shí),抗EspA單克隆抗體1H10能夠與E2(40～120aa)、E3(90～192aa)、E4(67～192aa)、E5(77～192aa)和E6(100～192aa)目的蛋白發(fā)生特異性抗原抗體反應(yīng);單克隆抗體2A3與目的蛋白E4(67～192aa)和E5(77～192aa)發(fā)生特異反應(yīng);單克隆抗體3E5與目的蛋白E2(40～120aa)發(fā)生特異反應(yīng)。初步證實(shí)單克隆抗體1H10所識(shí)別的抗原表位位于EspA蛋白N端第100～120位氨基酸內(nèi),而2A3、3E5所識(shí)別的抗原表位疑為構(gòu)象型表位。 4.采用步移合成表位多肽方法,分別合成E_(67～90aa),E_(70～95aa),E_(75～100aa),E_(80～105aa),E_(85～110aa),E_(90～115aa)和E_(100～120aa)7條多肽。通過ELISA和Dot blot方法證實(shí),單克隆抗體1H10所對(duì)應(yīng)的B細(xì)胞抗原表位為EspA蛋白N端第100～120位氨基酸,2A3、3E5不與任何一段多肽結(jié)合,確定兩者均識(shí)別構(gòu)象型表位。 5.合成的表位多肽E_(100～120)能夠競(jìng)爭(zhēng)抑制單克隆抗體1H10與EspA的抗原抗體反應(yīng),且抑制效應(yīng)具有劑量依賴性,當(dāng)EspA蛋白濃度為2μg/ml、單克隆抗體濃度為12μg/ml、表位多肽E_(100～120)濃度分別為0.02、0.04、0.2、1、2μg/ml時(shí),根據(jù)OD值的變化情況,按公式計(jì)算抑制率,抑制率分別為7%、27%、58%、84%和96%。 6.應(yīng)用戊二醛將表位多肽E_(100～120)與載體蛋白BSA進(jìn)行偶聯(lián),制備偶聯(lián)物,并將其免疫BALB/c小鼠,產(chǎn)生的抗血清其效價(jià)高達(dá)1:256 00,并且表位與載體蛋白偶聯(lián)物免疫后的抗血清能夠與天然O157菌株的EspA發(fā)生特異性抗原抗體反應(yīng),進(jìn)一步證實(shí)E_(100～120)具有免疫原性,可以作為O157疫苗的候選抗原成分。 7.通過FAS實(shí)驗(yàn)對(duì)EspA生物功能進(jìn)行了初步研究,表明多肽抗血清和單克隆抗體均能夠阻斷肌動(dòng)蛋白的聚集,也就更進(jìn)一步證明EspA在A/E損傷的產(chǎn)生中起到非常重要的作用。 結(jié)論 1.采用雜交瘤技術(shù)制備得到了3株抗EspA單克隆抗體,并對(duì)3株單抗的生物學(xué)特性進(jìn)行了鑒定。 2.以單抗1H10篩選到一個(gè)EspA的優(yōu)勢(shì)B細(xì)胞抗原表位,位于EspA第100-120個(gè)氨基酸,該表位表現(xiàn)出良好的免疫原性和免疫反應(yīng)性。 3.單抗2A3、3E5可能分別針對(duì)兩個(gè)不同的構(gòu)象型表位,考慮通過解析單抗與EspA的抗原抗體復(fù)合物結(jié)構(gòu)來確定該兩株單抗針對(duì)的B細(xì)胞抗原表位。
[Abstract]:Enterohemorrhagic Escherichia coli (Enterohemorrhagic Escherichia, coli, EHEC) O157:H7 (i.e. O157) is an important pathogen of infectious disease and bacteria. Since 1982 was identified as the pathogen since 20 years, all over the world including China have different scale outbreak of.EHEC infection of O157:H7 can make people suffering from diarrhea. Hemorrhagic colitis (hemorrhagic colitis, HC), but also in the 5 to 10% of cases caused hemolytic uremic syndrome (hemolytic uremic, syndrome, HUS) and thrombotic thrombocytopenic purpura (thromboticthrombocytopenic purpura, TTP) and other serious complications, severe cases can lead to death due to.O157 infection with the outbreak trend, pathogenicity fatal and strong antibiotic treatment may exacerbate the disease risk characteristics, has become a global public health problem.
The adhesion of EHEC on intestinal epithelial cells is the first step in the development of the disease. The main structural unit of O157 mediated by bacteria colonization is a type III secretion system (type- secretion system, TTSS).EspA (Esp E.coli secreted protein) is a hollow three-dimensional pipeline structure is an important component of intestine composition of Escherichia coli O157:H7 type III secretion system. The transmembrane protein Tir and other type III secretion system is through the channel into the host.EspA gene full-length 579bp protein expression, molecular weight is about 25kDa, the expression of EspA on the surface of O157, and present in the form of polymers, has strong immunogenicity and play an important role in the pathogenesis of O157.
This study through the preparation of anti EspA monoclonal antibody, and the antibody as a research tool to identify EspA B cell epitopes and immunological properties, and its role in colonization in providing basis for studying the biological function of EspA, and to elucidate the pathogenic mechanism of O157:H7 and find the means of prevention, control and treatment of O157:H7 infection.
Method
1. monoclonal antibodies against EspA were prepared and purified. Monoclonal antibodies against EspA were prepared by traditional hybridoma technology, and mAbs were purified by saturated ammonium sulfate (SAS) precipitation and HiTrap rProtein A column.
2. monoclonal antibody identification of biological activity in a non competitive ELISA method for the determination of monoclonal antibody affinity constant; preliminary identification by combining addition experiments on McAb recognized epitopes; specific binding with Western monoclonal antibody and identification of blot recombinant protein; characteristics combined with indirect immunofluorescence assay and monoclonal antibody O157 Sakai strain EspA protein.
The prediction of 3.EspA epitopes is based on bioinformatics analysis of EspA protein's hydrophilicity, antigenicity and surface accessibility. Combined with EspA protein function, structural stability and selectivity, EspA protein is divided into 6 segments: E1, E2, E3, E4, E5 and E6..
Construction and expression of E1 from EHEC O157:H7 Sakai amplification, the genome by the method of PCR 4.EspA epitope, E2, E3, E4, E5 and E6 gene fragments were cloned into the prokaryotic expression vector pET-28a and pET-22b, by enzyme digestion and sequencing test positive recombinants. The positive recombinant transformed host Escherichia coli BL21, Tris-Tricine PAGE expression induced by IPTG, electrophoresis to determine the expression of the target protein.
The 5.EspA screening of B cell with Western blot method for the identification of 6 fragments of monoclonal antibody and EspA were determined in EspA reaction, the epitope region, this region will adopt step synthesis of 7 polypeptides by Dot blot and ELISA double identification, B cell epitope of monoclonal antibody for precise positioning.
6. epitope identification of the immunological properties of B cell and EspA coupling carrier protein BSA by glutaraldehyde, the conjugates of BALB/c mice immunized single table group and BSA control group. At the same time in 0,14,28,31 days of immunization, the dose is about 100 g/ / time. 4 days after the last immunization, serum titer ELISA antibody levels were determined. And by Western blotting and immunofluorescence method to detect antiserum could recognize the recombinant and natural EspA, in order to evaluate the immune function by the antiserum prepared a table.
The biological function of 7.FAS and experimental evaluation of monoclonal antibody epitopes were detected by immunofluorescence and monoclonal antibody epitopes blocking host cell activation ability protein aggregation, and comparison.
Result
1. 3 strains of anti EspA monoclonal antibody were prepared by traditional hybridoma technology, and the monoclonal antibody was purified with HiTrap rProteinA column. The purity of the monoclonal antibody was over 90%
2., by immunoblotting, ELISA, immunofluorescence and combining experiments, the specificity and affinity of antibodies, the immunoreactivity of natural proteins, and the recognition of antigen epitopes have been confirmed.
3.閲囩敤鐢熺墿淇℃伅瀛﹀拰琛ㄤ綅浣滃浘鐩哥粨鍚堢殑鏂規(guī)硶搴旂敤鍒跺鐨勫崟鎶楅壌瀹欵spA鎶楀師B緇嗚優(yōu)琛ㄤ綅.PCR鏂規(guī)硶浠嶰157鍩哄洜緇勪腑鍒嗗埆鎵╁鍑篍1(198bp),E2(240bp),E3(306bp),E4(375bp),E5(345bp)鍜孍6(270bp)鐗囨鐨勭⒈鍩哄簭鍒，

本文編號(hào)：1471937