肠出血性大肠杆菌（EHEC）O157：H7 EspA B细胞表位的鉴定及免疫学性质研究

发布时间：2018-01-29 00:06

  本文关键词： 肠出血性大肠杆菌O157:H7 单克隆抗体 表位　出处：《第三军医大学》2007年硕士论文　论文类型：学位论文

【摘要】： 肠出血性大肠杆菌(Enterohemorrhagic Escherichia coli,EHEC)O157:H7(本文简称O157)是一种重要的人畜共患传染病病原菌。自1982年被确认为致病菌以来的20多年中,世界各地包括中国都有不同规模的暴发流行。EHEC O157:H7感染可使人患腹泻、出血性结肠炎(hemorrhagic colitis,HC),还可在5～10%的病例中引发溶血性尿毒综合征(hemolytic uremic syndrome,HUS)及血栓性血小板减少紫癜(thromboticthrombocytopenic purpura,TTP)等严重并发症,严重者可导致死亡。O157的感染因具有暴发流行趋势、强烈的致病性与致死性和抗生素治疗可加剧病情的危险性等特点,已经成为全球性的公共卫生问题。 EHEC与宿主肠道粘膜上皮细胞的粘附是疾病发展的第一步。介导O157细菌粘附定植的主要结构单元是Ⅲ型分泌系统(type-Ⅲsecretion system,TTSS)。EspA(Esp为E.coli secreted protein缩写)是一个中空的三维立体管道结构,是组成肠出血性大肠杆菌O157:H7Ⅲ型分泌系统的重要成分。跨膜蛋白Tir以及其他Ⅲ型分泌系统都是通过此通道进入宿主的。EspA基因全长579bp,表达的蛋白分子量约为25kDa,EspA在O157表面表达,并以多聚体形式存在,具有很强的免疫原性,在O157致病过程中发挥重要作用。 本研究拟通过制备抗EspA的单克隆抗体,并以该单抗作为研究工具鉴定EspA B细胞表位及其免疫学性质,为研究EspA的生物学功能及其在粘附定植中作用提供依据,并以之来阐明O157:H7的致病机理,寻找预防、控制和治疗O157:H7感染的手段。 方法 1.抗EspA单克隆抗体制备和纯化应用传统杂交瘤技术制备抗EspA的单克隆抗体,用饱和硫酸铵(saturated ammonium sulfate,SAS)沉淀法和HiTrap rProtein A柱纯化单抗。 2.单抗生物学活性鉴定以非竞争ELISA法测定单抗的亲和常数;以结合相加实验对单抗识别的表位进行初步鉴定;以Western blot鉴定单抗与重组蛋白结合的特异性;以间接免疫荧光检测单抗与O157 Sakai菌EspA蛋白结合的特性。 3.EspA表位的初步预测通过生物信息学对EspA蛋白的亲水性、抗原性和表面可及性等进行分析并结合EspA蛋白功能、结构稳定性,选择性的将EspA蛋白分成6段:E1,E2,E3,E4,E5和E6。 4.EspA表位的构建表达采用PCR方法从EHEC O157:H7 Sakai基因组中扩增E1,E2,E3,E4,E5和E6目的基因片段,分别构建到原核表达载体pET-28a和pET-22b中,通过酶切鉴定及送检测序阳性重组子。将阳性重组子转化宿主菌大肠杆菌BL21,IPTG诱导表达,Tris-Tricine PAGE电泳确定目的蛋白表达。 5.EspA B细胞表位的筛选以Western blot鉴定方法将单抗与EspA的6个片段分别反应,初步确定针对EspA的表位区域,将这个区域采用步移合成法合成7条多肽,通过Dot blot和ELISA双重鉴定,精确定位单克隆抗体所针对的B细胞抗原表位。 6.表位的免疫学性质鉴定将EspA B细胞表位与载体蛋白BSA应用戊二醛偶联,将偶联物免疫BALB/c小鼠,同时做单一表位组和BSA对照组。分别于0、14、28,31天免疫接种,剂量约为100μg/只/次。末次免疫4天后,ELISA检测血清效价,测定抗体水平。并通过免疫印迹及免疫荧光方法检测抗血清能否识别重组及天然EspA,以此评价由表位制备的抗血清的免疫功能。 7.FAS实验评价单抗及表位多抗的生物学功能通过免疫荧光实验分别检测单抗及表位多抗阻断宿主细胞激动蛋白聚集的能力,并将两者作比较。 结果 1.通过传统的杂交瘤技术制备得到3株抗EspA单克隆抗体,并用HiTrap rProteinA柱对单克隆抗体进行纯化,纯度达90%以上 2.通过免疫印迹、ELISA、免疫荧光、结合相加等实验对抗体的特异性、亲和力、与天然蛋白的免疫反应性、识别抗原表位等都得到了证实。 3.采用生物信息学和表位作图相结合的方法应用制备的单抗鉴定EspA抗原B细胞表位。PCR方法从O157基因组中分别扩增出E1(198bp)、E2(240bp)、E3(306bp)、E4(375bp)、E5(345bp)和E6(270bp)片段的碱基序列,并分别成功构建到原核表达载体pET28a和pET22b上,分别经IPTG诱导,Tris-Tricine PAGE证实目的蛋白的表达,并经免疫印迹证实,抗EspA单克隆抗体1H10能够与E2(40～120aa)、E3(90～192aa)、E4(67～192aa)、E5(77～192aa)和E6(100～192aa)目的蛋白发生特异性抗原抗体反应;单克隆抗体2A3与目的蛋白E4(67～192aa)和E5(77～192aa)发生特异反应;单克隆抗体3E5与目的蛋白E2(40～120aa)发生特异反应。初步证实单克隆抗体1H10所识别的抗原表位位于EspA蛋白N端第100～120位氨基酸内,而2A3、3E5所识别的抗原表位疑为构象型表位。 4.采用步移合成表位多肽方法,分别合成E_(67～90aa),E_(70～95aa),E_(75～100aa),E_(80～105aa),E_(85～110aa),E_(90～115aa)和E_(100～120aa)7条多肽。通过ELISA和Dot blot方法证实,单克隆抗体1H10所对应的B细胞抗原表位为EspA蛋白N端第100～120位氨基酸,2A3、3E5不与任何一段多肽结合,确定两者均识别构象型表位。 5.合成的表位多肽E_(100～120)能够竞争抑制单克隆抗体1H10与EspA的抗原抗体反应,且抑制效应具有剂量依赖性,当EspA蛋白浓度为2μg/ml、单克隆抗体浓度为12μg/ml、表位多肽E_(100～120)浓度分别为0.02、0.04、0.2、1、2μg/ml时,根据OD值的变化情况,按公式计算抑制率,抑制率分别为7%、27%、58%、84%和96%。 6.应用戊二醛将表位多肽E_(100～120)与载体蛋白BSA进行偶联,制备偶联物,并将其免疫BALB/c小鼠,产生的抗血清其效价高达1:256 00,并且表位与载体蛋白偶联物免疫后的抗血清能够与天然O157菌株的EspA发生特异性抗原抗体反应,进一步证实E_(100～120)具有免疫原性,可以作为O157疫苗的候选抗原成分。 7.通过FAS实验对EspA生物功能进行了初步研究,表明多肽抗血清和单克隆抗体均能够阻断肌动蛋白的聚集,也就更进一步证明EspA在A/E损伤的产生中起到非常重要的作用。 结论 1.采用杂交瘤技术制备得到了3株抗EspA单克隆抗体,并对3株单抗的生物学特性进行了鉴定。 2.以单抗1H10筛选到一个EspA的优势B细胞抗原表位,位于EspA第100-120个氨基酸,该表位表现出良好的免疫原性和免疫反应性。 3.单抗2A3、3E5可能分别针对两个不同的构象型表位,考虑通过解析单抗与EspA的抗原抗体复合物结构来确定该两株单抗针对的B细胞抗原表位。
[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.
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