幽门螺杆菌融合蛋白VacA-HpaA卵黄抗体的制备及体外试验

发布时间：2018-03-04 11:50

本文选题：幽门螺杆菌　切入点：空泡毒素　出处：《重庆医科大学》2006年硕士论文　论文类型：学位论文

【摘要】： 第一部分幽门螺杆菌融合蛋白VacA-HpaA卵黄抗体的制备目的:采用基因工程技术大量表达幽门螺杆菌(Helicobacter pylori, H.pylori)细胞空泡毒素(vacuolating cytotoxin antigen A,VacA)与粘附素(Helicobacter pylori adhesin A ,HpaA)的融合蛋白(VacA-HpaA),鉴定其抗原性与免疫原性后,以此融合蛋白作为抗原,制备高效价的VacA-HpaA IgY。方法:通过大量培养重组菌pQE30-VacA-HpaA-DH5a,诱导表达获得融合蛋白VacA-HpaA Ni2+-NTA树脂纯化。用Western blot鉴定融合蛋白VacA-HpaA对VacA和HpaA的抗原特异性,用融合蛋白免疫兔,获得多克隆抗血清,采用间接ELISA法检测其分别针对VacA和HpaA的免疫原性。将纯化的融合蛋白免疫鸡,水稀释结合氯仿有机沉淀法提取IgY。ELISA法测定抗体产生的时间-效价变化。硫酸铵沉淀法纯化浓缩VacA-HpaA IgY,SDS-PAGE分析纯度,Bradford法测定蛋白含量,Western blot检测其分别对VacA和HpaA的特异抗原性,ELISA法检测效价。
[Abstract]:Preparation of VacA-HpaA yolk antibody of Helicobacter pylori fusion protein. Objective: to express the fusion protein VacA-HPA of Helicobacter pylori (H.pylori) vacuolating cytotoxin antigen AcaA) by genetic engineering, and identify its antigenicity and immunogenicity by using the fusion protein VacA-HpaA, which is a fusion protein of Helicobacter pylori adhesin A (HpaA), and to identify the antigenicity and immunogenicity of the fusion protein. Preparation of high titer VacA-HpaA IgY. Methods: the fusion protein VacA-HpaA Ni2-NTA was purified by mass culture of pQE30-VacA-HpaA-DH 5a. The antigenicity of the fusion protein VacA-HpaA to VacA and HpaA was identified by Western blot, and the polyclonal antiserum was obtained by immunizing rabbits with the fusion protein. The immunogenicity of VacA and HpaA was detected by indirect ELISA method. The purified fusion protein was used to immunize chickens. Water dilution combined with chloroform organic precipitation method was used to extract IgY.ELISA to determine the time-titer change of antibody production. Purification and concentration of VacA-HpaA IgYPS-PAGE by ammonium Sulfate precipitation Purity determination of protein content by Bradford method and Western blot Detection of its specific Antibody to VacA and HpaA. The titer was detected by Elisa.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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