幽门螺杆菌重组VacA单克隆抗体的制备及初步应用

发布时间：2018-11-20 13:41

【摘要】：研究目的 : 通过细胞工程的方法制备重组幽门螺杆菌(Helicobacter pylori,H.pylori)细胞空泡毒素抗原(vacuolating cytotoxin antigen,VacA)的单克隆抗体(简称单抗),并初步研究其应用。为制备检测 VacA 的试剂盒及探讨 VacA 的致病机制奠定基础。方法:本实验分两部分进行一、重组 VacA 的表达与纯化:在已构建的基因工程(pQE30-V-DH5α)基础上,大量诱导表达获得重组蛋白 VacA。用 SDS-PAGE 分析表达形式,Western blot 鉴定其抗原性, Ni2+-NTA 树脂纯化表达蛋白,SDS-PAGE 鉴定其纯度,Bradford 法测定蛋白浓度。用重组蛋白建立检测小鼠 VacA 抗体的间接 ELISA,评价重组蛋白免疫原性。二、 VacA 单克隆抗体的制备和初步应用:1.以纯化所得 VacA 为抗原免疫 BALB/c 小鼠,40 天后间接 ELISA 测小鼠尾静脉血以检测有无抗体产生;2.将脾细胞与骨髓瘤细胞用 50%聚乙二醇(PEG)融合,HAT 培养基培养选择出融合细胞,用建立的间接 ELISA 法筛选分泌VacA 抗体的细胞;3. 有限稀释法克隆化培养,间接 ELISA 法检测能稳定分泌抗 VacA 抗体的阳性细胞株,b.v 公司的抗体亚型检测试剂盒测定抗体类别;4.用间接 ELISA 法测定体外传代培养的 zhD4 细胞株分泌抗体的稳定性;5. 大量制备 zhD4 细胞株小鼠腹水后,饱和硫酸铵沉淀法初步纯化,间接 ELISA 法测定其效价,Bradford 法测定蛋白含量;6.用制备的单克隆抗体建立初步应用于胃粘膜中 VacA 的检测。
[Abstract]:Study on: preparation of vacuolating toxin antigen (vacuolating cytotoxin antigen, of Helicobacter pylori,H.pylori cells by the method of trapping through the process of fine cellularization in the preparation of the heavy group of Bacillus hirsurea (Helicobacter pylori,H.pylori) cell vacuolating toxin antigen (vacuolating cytotoxin antigen,) The monoclonal antibody (McAb) of VacA and its application were studied. To prepare the kit for detecting VacA and to explore the pathogenesis of VacA. Methods: this experiment was divided into two parts: the expression and purification of recombinant VacA: based on the constructed gene engineering (pQE30-V-DH5 伪), the recombinant protein VacA. was induced to express in large quantities. Its antigenicity was identified by, Western blot, Ni2-NTA resin was used to purify the expressed protein, the purity was identified by SDS-PAGE, and the protein concentration was determined by Bradford method. The immunogenicity of the recombinant protein was evaluated by indirect ELISA, using recombinant protein to detect mouse VacA antibody. 2. Preparation and preliminary application of VacA monoclonal antibody: 1. The purified VacA was used as antigen to immunize BALB/c mice. After 40 days, indirect ELISA was used to detect the production of antibodies in the tail vein of mice. Spleen cells and myeloma cells were fused with 50% polyethylene glycol (PEG). The fusion cells were selected in HAT medium. The cells secreting VacA antibody were screened by indirect ELISA method. 3. The indirect ELISA assay was used to detect the positive cell lines secreting anti-VacA antibody stably, and the antibody subtype detection kit of B. v company was used to detect the antibody types. 4. Indirect ELISA assay was used to determine the stability of secreted antibodies of zhD4 cell lines cultured in vitro. After the zhD4 cell line ascites were prepared in large quantities, saturated ammonium sulfate precipitation method was used to purify the mouse ascites, indirect ELISA method was used to determine its titer, and Bradford method was used to determine the protein content. 6. The monoclonal antibody was used for the detection of VacA in gastric mucosa.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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