预防幽门螺杆菌感染的蛋黄抗体的研制

发布时间：2018-09-11 10:44

【摘要】： 目的:采用基因工程技术大量表达重组幽门螺杆菌(Helicobac ter pylori,H.pylori)鞭毛粘附素(HpaA),用此重组蛋白作为抗原,制备高效价的抗HpaA的鸡蛋黄抗体IgY,为研制H.pylori预防性抗体制剂奠定基础。方法: 1.诱导重组质粒pQE30-hpaA在大肠杆菌DH5a中表达rHpaA,SDS-PAGE分析其表达形式。 2.重组蛋白经Ni~(2+)-NTA树脂纯化后,采用Bradford法测定蛋白浓度。 3.用rHpaA免疫30周龄立克体鸡,以水稀释法联合盐析法提取HpaA-IgY,纯化浓缩后用Bradford法测定其含量,SDS-PAGE电泳检测纯度,Western blot鉴定抗原特异性。 4. ELISA测定效价和巴氏消毒后的活性。结果: 1. SDS-PAGE电泳显示pQE30-hpaA-DH5a表达产物相对分子量为30 000。 2. SDS-PAGE分析表达形式,rHpaA主要存在于细菌裂解液上清中,约占70%,部分存在于包涵体中,经Ni~(2+)-NTA树脂对上清进行纯化后,纯度约为90%,Bradford法测得蛋白质含量为0.95mg/ml。 3. HpaA-IgY提纯后,Bradford法测得含量为24.6mg/ml,纯度约为90%,Western blot显示在相对分子量30 000处出现单一条带,ELISA效价为1:12800。
[Abstract]:Aim: to prepare the high titer HpaA yolk antibody IgY, for the preparation of H.pylori prophylaxis antibody preparation using the flagellin (HpaA), of recombinant Helicobacter pylori (Helicobac ter pylori,H.pylori) expressed in large quantities by genetic engineering technique and the recombinant protein was used as antigen to prepare the high titer chicken egg yolk antibody (IgY,). Methods: 1. The expression of recombinant plasmid pQE30-hpaA in Escherichia coli DH5a was analyzed by rHpaA,SDS-PAGE analysis. 2. 2. The recombinant protein was purified by Ni~ (2) -NTA resin, and the protein concentration was determined by Bradford method. RHpaA was used to immunize 30 week-old ricket-type chickens. HpaA-IgY, was purified and concentrated by water dilution method and salt-out method. The content of HpaA-IgY, was determined by Bradford method. SDS-PAGE electrophoresis was used to detect the purity and the specificity of the antigen was identified by Western blot. 4. The titer and pasteurized activity were determined by ELISA. Results: 1. SDS-PAGE electrophoresis showed that the relative molecular weight of the pQE30-hpaA-DH5a expression product was 30000.2. The expression form of SDS-PAGE was mainly found in the supernatant of bacterial lysate, accounting for about 70%, and partly in the inclusion body. The supernatant was purified by Ni~ (2) -NTA resin. The protein content measured by Bradford method was 0.95 mg / ml 路ml. 3. After purification by HpaA-IgY, the content was 24.6 mg / ml, and the purity was about 90%. Western blot showed that a single band Elisa titer was 1: 12800 at the relative molecular weight of 30 000.
【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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