预防幽门螺杆菌感染的蛋黄抗体的研制

【作者】 黄进；

【导师】 杨致邦；

【作者基本信息】 重庆医科大学 ， 病原生物学， 2006， 硕士

【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Ni²⁺-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Ni²⁺-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。更多还原

【Abstract】 Objective: To observe anti-rHpaA IgY from eggs yolk of hens immunized with recombinant Helicobacter pylori HpaA protein by gene-engineering.Methods: Recombinant Helicobacter pylori HpaA protein were aquired by gene-engineering. Laying hens were immunized with recombinant. rHpaA.IgY was isolated by using the water dilution method (WD) ,and their concentration and purity were measured respectively with Bradford method and SDS-PAGE. The specificity of IgY and titer of IgY was assayed with Western blot and ELISA respectively. The titer of post-pasteurized was assayed with ELISA.Results: The concentration and purity of IgY were 24.6mg/ml and 90 % respectively. The titer of IgY was 1:12800,and the titer of post-pasteurized did not decrease. Its specificity was confirmed by Western blot.Conclusion: rHpaA-IgY with high concentration, high purity, high titer, pasteurized-stability and specificity was successfully preparated.更多还原