核酸免疫制备鼠抗棉铃虫FK506结合蛋白的多克隆抗体.pdfVIP

生物技术通报 ·研究报告· BIOTECHNOLOGY BULLETIN 年第 期 2014 12 核酸免疫制备鼠抗棉铃虫 FK506 结合蛋白的 多克隆抗体 朱燕  张富春  马纪  黄丽娜  刘小宁 （新疆大学生命科学与技术学院 新疆生物资源基因工程重点实验室，乌鲁木齐 830046） 摘 要 ： 利用电注射基因导入法制备小鼠抗棉铃虫FK506 结合蛋白 （FKBP12）的多克隆抗体，并通过Western blot 验证抗 体与抗原的特异性结合。构建重组质粒pcDNA3-FKBP12 并测序验证序列的正确性，利用基因导入法将重组质粒pcDNA3-FKBP12 注射到小鼠体内，免疫4 次后用ELISA 检测法确定抗体的效价，并对抗体的特异性结合进行Western blot 以及免疫组化检测。结果 表明，小鼠抗FK506 结合蛋白血清的效价达到1 ∶25 600，Western blot 结果显示此抗血清能与融合蛋白His-FKBP12 结合，免疫组 化结果表明在棉铃虫3 龄幼虫不同组织中FKBP12 均有表达。说明电注射基因导入法制备的小鼠抗FK506 结合蛋白的抗体在滴度 和特异性结合方面都达到预期要求。 关键词 ： 棉铃虫 FK506 结合蛋白 多克隆抗体 DOI ：10.13560/ki.biotech.bull.1985.2014.12.023 Preparation of the Polyclonal Antibody Against Helicoverpa armigera - FK506 Binding Protein by Immunizing with pcDNA3 FK506BP ’ Zhu Yan Zhang Fuchun Ma Ji Huang Li na Liu Xiaoning （Xinjiang Key Laboratory of Biological Resources and Genetic Engineering ，College of Life Science and Technology ， Xinjiang University ，Urumqi 830046 ） Abstract: It was to prepare the polyclonal antibody against FK506 binding protein, which was to be to verified the specificity of the antibody by Western blot. Firstly, recombinant expression plasmid pcDNA3-FKBP12 was constructed and sequenced, and the recombinant plasmid pcDNA3-FKBP12 was then transfered into Kunming mice four times. Finally, ELISA assay, Western blot analysis and immunohistochemistry（IHC）were used to determine the titer of polyclonal antibody of FK506 binding protein and its spec