裂殖酵母Hsp90多克隆抗体的制备与鉴定.docVIP

  裂殖酵母 Hsp90 多克隆抗体的制备与鉴定 李文珠 1，李晓彤 2，张连茹 1，靳全文 1 (1. 厦门大学生命科学学院，福建厦门，361005; 2. 厦门大学生命科学学院杂交瘤与抗体中心，福建厦门，361005) 摘要：为制备兔抗裂殖酵母 Hsp90 多克隆抗体，在通过 PCR 获得了裂殖酵母 Hsp90 (Swo1) 基因后，构建了 pMALc2x-Swo1 表达载体，可用于表达编码正确氨基酸序列的目的基因。 转化大肠杆菌 BL21(DE3)，IPTG 诱导表达，Amylose Resin 柱纯化。目的蛋白表达量占菌体 总蛋白的 30%以上。纯化后，蛋白纯度达 95%以上。纯化后的 MBP-Swo1 融合蛋白抗原加 福氏完全佐剂背部皮内注射首次免疫新西兰大白兔，第 28 天用 MBP-Swo1 融合抗原加福氏 不完全佐剂同样剂量加强免疫，第 35 天时再次免疫。第 49 天心脏采血。收集血清后，用免 疫印迹(western blot)检测 Swo1 多克隆抗体的特异性。免疫印迹检测结果显示该抗体能够特 异性识别内源性的裂殖酵母 Hsp90/Swo1 蛋白，但并不识别人类细胞中的 Hsp90α/β。 关键词：裂殖酵母；Hsp90/Swo1；多克隆抗体 中图分类号：Q291 Preparation and identification of polyclonal antibody raised against heat shock protein 90 of fission yeast Li Wenzhu1, Li Xiaotong2, Zhang Lianru1, Jin Quanwen1 (1. School of Life Sciences, Xiamen University, Xiamen Fujian, 361005; 2. Hybridoma and Antibody Center, School of Life Sciences, Xiamen University, Xiamen Fujian, 361005, China) Abstract: To prepare the polyclonal antibody of Hsp90 (Swo1) of fission yeast Schizosaccharomyces pombe, the encoding gene swo1+ was amplified from the yeast genome and then inserted into expression vector pMALc2x. The resulted plasmid pMALc2x-Swo1 was transformed and expressed in E. coli BL21(DE3). The expressed fusion protein was purified through Amylose Resin column. The proteins were expressed mainly as secretion with the yield of more than 30% of total bacterial proteins. After purification, the purity of the proteins was about 95%. The New Zealand white rabbits were immunized with Freund’s complete adjuvant plus furified MBP-Swo1 fusion antigen through the back skin intradermal injection for the first time. The same dose of Freund’s incomplete adjuvant plus MBP-Swo1 was injected to strengthen the immunity after 28 and the 35 days respectively. After 49 days, blood sample was collected from heart, and antisera were extracted. The specificity of Swo1 polyclonal antibody was examined by Western blot. It showed that the antibody obtained had a high specificity to detect endogeneous Swo1 f