裂殖酵母Hsp90多克隆抗体的制备与鉴定

Preparation and identification of polyclonal antibody raised against heat shock protein 90 of fission yeast

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(westernblot)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。

To prepare the polyclonal antibody of Hsp90 （Swol） of fission yeast Schizosaccharomyces pombe, the encoding gene swol^＋ was amplified from the yeast genome and then inserted into expression vector pMALc2x. The resulted plasmid pMALc2x-Swol 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 purified MBP-Swol fusion antigen through the back skin intradermal injection for the first time. The same dose of Freund＇s incomplete adjuvant plus MBP-Swol 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 Swol polyclonal antibody was examined by Western blot. It showed that the antibody obtained had a high specificity to detect endogeneous Swol from fission yeast, but it could not recognize Hsp90α/β in human cells.