【摘要】 目的制备绿色荧光蛋白(GFP)兔多克隆抗体。方法选取GFP免疫原性较强、保守性低的98～117位氨基酸序列,利用F-moc法固相合成该段序列,经高效液相色谱纯化,MALDI-TOF-MS鉴定合成多肽的分子量后,与匙孔血蓝蛋白(KLH)偶联,免疫新西兰雄兔,制备抗血清,并进行Western blot鉴定。结果合成的GFP多肽经MALDI-TOF-MS分析,分子量为2497.9,与理论值相符。1∶200稀释的抗血清与转染pEGFP-N1质粒的HeLa细胞表达的增强型GFP在相对分子质量27000处有清晰的杂交信号出现。结论已成功制备了特异性较好的GFP兔多克隆抗体。更多还原

【Abstract】 Objective To prepare rabbit polyclonal antibody against green fluorescent protein(GFP). Methods The immunogenic and lowly conversed amino acids 98-117 of GFP was synthesized by F-moc solid phase synthesis, purified by HPLC and determined for relative molecular mass by MALDI-TOF-MS. Couple the synthetic polypeptide to keyhole limpet hemocyanin (KLH) and immunize male New Zealand rabbits with the prepared conjugate. Collect the antisera and identify by Western blot. Results The molecular mass of synthetic GFP polypeptide was 2 497. 9, which was consistent with the theoretical value. Western blot showed a clear reaction band, with relative molecular mass of about 27 000, of 1 ∶ 200 diluted rabbit antisera against the enhanced GFP expressed in HeLa cells transfected with plasmid pEGFP- N1. Conclusion The rabbit anti-GFP polyclonal antibody with good specificity was successfully prepared.更多还原