利用聚丙烯酰胺凝胶电泳纯化的微量蛋白制备抗血清

Preparation of antiserum using trace protein purified by SDS-PAGE as immunogen

目的 以SDS PAGE纯化的微量蛋白制备抗人层粘连蛋白受体前体蛋白 (LRP)的血清。方法 以SDS PAGE分离纯化原核表达的人LRP与 6×组氨酸的融合蛋白 ,切下并粉碎融合蛋白所在凝胶块。将含 10 μg融合蛋白的凝胶颗粒与弗氏佐剂混匀后 ,免疫BALB/c小鼠。以Westernblot检测所获抗血清的滴度和特异性。结果 以胃癌细胞总蛋白为靶抗原时 ,Westernblot显示 ,所获抗血清仅识别 1条Mr 约 4 2 0 0 0的蛋白带 ,将抗血清做 1∶2 0 0 0稀释后 ,仍能清晰地显示其靶抗原所在位置。结论 以聚丙烯酰胺凝胶颗粒为载体 。

Aim To prepare antiserum to human laminin receptor precursor (LRP) using trace protein purified by SDS PAGE as immunogen. Methods Fusion protein of human LRP with 6×histidine fag expressed in prokaryotic cells was purified by SDS PAGE. The gel slice corresponding to fusion protein was cut, crushed, and emulsified with Freund's adjuvant. BALB/c mice were each immunized with emulsified mixture corresponding to 10 μg fusion protein. Titer and specificity of antiserum were evaluated by Western blot. Results Using total protein of gastric cancer cells as target antigen, Western blot analysis showed that the antiserum could recognize a single protein band with M r of 42 000, and that even if being diluted as 1∶2 000, the antiserum still reacted to its target antigen protein. Conclusion The antiserum against LRP can be prepared successfully by using trace protein as immunogen.