兔抗人肥大细胞羧肽酶抗体的制备与鉴定

Preparation and characterization of rabbit antibody against human mast cell carboxypeptidase

目的:以原核表达的人肥大细胞羧肽酶(hMC-CP)免疫家兔,制备兔抗hMC-CP多克隆抗体并进行鉴定。方法:在大肠杆菌中诱导表达重组hMC-CP,经Ni-NTA-Agarose柱层析纯化后,以其为免疫原制备兔抗hMC-CP多抗,并以间接ELISA测定抗体效价,以Western blot鉴定抗体的特异性。结果:成功地表达并纯化了hMC-CP,以纯化的重hMC-CP组免疫家兔成功制备了兔抗hMC-CP抗体,ELISA结果显示效价达1∶6 400,Western blot分析表明该抗体能特异结合hMC-CP。结论:以纯化的重组hMC-CP为免疫原,成功地制备了效价、特异性较强兔抗hMC-CP抗体,为进一步建立简便的hMC-CP检测方法及进一步研究hMC-CP生物学功能打下了良好的基础。

AIM： To prepare antibody against human mast cell carboxypeptidase （hMC-CP） by using recombinant hMC-CP expressed in E. coli, and to characterize the antibody. METHODS： hMC-CP was expressed in E. coli with L-Arabinose induction and purified through Ni-NTA column. The purified hMC-CP as immunogen was used to immunize rabbit. The titer and the specificity of the rabbit anti-hMC-CP antibody was analyzed by indrect ELISA and Westem blot respectively. RESULTS： The hMC-CP was successfully expressed and purifed. The polyconal anit-hMC-CP antibody was prepared by immunizing rabbit using the purified recombinant protein, The titer of the generated antiserum was l：6 400 by ELISA. Westem blot analysis showed that this antiboday could bind with hMC-CP specifically. CONCLUSION： The rabbit anti-hMC-CP antibody with high titer and high specificity has been prepared by using purified recombinant hMC-CP as immunogen, which lays the foundation for further research on detection and function of hMC-CP.