抗人ASGPR大亚基异构体蛋白H1b多克隆抗体的制备和鉴定

Preparation and identification of polyclonal antibody against protein H1b: the variant of major subunit of human ASGPR

目的:制备小鼠抗人ASGPR大亚基异构体蛋白H1b的多克隆抗体,并鉴定其特异性。方法:合成H1b特异性多肽,以匙孔血蓝蛋白(KLH)作为载体构建多肽免疫原,通过致敏小鼠,制备小鼠抗人H1b蛋白的多克隆抗体;ELISA法检测抗体效价,通过Western blot和免疫组织化学检测,鉴定抗体的特异性与适用范围。结果:得到小鼠抗人H1b蛋白的多克隆抗体,效价达1∶105。纯化后的抗体用于Western blot可高特异性识别真核表达的H1b蛋白,并可用于免疫组织化学实验。结论:成功制备小鼠抗人H1b蛋白的多克隆抗体,该抗体具有较高的效价以及特异性,能区分ASG-PR大亚基的两种剪接异构体,从而为进一步研究H1b蛋白的生理功能及其在人类疾病中的意义提供了有利工具。

AIM： To prepare and identify mouse polyclonal antibody against protein Hlb, which is the variant of major subunit of human ASGPR. METHODS： H1b specific peptide was synthesized and coupled with keyhole limpet hemocyanin （KLH） for immunization. Then Hlb-KLH conju- gation was injected into mouse subcutaneously to produce polyclonal antibody. ELISA assay was used to detect the titer of the antibody. Antibody was also identified by Western blot and immunohistochemistry assays. RESULTS： Mouse antibody against Hl b was prepared after injection of H1b- KLH conjugation. The titer of H1 b antibody was about 1：105. Western blot confirmed its high specificity. This antibody could also be used for immunohistochemistry analysis. CONCLUSION： The successful preparation of the polyclonal antibody against protein H1 b, which can discriminate the two variants of the major subunit of ASGPR with high specificity, will provide an efficient reagent for further study of the physiologic functions of HI b and its role in the pathogenesis of human disease.