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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。
Objective: To prepare a polyclonal antibody against mouse anti-human ASGPR isoform H1b and identify its specificity. Methods: The H1b specific polypeptide was synthesized and the immunogen of polypeptide was constructed with keyhole limpet hemocyanin (KLH) as carrier. The mouse anti-human H1b protein polyclonal antibody was prepared by sensitized mice. The antibody titer was detected by ELISA Western blot and immunohistochemistry to identify the specificity and scope of the antibody. Results: The mouse anti-human H1b protein polyclonal antibody titer of 1:10. The purified antibodies were used in western blot to identify the eukaryotic expression of H1b protein in high specificity and can be used in immunohistochemistry. CONCLUSION: The polyclonal antibody of mouse anti-human H1b protein has been successfully prepared, which has high potency and specificity and can distinguish two splice isoforms of ASG-PR large subunit. Therefore, Physiological function and its significance in human disease provides an advantageous tool.