论文部分内容阅读
目的:制备抗人m_3受体肽链的单克隆抗体。方法:人工合成人m_3受体亚型特异的细胞外氨基端肽链段,与血蓝蛋白偶联,免疫Balb/c小鼠,制备m_3受体的单克隆抗体。用PEG-6000二步沉淀法与Sephadex G-50柱层析分离纯化抗体。用ELISA、免疫组织化学和放射配基法鉴定抗体的特异性。结果:抗体与m_3肽链、大鼠唾液腺及大脑皮质膜蛋白发生特异性的结合,不与m_4肽链发生结合。抗体能抑制~3H-QNB与大鼠唾液腺膜蛋白的结合,不抑制~3H-QNB与大鼠心肌的结合,也不抑制~3H-PZ与大鼠大脑皮质膜蛋白结合。免疫组织化学结果显示唾液腺呈棕色阳性反应,而心肌呈阴性反应。结论:本研究制备的人m_3受体单克隆抗体具有高的特异性,可专一性识别m-3受体。
Objective: To prepare monoclonal antibodies against human m_3 receptor peptide. Methods: The extracellular amino - terminal peptide fragment of human m_3 receptor subtype was artificially synthesized and conjugated to hemocyanin to immunize Balb / c mice to prepare m_3 receptor monoclonal antibody. The antibodies were isolated and purified by PEG-6000 two-step precipitation and Sephadex G-50 column chromatography. Antibody specificity was identified by ELISA, immunohistochemistry and radioligand methods. Results: Antibody binds specifically with m_3 peptide chain, rat salivary gland and cerebral cortex membrane protein without binding to m_4 peptide chain. Antibodies could inhibit the binding of ~ 3H-QNB to rat salivary glandular protein, inhibit the binding of ~ 3H-QNB to rat myocardium, and also inhibit the binding of ~ 3H-PZ to rat cerebral cortical membrane protein. Immunohistochemistry results showed that the salivary glands showed a brown positive reaction and a negative myocardial reaction. Conclusion: The m_3 receptor monoclonal antibody prepared in this study has high specificity and can specifically recognize m-3 receptor.