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目的:制备RAI16蛋白的合成肽多克隆抗体,并进行初步鉴定和应用,为研究RAI16蛋白的功能及作用机制获得重要的实验工具。方法:应用Fmoc法化学合成RAI16蛋白N端第44~55位氨基酸的多肽,经C18的RP-HPLC纯化后,通过高碘酸钠法将纯化的RAI16蛋白的多肽与KLH交联;皮下注射抗原免疫新西兰纯种大耳白兔,加强免疫得到抗血清,应用蛋白G纯化获得多克隆抗体。对纯化的抗体进行ELISA、免疫组化、Western blot等初步鉴定和应用。结果:化学合成RAI16蛋白N端第44~55位氨基酸的多肽,纯化后多肽纯度为96%,达到免疫用抗原标准。多肽与KLH交联,用于免疫动物。经纯化后的抗体效价为1∶125 000。该多肽抗体可特异识别人脾脏组织中相对分子质量(Mr)约为55 000的RAI16蛋白。结论:所制备的多克隆抗体能与天然RAI16蛋白发生特异性反应,可应用于ELISA、免疫组化、免疫沉淀和Western blot等实验,为确定RAI16蛋白的组织分布和亚细胞定位、研究RAI16蛋白的功能及作用机制提供了重要的实验工具。
OBJECTIVE: To prepare polyclonal antibody against synthetic RAI16 protein and preliminary identification and application of the recombinant RAI16 protein to gain important experimental tools for studying the function and mechanism of RAI16 protein. Methods: The amino acids 44-55 of N-terminal of RAI16 protein were chemically synthesized by Fmoc method. After purified by RP-HPLC of C18, the purified RAI16 protein was cross-linked with KLH by sodium periodate method. Subcutaneous injection of antigens Immunized New Zealand purebred white rabbits, boosted to obtain antiserum, polyclonal antibodies were obtained by protein G purification. The purified antibodies were identified by ELISA, immunohistochemistry, Western blot and other applications. Results: The amino acids 44 to 55 of N-terminal of RAI16 protein were chemically synthesized, and the purity of purified polypeptide was 96%, which reached the standard of antigen for immunization. The polypeptide is cross-linked to KLH for immunization of animals. The purified antibody titer is 1: 125 000. The polypeptide antibody specifically recognizes RAI16 protein with a relative molecular mass (Mr) of about 55,000 in human spleen tissue. CONCLUSION: The prepared polyclonal antibody can react specifically with natural RAI16 protein and can be used in ELISA, immunohistochemistry, immunoprecipitation and Western blot. To determine the tissue distribution and subcellular localization of RAI16 protein, we studied the expression of RAI16 protein The function and mechanism of action provides an important experimental tool.