RAI16蛋白合成肽多克隆抗体的制备及初步应用

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.

Preparation and preliminary application of a rabbit anti-human polyclonal antibody against NH2-terminal peptides of RAI16

AIM: To prepare a rabbit anti-RAI16 polyclonal antibody and then to identify and apply the rabbit polyclonal antibody against NH2-terminal peptides of RAI16. METHODS: The peptide (aa.44 to 55)of human RAI16 NH2-terminus was synthesized by standard Fmoc.The synthesized peptide was purified by reversed phase high-performance liquid chromatography (RP-HPLC) and cross-linked with keyhole limpet hemocyanin (KLH)by sodium metaperiodate. The rabbits were immunized with the conjugated peptide 4 times(400 mug/rabbit).The polyclonal antibody was purified by Protein G from the collected antiserum. Then it was identified and employed for ELISA, immunohistochemisty and Western blot et cetera. RESULTS: The NH2-terminal peptide of human RAI16 with the purity of 96% was prepared.The titer of the purified polyclonal antibody was 1:125 000. Western blot results showed that the antibody could recognize the protein with molecular weight of 55 000 in the total lysates of human spleen. CONCLUSION: The polyclonal antibody against NH2-terminal peptide generated and identified in the present research can correspond specifically to natural RAI16 protein and therefore it can be employed in ELISA, immunohistochemisty, immunoprecipitation and immunoblotting. The successful preparation of the polyclonal antibody is helpful not only for the study of the tissue distribution and subcellular localization of RAI16, but lso for the research into the function and mechanism of human RAI16.