【摘要】 目的:制备抗KIAA0649多克隆抗体并鉴定抗体的特异性,同时初步鉴定KIAA0649蛋白在细胞内的定位及其功能结构域。方法:通过TMHMM和DNAstar等软件对KIAA0649的蛋白质序列进行分析,选取了3段序列合成多肽,将3段多肽混合后对新西兰兔进行免疫,通过酶联免疫吸附实验(enzyme-linked immuno-sorbnent assay,ELISA)测定血清中抗多肽的滴度,当抗血清的滴度达到1∶105(体积比)时取血清,通过免疫亲和层析柱对抗体进行纯化。在U2OS细胞中表达Flag-KIAA0649或通过小干扰RNA沉默内源的KIAA0649,提取细胞蛋白,电泳后转膜,通过Western blot分析上述制备的多克隆抗体的特异性;将表达Flag-KIAA0649的细胞在玻片上固定,用抗Flag抗体和抗KIAA0649抗体进行双色免疫荧光分析,通过免疫荧光对KIAA0649抗体进行鉴定。利用PCR克隆构建Flag-KIAA0649全长表达质粒和系列Flag-KIAA0649缺失突变体,转染细胞后通过免疫荧光对KIAA0649在细胞内的定位进行研究。结果:得到的纯化KIAA0649多克隆抗体特异性地识别内源及外源KIAA0649蛋白,可用于免疫荧光和Western blot分析;全长KIAA0649蛋白在细胞核内显示特定的分布方式;KIAA0649的缺失突变体中,含有PENF motif的突变体与全长KIAA0649蛋白在细胞核内的分布形式一致,推测该结构域可能为KIAA0649的功能结构域。结论:通过上述方法制备的KIAA0649多克隆抗体具有较高的特异性,可应用于KIAA0649的功能研究,KIAA0649在细胞内定位的研究对于阐明KIAA0649的功能机制具有重要意义。更多还原

【Abstract】 Objective:To prepare and characterize the polyclonal antibody against KIAA0649 and identify the localization and the functional motif of KIAA0649.Methods: Three polypeptides were synthesized based on the bioinformatics analysis of KIAA0649 protein.New Zealand rabbits were immunized with the mixture of the three KIAA0649 peptides coupled with keyhole limpet hemocyanin(KLH).The titer of the antisera was detected with ELISA.The antisera were purified with immuno-affinity chromato-graphy when the titer reached 1∶105.Western blot was performed with the purified antisera on the cell lysates of U2OS cells transfected with either Flag-KIAA0649 or KIAA0649-targeting siRNA.Immunofluorescence was performed with the purified antisera and anti-Flag antibody on the cells transfected with Flag-KIAA0649.A series of Flag-KIAA0649 deletion mutants was constructed by PCR cloning.The cellular compartmentation of full-length Flag-KIAA0649 and its deletion mutants were analyzed with immunofluorescence.Results: The results of Western blot and immunofluorescence demonstrated that the antisera from the KIAA0649 polypeptides-immunized rabbits specifically recognized endogenous and exogenous KIAA0649.The full-length Flag-KIAA0649 displayed specific nuclear foci.The Flag-KIAA0649 deletion mutant containing PENF motif showed the same nuclear foci as the full length of Flag-KIAA0649,suggesting that the PENF motif could be the minimum functional motif of KIAA0649.Conclusion: We have obtained anti-KIAA0649 polyclonal antibody which will be useful for further investigation.The PENF motif could be the minimum functional domain of KIAA0649.更多还原