CAJ | 학술논문

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1:10~5(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或通过小于扰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.
목적:제비항KIAA0649다극륭항체병감정항체적특이성,동시초보감정KIAA0649단백재세포내적정위급기공능결구역.방법:통과TMHMM화DNAstar등연건대KIAA0649적단백질서렬진행분석,선취료3단서렬합성다태,장3단다태혼합후대신서란토진행면역,통과매련면역흡부실험(enzyme-linked immuno-sorbnent assay,ELISA)측정혈청중항다태적적도,당항혈청적적도체도1:10~5(체적비)시취혈청,통과면역친화층석주대항체진행순화.재U2OS세포중표체Flag-KIAA0649혹통과소우우RNA침묵내원적KIAA0649,제취세포단백,전영후전막,통과Western blot분석상술제비적다극륭항체적특이성;장표체Flag-KIAA0649적세포재파편상고정,용항Flag항체화항KIAA0649항체진행쌍색면역형광분석,통과면역형광대KIAA0649항체진행감정.이용PCR극륭구건Flag-KIAA0649전장표체질립화계렬Flag-KIAA0649결실돌변체,전염세포후통과면역형광대KIAA0649재세포내적정위진행연구.결과:득도적순화KIAA0649다극륭항체특이성지식별내원급외원KIAA0649단백,가용우면역형광화Western blot분석;전장KIAA0649단백재세포핵내현시특정적분포방식;KIAA0649적결실돌변체중,함유PENF motif적돌변체여전장KIAA0649단백재세포핵내적분포형식일치,추측해결구역가능위KIAA0649적공능결구역.결론:통과상술방법제비적KIAA0649다극륭항체구유교고적특이성,가응용우KIAA0649적공능연구,KIAA0649재세포내정위적연구대우천명KIAA0649적공능궤제구유중요의의.
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 chromatography when the titer reached 1:10~5. 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 FlagKIAA0649. A series of Flag-K1AA0649 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-K1AA0649 displayed specific nuclear foci. The Flag-KIAA0649 deletion mutant containing PENF motif showed the same nuclear foci as the full length of Flag-KJAA0649, 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 motifcould be the minimum functional domain of KIAA0649.