采用杆状病毒/昆虫表达系统纯化蛋白制备抗hUTP14a多克隆抗体

Preparation of Polyclonal Antibody to hUTP14a Using Purified Baculovirus/Insect Expression System

为制备兔抗人hUTP14a多克隆抗体并鉴定其抗体的特异性,本研究通过杆状病毒/昆虫表达系统制备并纯化Flag-hUTP14a-his蛋白,免疫新西兰家兔。ELISA方法检测免疫兔的抗血清滴度达到1∶10~5(体积比)时取血清,并通过Protein A免疫亲和层析柱纯化抗体。在人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)中过表达Flag-hUTP14a,或用小干扰RNA(small interference RNA, siRNA)沉默内源的hUTP14a蛋白表达后,提取细胞总蛋白质。经Western印迹分析制备的多克隆抗体的特异性;通过细胞免疫化学染色、细胞免疫荧光、免疫组织化学染色和免疫沉淀(immunoprecipitation, IP)实验对hUTP14a抗体的特异性进行鉴定。研究证实,制备的抗hUTP14a多克隆抗体能够特异性识别内源及外源表达的hUTP14a蛋白。该抗体可以用于细胞免疫荧光、细胞免疫化学染色、免疫组织化学染色、Western印迹及免疫沉淀等技术,为进一步研究hUTP14a的生物学功能提供了特异性抗体。

To prepare specific antibody against hUTP14 a, Flag-hUTP14 a-his protein was expressed and purified from the baculovirus/insect expression system. The purified Flag-hUTP14 a-his protein was used to immunize New Zealand rabbits. The sera were collected from the immunized rabbits when the titer of the anti-sera reached 1∶10~5(volume ratio) measured by ELISA. IgG from the rabbit anti-sera were purified by protein A immuno-affinity chromatography. Western blot was performed with the purified IgG from the anti-sera on the cell lysates of human umbilical vein endothelial cells(HUVEC) transfected with either Flag-hUTP14 a or hUTP14 a-specific siRNA. The purified IgG specifically recognizes endogenous hUTP14 a and ectopic Flag-hUTP14 a, demonstrating that polyclonal anti-hUTP14 a antibody were obtained. It was confirmed that the anti-hUTP14 a antibody could be used in the immunocytochemistry, immunofluorescence(IF), immunohistochemistry staining(IHC) and immunoprecipitation(IP). We have thus obtained a specific rabbit anti-human hUTP14 a polyclonal antibody that can be used in the further study of the biological functions of hUTP14 a.