小鼠KCTD10特异性抗体制备及其在小鼠背神经上皮和背根神经节中的表达  

Preparation of Mouse KCTD10 Antibody and Expression Analysis of KCTD10 in Neuroepithelium of Neural Tube and Dorsal Root Ganglion

作　　者：孙吉康[1] 张波[2] 张健[1] 周建林[1] 

机构地区：[1]湖南师范大学生命科学学院 [2]湖南中医药大学,长沙410007

出　　处：《生物工程学报》2007年第6期1011-1016,共6页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No30571005);教育部创新团队(NoIRT0445);湖南省科技厅(No05FJ4016)资助~~

摘　　要：KCTD10蛋白是一个TNF-α诱导表达的蛋白,能与DNA聚合酶δ的小亚基和PCNA相互作用。但其具体功能尚不清楚。为了研究KCTD10的功能,用小鼠KCTD10融合蛋白免疫新西兰大白兔获得兔抗鼠KCTD10多克隆抗体。由于小鼠KCTD10蛋白与小鼠PDIP1蛋白和TNFAIP1蛋白有较高的蛋白序列相似性,KCTD10抗体同PDIP1蛋白、TNFAIP1蛋白有抗体交叉反应。将同源蛋白PDIP1和TNFAIP1的部分降解片段与抗KCTD10血清在4℃下孵育3h从而消除非特异性抗体,成功地消除了KCTD10多克隆抗体对PDIP1蛋白和TNFAIP1蛋白的交叉反应。用此抗体做小鼠胚胎及胚胎切片的免疫组化,发现KCTD10蛋白在E12.5d的小鼠胚胎背根神经节以及神经管神经上皮中有明显的表达。该结果提示KCTD10可能在小鼠的神经管神经上皮和背根神经节发育中发挥作用。KCTD10 is a TNF-α inducible protein that can interact with the small subunit of DNA polymerase δ and PCNA. In order to study the function of KCTD10, we prepared the rabbit anti-mouse KCTD10 polyclonal antibody by using the His-tagged recombinant mouse KCTD10 protein to immune New Zealand white rabbit. Mouse KCTD10 shares significant similarity with PDIP1 （polymerase delta-interacting protein 1 ） and TNFAIP1 （tumor necrosis factor alpha-induced protein 1 ） protein, and then KCTD10 polyclonal antiserum possesses cross-reactivity with PDIP1 protein and TNFAIP1 protein. The partially digested fragments of homogeneous proteins PDIP1 and TNFAIP1 were mixed and incubated with anti-KCTD10 antiserum at 4℃ for 3 h to deplete unspecific antibodies. Through this method, we removed successfully the cross-reactivity of anti-KCTD10 antibody with PDIPI and TNFAIP1 and obtained specific anti-KCTD10 antibody. Then, the anti-KCTD10 antibody was used in immunohistochemistry experiments of mouse. The results of immunohistochemistry on whole-mount embryo and paraffin section demonstrated that KCTD10 is highly expressed in neuroepithelium of neural tube and dorsal root ganglion of 12.5 d embryos. These results suggest that KCTD10 may play roles in the development of neuroepithelium of neural tube and dorsal root ganglion.

关 键 词：KCTD10 抗体交叉反应 免疫组化 神经管神经上皮 背根神经节 

分 类 号：Q954.4[生物学—动物学]