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
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国家自然科学基金(No. 30571005)、教育部创新团队(No. IRT0445)和湖南省科技厅(No. 05FJ4016)资助。


Preparation of Mouse KCTD10 Antibody and Expression Analysis of KCTD10 in Neuroepithelium of Neural Tube and Dorsal Root Ganglion
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This work was supported by the National Natural Science Foundation of China (No. 30571005),Changjiang Scholars and Innovative Research Team in University (No. IRT0445) and Science & Technology Department of Hunan Province (No. 05FJ4016).

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    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3 h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。

    Abstract:

    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 PDIP1 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.

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孙吉康,张波,张健,周建林. 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[J]. 生物工程学报, 2007, 23(6):

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