人Dcp2基因克隆表达、多克隆抗体的制备及其应用

Cloning,expression and preparation of polyclonal antibody for human Dcp2 gene

mRNA在细胞质内的稳定性是调控基因表达的重要方式.细胞以mRNA降解的方式对过时的、突变有害的mRNA进行及时清除,以保证基因的正确表达和细胞正常的生命活动.其中脱帽酶Dcp1/Dcp2在mRNA降解中起到主要作用,但对其降解mRNA的详细作用机制了解甚少.到目前为止,尚没有该基因市售的抗体出现,阻碍了对该基因机理的研究.目的是利用PCR方法,从人的胎肝文库中克隆到Dcp2基因,制备其多克隆抗体血清.实验结果显示,通过溴化氰偶联柱子对该多克隆抗体血清经纯化得到的多克隆抗体,用于Western Blotting试验,可以检测到该蛋白内源性的表达;用于激光共聚焦定位实验显示,该内源性蛋白定位在细胞核内;此抗体同时可用于免疫沉淀实验.因此Dcp2基因和多克隆抗体的获得,为进一步研究该基因的功能创造了条件.

The degradation of eukaryotic mRNAs plays important roles in the modulation of gene expression, and quality control of mRNA biogenesis. Dcp1/Dcp2 is a major component of mRNA decay factors. However, the more detailed mechanisms of Dcp1/Dcp2 in mRNA degradation are unknown. In this study, our object is to amplify Dcp2 gene from human brain eDNA library, and then to prepare rabbit-anti-human Dcp2 polyclonal antibodies. Our results showed that anti-Dcp2 rabbit polyclonal antibody was obtained. The antibody could detect the endogenous Dcp2 expression and its cell localization by both Western Blotting and immunohistochemistry, and Dcp2 protein also could be enriched by immunoprecipitation with this antibody. Therefore, this work provides a useful tool for further functional study of Dcp2 gene.