人源性基因calsenilin/KChIP3/DREAM单克隆抗体的制备及鉴定

Preparation and Identification of Monoclonal Antibody Against Human calsenilin/KChIP3/DREAM

Calsenilin/KChIP3/DREAM,是脑中高表达蛋白,最初发现是因其与presenilin和钙离子结合而得名。作为转录因子抑制因子,该基因在细胞核内具有多种功能。该基因在钙离子作用下细胞核内常常与c-fos、prodynorphin等基因的启动子下游的特异性DRE位点相结合,调节这些基因的表达。另一方面,作为钾离子通道结合蛋白,该基因具有4种isoforms,其中KChIP1广泛存在于各种组织中而KChIP2只在心脏中特异表达,KChIP3和KChIP4则在脑中显示较高的表达。4种基因在C-端结构非常相象,N-端则显示多样性。除此之外,和许多基因相似,calsenilin经PKC、CKI、PKA等激酶作用可产生多位点的磷酸化,其中主要位点Ser63的磷酸化可以阻止caspase-3对该基因的降解作用。另一方面,Calsenilin作为转录因子激动因子结合于维生素D和视黄酸效应因子启动子上游促进转录的进行。到目前为止,Calsenilin/KChIP3/DREAM在细胞核内具有双重基因表达调控作用,即当结合于启动子上游时显示正调控而当结合在启动子下游时显示负调控。为了更加深入研究calsenilin的功能及寻找新的受其调控的基因,首先制备可特异性识别的单克隆抗体。利用RT-PCR技术,从人脑中提取RNA扩增calsenilin全基因,克隆于pGEX-4T-2原核细胞表达载体中,经IPTG诱导表达、Gluthathion Sepharose 4B纯化得到GST-calsenilin/DREAM/KChIP3重组蛋白,并免疫小鼠。通过PEG细胞融合得到单克隆抗体。经细胞免疫染色及Western blotting检测显示说明本实验得到单克隆抗体可以用来进行细胞免疫染色及Western blotting等检测。该抗体的成功制备,为今后对calsenilin/DREAM/KChIP3调控基因表达的更深入研究提供了有效工具,也填补了国内尚无该基因单克隆抗体资源的空白。

Calsenilin/KChIP3/DREAM, which was originally identified as a presenilin and calcium binding protein expressed at higher levels in nerve system, plays multiple funtional roles as a transcriptional repressor in the nucleus. It binds to the downstream regulatory element （DRE） of prodynorphin or c-fos and its transcriptional repression is modulated by intracellular calcium release. To understaning the functional mechanism of calsenilin/KChIP3/DREAM in aging and oxidative stress response, the functional role in the phosphorylation of calsenilin and relationship between transcriptional repression and phosphorylation of calsenilin in nerve system, the monoclonal antibody against human calsenilin was generated by tranditional methods： the human calsenilin was cloned into pGEX-4T-2 vector and expressed by IPTG induction. The purified GST-calsenilin fusion protein was used to immunize mice. PEG-1500 reagent was used to fuse cells. Confocal micrograph results indicate that the monoclonal antibody against calsenilin workes well for Immuno-histochemical analysis, so the overexpressed calsenilin was able to observed in ER/ Golgi complex of H4 neuroglioma cells. Western blot analysis confirmed the monoclonal antibody against human calsenilin reacted on overexpressed protein calsenilin. However, immunoprecipitation analysis （the dada not shown here ） indicated that the specificity generated monoclonal antibody did not specifically recognize the endogenous or exogenous calsenilin and did not recognize the N-terminus of calsenilin. The successful developed mouse monoclonal antibody against huamn calsenilin could be utilized as a useful reagent for the analysis of biochemical, structural, and functional properties.