具腦部組織特異表現的蛋白激脢 msbk 基因之選殖與特性分析

1. 具腦部組織特異表現的蛋白激脢msbk 基因之選殖與特性分析 我們從老鼠腦部 cDNA 中選殖了一 msbk 基因，與斑馬魚蛋白激脢 bsk146 基因及大鼠 sbk 基因同源，會轉譯出一 417 個胺基酸之serine/threonine 之蛋白激脢，其 C 端上有一富含有 proline 的區域，內有可和SH3 ( Src homology 3 ) domain 結合的序列以及具有細胞核定位訊息 ( nuclear locaization signal，NLS ) 的序列，我們構築了3種基因片段，msbk-FL ( msbk基因全長 ) ， msbk —KR ( ATP 結合位置變異 ) ， sbk—D ( 去除 C 端 ) 並選殖到 HA-yun 表現載體上，分別去轉染 COS1 、 C6 、 NG108-15 和 HepG2 細胞株，利用免疫染色法，觀察到 mSBK-FL 在 NG108-15 和 HepG2只會在細胞質表現，在 COS1 和 C6 細胞則是細胞質和細胞核皆存在， mSBK-KR 及 mSBK-D 就只在細胞質表現，顯示出此蛋白激脢之表現存在著細胞差異，且推測磷酸化與否及 C 端的 NLS序列、 SH3 domain 結合序列會影響在細胞中表現的位置。為了進一步分析此基因在生物體內的功能，我們去選殖出含 msbk 之老鼠基因體片段約 15kb，並將 msbk 之 exon1 置換成 GK-Neo ，製成基因剔除老鼠之目標載體，以便日後生產出 msbk 剔除老鼠；最後我們又選殖出約 5.5kb 之 msbk 5’ 端之基因體片段，利用 pCAT-basic 載體去進行啟動子活性分析，結果顯示啟動子約位在 - 613 ~ + 240 的地方，而在所使用的三種細胞中，以在 NG108-15 中 msbk 啟動子的活性為最強。

2. Molecular cloning and characterization of a protein kinase msbk • We have isolated a mouse gene msbk , which is homologous to zebrafish bsk146 gene and rat sbk gene , from mouse brain cDNA . It encodes a 417 amino acid residues as a serine/threonine protein kinase . It’s C-terminal end contains a proline-rich region , which included a SH3 bind core and a nuclear localization signal ( NLS ) . We have constructed msbk-FL ( wild type msbk ) , msbk-KR ( ATP binding site mutation form ) , msbk-D ( C-terminal end deletion form ) and cloned in HA-yun vector . Then we transfected these plasmids into COS1、C6、NG108-15 and HepG2 cell lines . By immunostaining , we observed that mSBK-FL only expressed in cytoplasm of NG108-15 and HepG2 cells . But in COS1 and C6 cells , mSBK-FL expressed both in cytoplasm and nucleus . And mSBK-KR and mSBK-D only expressed in cytoplasm of these cell lines. It may reveal that mskb expressed differently in distinct cell lines . And we can guess that phosphorylation and C-terminal end , contained a NLS and SH3 binding core , may affect the cellular localization of mSBK . In order to analyze the function of msbk in vivo , we also cloned a mouse genomic fragment about 15kb including msbk gene . We replaced the msbk exon1 with GK-Neo . Then we cloned this genomic fragment into a pBSK-sk+ vector to be a targeting vector for msbk knock-out mice . Finally we have cloned a msbk 5’flanking sequence about 5.5 kb . By CAT-basic vector , we performed promoter assay and found that the msbk promoter was located at -613 ~ +240 . Of these three kinds of cell lines that we used , the msbk promoter activity in NG108 cells was the strongest