老鼠細胞週期蛋白激?CHK1之基因選殖

Abstract

蛋白激?在細胞內生長、分化、增殖、訊息傳遞、細胞週期的控制等各方面扮演極為重要的角色。當這些蛋白激?因為基因放大，突變，病毒活化而失去了控制時，常常會引起腫瘤的發生。 本實驗使用 CT-26 細胞株和腫瘤轉移組織所得到total RNA 反轉錄成的cDNA，以針對蛋白激?催化區域中最保守的第七（Ⅶ）和第九（Ⅸ) 次結構區所設計 degenerate 的引子（primer)，進行聚合?鏈反應，調查蛋白激?表現情形，建立癌症研究最基本資料。經過選殖，核酸序列分析，我們看到34種不同的蛋白激?表現。 在 CT-26 細胞株方面，我們找到兩個新的蛋白激?DNA片段，分別稱為PK1與PK2。我們利用5'- 和 3'- RACE 方法，得到PK1 cDNA完整的DNA序列。PK1全長含3051個核甘酸，可轉譯成一個含有476個胺基酸的蛋白質。比較整個蛋白激?的胺基酸序列，PK1與分裂酵母CHK1、線蟲（Caenorhabditis elegans）及人身上所找到相對應之PSK （來自中研院生醫所林文昌博士未發表之資料），分別為28.43%、37.47%及94.12%的相同程度；如果進一步比較催化區域胺基酸序列，則相同程度提高到96.67%（人）、47.24%（線蟲）和35.92%（分裂酵母CHK1）。因此，我們把 PK1、人及線蟲相對應之PSK，認為是小白鼠、人和線蟲的CHK1。 為了調查小白鼠CHK1(PK1)基因在老鼠大腦、肝臟、胰臟、小腸、畢丸、CT-26細胞株及腫瘤組織中的表現情形，我們進行反轉錄?--聚合?連鎖反應。我們的結果指出：CHK1(PK1)mRNA在小白鼠中各正常組織表現大致相等，畢丸部份較高，而腫瘤組織、CT－26細胞株表現量較低。這樣的結果，有待我們深入探討 CHK1(PK1) mRNA在生物體中所扮演的角色。 為了能製備小白鼠CHK1(PK1)多株抗體血清，我們在大腸桿菌M15中，表現帶有六個組氨酸（His）的小白鼠 CHK1(PK1)融合蛋白。最後，只有在催化區域中的一段，能被大量表現；我們將它製成抗原，對兔子進行免疫反應，得到小白鼠CHK1(PK1)多株抗體血清。利用此多株抗體血清，對老鼠各組織—大腦、心臟、肝臟、胰臟、腎臟、睪丸及儲精囊的萃取液進行西方墨點轉印法分析。結果顯示，我們所得到小白鼠CHK1(PK1)多株抗體血清並不能偵到到單一的訊號，相反的，它可能可以辨識其他與小白鼠的CHK1(PK1)催化區域相似性高的蛋白激?，因此得到很多在其他位置的訊號。所以，製作特異性高的抗體可能是我們未來的一個工作。

The superfamily of protein kinases a play very important role in cellular functions. They are involved in many biochemical events, including signal transduction, cell cycle regulation and cell differentiation. We have used the polymerase chain reaction to identify protein kinases that have been linked to the pathogenesis and progression of the cancer. Degenerate primer from the conserved catalytic domain of protein kinase genes were used to amplify and clone partial cDNA sequences from a murine colon cancer cell line CT-26 and Balb/c mouse tumor tissue. When the reaction products were sequenced, 34 distinct protein kinases were found, of which two are novel to date and have provisionally been named PK1,PK2. By using 5'- and 3'- RACE, the full length cDNA was obtained. The PK1 cDNA contains 3051 bp, encoding a 476 amino acid protein. By comparing with amino acid sequences from other species, PK1 is 28.43%,37.47%,94.12% identical to fission yeast (Saccharomyces prombe) CHK1, Caenorhabditis elegans serine/threonine kinase, human CHK1 homolog, respectively. When only the amino acid sequences of the putative kinase catalytic domain were compared, mouse PK1 displays 35.9% and 47.2% identity to fission yeast CHK1 and C. elegants serine/threonine kinase. Therefore, mouse PK1 may be a homolog of fission yeast CHK1. Thus, PK1 is renamed as the mouse CHK1. Expression of PK1 gene in normal tissues, tumor tissues and CT-26 cell line were examined by RT-PCR. Our data indicated that gene level expression of mouse CHK1 is slightly higher, whereas its expression in CT-26 cell line and tumor tissue are lower. The significance of lower expression of mouse CHKl gene in tumor cell line and tumor tissues need to be further investigated. To prepare specific antibody for mouse CHK1, we generated several His-tag fusion proteins, and expressed in E.coli. However, only one fusion protein containing amino acid residues 119 to 215 could be overexpressed in E.coil. The resulting polyclonal antibody was then used for Western blotting analysis. A protein band with molecular weight of 52 kDa was recognized in brain, heart and liver. Several other protein bands in varity of tissues were detected as well. Therefore, the polycolonal antibody is not specific for mouse CHK1. Peptide antibody will be generated and investigated in the future.