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標題: | 探討介白質-15異構蛋白的特性與功能 Biochemical and functional analysis of an IL-15 alternative splice variant |
作者: | Chih-Hsiu Wang 王之秀 |
指導教授: | 顧家綺(Chia-Chi Ku) |
關鍵字: | 介白質-15,異構蛋白,介白質-15受體,內質網, interleukin-15,isoform,IL-15 receptor,endoplasmic reticulum, |
出版年 : | 2010 |
學位: | 碩士 |
摘要: | 介白質-15 (interleukin-15, IL-15) 為多效性的細胞激素,在免疫細胞的功能及恆定性扮演重要的角色。IL-15蛋白的表現已知在轉錄與轉譯的過程都必須受到嚴格的調控,究竟已報導的IL-15選擇性剪接異構體是否也參與此一調控的過程仍不清楚。
本論文首先分析IL-15異構體的生化特性,我們將原型IL-15與IL-15ΔE7的cDNA選殖到表達質體,藉由N端的IL-2 leader peptide增加分泌性與C端的FLAG為蛋白標記,經由轉染到COS-7細胞株中探討兩者在蛋白質表現、分泌性、細胞表現位置與生物活性等方面的差異。利用西方墨點法結合內糖苷酶 (endoglycosidase H, Endo H) 分析,雖然IL-15異構體和原型IL-15都具有Endo H的敏感性,但是醣類修飾的程度可能不同;進一步以酵素結合免疫吸附分析 (ELISA) 測得IL-15異構體在細胞中或分泌至細胞外的量都相對較低;而且利用免疫螢光染色觀察到大部分的IL-15異構體都累積在內質網。分泌出細胞外的IL-15異構蛋白不僅不具有生物活性,還能抑制原型IL-15的生物活性。接著我們針對IL-15 受體的 alpha 次單元 (IL-15Rα) 探討IL-15異構體的作用機制,利用流式細胞分析技術發現雖然原型IL-15能增加IL-15Rα在細胞表面的表現,但是IL-15異構體卻能抑制IL-15增強IL-15Rα表現的作用;另外,將IL-15異構體與IL-15Rα共同轉染至細胞中,發現IL-15Rα會累積在細胞中而無法運輸至細胞膜上。這些結果暗示IL-15異構體可能藉由降低IL-15Rα在細胞膜的表現量而抑制trans-presentation的訊息傳遞。 總之,本實驗發現,累積在內質網的IL-15異構體無法有效分泌至細胞外,一旦分泌出細胞外,極低量的IL-15異構體可能藉由降低IL-15Rα在細胞膜上的表現而影響原型IL-15的訊息傳遞。這些現象將有助於未來進一步了解IL-15在體內的調控機制。 Interleukin 15 (IL-15) is a pleiotropic cytokine which plays an essential role in innate and adaptive immune cell function and homeostasis. Expression of IL-15 protein is known to be tightly controlled at transcriptional and translational levels. Whether alternative pre-mRNA splicing is also involved in regulating IL-15 function is not clear. In this study, the biochemical properties and biological functions of IL-15ΔE7 which is encoded from an alternatively spliced mRNA having partial deletion in the exon 7 of IL-15 gene were assessed in vitro. The full length of IL-15 and IL-15ΔE7 cDNAs encoding the mature protein was cloned individually in an expression plasmid flanked with IL-2 leader peptide at the N-terminus and a FLAG tag at the C-terminus and were expressed in COS-7 cells by transient transfection method. Both IL-15 and IL-15ΔE7 were sensitive to Endo H treatment. However, their banding patterns on SDS-PAGE were different by Western blotting, suggesting the post-translational modifications on these proteins were different. Most of the IL-15ΔE7 protein retained intracellularly and accumulated in the ER as identified by colocalization with ER chaperone calnexin. Very few IL-15ΔE7 protein was secreted and failed to support HT-2 cell proliferation as well as inhibited the biological activity of IL-2 and IL-15 in a dose dependent manner. In addition, flow cytometric analysis showed that the exogenous addition of IL-15ΔE7 reduced the upregulation of surface IL-15 receptor α (IL-15Rα) induced by IL-15. Co-transfection of IL-15ΔE7 and IL-15Rα led to the intracellular accumulation of IL-15Rα which might inhibit the trans-presentation by limiting expression of IL-15Rα on the cell surface. In summary, IL-15ΔE7 was accumulated in the ER without efficient secretion. Once secreted, the extremely low amount of IL-15ΔE7 may have a regulatory role for IL-15 signaling by limiting IL-15Rα surface expression. The functional mechanism as well as the biological significance of this alternative splice variant remain to be clarified. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47248 |
全文授權: | 有償授權 |
顯示於系所單位: | 免疫學研究所 |
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