"點帶石斑(Epinephelus coioides)之glycoprotein130 (gp130)基因選殖,細胞外片段重組蛋白表現及抗血清製作"

Abstract

gp130是非常重要的穿膜蛋白，可傳遞IL-6所誘發的免疫訊息傳遞。當IL6與IL-6r結合時，會進一步跟膜上的gp130結合形成複合物。該複合物可經由磷酸化進而活化下游的STAT-3訊號，隨後的信號傳遞誘發下游基因開始進行傳遞免疫訊息。在本篇研究中，點帶石斑於gp130全長序列首先經由circular DNA/RNA as template RACE方法進行選殖。其circular DNA/RNA as template RACE與市售的RACE試劑盒相比，circular DNA/RNA as template RACE可以降低RNA降解程度，並降低其時間及金錢成本。取得點帶石斑gp130開放閱讀框序列，全長約2694bp，可編碼899個氨基酸。經由MEGA5軟體，分析其gp130與其他魚類之gp130以及哺乳類之gp130氨基酸序列進行多序列分析，經親緣關係樹分析，將與gp130同一家族的其他蛋白－colony-stimulating factor receptor (CSFR)、Interleukin-31 Ra (IL-31Ra)、leukemia inhibitory factor receptor (LIFR) 以及oncostatin M receptor (OSM) 當作外群進行分析。並分析其穿膜區，膜內區和膜外區的位置。結果發現所選殖基因與gp130最為接近。為了進一步探討其gp130的生物功能為何，將其gp130膜外區與載體連接並轉殖入大腸桿菌中進行重組蛋白表現。接著進行點帶石斑的抗血清製備。在這項研究中，我們已製備許多重要的基礎研究，這些研究及抗血清製備將有助於未來使用免疫共沉澱的方法，以探討IL-6，IL-6R和gp130三者蛋白之間的相互作用。

Glycoprotein130 is an essential transmembrane protein, it plays a critical role in IL-6 immune signal transduction. When IL6 combined with IL-6R, and the combined protein were further bind with gp130 on the target cell membrane, forming a complex. The complex activating the signaling transduction by phosphorylated signal transducer and activator of transcription 3; STAT-3, the subsequent signal transduction will turn on downstream genes which induce immune reactions. In this research, full length grouper gp130 sequence was first cloned through circular DNA/RNA as template RACE. Compare with commercial RACE kit, circular DNA/RNA as template RACE can avoid RNA degradation, more convenience and more time and money save. The full length 2694 bp grouper gp130 open read frame sequence was obtained, which estimated encode an 899 amino acid protein. Secondary, the Phylogenetic analysis was performed by multiple alignment of grouper gp130 with available fish gp130 and mammal gp130 amino acid sequence.by using MEGA5 software, leukemia inhibitory factor receptor (LIFR) and oncostatin M receptor (OSM) was using as the out group in the analysis. In the analysis all contain to segregate the transmembrane domain, intramembrane domain and outer membrane domain. In order to further verified the possible biofunction of gp130, gp130 outer membrane domain was further subcloning in to a E. coli expression plasmid and the recombinant protein was expression. Then the antiserum against grouper was also prepared. In this study, we prepared all the important tools, that will help to discover the interactions of IL-6, IL-6R and gp130 proteins in the future.