陳弘成臺灣大學：漁業科學研究所沈子耘Shen, Tzu-YunTzu-YunShen2007-11-282018-07-062007-11-282018-07-062006http://ntur.lib.ntu.edu.tw//handle/246246/59342本研究主要目的是建立Bac-to-Bac baculovirus表現鱧魚(Channa maculata)促性腺激素(Gonadotropin, GTH)重組蛋白，選殖鱧魚促性腺激素兩次單元之cDNA至pFastBac Dual載體的兩個啟動子下游，轉殖至DH10 Bac勝任細胞產生Bacmid，再感染Sf9及High 5昆蟲細胞生成鱧魚促性腺激素重組蛋白。預期生成之兩次單元重組蛋白於表現過程能形成正確構型，與醣基化，供進一步生化特性，生物活性及蛋白質晶體結構研究。脊椎動物腦下垂體可分泌兩型促性腺激素，一為促黃體激素(luteinizing hormone, LH)，另一為促濾泡激素(follicle stimulating hormone, FSH)，控制性腺(精巢與卵巢)之發育，生長及生理功能。。FSH和LH各具有兩個次單元體α、β-subunit，對同一種別α-subunit是共用且相同的，但是β-subunit不同且決定其激素專一性。研究顯示脊椎動物之LH-b及FSH-b次單元皆有12個半胱胺酸(cysteines)，其位置非常保守，其所形成的六對雙硫鍵，對於GTH分子整體結構扮演重要角色。本研究已選殖鱧魚腦下垂體GTH cDNAs，發現FSH-β次單元之第三個半胱胺酸並不存在，取而代之在N端有一多的半胱胺酸，由進一步資料分析發現鱧魚相近魚種亦有此特性。從人類FSH及hCG蛋白質晶體結構顯示第三個半胱胺酸和與十二個半胱胺酸所形成雙硫鍵，將a-次單元纏繞扮演如”seat-belt”功能，於兩次單元交互作用扮演非常重要角色，為探討shfFSH-b第三半胱胺酸之功能，本研究以baculovirus表現系統表達鱧魚兩促性腺激素重組蛋白，以供將來進一步晶體結構之研究。The aim of this study is to establish Bac-to-Bac Baculovirus protein express system to over-express the recombinant snakehead fish gonadotropins (shfGTHs). We cloned two subunits of shfGTH cDNAs (α and β) into two distinct promoters of pFastBac Dual vector, transformed into DH10 Bac competent cells to produce the Bacmid, and then infected High 5 insect cells to produce the recombinant shfGTHs. The expressed proteins, hopefully folded correctly and glycosylated, will be used for the studies of biochemical characterization, biological activity assay, and crystal structural analysis. Pituitary glands of vertebrates can synthesize and secret two gonadotropins to control the development, growth, and functions of gonad: one is luteinizing hormone (LH), and the other is follicle-stimulating hormone (FSH). Both LH and FSH are composed of two subunits, α- andβ-subunit: α-subunits are common in a species; whileβ-subunits are different and determine the hormonal specificities. The studies of vertebrate FSH and LH showed that twelve cysteines residues are all conserved in their b-subunits to form six disulfide bonds, expect some fish FSHs. Our previous molecular cloning of snakehead fish GTH cDNAs revealed that the third cysteine residues is absent in shfFSH-β; instead, a positionally shifted cysteine is present at the N-terminus, as found in some phylogenetic related fish. The crystal structures of hFSH and hCG showed that the 3rd and 12th cysteines forms an important disulfide bond to wrap theα-subunit, as “seat-belt”. To investigate the role of the 3rd cysteine in shfFSH-β, we expressed the recombinant shfFSH and shfLH by baculovirus system for future crystal structure analysis.誌謝………………………………………………………………………….……..i 中文摘要……………………………………………………………………….….iii 英文摘要………………………………………………………………………..….v 目錄………………………………………………………………………….…….vii 圖表目錄………………………………………………………………………..…ix 第一章 諸論 1 脊椎動物腦下垂體與促性腺激素 1 魚類促性腺激素 4 鱧魚促性腺激素 5 重組促性腺激素蛋白質之表現系統 6 本研究目的 8 第二章 鱧魚促性腺激素基因選殖與Baculovirus載體建構 9 前言 9 材料與方法 10 I. 鱧魚促性腺激素 (GTH) cDNA選殖 10 II. 鱧魚GTH表現蛋白pFastBac Dual 載體的建構 13 III. shfFSH和shfLH Bacmid載體的建構 22 結果與討論 25 I. 鱧魚FSH及LH的基因選殖 25 II. pFastBac Dual載體建構 25 III. shfFSH和shfLH Bacmid載體建構 29 第三章 鱧魚促性腺激素重組蛋白質之表現與特性分析 31 前言 31 材料與方法 32 I. Sf9及High 5昆蟲細胞培養 32 II. 以Sf9昆蟲細胞產生shfFSH及shfLH Bacmid之Baculovirus 32 III. shfFSH及shfLH蛋白質表現與純化 34 IV. shfFSH及shfLH蛋白質分析 36 結果與討論 37 I. shfFSH及shfLH的Bacmid載體感染至Sf9細胞 37 II. shfGTH蛋白質表現 37 III. shfGTH蛋白質分析 38 IV. Bac-to-Bac baculovirus系統 39 V. shfFSH及shfLH模型結構分析 40 第四章 綜合討論 42 研究成果 42 結論 43 未來研究方向 43 參考文獻 45 圖表目錄 Figure 1-1. 脊椎動物之GTH-b演化樹圖(Chatterjee et al., 2005)。 51 Figure 2-1. shfGTH cDNA 示意圖。 52 Figure 2-2. 以PCR擴增HBM-shfGTH-α cDNA片段。 53 Figure 2-3. HBM-shfGTH-α/pFastBac Dual1321660 bytesapplication/pdfen-US鱧魚腦下垂體促性腺激素促黃體激素促濾泡激素Snakehead fishPituitarygonadotropinLHFSHotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/59342/1/ntu-95-R93b45003-1.pdf