結論與未來展望

1. 本論文之目的為試圖利用大腸桿菌製造去醣基 hFSH α- 和β-次單元體，加以組合成去醣基hFSH 變異體，並和野生型 hFSH 做活性比較和競爭作用，期望去醣基 FSH 變異體對於卵巢癌細胞的訊息傳遞有所影響，因而間接降低或抑制癌細胞的增生。因此首要工作為放大hFSHA 和 hFSHB 此兩段基因，故利用聚合酶連鎖反應 作為選殖並放大此兩段cDNA 作為整個架構之啟始。以 commercial

“human Pituitary Gland Marathon-Ready cDNA” 作為選殖 hFSH 次單元體基因的模版，另設計合適之引子後以最適當的PCR 反應程式放大之。其結果經由0.8 %瓊脂洋菜膠體電泳染色後可以清楚觀察到放大之後的cDNA 片段確實存在。

2. 接著將此兩段 cDNA 分別構築到 yT&A 選殖載體中，之後以大腸桿菌 DH5α 之轉形殖入載體，作為長久保存。微量製備出的 hFSHA-yT&A 和 pET30a 兩質體以 BamHⅠ限制酶進行切割；而 hFSHB-yT&A 和 pACYCDuet-1 兩質體則以 HindⅢ限制酶進行切割反應，之後成功利用T4 DNA ligase 將 “insert DNA” 和 “表現載體” 接合並轉形到大腸桿菌中。經限制酶和 DNA 定序之鑑定後可以確認 hFSHA-pET30a 轉形至 NovaBlue (DE3) ，而 hFSHB-pACYCDuet-1 轉形至 NovaBlue (DE3) 、BL21 (DE3) 、 BL21 trxB (DE3) 和 Origami (DE3) 四株菌體內。

3. 在其個別表現方面，SDS-PAGE 染色結果觀察到去醣基 hFSH α-次單元體的蛋白質帶存在於約 20 kDa 之位置，且無論利用抗 6x

His 之一級抗體或者抗 FSH 之一級抗體皆顯示著與 SDS-PAGE 同樣之蛋白質帶，故已成功表現出去醣基 hFSH α-次單元體。但在 NovaBlue (DE3) 、BL21 (DE3) 、BL21 trxB (DE3) 和 Origami (DE3) 四株菌中皆無法明顯從 SDS-PAGE 染色結果觀察到去醣基 hFSH β-次單元體存在，進一步利用抗 6x His 作為一級抗體的西方墨點法之結果卻能明顯看出β-次單元體的微量存在，且位於 18 kDa 之位置。

4. α、β 兩個次單元體分別利用 Ni-NTA 方式純化，通過 Ni-NTA 管柱的兩次單元體沖提部分進行 SDS-PAGE 和西方墨點法鑑定之。

雖然純化完的 SDS-PAGE 結果依舊混雜著其他蛋白質帶，但在西方墨點法呈色後卻可觀察到單一個蛋白質帶，即表示確實有純化到我們所要的α、β 兩次單元體，選擇純度 80 %以上的沖提部分再一次純化並進行透析再折疊。

5. 再折疊完 BCA 定量 α、β 兩個次單元體後，可知由 500 ml LB 培養液可獲得約2.5 mg 的純 α-次單元體，同理在 1000 ml LB 培養液中約只能純化出0.5 mg 較純的 β-次單元體，兩者表現率相差了十倍。接著便進行次單元體的雙體化反應，根據定量的結果調整相同體積下兩者濃度相等，一比一混和的 α、β-次單元體能藉由在相同環境下彼此的碰撞吸附，進行非共價鍵結合。經由還原態與非還原態的 SDS-PAGE 染色和西方墨點法鑑定後可判定去醣基的 hFSH 兩次單元體順利完成雙體化反應，也就是成功重組出去醣基 hFSH 變異體。

6. 另關於原核表現系統製造出的醣蛋白激素可由 hCG 之大腸桿菌表現證實表現出來的激素並無接合醣基支鏈 [50, 51] ，且分析圖 26 至圖28 之西方墨點法和 SDS-PAGE 結果的蛋白質大小可知其分子量單純為多胜肽部分，並無包含醣基支鏈，因此本論文之 hFSH 變異體確為無醣基形式存在。

7. MTT 細胞增生分析活性影響和 TMB 反應分析親和力強度之結果，可與先前研究加以比較，歸納出以下幾點結論。一、訊息傳遞活性方面，利用直接點突變之方式加以改變 α-次單元體 Asn52 成為Gln [31] ，如此除去 Asn52 之醣基部分，發現嚴重降低 FSH 74

%訊息傳遞活性。至於突變掉 α-次單元體 Asn52 和 Asn78 兩個位置亦只剩下40 %的活性 [31] 。此外，雙突變 FSH β-次單元體 Asn7 和 Asn24 亦會減少 30 %的訊息傳遞活性 [31]，另突變 βAsn7、

βAsn24、βAsn7,24 則為野生型之訊息傳遞活性的 48 %、41 %和 38 % [30] 。二、受體鍵結親和力方面，關於突變 αAsn52 之後的受體鍵結親和力卻是些微增加 [32] 或甚至增強 2~3 倍，但缺少 αAsn78、βAsn7 或 βAsn24 之醣基對於鍵結強度卻無影響 [30, 34] 。不過另一研究顯示單獨缺少 αAsn78 醣基的鍵結強度卻增強 72 % ，但移除其它醣基對於鍵結強度則無顯著影響 [31] 。三、

本論文活性分析結果顯示 NG-hFSH 降低 30 %活性，若野生型額外添加 NG-HFSH 則可降低 7 %活性，此結果與直接突變 αAsn52 或兩個以上醣基部分相比，降低能力並非如此強烈。另外 hFSH α、hFSHβ、「hFSHα+ hFSHβ」、NG-hFSH 之受體鍵結親和力則分別增強48 %、34 %、83 % 和 52 %。以上數據顯示本論文發現去除醣基的重組hFSH 能對 SKOV-3 產生降低增生之影響，並壓

抑野生型hFSH 對其增生的活性效果，更間接證實具有抑制卵巢癌細胞的訊息傳遞能力。並且相對於野生型的受體鍵結，去除醣基的hFSH 兩次單元體或雙體皆可提升與受體的親和力。相較於點突變去醣基方式，直接以菌體重組出NG-hFSH 有著大量快速製造之優點，另只降低30 %活性對於影響正常細胞的副作用來說或許反而有益，不至於如同單獨去除 αAsn52 醣基而嚴重降低 74 %導致阻斷正常生理活性，但NG-hFSH 實際對於正常細胞的活性還得進一步探討。

8. 綜合來說，本論文成功利用大腸桿菌表現系統重組出去醣基 hFSH 變異體，發展出原核表現系統製造去醣基人類濾泡刺激素之技術，未來如何克服 β-次單元體表現微量之問題將是研究重點所在，以合成出最適產量的去醣基hFSH 變異體。之後的研究將可著重於正常卵巢顆粒細胞和其他卵巢癌細胞株的生物活性和受體親和力分析，以深入探討去醣基hFSH 變異體與野生型 hFSH 對於正常顆粒細胞的受體競爭關係。另其對於男性生殖生理之關係亦是相當有趣的課題，去醣基 hFSH 變異體對於男性正常 Sertoli 細胞和前列腺癌細胞能產生何種影響皆可深入研究。未來期望去醣基濾泡刺激素能發展為卵巢癌抑制劑或扮演壓抑卵巢癌細胞發育之重要角色。

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圖13. 質體 hFSHA-yT&A、hFSHB-yT&A 切割反應之電泳結果。

Lane 1：hFSHA-yT&A 之 BamHⅠ切割產物。

pACYCDuert-1 4008 bp

hFSHB, 375 bp

hFSHB-pACYCDuet-1 4383 bp

(A) (B)

(C) (D)

圖 14. 分別以限制酶鑑定轉形進表現宿主 Nova blue (DE3) 之兩質體：hFSHA-pET30a 和 hFSHB-pACYCDuet-1。圖 A：以 BamHⅠ 切割hFSHA-pET30a 質體。圖 B：以 EcoRV 切割 hFSHA-pET30a

在文檔中去醣基人類濾泡刺激素之菌體表現及其對於卵巢癌細胞增生影響之研究 (頁 83-114)

第六章 參考文獻

(A) (B)

(C) (D)

第六章參考文獻