Production of insulin-like growth factor II feed yeast

本研究是以飼料酵母菌Candida utilis BCRC 21645作為表現外源蛋白質重組吳郭魚類胰島素生長因子之宿主，載體pGAPZB屬於Pichia pastoris表現系統，目前商業上並無法取得屬於C. utilis表現系統之載體，P. pastoris與C. utilis之菌種親源性較為接近，因此將IGF2基因插入載體pGAPZB中，命名為pGAPZB-IGF2，作為本研究之表現質體。 由前人所構築之C. utilis轉形株無法正常表現吳郭魚重組蛋白質IGF2，經由PCR檢測，發現其structure gene部分即IGF2基因仍正常存在於染色體上，但在GAP promoter的前半段序列可能發生遺失或是序列變異，因此重新構築菌種。 重新構築之C. utilis轉形株，其表現載體pGAPZB-IGF2雖然正確嵌入異源宿主C. utilis之染色體，但是轉形效率明顯相較於已知研究為低，目前轉形菌株中並不能有效率的篩選出表現外源蛋白質IGF2的菌種，結論是利用異源的載體來構築表現菌種，由於該載體上不帶有同源的啟動子或是其他相同的序列，造成轉形效率偏低，同時無法有效的表現目標的外源蛋白質，並不是一個理想的表現系統；未來即使經過長時間的篩選，可能篩選到表現量較理想之轉形株，但是其嵌入基因之穩定性不佳，將不利於研究之持續進行，故此表現質體之改良為未來研究之主要發展方向。Feed yeast Candida utilis BCRC 21645 was used as a host for tilapia insulin-like growth factor 2 expression. An expression plasmid pGAPZB-IGF2 was constructed by inserting igf2 gene into a commercial vector pGAPZB belonging to Pichia pastoris expression system, because a commercial vector for C. utilis was not available and P. pastoris is more relative to C. utilis than Saccharomyces cerevisiae. The expression plasmid pGAPZB-IGF2 was transferred into C. utilis for the expression of tilapia igf2 gene. The recombinant IGF2 was produced as soluble protein, and its content was as high as 6.7% of the soluble protein (Hu, 2004). After six months of storage, IGF2 was not produced in C. utilis/pGAPZB-IGF2 . After PCR analysis, we found that a fragmaent of GAP promoter was lost in chromosome DNA of transformants. The expression plasmid pGAPZB-IGF2 was prepared from Escherichia coli BL21(DE3) and linearized by Bgl II. Linearized pGAPZB-IGF2 was used to transform C. utilis by electroporation . The maximum transformation efficiency obtained was 16 transformants per μg of linearized pGAPZB-IGF2 at a field strength of 7.5 kv cm-1, an internal resistance of 800 Ω and 10 μg plasmid DNA. Although the DNA sequencing of PCR analysis demonstrated that linearized pGAPZB-IGF2 was successfully insert into chromosome DNA of C. utilis, igf2 gene was not expressed in transformants. There is no homologous sequence in pGAPZB-IGF2 result in the low transformation efficiency. We didn’t comfirm if the GAP promoter of P. pastoris work in C. utilis. The difference of GAP promoter between C. utilis and P. pastoris or the codon usage of structure gene in host may be concerned to explain why no igf2 gene expression in transformants. The transformants constructed before may be an exception of unstable insertion of igf2 gene, the expression of igf2 gene was not initialized by GAP promoter in expression plasmid pGAPZB-IGF2. We suggested to construct an expression plasmid with the homologous DNA sequence of C. utilis such as GAP promoter, rDNA gene or URA3 gene for the improvement of transformation efficiency and the expression of heterologous gene.目錄 中文摘要…………………………………………….…………….……….…..……… I 英文摘要…………………………………………….………………….….…..……….II 目錄…………………………………………….…………………….…..…………….IV 表次…………………………………………….…………………….…..…………..VIII 圖次………………………………………….…………………….…..…………….....IX 縮寫表…………………………………………….…………………….…..…………..X 第一章 前言…………………………………………...….…………………….…..…1 一、 類胰島素生長因子.……………………………………………...……..….…1 二、 酵母菌異源性重組蛋白質之表現.………………………….……….………5 1. 酵母菌表現系統……………...……………………….………………...…5 2. 質體之穩定性……………...………………………….……………...……6 3. 後轉譯修飾作用……………...……………………….………………...…7 4. Candida utilis表現系統………………………………..…..………………8 5. 酵母菌之轉形……………...…………………………….…………...……9 三、 研究動機………..……………...……………....……………...…………...…10 四、 實驗架構………..……………...…………..………..………...…………...…12 第二章 材料與方法…………………...……………….………………………...…..13 一、 研究材料…..……..……………...…………………………...…………...…13 1. 菌株與質體…..……………...……………………………..…………..…13 2. 培養基…..……………...………………………………….……...………13 3. 藥品與試劑…..……………...……………………………..…………..…13 4. 儀器設備…..……………...…………………………….…………...……16 5. 緩衝液…..……………...…………………………………….…...………17 6. 質體抽取試劑…..……………...………………………….……………...18 7. DNA瓊脂膠體電泳試劑…..……………...…………….………………...18 8. 蛋白質膠體電泳試劑…..……………...…………………….…………...18 9. 蛋白質分子量標準品…..……………...…………………….…...………20 10. 常用之Tricine SDS-PAGE膠體溶液…...………..…………………….…20 二、 實驗方法…..……..……………...…………………………………...…...…21 1. 前人所構築C. utilis轉形株之IGF2表現…...…..………………...………21 2. 酵母菌染色體DNA之抽取….…………...…………………………….…21 3. 嵌入基因之PCR確認………...…………………………….………….….22 4. DNA瓊脂膠體電泳………...……………………………….………...…..22 5. 質體pGAPZB-IGF2之大量抽取………...……………………………….23 6. Bgl II 限制酶切及RNase處理………………………………………...…25 7. C. utilis細胞之轉形前處理…………………………….…………...…….25 8. C. utilis之電衝轉形法最適條件探討……………….……………………25 9. C. utilis轉形株復原期……………………...……………...…...…………26 10. C. utilis轉形株之篩選……………………...……………….………...…..26 11. 酵母菌破菌條件之探討…………………………………..………….…..26 12. 酵母菌轉形株之菌種保存………………………...……...……………...27 13. 酵母菌轉形株之培養………...…………………………...…...…………27 14. 酵母菌之破菌…………………………...………………...…...…………27 15. 酵母菌IGF2之表現…………………………………….…………………28 1) 培養時間對IGF2表現量之探討…………………………………....…28 2) 碳源對IGF2表現量之探討………………………………………....…28 3) 氮源對IGF2表現量之影響……………………………………..……..28 4) 糖蜜之蔗糖當量濃度對IGF2表現量之影響………...….…..….…….29 16. 酵母菌IGF2之純化……………...…………...…....……………….……..29 17. 分析方法…………………………………………….……..……….….…30 1) 菌體濃度測定..……………...………………………………….…...…30 2) 蛋白質濃度測定..……………...…………………………….………...30 3) 蛋白質色帶之強度分析..……………...…………………….………...30 4) 總糖濃度之測定..……………...………………………….…………...30 5) 葡萄糖濃度之測定……………...……………………………………..31 第三章 結果與討論….……..………………………………………...……………...32 一、 前人研究結果….……..……………...……………………………………...32 1. C. utilis轉形株之IGF2表現…………………………...……………...…..32 2. C. utilis轉形株之PCR檢測…………………………………...…………..32 二、 IGF2飼料酵母菌之重新構築….....…….……..………………….…………39 1. 質體pGAPZB-IGF2之大量製備……………………...……………...…..39 2. 酵母菌之電衝轉形法最適條件探討………………………...…………..39 1) 電場強度對轉形效率之影響.…………………………………..…...…39 2) 內電阻對轉形效率之影響……………….………………….….……...39 3) DNA量對轉形效率之影響………...…………………….…..…….…..43 4) 不同生長階段之酵母菌細胞對轉形效率之影響……………………..43 3. C. utilis轉形株之篩選………...…………………..………………………43 4. C. utilis轉形株之PCR鑑定…………………..…………………...………43 5. 最適破菌條件之探討………………………………...……………..……49 1) Lyticase活性對破菌效率之影響………………………………...…….49 2) Lyticase反應時間對破菌效率之影響……………………………...….49 3) 玻璃珠量對破菌效率之影響…….…………………………………….51 4) 研磨時間對破菌效果之影響…….…………………………….………51 6. 酵母菌轉形株之培養………………………………………….………….51 7. 酵母菌轉形株之IGF2表現…………………………………………..……51 1) 培養時間對IGF2表現量之影響………………….………………....…51 2) 碳源對IGF2表現量之影響………………………………….……....…54 3) 氮源對IGF2表現量之影響………………………………...……..…....54 4) 糖蜜之蔗糖當量濃度對IGF2表現量之影響…………………….…....54 8. 酵母菌IGF2之純化……………...………………….…..…....……….…..54 第四章 結論…..……..……………...…………………………………………...…...61 第五章 參考文獻…..……..……………...……………………………………......…6