類胰島素生長因子第二型飼料酵母之生產

Abstract

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.