提升C型肝炎核心蛋白抗原活性與新果寡糖之二階段醱酵生產

Abstract

本論文第一部分敘述將帶有C型肝炎病毒核心蛋白抗原片段的重組質體轉型入高突變性的大腸菌株，經過數代培養，藉以篩選具高抗原性的核心蛋白突變株，再量化此突變蛋白作為檢測抗原，期能提高現行偵測C型肝炎病毒抗體的零敏度。首先，將帶有核心抗原基因片段（1至123胺基酸）質體pET-Wt轉型至具高突變性的大腸桿菌XL1-Red，經10次繼代培養後抽取質體轉型至大腸桿菌BL21(DE3)，挑選616個單一菌落分別培養後以C型肝炎患者血清中多株抗體進行免疫分析後，篩選得到對抗體之反應活性約高2-3倍之5株菌；接著，分別抽取質體定序並轉型大腸桿菌BL21(DE3)來大量表現具突變胺基酸的重組蛋白，再將純化後的重組蛋白以免疫分析活性。結果顯示M3b突變株（W84S，P110S和V129L）比野生株抗原活性增加66%，結合力和親和力分別為0.96以及113M-1，而以M3b為檢測抗原可減少約三分之一的抗原使用量。 第二部分敘述以法夫酵母菌和麵包酵母菌利用蔗糖二階段醱酵生產新果寡糖（果糖基以26鍵結至蔗糖之fructofuranosyl殘基）之最佳化條件。第一階段以法夫酵母菌醱酵，最適之蔗糖濃度為450 g/L，其醱酵條件為23ºC、200 rpm、1 vvm及pH 7，使大部分的蔗糖藉由6G-fructofuranosidase（6G-FFase）的催化作用轉化為新果寡糖以及其他糖類，接著提升至pH 8及60ºC處理一小時後，進入第二階段：接種入麵包酵母菌，設定醱酵反應條件為30ºC、300 rpm、1 vvm及pH 7。兩階段醱酵64小時後，可取得約占乾重94.9%之高純度果寡糖糖漿，換算1公克蔗糖可得0.58公克之果寡糖。 總結以新果寡糖用於C型肝炎病情控制的展望。已有文獻指出C型肝炎治療輔以果寡糖以增進益生菌生長來降低肝炎性腦病變之臨床研究。未來可比較新果寡糖與1F-果寡糖在上述研究之成效。

Part 1 of the thesis describes the screening for the recombinant HCV core peptide with high antigenicity in the Escherichia coli with high mutation rate to enhance the current diagnostic sensitivity for anti-HCV antibody. First, the plasmid pET-Wt which containing domain I of HCV core (amino acid residues 1 to 123) was transformed into the E. coli mutator XL1-Red. After subculturing for 10 generations, the plasmids were isolated and transformed into E. coli BL21(DE3). A total of 616 single colonies were isolated, cultured and then subjected to immunoassay using the polyclononal antibody isolated from HCV patient’s serum. Five mutants with higher sensitivity compared to wild type strain were obtained. The mutation sites were identified by sequencing and the mutant proteins were overexpressed in E. coli BL21(DE3). The recombinant proteins were purified and their antigenicity determined by immunoassay. Compared to the wild type antigen, the mutant M3b antigen (W84S, P110S and V129L) exhibited an increase of 66% antigenicity with a binding capacity of 0.96 and affinity of 113M-1. The one-third decrease of the production demand suggests that M3b is a potential substitute for anti-HCV antibody detection. Part 2 describes a two-stage fermentation process for optimal production of the neo-fructooligosaccharides (neo-FOS) that contains fructosyl 2,6-linkage bound to the fructofuranosyl residue of sucrose. The first-stage fermentation was performed with Xanthophyllomyces dendrorhous at 23ºC, 200 rpm, 1 vvm and pH 7 in the culture containing optimal sucrose concentration 450 g/L, in which most sucrose was converted into neo-FOS and other saccharides by 6G-fructofuranosidase (6G-FFase). After the culture was switched to pH 8 and 60ºC for 1 h, the second-fermentation was started with Saccharomyces cerevisiae at 30ºC, 300 rpm, 1 vvm and pH 7. FOS syrup with a purity of up to 94.9% on a dry weight basis was obtained after 64 h of the two-stage fermentation. One gram sucrose yields 0.58 g of FOS. Perspectively, the potential use of neo-fructooligosaccharides for the treatment of hepatitis C virus infection is discussed. Using FOS as prebiotics to retard the progression of hepatic encephalopathy (HE) in hepatitis C virus infected patient had shown beneficial effect. Future investigations and comparison of the prebiotic effect of neo-FOS and 1F-FOS for the treatment of HE could be carried out.