臺灣博碩士論文加值系統

樟芝具有我國特殊地域意義，是民間流傳已久的珍貴菇類。由於樟芝來源取得不易，因此可以利用蛋白質表現技術來表現樟芝中的有效成分，並解決市場需求。本實驗室已自樟芝菌絲體中純化出免疫調節蛋白ACA1，同時並發現此蛋白對小鼠具免疫調節活性，已進一步選殖出其核酸序列。本實驗進一步將此序列進行異體表現，以求大量生產重組ACA1蛋白。以GST融合蛋白表現系統表現ACA1可得一40 KDa大小的融合蛋白，但在切解融合蛋白GST後，重組ACA1的產量偏低。以pET32a(+) 表現系統成功地表現並純化出重組融合蛋白HIS-ACA1及無融合部分之純rACA1蛋白，每公升發酵液之產量各達10 mg與4 mg。帶有融合標記蛋白(HIS tag)的融合ACA1分子量為20 KDa，而純ACA1蛋白分子量為14 KDa。rACA1的等電點約為5.0。體外試驗結果顯示，重組ACA1蛋白可刺激小鼠巨噬細胞RAW264.7產生一氧化氮及TNF-α，同時可刺激小鼠脾細胞增生，具有劑量效應，且重組ACA1在熱處理、酸處理及乾燥處理後仍保有有高度活性。另以重組ACA1製備的四株單株抗體可專一地辨識到ACA1蛋白。除了建立兩個大腸桿菌表現系統外，wildtype aca1基因及yeast-favored aca1基因也經構築至持續性的表現載體pYEX-S1上，並在酵母菌中進行表現。結果顯示，在C端加上六個histidine氨基酸作為His tag的aca1基因表現狀況都比N端His tag的表現狀況來得高，但兩種aca1基因在酵母菌中的表現量並無顯著不同。綜合上述結果顯示，本實驗已建立了重組ACA1之原核與真核表現系統，所生產出的重組ACA1也證實與天然ACA1有相似的免疫調節效應。另外由於重組ACA1在仿工業加工處理下仍可保持良好活性，此可表現樟芝蛋白ACA1的食品級酵母菌在飼料業或食品業上具有高度應用潛力。

Antrodia cinnamomea is a well-known traditional Taiwanese herbal medicine which attracted a lot of attention these years due to its curative effects towards various diseases. Although sources of A. cinnamomea are highly limited in nature, overexpressing the bioactive components found in A. cinnamomea might be the answer to the markets’ demands. Immunomodulatory protein ACA1 has been previously isolated from the mycelia of A. cinnamomea by our laboratory. The bioactivities of ACA1 toward murine immune system have been verified and the gene encoding ACA1 has been cloned. To obtain the protein in large quantity, recombinant ACA1 was expressed in both prokaryotic and eukaryotic systems. Recombinant ACA1 expressed in GST fusion system yielded a soluble fusion protein GST-ACA1, which exhibited a molecular weight near 40 KDa. However, the pure rACA1 obtained after GST tag removal was technically too difficult to be collected. rACA1 expressed by pET-32a(+) expression vector obtained a satisfying quantity of both fusion HIS-ACA1 and pure rACA1, which were approximately 10 mg and 4 mg per liter culture respectively. Fusion HIS-ACA1 had a molecular weight of 20 KDa, and rACA1 had a molecular weight of 14 KDa. The isoelectric point of rACA1 was 5.0. In vitro studies revealed that rACA1 could effectively stimulate murine macrophage RAW264.7 to produce nitric oxide and TNF-α at low concentrations, and the responses increased in a dose-dependent manner. rACA1 could also stimulate the proliferation of murine splenocytes. The stability of rACA1 under various environmental stresses was demonstrated, and rACA1 was found to be stable under heat, acid, and dehydration treatments. Four monoclonal antibodies against rACA1 have also been established, and specific recognitions toward rACA1 were verified. In addition to being expressed in two bacterial systems above, recombinant ACA1 was also expressed in the eukaryotic, yeast Sacchromoyces cerevesiae expression system. Both wildtype aca1 gene and yeast-favored aca1 gene inserted in the constitutive expression vector pYEX-S1 had similar expression levels; the expression levels of C-terminal His-tagged ACA1s were found to be significantly higher than that of N-terminal His tagged ACA1s. In conclusion, recombinant ACA1 has been successfully expressed in both E.coli and yeast expression systems, and the purified rACA1 was proven to have immunomodulatory effects similar to that of natural ACA1. With good stability under various simulated conditions mimicking the industrial processing procedures, recombinant ACA1 expressed in the food-grade expression host S. cerevisiae could therefore possibly be utilized directly in animal feed or food in the near future.

Content
Figures and Tables V
Abstract 1
摘要 3
Chapter 1 Literature Review 4
1.1 INTRODUCTION OF ANTRODIA CINNAMOMEA, “NIU-CHANG-CHIH” 4
1.1.1 Classification, Nomenclature, and the Morphology of Antrodia cinnamomea 4
1.1.2 Chemical composition, compound identification, and cultivation studies of Antrodia cinnamomea 6
1.1.3 Bioactivities of Antrodia cinnamomea against various diseases 9
1.1.4 Immunomodulatory protein ACAs from Antrodia cinnamomea 17
1.2 PROTEIN EXPRESSION 20
1.2.1 Prokaryotic expression system 20
1.2.2 Eukaryotic expression system 32
1.3 RESEARCH OBJECTIVE 37
Chapter 2 Materials and Methods 39
2.1 CHEMICALS, REAGENTS, AND EQUIPMENTS 39
2.2 GENERAL PROTEIN ANALYSIS METHODS 45
2.2.1 Denaturing discontinuous polyacrylamide gel electrophoresis (SDS-PAGE) 45
2.2.2 Western Blotting 47
2.2.3 Protein concentration estimation 49
2.2.4 Polymerase chain reaction (PCR) 50
2.3 LARGE-SCALE EXPRESSION AND PURIFICATION OF RECOMBINANT ACA1 IN E. COLI 51
2.3.1 Large-scale expression of GST-ACA1 fusion protein 51
2.3.2 Large-scale expression of HIS-ACA1 fusion protein 55
2.4 MONOCLONAL ANTIBODY PREPARATION 60
2.5 BIOACTIVITIES OF RACA1 AND FUSION RACA1 UNDER DIFFERENT CONDITIONS 69
2.5.1 Sample preparation 69
2.5.2 Isoelectric Focusing (IEF) 70
2.5.3 LAL test 71
2.5.4 rACA1 stimulates macrophage RAW264.7 to produce NO and TNF-α 72
2.5.5 rACA1 simulates murine spleen cells proliferation measured by BrdU labeling assay 75
2.5.6 Cell viability and proliferation measured by MTT assay 77
2.5.7 Statistical analysis 78
2.6 EXPRESSION OF RECOMBINANT ACA1 IN YEAST 79
2.6.1 Modification of ACA1 codon according to yeast codon usage table 82
2.6.2 Plasmid constructs for yeast expression 83
2.6.3 Transformation of aca1-pYEX serial expression vectors in yeast 85
2.6.4 Yeast colony PCR 86
2.6.5 Storage of yeast strains 87
2.6.6 Expression of rACA1 in yeast 87
2.6.7 Detection of rACA1 in yeast 87
Chapter 3 Results 88
3.1 EXPRESSION OF RACA1 IN BACTERIAL SYSTEMS 88
3.1.1 Expression of rACA1 by GST fusion system 88
3.1.2 Expression of rACA1 by HIS fusion system 92
3.2 ESTABLISHMENT OF RACA1 MONOCLONAL ANTIBODIES 94
3.2.1 Immunization and cell fusion 94
3.2.2 Screening for positive hybridoma cell lines against recombinant ACA1 94
3.2.3 Ascites production in mice and purification of monoclonal antibodies C1, C2, C3, and C4 95
3.2.4 Recognition of mAb C1, C2, C3, and C4 against rACA1 and fusion HIS-ACA1 95
3.3 STUDIES ON THE BIOACTIVITIES AND STABILITY OF RACA1 UNDER VARIOUS CONDITIONS 96
3.3.1 Isoelectric point of rACA1 96
3.3.2 Endotoxin concentration estimation (LAL test) 96
3.3.3 Bioactivities and stability of rACA1 and fusion HIS-ACA1 toward murine macrophage RAW264.7 97
3.3.4 Bioactivities and stability of rACA1 and fusion HIS-ACA1 toward murine splenocytes 98
3.3.5 Influence of refolding methods on the activities of HIS-ACA1 fusion protein toward murine splenocytes 99
3.4 EXPRESSION OF RECOMBINANT ACA1 IN YEAST SYSTEM 101
3.4.1 rACA1 codon replacement according to yeast codon usage table 101
3.4.2 Construction of a secretion leader sequence-deleted expression vector pYEX 101
3.4.3 Construction of yfaca-pYEX expression vector 102
3.4.4 Construction of wtaca-pYEXplus expression vector by overlapping PCR technique 103
3.4.5 Growth rate and morphology of yeast strains DY150 and DBY747 103
3.4.6 Expression of yeast expression plasmids wtaca-pYEXplus and yfaca-pYEX 104
3.4.7 Construction of His-tagged ACA1 expression vectors 104
3.4.8 Expression and detection of His tag ACAs in yeast strains DY150 and DBY747 105
Chapter 4 Discussion 107
4.1 DISCUSSION 107
Chapter 5 Conclusion 114
5.1 CONCLUSION 114
References 115

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