茯苓表現序列標誌基因庫之建構分析與免疫調節蛋白基因之選殖

Abstract

許多的食、藥用蕈類含有多種固醇類、三類、核酸、多醣等藥用活性或潛力的成分，具有抗腫瘤與增進免疫力的功能。而近年的研究發現茯苓(Wolfiporia cocos)與樟芝(Taiwanofungus camphorate)的蛋白質亦具免疫調節活性，亦可歸為真菌免疫調節蛋白(fungal immunomodulatory proteins, FIPs)。茯苓是一種在中藥方劑裡應用範圍最廣的真菌。然而，其生物活性成份有關知識仍有限，且基因體研究尚在起步階段。因此，本研究欲自茯苓菌絲體中尋找可調節免疫系統功能的蛋白質，並建立一個茯苓表現序列標誌(express sequence tag, EST)基因庫。在分析液態培養茯苓菌絲體cDNA基因庫的過程中，發現一個與樟芝免疫調節蛋白(Aca1)胺基酸序列相似的基因(相似度達50%)。透過表現載體轉殖到大腸桿菌中，經誘導表現所純化出來的茯苓與樟芝重組蛋白質，皆能被茯苓蛋白單株抗體辨識。推測此蛋白質，是可能的茯苓免疫調節蛋白的成員之一。Most edible and medical fungi contain ingradents, such as steroids, triterpenes, nuclear acids, and polysaccharides. These compounds are commonly considered to have pharmaceutical activities or potentials, and therefore function in arresting the growth of tumer cells or stimulating immune systems. Few proteins have recently isolated from Wolfiporia cocos and Taiwanofungus camphorate with immunomodulatory activity, and are described as fungal immunomodulatory proteins (FIPs). Wolfiporia cocos is the most popular fungal ingredient in Chinese prescription, however, knowledge regarding to its bioactive compounds and genomic information is limited. Thus, I would like to explore FIP candidates, and establish a express sequence tag (EST) library from Wolfiporia cocos in this study. A potential FIP gene was characterized from 1-week-old Wolfiporia cocos cDNA library. This gene encodes a protein sharing 50% sequence similarity to Taiwanofungus camphorate FIP (Aca1), and is therefore named as WcFIP1. The recombinant WcFIP1and Aca1 was expressed in E. coli, and their identity were confirmed by western analysis. We concluded that the newly isolated WcFIP1 is one of the fungal immunomodulatory proteins in Wolfiporia cocos.