人乙型肝炎病毒聚合酶在酿酒酵母中的表达

　　【摘要】 目的 尝试在酵母中表达人乙型肝炎聚合酶蛋白(HBV-Pol)，并用镍基颗粒队蛋白粗提物进行部分纯化。方法 将全长HBV-Pol基因用PCR方法扩增并连接到在酿酒酵母高效表达的质粒中。转化株于30℃培养3 d后进行诱导表达16 h，4℃下收集蛋白粗提物并用镍基颗粒进行提纯。用免疫印迹技术检测表达结果。结果 重组的HBV-Pol存在于该型酵母的蛋白粗提物中，该蛋白产物可被Nickel-particle提取。结论 本研究为实现HBV-Pol在S.c.中的高水平表达提供了可靠依据，也便于HBV-Pol的生化特性研究以及蛋白纯化和抗体制备的进一步开展。

　　【关键词】 乙型肝炎病毒; 聚合酶;酿酒酵母

　　Expression of Human Hepatitis B Virus Polymerase in Saccharomyces CerevisiaeYU Yang, HONG Seong Tchool

　　(Department of Microbiology and Research Center for Industrial Development of Biofood Materials,

　　Chonbuk National University, Medical School, Jeonju 561-756, Republic of Korea)Abstract:Objective Attempt to express HBV-Pol in yeast expression system and then partially purify the rough extract with nickel-based particle. Methods Full length HBV-Pol gene was amplified and was introduced into a high effective expression plasmid of Saccharomyces cerevisiae(S.c.).The transformant was cultured at 30℃ for 3 days, and then induction was carried out for another 16 hours at 30℃, and then induction was carried out for another 16 hours at 30℃. Cell lysis and purification were preformed at 4℃. Target protein was detected in harvested rough extract with western blot. Results The recombinant HBV-Pol was present in rough extract of yeast and could be purified with nickel particle. Conclusions Results of this study prove that HBV-Pol can be expressed in S.c. yeast and might facilitate the biochemical study, protein purification and antibody preparation of HBV-Pol.

　　Key words： hepatitis B virus; polymerase; Saccharomyces cerevisiae

　　乙型肝炎病毒(Hepatitis B virus，HBV)感染是全球性公众健康问题。目前世界上约有3.5 亿乙肝病毒携带者，其中很大比例将会发展成严重的肝脏疾病例如肝硬化、肝细胞癌及其它综合征[1]。

　　乙肝病毒染色体仅有3 200个左右的碱基对，属于hepadnaviridae 家族，有极强的肝细胞靶向性[2]。在乙肝病毒生命周期很重要的一步就是它利用自身编码的逆转录酶进行将其染色体复制。乙肝病毒聚合酶蛋白(HBV polymerase, HBV-Pol)是一种多功能酶，具有蛋白始动活性[3-5]、DNA聚合酶活性、反转录酶活性[6]及RNA酶H活性[7]。它包括了三个结构域，从氨基末端到羰基末端依次是末端蛋白(terminal protein，TP)，聚合酶/逆转录酶(RT)及RNA酶H(RH)。其中末端蛋白与其它两个域间由一空白序列隔开。

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