【作者】 张玲；

【导师】 王武；

【作者基本信息】 江南大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 胆固醇氧化酶(EC1.1.3.6 COD)在食品加工、医疗检测、生物抗虫等方面的作用日益受到人们的重视,并且显示出巨大的应用潜力。本文主要研究短杆菌Brevibacterium sp. DGCDC-82胆固醇氧化酶在大肠杆菌和在巴斯德毕赤酵母中的表达。用PCR方法从Brevibacterium sp. DGCDC-82染色体DNA中扩增出含信号肽序列的胆固醇氧化酶结构基因,插入大肠杆菌表达载体pET28a(+)中,构建成重组质粒pET28a-COD(s+)。以pET28a-COD(s+)为底物,扩增出不含信号肽的胆固醇氧化酶结构基因,构建成重组质粒pET28a-COD(s-)。两种重组载体转化大肠杆菌BL21(DE3)均获得活性表达,不含信号肽基因的转化子酶活较高。在转化子中,两种结构基因与pET28a(+)携带的His-tag融合表达,经IPTG诱导后均表达出分子量约为55kD的蛋白质,目的蛋白大都为没有活性的包涵体,占细胞总蛋白的50%以上。将不含信号肽的胆固醇氧化酶基因克隆至含trp/lac双启动子的表达载体pTrc99a中,并在大肠杆菌JM109中进行了IPTG诱导表达,并分别考察了诱导温度、时间、IPTG浓度等因素对重组菌表达的胆固醇氧化酶的影响。在优化条件下,该胆固醇氧化酶的酶活可以达到700 U/L。酶学特性分析表明其反应的最适pH为7.5,最适温度为40℃。将不含信号肽的胆固醇氧化酶结构基因插入巴斯德毕赤酵母分泌型表达载体pPIC9K的EcoR I和Not I位点,构建成pPIC9K-COD,重组质粒以Sac I线性化后电转化巴斯德毕赤酵母GS115。甲醇诱导条件下,重组胆固醇氧化酶仅有少量存在于细胞破碎液的上清液中,并没有在毕赤酵母中获得分泌表达。更多还原

【Abstract】 The significant functions of cholesterol oxidase (EC 1.1.3.6, COD) in food processing, medical assay and biological pest-resistance were recognized gradually and showed potentially use. This research is mainly focused on expression of Cholesterol oxidase gene from Brevibacterium sp. DGCDC-82 in Escherichia coli and expression of the gene in eukaryotes.The structure gene of Brevibacterium sp. DGCDC-82 Cholesterol oxidase with signal sequence was amplified from genomic DNA of Brevibacterium sp. DGCDC-82 by PCR technique. The recombinant vector, pET28a-COD(s+), was constructed by inserting the amplified fragment(about 1.5kb)into the expression vector pET28a(+). The DNA fragment encoding mature peptide of COD was amplified from pET28a-COD(s+) and was then inserted into pET28a(+) to construct pET28a-COD(s-).Both recombinant plasmids were transformed into E.coli BL21(DE3) respectively. Promoted and controlled by T7lac promoter, the fusion protein containing His-tag and the peptide of COD with or without signal peptide could be expressed within the cell of recombinant E.coli. The active recombinant enzyme was tested after the two recombinants induced by IPTG.. The expression products of full gene and mature enzyme gene were analyzed by SDS-PAGE indicating that about 55kD protein was obtained, which accounted for about 50% of the cell total protein.The mature enzyme gene of COD from Brevibacterium sp. DGCDC-82 was inserted into a prokaryotic expression vector pTrc99a. The vector was controlled by trp/lac promoter. A recombinant plasmid pTrc99a-COD was obtained and then transformed into the expression host JM109. The influences of induction conditions such as IPTG concentration, the induction temperature and time of induction on the expression of the recombinant protein were investigated. Under optimal condition, the enzyme activity reached 700U/L.The enzymatic analysis of the cholesterol oxidase revealed that its optimal pH and temperature was 7.5 and 40℃, respectively.The structure gene of COD without signal sequence was into the site EcoR I、Not I of pPIC9K, a secreting expression vector of Pichia pastoris. The plasmid pPIC9K-COD was linear with Sal I and transform P.pastoris GS115. Inducting with methanol, The active COD enzyme expressed from the recombinant P.pastoris was found only exsisting in the supernatant of the crude cell extract, but not secreted into culture medium.更多还原