文章摘要
蒋霞云,袁襄南,魏福卫,周培根,邹曙明.总状毛霉甲壳素脱乙酰酶(CDA2)全长cDNA的克隆及原核表达[J].上海海洋大学学报,2011,20(1):44-49
总状毛霉甲壳素脱乙酰酶(CDA2)全长cDNA的克隆及原核表达
Complete cDNA cloning and prokaryotic expression of chitin deacetylase CDA2 from Mucor racemosus
  
DOI:
中文关键词: 甲壳素脱乙酰酶  甲壳素脱乙酰酶2基因  cDNA克隆  总状毛霉  原核表达
英文关键词: chitin deacetylase  cda2  cDNA cloning  Mucor racemosus  prokaryotic expression
基金项目:上海海洋大学博士启动基金项目(080219);上海海洋大学优秀青年教师基金(080236)
作者单位
蒋霞云 上海海洋大学 食品学院 
袁襄南 上海海洋大学 农业部水产种质资源与利用重点开放实验室 
魏福卫 上海海洋大学 食品学院 
周培根 上海海洋大学 食品学院 
邹曙明 上海海洋大学 农业部水产种质资源与利用重点开放实验室 
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中文摘要:
      采用快速扩增cDNA末端(RACE)克隆技术,通过设计甲壳素脱乙酰酶简并引物,从总状毛霉Mucor racemosus菌丝体中克隆了CDA2的基因(EF468349)及其全长cDNA(DQ678929)序列。与已获得的总状毛霉菌 cda1基因相比, cda2基因中不含内含子。总状毛霉 cda2全长cDNA为1 378 bp,包含23-bp 5’端非翻译区、1 254 bp开放阅读框和101-bp 3’端非翻译区,编码一条由418个氨基酸残基组成的多肽链,其N端包含一段21个氨基酸残基组成的信号肽,在150~272氨基酸残基区存在一个多糖脱乙酰酶保守结构域。通过构建pET28a-cda2表达载体和进行原核表达研究,结果显示:原核表达产生的重组蛋白CDA2的分子量约为46 ku,表达形式以包涵体为主,纯化获得的重组蛋白CDA2具有甲壳素脱乙酰酶催化活性。本研究为进一步研究总状毛霉CDA2的结构和功能奠定了基础。
英文摘要:
      The complete cDNA (GenBank accession number DQ678929) and its corresponding gene (EF468349) of chitin deacetylase CDA2 from Mucor racemosus mycelium had been cloned by rapid amplification cDNA end (RACE) with specific degenerate primers. In contrast to previously obtained gene cda1, cda2 contained no intron sequence. It consisted of 1 378 bp nucleotides, comprising 23-bp 5’ untranslated region (UTR), 1 254 bp open reading frame (ORF) and 101-bp 3’ UTR. The ORF encoded 418 amino acid (a.a.) residues including a 21 a.a. N-terminal signal peptide and a conserved polysaccharide deacetylase domain were located in an area of 150-272 a.a. residues. The results from subsequent construction of expressional vector pET28a-cda2 and prokaryotic expression revealed that molecular weight of recombinant protein CDA2 was about 46 ku and it was mainly found in inclusion bodies. The purified CDA2 showed chitin deacetylating activities. This work is necessary for further structural and functional exploration in chitin deacetylase CDA2 from M. racemosus.
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