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密码子优化烟曲霉壳聚糖内切酶基因在毕赤酵母中的高效表达
引用本文:裘梁,杨萍,游清徽,董得刚,乔贝贝,王曼莹.密码子优化烟曲霉壳聚糖内切酶基因在毕赤酵母中的高效表达[J].安徽农业科学,2012,40(26):12769-12771.
作者姓名:裘梁  杨萍  游清徽  董得刚  乔贝贝  王曼莹
作者单位:江西中医学院基础医学院生物技术研发中心,江西南昌,330096;江西师范大学生命科学学院,江西南昌,330022
基金项目:江西省教育厅青年科学基金(GJJ11195)
摘    要:目的]烟曲霉(Aspergillus fumigatus)壳聚糖内切酶基因(Chitosanase,EC.3.2.1.132)(csn)的高效表达研究。方法]根据毕赤酵母密码子的偏爱性对壳聚糖内切酶的基因进行了优化,利用连续延伸PCR方法扩增全长csn基因,构建重组质粒pPIC9k-csn,将重组质粒pPIC9k-csn线性化后转化毕赤酵母GS115,分析重组蛋白的表达,测定其酶活性,并对其酶解产物成分进行分析。结果]实现了密码子优化后的壳聚糖内切酶的高效表达,重组菌株比野生菌株所产的壳聚糖内切酶活性提高了近20倍。结论]该研究成功实现了烟曲霉壳聚糖内切酶基因在毕赤酵母中的高效表达,为工业化大规模生产壳聚糖酶奠定了基础。

关 键 词:密码子优化  壳聚糖内切酶  毕赤酵母  连续延伸PCR

High-level Expression of Codon-optimized Chitosanase Gene from Aspergillus fumigatus in Pichia pastoris
Institution:QIU Liang et al(Biotechnology Center,College of Basic Medical Sciences,Jiangxi University of Traditional Chinese Medicine,Nanchang,Jiangxi 330096)
Abstract:Objective] To study the high-level expression of chitosanase(EC.3.2.1.132)(csn) from Aspergillus fumigatus.Method] Chitosanase gene was optimized according to the codon bias of Pichia pastoris,and then the full-length csn gene was amplified through successive extension PCR,a recombinant plasmid pPIC9k-csn was constructed,linearized and transformed into Pichia pastoris GS115.The expression of recombinant protein was analyzed,its enzyme activity was determined,and the components of its enzymatic digestion products were analyzed.Result] The high-expression of codon-optimized chitosanase gene was realized,and the recombinant chitosanase activity reached was almost 20 times as much as the chitosanase from wild-type strain Aspergillums fumigates.Conclusion] The study successfully realizes the high-level expression of chitosanase gene from Aspergillums fumigates,laying a foundation for the industrialized production of chitosanase.
Keywords:Codon optimization  Chitosanase  Pichia pastoris  Successive extension PCR
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