| 海参溶菌酶基因克隆及在毕赤酵母中的表达与纯化 |
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| 引用本文： | 谷跃峰,丛丽娜,骆宁.海参溶菌酶基因克隆及在毕赤酵母中的表达与纯化[J].大连轻工业学院学报,2010(5). |
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| 作者姓名： | 谷跃峰 丛丽娜 骆宁 |
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| 作者单位： | 大连工业大学生物与食品工程学院; |
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| 基金项目： | 国家自然科学基金资助项目(31072224); 辽宁省教育厅创新团队项目(LT2010012); 大连市科技计划项目(2009J31SC034) |
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| 摘 要： | 研究构建并筛选了具有抑菌活性的重组海参溶菌酶(Stichopus japonicuslysozyme,SJL)的毕赤酵母工程菌。从海参肠组织中提取总RNA,根据已经测得的SJL的基因序列(GenBank accession No.EF036468)设计引物,以总RNA为模板经RT-PCR获得目的基因,并将该基因与表达载体pPIC9K连接,构建重组质粒pPIC9K-SJL,再转化至毕赤酵母GS115中。利用MD、MM和含抗生素G418的YPD平板培养基筛选出表型为His+Muts的高拷贝阳性菌株。挑选具有抑菌活性的菌株进行甲醇诱导表达液体发酵,其上清液经硫酸铵沉淀、透析后得到溶菌酶粗品,再经CM52-纤维素阳离子交换柱进一步纯化,得到电泳纯的溶菌酶。结果显示,共筛选到7株有抑菌活性的重组酵母菌株,诱导72 h时表达量最高。SJL在毕赤酵母中得到成功表达。
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| 关 键 词： | 海参溶菌酶 毕赤酵母 表达 纯化 |
Cloning and expression of lysozyme from Stichopus japonicus in Pichia pastoris |
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| GU Yue-feng,CONG Li-na,LUO NingSchool of Biological & Food Engineering,Dalian Polytechnic University,Dalian ,China.Cloning and expression of lysozyme from Stichopus japonicus in Pichia pastoris[J].Journal of Dalian Institute of Light Industry,2010(5). |
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| Authors: | GU Yue-feng CONG Li-na LUO NingSchool of Biological & Food Engineering Dalian Polytechnic University Dalian China |
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| Affiliation: | GU Yue-feng,CONG Li-na,LUO NingSchool of Biological & Food Engineering,Dalian Polytechnic University,Dalian 116034,China |
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| Abstract: | The expression of lysozyme from sea cucumber Stichopus japonicus in Pichia pastoris was studied.The target gene was obtained by RT-PCR from the total RNA of Stichopus japonicus,cloned into the expression vector pPIC9K,and transformed into Pichia pastoris GS115.The phenotype His+Muts for high-copy positive strains were screened out by MD,MM and YPD containing antibiotics G418.As a result,seven genetically engineered bacteria were screened by methanol-induction which could express lysozyme activity.After the ... |
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| Keywords: | Stichopus japonicus lysozyme Pichia pastoris expression purification |
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