海参溶菌酶基因克隆及在毕赤酵母中的表达与纯化

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。

Cloning and expression of lysozyme from Stichopus japonicus in Pichia pastoris

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 fermentation,the filtrate was collected and dealt with ammonium sulfate,and the preliminary purification of lysozyme was obtained by dialysis.The further purification of lysozyme was done through CM52-cellulose cation exchange column,and the electrophoretic pure lysozyme was obtained.The results show that the lysozyme of Stichopus japonicus can be expressed in Pichia pastoris expression system.