黑曲霉外源表达系统对Dactylellina cionopaga基因PrD Ⅰ的表达

Expression of PrD Ⅰ of Dactylellina cionopaga in Aspergillus niger

目的:Dactylellina cionopaga是产生黏性分枝的捕食线虫真菌,进行其基因外源表达是鉴定该菌侵染相关因子的途径之一。方法:该文构建了含有组氨酸标签的黑曲霉表达载体pGT21M-HIS,对D.cionopaga中与侵染机制相关的丝氨酸蛋白酶基因PrD Ⅰ进行了外源表达。结果:RT-PCR鉴定结果显示,PrDⅠ在黑曲霉201中获得了转录。SDS-PAGE和酶活分析结果表明,经亲和层析纯化的重组蛋白分子量约为45kDa,在pH 5.0的条件下具有蛋白水解活性。纯酶液的蛋白浓度为30μg/ml。结论:构建的带有组氨酸标签的表达载体pGT21M-HIS能够用于基因在黑曲霉表达系统中的外源表达,并为目的蛋白的纯化和鉴定提供了极大便利。黑曲霉外源表达系统在表达丝状真菌基因方面相对其他表达系统具有优势。

Objective:Dactylellina cionopaga is an adhesive column-producing nematophagous fungus.To express the genes exogenously is one way of identifying the infection-related factors of the fungus.Method:In this study,a vector pGT21M-HIS with a His-tag was constructed for the heterologous expression in Aspergillus niger.The serine proteinase-encoding gene PrD Ⅰ of D.cionopaga,which was related to the infection of nematodes,was expressed with the vector.Result:The result of RT-PCR presented that the target gene was transcripted in A.niger 201.SDS-PAGE analysis and enzyme activity assay shown that the molecular weight of the recombinant protein purified with affinity chromatography was about 45kDa and possessed the proteinase activity at pH 5.0.The concentration of the purified protein in the enzyme solution was 30μg/ml.Conclusion:This expression indicated that the constructed pGT21M-HIS could be used for the foreign expression of genes in A.niger,which provided the convenience for the purification and identification of target protein.A.niger expression system is advantageous in contrast to other expression systems so far as the expression of filamentous fungi genes concerned.