金针菇免疫调节蛋白基因fip-fve在毕赤酵母GS115中诱导型和组成型表达

Inducible and constitutive expression of fip-fve from Flammulina velutipes in Pichia pastoris GS115

摘要:

为获得稳定来源并且具有生物学活性的重组金针菇免疫调节蛋白 (Fip-fve)，将fip-fve基因转至毕赤酵母GS115中进行诱导型和组成型表达。用PCR方法从金针菇子实体基因组DNA中扩增fip-fve基因，连接至pPIC9构建诱导型表达载体pPIC9-FIP-fve，从毕赤酵母基因组DNA中扩增三磷酸甘油醛脱氢酶启动子 (pgap)，替换pPIC9-FIP-fve上乙醇氧化酶启动子 (paox1) 构建组成型表达载体pPIC9-PGAP-FIP-fve。将线性化的两种表达载体用PEG法转化毕赤酵母GS115，经组氨酸缺失培养基筛选和酵母菌落PCR鉴定后进行表达。结果表明，重组Fip-fve在以甲醇 (1%，V/V) 为碳源进行诱导型表达4 d达到最高，粗蛋白表达量为158.2 mg/L，在以葡萄糖 (10%) 和甘油 (1%，V/V) 为碳源进行组成型分别在表达第4天和第5天达到最高，粗蛋白分别为46.3 mg/L和29.5 mg/L。SDS-GAGE及Western blotting证明重组Fip-fve已正确表达，血细胞凝集活性检测初步证明重组Fip-fve具有良好生物学活性。

Abstract:

We transformed the fip-fve gene into Pichia pastoris GS115 for inducible and constitutive expression to obtain feasible bioactvie recombinant Fip-fve. The fip-fve gene was cloned from Flammulina velutipes fruting body by PCR and ligated to pPIC9 to construct inducible expression vector pPIC9-FIP-fve, and promotor pgap was used to replace the paox1 to construct constitutive expression vector pPIC9-PGAP-FIP-fve. These two vectors were used to transform P. pastoris by PEG method. The fip-fve was expressed after histamine-absence screening and yeast colony PCR. The inducible expression level reached 158.2 mg/L at the fourth day and the constitutive expression level was 46.3 mg/L and 29.5 mg/L using glucose and glycerol, respectively. The SDS-PAGE and Western blot both proved the correctness of rFip-fve, and the hemagglutination test indicats the rFip-fve's bioactivity.