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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-PAGE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。
To obtain a stable source of biologically active recombinant Flammolin Immunofluorescent protein (Fip-fve), the fip-fve gene was transferred into Pichia pastoris GS115 for inducible and constitutive expression. The fip-fve gene was amplified by PCR from genomic DNA of fruiting bodies of Vicia faba and ligated into pPIC9 to construct inducible expression vector pPIC9-FIP-fve. The glyceraldehyde triphosphate dehydrogenase promoter was amplified from Pichia pastoris genomic DNA pgap) was used to construct the constitutive expression vector pPIC9-PGAP-FIP-fve by replacing the alcohol oxidase promoter (paox1) on pPIC9-FIP-fve. The two linearized expression vectors were transformed into Pichia pastoris GS115 by PEG method, and then were screened by histidine-deficient culture medium and identified by yeast colony PCR. The results showed that the recombinant Fip-fve reached the highest level after 4 days induction with methanol (1%, V / V) as carbon source, and the crude protein was 158.2 mg / L. After treated with glucose (10% 1%, V / V) as the carbon source, reached the highest on the 4th and 5th day of expression, respectively. The crude protein was 46.3 mg / L and 29.5 mg / L, respectively. SDS-PAGE and Western blotting confirmed that the recombinant Fip-fve was correctly expressed. The detection of hemagglutination activity preliminarily demonstrated that the recombinant Fip-fve has good biological activity.