白蛋白融合α-2b干扰素在毕赤酵母中的表达  

Expression of HSA-IFNα-2b Gene in Pichia pastoris

作　　者：张伍魁[1] 范清林[2] 邹文艺[2] 仲皙[3] 宋礼华[1] 

机构地区：[1]安徽医科大学基础医学院,合肥230032 [2]安徽安科生物工程股份有限公司,合肥230088 [3]中国药科大学,南京210009

出　　处：《中国生物工程杂志》2005年第12期45-49,共5页China Biotechnology

摘　　要：α2b干扰素的重组表达质粒pPIC9KHSAIFNα2b经限制性内切酶SalI酶切线性化后电转化巴斯德毕赤酵母菌(P.Pastoris)SMD1168,将阳性转化子进行PCR鉴定和Mut表型鉴定并用甲醇诱导表达,WesternBlot结果表明白蛋白融合IFNα蛋白与IFNα抗体具有结合能力,Wish细胞VSV病毒系统鉴定表达蛋白具有抗病毒生物活性。在摇瓶培养条件下,甲醇在0.5%～4.0%的范围,随着浓度的升高蛋白表达量也升高,在其他因素固定时,菌体起始OD值在5～80的范围内,随着OD值的升高表达量反而下降。成功表达出具有高生物学活性的白蛋白融合干扰素蛋白(HSAIFNα2b),为进一步应用打下基础。The competent Pichia pastoris was transformed by the recombinant plasmid pPIC9k-HSA-IFNα- 2b that was cut with restriction endonucleases SalI through electroporation. The positive recombinant Pichia pastoris clones were screened and identified by PCR and Mut phenotype. The target protein secreted by the recombinant Pichia pastoris was induced by methanol. Western-Blot showed the rHSA-IFNα-2b combining activity to McAb of IFNα-2b. The anti-virus activity of rHSA-IFNα-2b was identified by Wish-VSV system. In flask fermentation, the expression was increased in higher methanol concentration within 0. 5% - 4.0%, and decreased when the start OD600 of recombinant Pichia pastoris changed from 5 to 80. The high-bioactivity protein, rHSA-IFNα-2b was successfully expressed.

关 键 词：白蛋白融合α-2b干扰素 巴斯德毕赤酵母茵 Wish细胞-VSV病毒系统 

分 类 号：Q786[生物学—分子生物学] Q512.1