复合干扰素突变体在毕赤酵母中的表达、纯化及活性分析  

Expression, Purification and Activity Analysis of Consensus Interferon Mutant Produced in Pichia pastoris

作　　者：于长明[1] 付玲[1] 齐连权[1] 张晓鹏[1] 于婷[1] 来大志[1] 王海涛[1] 陈薇[1] 

机构地区：[1]北京微生物流行病研究所,北京100071

出　　处：《中国生物化学与分子生物学报》2003年第3期317-320,共4页Chinese Journal of Biochemistry and Molecular Biology

摘　　要：根据毕赤酵母密码子偏性合成了复合干扰素突变体基因 ,克隆至分泌型酵母表达载体pMEX9K ,将重组载体pMEX CIFNm用SacⅠ线性化后 ,转化毕赤酵母GS115 .转化子经诱导后 ,培养上清有抗病毒活性的蛋白产生 .经过离子交换 ,疏水层析 ,凝胶过滤三步层析纯化 ,得到了纯度大于95 %的重组复合干扰素突变体 ,经N端氨基酸序列分析表明 ,该蛋白N端序列与理论值一致 ,质谱测定分子量为 19 3kD ,与理论值一致 .用细胞病变抑制法测定其活性 ,并结合Lowry法蛋白定量计算其比活性为 6× 10 8IU mg ,与复合干扰素的比活相当 .An artificial gene for consensus interferon mutant was synthesized by using favored codons of the yeast Pichia pastoris . The gene was cloned into the secretory expression vector pMEX9K, and the recombinant vector pMEX CIFNm was linearized and transformed into GS115 for expression. The culture supernatant of transformants had the antiviral activity after inducement with methanol. The purity of recombinant protein reached over 95% through the steps of ion exchange, hydrophobic interaction, size exclusion chromatography. The first 5 amino acid sequence of the N terminal was consistent with the theoretical sequence. Its molecular weight measured by MS agree with the theoretical value 19 3 kD. The specific activity of protein was similar to consensus interferon(infergen)——6×10 8 antiviral units/mg when assayed by the antiviral activity of WISH cells challenged with VSV virus and Lowry protein assay.

关 键 词：毕赤酵母 表达 纯化 活性 复合干扰素突变体基因 

分 类 号：Q786[生物学—分子生物学] Q511