复合干扰素在毕赤酵母中的分泌表达、纯化及活性分析  

Secretive expression, purification and activity assay of consensus interferon in Pichia pastoris

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

机构地区：[1]军事医学科学院微生物流行病学研究所,北京100071

出　　处：《免疫学杂志》2004年第2期113-116,共4页Immunological Journal

摘　　要：目的　在毕赤酵母中高效分泌表达复合干扰素。方法　根据毕赤酵母密码子偏性合成了复合干扰素基因 ,克隆至分泌型酵母表达载体pMEX9K ,线性化后转化毕赤酵母GS115 ,筛选高表达菌株。诱导后的培养上清经过离子交换 ,疏水层析 ,凝胶过滤三步层析纯化 ,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。结果　SDS PAGE分析显示 ,重组蛋白以可溶性分子的形式存在于上清液中 ,经纯化后纯度达到 96 % ,其N端氨基酸序列与理论值一致 ,比活与干复津相当。结论　复合干扰素在毕赤酵母中获得高效分泌表达 。Objective To obtain high-level secretive expressed consensus interferon in Pichia pastoris. Methods An artificial gene for consensus interferon was synthesized by using Pichia pastoris favored codons, then cloned into the secretory expression vector pMEX9K. The recombinant vector was linearized and transformed into GS115 for expression. The induced culture supernatant was purified through ion exchange, hydrophobic interaction, size exclude chromatography. The specific bioactivity of the recombinant protein was assayed by antiviral activity of WISH cells challenged with VSV virus and Lowry protein quantitative analysis. Results SDS-PAGE analysis suggested the recombinant protein was secreted into culture medium. The purity of recombinant protein reached over 96% through three steps of purification. The amino acid sequence of the N-terminal was completely the same with the theoretical sequence, and the specific bioactivity was similar to infergen. Conclusion High-level expression of secreted consensus interferon has been achieved in Pichia pastoris expression system.

关 键 词：复合干扰素 毕赤酵母 基因表达 

分 类 号：R346[医药卫生—基础医学]