毕赤氏酵母SMD1168/HuIFNα-2b分泌型表达的研究

Study on Pichia. Pastor is SMD1168 of Secretory Expression for huIFNα-2b

利用毕赤氏酵母SMD1168构建huIFNα 2b分泌型高效表达工程菌株 ,以提高huIFNα 2b表达量和生物比活性 ,增强临床治疗效果和降低临床应用的毒性副作用。将克隆修饰的huIFNα 2b基因插入pGAPZα A之GAP启动子下游位点 ,构建成含α factor信号肽的重组表达载体 ,转入毕赤氏酵母 (Pichiapastoris)SMD1168,构建成分泌型表达huIFNα 2b的酵母工程菌株。SDS PAGE分析表明 ,该工程菌株huIFNα 2b表达量占菌体分泌总量的 50 %以上 ,表达量为 10 0～ 12 0 μg ml;MTT法测定纯化样品的生物比活性为 6 69× 10 8IU mg蛋白～ 1 11× 10 9IU mg蛋白 ,并通过柱层析分离纯化了huIFNα 2b ,纯度达 99 7% ,回收率达 15%～ 2 0 % ;Westernblotting分析表明表达的huIFNα 2b具有与天然huIFNα

Pichia. Pastoris SMD1168 strain of high efficie nt and secreting expression for h uIFNα 2b was constructed. A expression vector containing α factor signal pep tide was constructed by inserting modified huIFNα 2b gene into GAP promoter do wnstream of pGAPZ α A, which was transferred into Pichia. Pastoris SMD1168 , for secreting expression huIFNα 2b. The result of SDS PAGE showed that huIFN α 2b possess over 50 percent of total quantity of the secreted proteins with t he expression value of 100～120 μg/ml, the bioactivity was 6.69×10 8IU/m g protein～1.11×10 9IU/mg protein identified by MTT method. huIFNα 2b was purified by column chromatography techniques, and the purification rate reached to 99.7 percent with the recovery rate of 15%～20%, western blot analysis showe d that the expressed huIFNα 2b had the same immunogenicity as natural one.