牛α干扰素在毕赤酵母中的高效分泌表达及活性鉴定  

High-level secretory expression of bovine alpha interferon in Pichia pastoris

作　　者：王延群[1] 张璐[1,2] 李玉明[1,2] 汪志艳[1] 金家民[1,2] 刘永刚[1] 王刚[1] 王淑杰[1] 姜成刚[1] 涂亚斌[1] 蔡雪辉[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/动物疫病诊断中心,黑龙江哈尔滨150001 [2]东北农业大学动物医学学院,黑龙江哈尔滨150030

出　　处：《中国预防兽医学报》2013年第10期845-848,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：黑龙江省自然科学基金(C201047);国家863计划(2011AA10A213)

摘　　要：为高效分泌表达牛α干扰素(boIFN-α),本研究通过人工合成boIFN-α基因,将目的基因克隆至表达载体pPIC9K中,构建重组质粒pPIC9K-boIFN-α,将其电转化于毕赤酵母菌株GS115,利用抗药选择压力G418筛选重组菌,对重组菌诱导表达,取上清进行SDS-PAGE和western blot检测,并优化重组菌的诱导表达条件。结果显示:筛选获得高效分泌表达boIFN-α的重组菌,其最佳诱导条件为:250 r/min,26℃培养,1%甲醇浓度诱导,诱导72 h。上清中目的蛋白表达量最高可达200μg/mL,本研究为boIFN-α在生产中的应用奠定了基础。To achieve high-level secretory expression of bovine interferon-α （bolFN-α） in Pichia pastoris, the bolFN-α gene was artificially synthesized and inserted into the secreted expression vector pPIC9K. The Sac I linearized resultant recombinant plasmid was transformed into P.pastoris GS115 by electroporation and the positive recombinant strains P.pastoris were screened with 0.25 mg/mL to 4 mg/mL of G418 which were capable of expressing bolFN-α in culture medium detected by SDS-PAGE and western blot. In addition, approximate 200 μg/mL of bolFN-α was secretory expression fi＇om recombinant P.pastoris under the optimized conditions of the fermentation by incubating at 26 ℃ for 72 hours with 250 r/min and inducing with 1% of methanol, respectively. Furthermore, the bolFN-α was able to efficiently inhibiting virus replication in recombinant vesicular stomatitis virus （VSV-GFP）/MDBK cells detection system. These results provided a basis for further application of bolFN-α.

关 键 词：牛IFN-α 毕赤酵母 分泌表达 

分 类 号：S852.4[农业科学—基础兽医学]