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

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.