鹅源副黏病毒融合蛋白在毕赤酵母细胞中的表达

Expression of Fusion Protein of Goose Paramyxovirus in Pichia pastoris

应用特异性引物,从鹅源副黏病毒NA-1株中扩增出F蛋白基因,PCR产物纯化后克隆入pGEM-T载体,得到重组质粒pT-F。用EcoRⅠ和NotⅠ双酶切pT-F,回收目的基因F片段,并将其定向克隆到pPICZαA中,构建重组质粒pPICZαA-F。用PmeⅠ酶切pPICZαA-F使其线性化,电击转化至感受态毕赤酵母GS115菌中。PCR法鉴定阳性重组子,10 mL/L甲醇诱导表达后,进行SDS-PAGE及Westernblot分析。结果表明,在酵母菌培养基上清中检测到相对分子质量为63 ku的重组蛋白,该重组蛋白可与NA-1株鹅源副黏病毒多克隆抗体发生特异性血清学反应。

To express F protein of NA-1 in Pichia pastoris,F gene was amplified by RT-PCR from NA-1 with a pair of specific primers.Then PCR product was purified and cloned into pGEM-T vector to obtain the plasmid pT-F.The gene fragment was recovered after the double enzyme digestion with EcoRⅠ and NotⅠ,then was subcloned into pPICZα A.The recombinant pPICZαA-F was linearized with PmeⅠ and then transformed into GS115 yeast cells for expression.The recombinant strains were screened by PCR technique.The expression products were identified by SDS-PAGE and Western blot.The results showed that there was a molecular weight of 63 ku protein specifically recognized by polyclonal antibody against NA-1.It suggested that the F protein of NA-1 was obtained in Pichia pastoris expression system.