伪狂犬病病毒闽A株gE基因去信号肽片段在Pichia pastoris中的表达

Expression of gE Gene Fragment Deleted Signal Peptide of Pseudorabies Virus Fa Strain in Pichia pastoris

伪狂犬病病毒囊膜糖蛋白E是一种在伪狂犬病根除计划中具有重要作用的糖蛋白 .将伪狂犬病病毒闽A株 gE基因去信号肽片段克隆到巴斯德毕赤酵母 (Pichiapastoris)表达载体pPICZαA中 ,获得的重组表达载体pPICZαA FL电击转化野生型酵母菌SMD116 8后 ,得到多株酵母工程菌SMD116 8/ pPICZαA FL .经高浓度ZeocinTM筛选、表型鉴定、工程菌的诱导表达及表达产物的鉴定 ,最后得到高效表达 gE基因去信号肽片段的酵母工程菌SMD116 8/ pPICZαA FL 7.工程菌 72h培养上清的SDS 聚丙烯酰胺凝胶电泳与蛋白质印迹结果显示 ,gE基因去信号肽片段表达产物大小约为 80ku ,比预期的 6 3 8ku大 .凝胶薄层扫描结合Bradford蛋白质总含量测定结果表明 ,表达产物占工程菌培养上清总蛋白的 13 4 9% ,表达量可达 11 7mg/L .间接ELISA结果表明重组表达产物具有良好的抗原性 。

The envelope glycoprotein E is a major glycoprotein of pseudorabies virus, which exerts important effect in pseudorabies eradication campaign. The gE gene fragment deleted signal peptide of PRV Fa strain was inserted into Pichia pastoris expression vector pPICZalphaA, the resulted recombinant expression vector transformed SMD1168 competent cells and obtained engineering Pichia pastoris strain SMD1168/pPICZalphaA-FL. After high concentration Zeocin(TM) screening, phenotype identification, inductive expression, SDS-PAGE and Western blot analysis of culture supernatant, an engineering Pichia pastoris strain SMD1168/pPICZalphaA-FL-7 in which gE gene fragment deleted signal peptide was expressed in high levels was obtained. SDS-PAGE and Western blot indicated that expression product of gE fragment deleted signal peptide in culture supernatant of SMD1168/pPICZalphaA-FL-7 was about 80 ku, a little larger than expected. Gel scanning and Bradford protein analysis results showed that expression product reached 11.7 g/L, or 13.49% of total culture supernatant protein in SMD1168/ pPICZalphaA-FL-7.