新城疫病毒F48E9株F-HN蛋白融合基因在毕赤酵母中的表达

F gene and HN gene co-express of Newcastle disease virus strain F48E9 in Pichia pastoris

利用重叠PCR技术拼接新城疫病毒F蛋白基因和HN蛋白基因,并将构建好的融合基因克隆到载体pET28a,经测序后融合基因插入表达载体pPIC9K中,在启动子AOXⅠ和α交配因子信号肽的作用下,分泌表达融合蛋白F-HN。重组质粒pPIC9K-F-HN经SacⅠ线性化后,电击转化毕赤酵母GS115,经G418筛选得到转化子。PCR扩增鉴定后,用甲醇诱导表达,SDS-PAGE分析结果表明,目的蛋白为分泌性表达,分子量约为86.5 ku;Western-blot结果表明融合蛋白具有良好的免疫反应性,为下一步研究高效亚单位基因工程苗奠定基础。

The fused gene （F-HN） of the newcastle disease virus F gene and HN gene were amplified without linker by Overlapping PCR technology. The spliced gene was clone into Piehia pastoris secretory vector pPIC9K. With the help of promoter AOX1 and mat α signal peptide, the F-HN gene was designed to secretory expression. Linearized by restriction enzyme Sac I , The recombinant plasmid pPIC9K-F-HN was transformed into Pichia pastoris GS115 by electroporation. The recombinant strains which were identified by G418 and PCR analysis were induced by methanol to express protein F-HN. The target protein was expressed in fermentation supernatant. Western blot analysis of the fusion protein suggested that F-HN combination protein had good reactionogenicity. It provids further research efficient subunits genetic engineering vaccine in foundation.