小菜蛾中肠氨肽酶N2在昆虫细胞中的表达

Expression of aminopeptidase N(APN2)from Plutella xylostella larval midgut in Sf9 cells

摘要: 利用昆虫细胞-杆状病毒表达系统(Bac-to-Bac baculovirus expression system)表达获得小菜蛾 Plutella xylostella(L.)中肠氨肽酶N2(aminopeptidase N,APN2)蛋白,成功建立了该蛋白在昆虫细胞中的表达体系。将对Cry1Ac毒素敏感和已产生抗性的小菜蛾中肠APN2基因插入表达载体pFAST Bac HTB中,并在草地夜蛾 Spodoptera frugiperda 细胞系(Sf9)中进行了重组表达。聚丙烯酰胺凝胶电泳(SDS-PAGE)及蛋白免疫印迹技术(Western-blotting)结果显示,在107 kDa处有1条特异性蛋白条带。研究表明,小菜蛾中肠APN2基因可在Sf9细胞中成功表达,这为继续研究其功能及抗性的关系奠定了基础。

Abstract: Using the insect cells-Bac-to-Bac baculovirus expression system to express APN2(aminopeptidase N)protein,the APN2 expression system in Sf9 cells was successfully established.APN2 gene from susceptible and resistant Plutella xylostella(L.)larval midgut were inserted into pFAST Bac HTB vector,expressed in insect cells Spodoptera frugiperda(Sf9)cells,respectively,with Bac-to-Bac baculovirus expression system.A 107 kDa protein was examined and confirmed by SDS-PAGE and Western-blotting.The results showed that P.xylostella larval midgut APN2 gene was successfully expressed in Sf9 cells which provided a foundation for further investigation on function of APN2 and its relationship with Bt resistance.