斜纹夜蛾ppie基因的克隆及表达载体构建

Cloning and expression vector construction of ppie gene of Spodoptera litura

肽基脯氨酰基顺反异构酶E (Peptidyl-prolyl cis-trans isomerase E, PPIE)是亲环蛋白家族的成员之一,具有肽基脯氨酰基顺反异构酶(Peptidyl-prolyl cis-trans isomerase, PPIase)活性,能与亲环蛋白抑制剂CsA特异性结合。PPIE具有两个结构域:N-端的RNA识别域(RNA Recognition Motif, RRM)和C-端的PPIase活性域。PPIE和其他亲环蛋白一样,具有蛋白质折叠功能。研究表明,哺乳动物细胞中的PPIE能抑制流感病毒复制,能与MLL PHD3相互结合。但昆虫细胞中PPIE的功能研究较少。本文以斜纹夜蛾Spodoptera litura为研究对象,克隆得到ppie基因,通过生物信息学分析发现,斜纹夜蛾ppie基因ORF全长876 bp,共编码291个氨基酸,具有亲环蛋白家族标签:YQGSlFHRIIPDFMCQGG。构建了真核表达载体pIZT/V5-His-ppie,结果证明,构建的真核表达载体能在昆虫细胞中表达,融合蛋白大小为36 kDa左右,与预测大小一致。还通过免疫荧光实验确定,PPIE定位于细胞核中,与前人研究报道一致。本文研究内容将为进一步研究斜纹夜蛾PPIE功能奠定基础。

Peptidyl-prolyl cis-trans isomerase E (PPIE) is a member of the cyclophilin family, which has the activity of peptidyl prolyl cis-trans isomerase (PPIase), can specifically bind to the cyclophilin inhibitor CsA. PPIE has two domains: The RNA recognition motif (RRM) at the N-terminus and the PPIase active domain at the C-terminus. Like other cyclophilins, PPIE has protein folding function. Studies had shown that PPIE in mammalian cells can inhibit influenza virus replication and bind to MLL PHD3. However, there were few studies on the function of PPIE in insect cells. In this study, Spodoptera litura was used as the research object, and we had cloned ppie gene. The results of bioinformatics showed that the ORF of the ppie gene was 876 bp in length, encoding 291 amino acids with the tag sequence of the cyclophilin family: YQGSlFHRIIPDFMCQGG. The eukaryotic expression vector pIZT/V5-His- ppie was constructed. The results showed that the vector could be expressed in insect cells. The fusion protein was about 36 kDa, which was consistent with the predicted size. It was also confirmed by immunofluorescence assay that PPIE was localized in the nucleus, consistent with previous studies. The content of this study will lay a foundation for the further study of PPIE function of Spodoptera litura.