Research On SpliInx1and SpliInx4Relationship With Innate Immune Suppression

Innexin is a highly conserved gene in invertebrate, different from the connexins of vertebrate. Innexins would expressed in the cell surface to form functional proteins hemichannel, and related to cell signaling pathway, embryonic development and nervous system differentiation. In this study, we got innexinl and innexin4Open reading Frame from the Spodoptera litura hemocytes transcriptom then designed primers. Then we demonstrated innexinl and innexin4belongs to the conversed S. litura innexin gene family with sequencing and alignment, named Splilnxl and Splilnx4. Splilnxl open reading frame is1086bp in length, encoding a361amino acid protein, SpliInx4is1116bp and encoding a371amino acid protein. Finally, we constructed expression vectors for prokaryotic and eukaryotic system, demonstrated Splilnxl and SpliInx4expressed in E.coli and lepidopteran insect cell lines with western blot. Then we detected the SpliInxl, SpliInx4expression in different tissues:hemocytes, nerve, fatbody, midgut, ovary and testis. And got hemocytes cDNA of S.litura with7days post-parasitization, detected the expression of inxs, p53, p110and CypD. We discussed performance and the role of Inxs, PI3K gene and apoptotic gene in innate immune suppression, and found the importance of SpliInx4in S.litura reproductive system development. Contructed stable expression cell lines of inxs, demonstrated inxs were expressed on cell membrane, inx3clearly expressed in cytoplasmic solubleprotein. Then we detected the phosphorylated attainment of inxs protein in stable expression cells with fluorescent probe, suggested inxs would be phosphorylated and the role in formation of hemichannel and transfer cell signaling. After that, transfected SpliInxl and SpliInx4into Sf9and Spli221cells, and produced apoptosis. Then contructed Bac virus vector of Splilnx2and SpliInx3, infected Sf9cells, found SpliInx3had strong dephosphorylation, phosphate ions closely associated with apoptosis.