The Of Spodoptera Litura Blood Cells Inx2 And Inx3 Gene Cloning And Function Of

Previous reports have suggested that there are functional and activity hemichannels in the hemocytes surface of insects, allowing direct intracellular and extracellular signal transduction and material exchanges. The research first discovered the proteins in vertebrates named connexin. whereas the channel proteins in invertebrates known as the innexin. A hemichannel was composed of six-innexin proteins, each containing four transmembrane domains (TM) which were linked with two extracellular loops and a cytoplasmic loop. In present study, we further characterized two novel members of the insect innexin family, Spli-inx2and Spli-inx3, from the hemocytes of Spodoptera litura. Sequence analysis showed that Spli-inx2encodes359amino acids residues, with a MW of41.5kDa, while Spli-inx3encodes386amino acids residues, with a MW of43.8kDa. The analysis also predicted that both of the two proteins have four transmembrane domains. The phylogenetic tree suggested that both the Spli-inx2and Spli-inx3genes were belonged to the conservative innexin genes family.In the prensent study, we successfully constructed vectors for Spli-inx2and Spli-inx3eukaryotic expression, named pIZT/V5-His-Spli-inx2and pIZTN5-His-Spli-inx3. After transfected Hi5, Sf9, Spli221and Bm12cell lines, we used fusion protein labeled V5antibody via western blot detection, and the results indicated that the SpliINX2and SpliINX3proteins could been expressed in all the four cells, and showed different physiological changes. Hi5cells stably expressing INX2were harvested by selection with zeocin, as well as INX3protein. Meanwhile, the cell membrane surface protein was extracted and detected by labeled V5antibody in western blot, and the result indicated that the Hi5-Inx2and Hi5-Inx3proteins could be correctly located on the cell membrane surface. Furthermore, immunofluorescence experiments showed that the SpliINX2and SpliINX3proteins were mainly distributed in the cytoplasm and cell membrane, and in-depth dye uptake experiments indicated that SpliINX2and SpliINX3formed the functional hemichannels. Spli221, the homologous cells with Spodoptera litura, failed to establish a stable expression cell lines. Sf9cells that expressed the SpliINX2and SpliINX3protein displayed features of apoptosis, while an obvious aggregation phenomenon in Bm12cells. The preliminary results showed that SpliINX2and SpliINX3protein were able to form hemichannel, which probably involve in the cell signaling pathway of apoptosis and aggregation.