Construction And Expression Of Eukaryotic Plasmid Of SpliInx2and SpliInx3

Innexin (Inx) protein can form hemichannel on the invertebrate cell membrane or it form gap junction channels between two neighboring invertebrate cells. Recent studies indicated that innexin protein may have multiple properties. According to the relevant literature that a variety of connexin genes deletion in the human cancer cells like HeLa and HCT116and so on, may cause abnormalities in the intercellular space and connection structure. Based on the background above, we wanted to confirm the innexin proteins involved in constructed functional activity of the hemichannel and gap junction, and related to cell adhesion and apoptosis, so we carryed out a series of research about Inx2and Inx3genes were heterologous expressed in293T, HCT116and HeLa cells, in order to explore the genes functions of Inx2and Inx3.We successfully constructed eukaryotic expression vectors of Splilnx2and Splilnx3, and named p3×FLAG-CMV-SpliInx2and p3×FLAG-CMV-SpliInx3, which used p3×FLAG-CMV-EGFP as a positive control, and transfected293T, HCT116and HeLa cells. Though transient transfected p3×FLAG-CMV-EGFP vector to these cells using fluorescence microscopy and Western blot to detected, so the results indicated that the EGFP genes succeed expressing in the HeLa, HCT116and293T cells and appear modified effect; Furthermore, we observed the features of apoptosis after Spliinx2and Spliinx3transfected HeLa cells in72hours. Afterwards used labeled FLAG antibody, SpliInx2and SpliInx3protein were identified in293T cells, in HCT116cells only detected SpliInx2protein, while SpliInx2and SpliInx3proteins all cannot detection. These preliminary findings show that the SpliINX2and SpliINX3proteins may involved in the cellular apoptosis signaling pathways activated. And found that the cell apoptosis is related to actatived PI3K/Akt signal pathway and then truncated Bid proteins, even invoked Caspase3proteins in the pathways.