The Screening Of Downstream Signaling Molecules After CacyBP/SIP Nuclear Translocation In Colon Cancer Cells

Objective: In colon cancer， the CacyBP/SIP can promote the cancer cellsdevelopment when it translocats into cell nucleus. To determine target proteininteracting with CacyBP/SIP after CacyBP/SIP translocated into nucleus inhuman SW480cells.Methods: Constructed the lentiviral vector pGC-FU-CBP-SBP-cacyBP-3XNuclear targeting sequences and enveloped lentivirus, then transfected the humanSW480cells. Stable expression of fusion protein CBP-SBP-CacyBP-3X Nucleartargeting sequences and endogenous CacyBP/SIP was confirmed by Westernblotting. And then Tandem Affinity Purification (TAP) and immunoprecipitation(IP) were used to depurate the protein complex interacting with CacyBP/SIP inhuman SW480cells, then analysis the protein complex using the massspectrometryRseults: The CBP-SBP-cacyBP-3X Nuclear targeting sequences lentiviralwas enveloped successfully. The virus titer was up to2×108TU/mL. Westernblotting shew confirmed the fusion protein expression at protein level in humanSW480cells nucleus. Tandem affinity purification (TAP) screening downstreamsignaling molecules didn’t obtain positive result. But the immunoprecipitation (IP)method suggested that actin may be a significant protein which can interact withCacyBP/SIP in SW480cells.Conclusions:1. Successful Constructed the lentiviral vector pGC-FU-CBP-SBP-cacyBP-3X Nuclear targeting sequences. The virus titer is upto2×10~8determined by quantitative PCR.2. The lentivirus of CBP-SBP-CacyBP-3X Nuclear targeting sequencestransfected the human SW480cells and the fusion protein expressed successfully.3. Actin may be one proein which can interact with CacyBP/SIP involved incolon cancer development.