Cloning And Expressing Of ICAT And Investigating It's Anti-Proliferation Activity

The abnormity of Wnt Signaling Transduction Pathway is one of themajor causes of the occurrence of many types of cancer cells. one of the components Pathway,beta-catenin/TCF-4, when activated,can facilitate cancerization,While the other Pathway component ICAT, which prevents the formation of the compound of beta-catenin/TCF-4, can negatively regulate the activity of the Wnt Pathway and thereby inhibit cancer. In this work,we cloned ICAT cDNA from normal human liver andconstructed pET-32a(+)-ICAT express vector.Then we expressed theproteins in prokaryote cells and tested ICAT's anti-proliferation biologicalactivity for further research of the function of ICAT and to pave a way tocure human tumors. First, Total RNA was extracted from normal human liver tissue. TheICAT cDNA was amplified from total RNA by RT-PCR, then cloned intopET-32a(+) and sequenced. Second, ICAT proteins was expressed in coliform after IPTGinduction. We purified these proteins inclusion body by using Ni2+ -NTAaffinity chromatography ,and renatured them by density gradual dialysis. Third,We investigated the proteins' activity of anti-proliferation totumor cells MCF-7 and MDA-MB-231 by MTT analysis.Then weconstructed the eukaryotic vector pcDNA3.1(+)-ICAT by subcloning, thentransfected it into tumor cells and let them express transiently, tested theeffect of ICAT gene expression on the proliferation of tumor cells. We have cloned human ICAT cDNA and constructed pET-32a(+)-ICAT Prokaryotic expression vectors successfully . The fusion proteinsexpressed in Escherichia coli BL21 were insoluble inclusion body, Solublefusion protein was obtained by Ni2+ -NTA affinity chromatography andrenaturing. The molecular weight of the purified protein were about28kDa. The purity of the product was up to 90 percent. The expressedproducts were identified as ICAT by Western blot. The transient expressionof ICAT gene by pcDNA3.1-ICAT can inhibit cancer cells proliferation invitro. The successful expression of human ICAT laid the foundation for theanti-tumor therapy.