The Related Research Between New Fusion Protein PTD-GRB2-SH2and Ras Signaling Transduction Of Breast Cancer

Growth factor receptor-bound protein2(Grb2) is combined with a centralSH2domain and two ends of the SH3domain, which activates receptor tyrosinekinase (RTK) in one end. And the other end connects with guanine nucleotideexchange factor (SOS) through the SH3domain, and further activatesdownstream signaling, then initiates the MAPK cascade to launch the tumorsignaling transduction processes.In this study, in order to investigate the potential function and position ofGrb2-SH2domain in breast cancer cell lines, we designed and constructed anovel fusion protein which contains a Grb2-SH2domain and the proteintransduction domain. The Grb2-SH2domain participates and inhibits the Rassignaling transduction through the protein transduction domain in the gene andthe protein levels. The constructed fusion proteins successfully penetrated intothe living cells through the PTD domain, and bind to their substrates competitively, thus influence proliferation signaling transduction and thebiological behavior of the breast cancer cells.The experiment is mainly divided into three parts:1. The prokaryotic fusion protein expression vector pET-16b-PTD-Grb2-SH2containing Grb2-SH2domain and the mutant contrast were constructed andvalidated through the enzyme digestion, DNA electrophoresis and plasmidsequencing, and then we induced the prokaryotic expression by IPTG treatment.After that we collected purification of the fusion protein pET-16b-PTD-GRB2-SH2through nickel column, and identified by the molecular weight which wasuniformed with expected by Western blotting.2. We co-incubated the purified protein with the breast cancer cell linesMCF-7and MDA-MB-231cells, and observed the tumor cell biologicalfunction through MTT, Transwell tumor migration experiment, FACS analysisand EdU proliferation detection and found that the prokaryotic fusion proteinPTD-GRB2-SH2have a convincing inhibition effects on proliferation andmigration of the breast cancer cell lines MCF-7cell and MDA-MB-231cell，buta convincing promotion on apoptosis.3. We applied Gateway system to construct the eukaryotic fusion proteinexpression vector plenti-PTD-Grb2-SH2contains Grb2-SH2domain and themutant contrast carrier, which validated through the enzyme digestion, DNAelectrophoresis and plasmid sequencing. After that we used three kinds oflentivirus packaging plasmid to transfect and collected the supernatant containsvirus particles, and then we infected T lymphoma Jurkat cell lines in order toestablish a stable transfection cell lines through the selection of infected cells bythe drug Blasticidin. At last Western blotting helped us to identify that modifiedJurkat cells could produce and secrete the object protein,thus proving the success of the eukaryotic fusion protein expression carrier construction.