Contruction And Identification Of Eukaryotic Expession Plasmid PcDNA3.1-GPC3

Objective: To construct eukaryotic expression plasmid pcDNA3.1-GPC3, and transfect pcDNA3.1-GPC3 into dendritic cells (dendritic cell, DC), and verify high expression of GPC3 of the transfected DC, as the solid foundation for our future gene thearapy experiment of the hepato-cellular carcinoma.Method: 1. Construction of recombinant plasmid pcDNA3.1-GPC3. To anti-transcript GPC3 gene's mRNA into cDNA by RT-PCR method,and amplify the cDNA of GPC3 gene,and clone the cDNA of GPC3 gene into eukaryotic expression vector pcDNA3.1 , then construct the recombinant plasmid of pcDNA3.1-GPC3,and sequence pcDNA3.1-GPC3 by restiction enzyme digestion. 2. Extraction of DC from mormal bone marrow cells of mice limbs. To remove granulocytes,T cells,B lymphocytes,NK cells,monocytes and macrophages by specific CD4,CD8a,CD45R monoclonal antibody and complement buffer and the half DC adhesion, then to culture DC in vitro by mouse granulocyte - macrophage colony-stimulating factor ( murine granulocyte marcophage-colony stimulating factor, GM-CSF) and murine interleukin -4 (murine interleukin-4, IL-4), and acquire high purity of matured DC. 3. To transfect recombinant plasmid pcDNA3.1-GPC3 into mouse DC by liposome method. 4. To identify high expression protein of GPC3 in DC that plasmid pcDNA3.1-GPC3 was transfected in .Results: (1) The cDNA of GPC3 gnene was cloned from the H22 tumor cells . (2) The eukaryotic expression plasmid pcDNA3.1-GPC3 was successfully constructed. (3) The eukaryotic expression plasmid pcDNA3.1-GPC3 was successfully transfected into DC, and the protein of GPC3 in the transfected DC was successfully identified.