| Objective:To construct rat eukaryotic expression vectors of pcDNA3.1(+)/GPC3N48-EGFP-GPC3C550containing Glypican-3(GPC3) gene cloned from rat fetal liver tissue and enhanced green fluorescent protein (EGFP) for further functional and therapeutical research on GPC3.Methods:①All the primers were designed according to published rat GPC3gene sequence. Appropriate restriction enzymes needed in the clone strategy were added to the primers by analysis all the restriction enzyme cutting sites of plasmids including pGEM-T, pcDNA3.1(+) and pGEM-T/EGFP.②Full length GPC3gene was amplified from total RNA extracted from rat fetal livers by using reverse transcription polymerase chain reaction (RT-PCR), then inserted into pGEM-T plasmid. The positive clones were chosen by blue-white screening. Plasmid of pGEM-T/RGPC3-Long isolated from positive bacterial clones was identified by PCR and DNA sequencing.③All plasmids were constructed including pGEM-T/GPC3ORF containing open read frame of GPC3gene, pGEM-T/GPC3N48with truncated GPC3gene N-terminus as well as pGEM-T/GPC3C550with truncated GPC3gene C-terminus using plasmid of pGEM-T/GPC3R-Long as a template.④The above plasmids and conserved plasmid of pGEM-T/EGFP were digested by double enzymes to get inserts of GPC3-N48, EGFP and GPC3-C550, then all inserts subcloned into plasmid of pcDNA3.1(+) orderly to construct plasmid of pcDNA3.1(+)/GPC3N48-EGFP-GPC3C550, which was identified by restriction enzyme digestion and DNA sequencing.Results:①Full-length of GPC3gene was amplified from rat fetal liver tissue successfully.②All recombinant plasmids including pGEM-T/RGPC3-Long, pGEM-T/GPC3ORF, pGEM-T/GPC3N48and pGEM-T/GPC3C550were constructed successfully.③Eukaryotic expression vector of pcDNA3.1(+)/RGPC3N48EGFP-GPC3C550was constructed and confirmed successfully.Conclusion:Eukaryotic expression vector of rat pcDNA3.1(+)/RGPC3N48-EGFP-GPC3C550with EGFP gene was constructed successfully for further investigation of the function of GPC3. |
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