Construction And Function Research Of Eukaryotic Expression Plasmid Encoding Tumor-specific Anchored α-GAL Mimic Epitope

ObjectiveIn order to combine the targeting gene therapy and autoimmunity enhancement therapy of lung cancer, to amplify the fusion gene ofα-gal mimic epitope sequences and GPI anchored sequence, then to construct the eukaryotic expression plasmid of the fusion gene which was modulated by tumor-specific survivin promoter, and to study the expression of tumor-specific anchoredα-gal mimic epitope and its cytolysis effect in A549 cells, which would provide a basis for further study of targeting gene therapy of tumor.Methods1. Lymphocytes were isolated from human peripheral blood and their total RNA was extracted. The gene fragment encoding GPI of CD24 was amplified by RT-PCR by using the obtained RNA as template, which was inserted into the pSGFP plasmid after double enzyme digestion to construct recombinant plasmid pSGFP-GPI. Then the plasmid was transfected into A549cells by Lipofectamine 2000, and its expression function was detected by observing the expression of enhanced green fluorescent protein (EGFP) through fluorescent microscope.2. The gal-GPI fusion gene was amplified by PCR, then the pSGFP-gal-GPI eukaryotic expression plasmid was constructed, which was modulated by tumor-specific survivin promoter. The recombinant plsmid was transfected into A549 cells. After 72 hours, the whole RNA of transfected A549 was extracted and the transcription level of gal-GPI fusion gene of pSGFP-gal-GPI plasmid in A549 was identified by RT-PCR.3. After the recombinant plasmid was transfected into A549 cells by Lipofectamine 2000, the GFP-gal-GPI fusion protein was detected under the fluorescence microscope, and the location and immuno- reactivity of GFP-gal-GPI fusion protein was studied by immunocytochemistry and Western blotting respectively. The A549 cells expressingα-gal mimic epitope reacted with humam serum to detect the cytolysis effect induced by complement.Results1. The results of restriction endonuclease analysis and DNA sequencing proved that the GPI gene fragment was correctly inserted into pSGFP plasmid. The enhanced green fluorescence was observed around the cell membrane of A549 cells transfected with pSGFP-GPI.2. The results of restriction endonuclease analysis and DNA sequencing proved that the gal-GPI gene fragment was correctly inserted into pSGFP plasmid. After being transfected into A549 cells, the gal-GPI fusion gene was transcribed successfully, which was confirmed by RT-PCR.3. The fluorescence of transfected A549 distributed around the cell membrane unevenly. The recombinant protein was observed around the cell membrane of A549 cells by immunocytochemistry, and the Western blotting confirmed that the protein was successfully expressed in A549 cells, which can react with human IgG in serum. The transfected A549 cells reacted with normal human serum (anti-gal) and caused the cytolysis effect induced by complement. Conclusions1. The eukaryotic expression plasmid pSGFP-GPI modulated by tumor-specific survivin promoter was constructed successfully and could express anchored EGFP around the cell membrane, which provides a basis for the construction of anchored tumor vaccine.2. The pSGFP-gal-GPI eukaryotic expression plasmid modulated by tumor-specific survivin promoter which can express anchoredα-gal mimic epitope on A549 cell was constructed successfully, which provide a basis for further study of targeting gene therapy and Immunotherapy of lung cancer and other tumor.