| ObjectiveFirstly, to construct a eukaryotic expression plasmid which can encode the enhanced fluorescent protein and been modulated by Survivin promoter from A549 cells, with which the CMV promoter was substituted. Then compare their transcriptional activities by transfected into A549 and MRC-5 cells. To construct pSGFP-SEA-PNP eukaryotic expression plasmid modulated by surviving promoter by using the technique of Gene SOEing. To identify the specific expression of sea-pnp fusion gene of pSGFP-SEA-PNP plasmid in A549 by RT-PCR. To study the superantigen effect of recombinant fusion protein expressed in A549 by stimulation test of PBMC and MTT test. By MTT test, to study the cell poisonous effect of fusion protein expressed in A549 by translating the hypotoxic F-ara-AMP to the highly toxic 2-F-A. It would lay the groundwork for the exploring strategy and mechanism of transcriptional targeting of lung cancer and other cancer gene therapy.Methods1. By PCR and gene cloning technique, the eukaryotic expression plasmid pSGFP(pEGFP-C1/Surp) were constructed, which has Green Fluorescin reporter gene (EGFP) and been modulated by Survivin promoter from A549.2. The recombinant plsmid and the pEGFP-C1 were transfected into A549 cells and MRC-5 cells with liposome. After 72 hours, the expressed green fluorescin target protein was observed under fluorescence microscope, then the expression level of green fluorescin modulated by Survivin promoter was analyzed by Imagepro-Plus6.0 software.3. The sea gene was amplified by PCR from genome of staphylococcus aureus ATCC25923, and the pnp gene was amplified by PCR from genome of escherichia coli JM109, then construct the sea-pnp fusion gene by the technique of Gene SOEing. Then insert the sea-pnp fusion gene into the pSGFP to construct the pSGFP-SEA-PNP. The recombinant plsmid was transfected into A549 cells. After 72 hours, extract the whole RNA of transfected A549 and identify the specific expression of sea-pnp fusion gene of pSGFP-SEA-PNP plasmid in A549 by RT-PCR.4. The pSGFP-SEA-PNP plasmid was transfected into A549 cells. After 72 hours, gather the supernatant and lysate of transfected A549 and prepare the human PBMC, then do the stimulation test of PBMC and MTT test, and analyze the SI (Stimulation index) to study the superantigen effect of recombinant fusion protein expressed in A549.5. The pSGFP-SEA-PNP plasmid was transfected into A549 cells. After 36 hours, add the culture medium with F-ara-AMP. After 96 hours, analyze the cell-killing rate by MTT test.Results1. The pSGFP(pEGFP-C1/Surp) plasmid were constructed succ- essfully, which promoter sequence gene were confirmed by restriction enzyme analysis and DNA sequencing. After the recombinate plasmid was transfected into A549 cells and MRC-5 cells, the green fluorescin target protein was observed in A549 cells, but there has no green fluorescence in MRC-5 control cells. As pEGFP-C1 plasmid control, the green fluorescin target protein was observed in transfected A549 and MRC-5 cells. Analyzing the level of green fluorescence by"Imagepro-Plus6.0 software", the activity of Survivin promoter is 65.15% of CMV promoter.2. The pSGFP-SEA-PNP plasmid was constructed successfully, which inserted sea-pnp fusion gene was confirmed by restriction enzyme analysis and DNA sequencing. After being transfected into A549 cells, the sea-pnp fusion gene was transcribed successfully, which was confirmed by RT-PCR.3.The lysate of A549 cells transfected by pSGFP-SEA-PNP plasmid can stimulate the PBMC activity and the supernatant has no this function, which confirmed by MTT test. The SI of lysate group, supernatant group and PHA control group is 1.290, 0.855 and 1.637respectively.4.The production of A549 which was transfected by pSGFP-SEA- PNP plasmid can translate the hypotoxic F-ara-AMP to the highly toxic 2-F-A which can kill the tumour cells. The killing rate of experimental group and conctrol group is 74.376% and 55.041% respectively.Conclusions1.The pSGFP(pEGFP-C1/Surp) plasmid was constructed successfully, and the Survivin promoter is tumor-specific, and has high transcriptional activities in lung cancer A549 cells.2.The pSGFP-SEA-PNP plasmid was constructed successfully, which sea-pnp fusion gene was modulated by survivin promoter. RT-PCR confirmed that the sea-pnp fusion gene was transcribed successfully.3.The expression product of transfected A549 cells can not only induce the PBMC activation, but also translate the hypotoxic F-ara-AMP to the highly toxic 2-F-A which can kill the A549 cells directly. |
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