| Purpose:Recombinant adenovirus vector of human SCL gene pDC315-SCL was constructed in order to research SCL gene function in ICC-like cells in high sugar environment.Methods:From containing purpose of the SCL gene of plasmid, Using PCR method for the purpose of the SCL gene, the aim vector pDC315-EGFP to cut with enzymes carry through switching, the product translate into the cell about feeling of states bacteria. To growing of the bacterial colony carry out to identify through the PCR, the PCR positive identify of the clones need to analysis and sequencing, a sequence of analysis is correct, cloning is a success for containing purpose of the plasmid of the SCL gene. HEK293 cells were co-transected with the constructed recombinant shuttle plasmid pDC315-EGFP/SCL and large adenovirus helper plasmid pBHGlox(delta)E1,3Cre in mediation of liposome. The obtained replication-defective recombinant adenovirus pDC315-SCL was propagated in HEK293 cells, purified pDC315-SCL plasmid and determined for virus titer. The distribution and efficiency of recombinant adenovirus mediated hSCL was observed by expression of green fluorescence protein (GFP) under the fluorescent microscope.Results:The length of specific fragment amplified by PCR was 1036bp, the recombinant plasmid pDC315-EGFP-SCL showed two correct bands by the PCR positive identify of the clones and sequencing results were expected, suggesting that SCL gene had been cloned into pDC315-EGFP vector. PCR analysis and Western Blot inspection confirmed that the human SCL gene was successfully inserted into the adenovirus vector. The titer of the recombinant adenovirus was 1×1010PFU/mL. The recombinant adenovirus had a high transfection efficiency up to 95%.Conclusion:. Eukaryotic expression vector pDC315-EGFP-SCL were successfully constructed. Recombinant adenovirus containing human SCL gene was successfully constructed by AdMax vector system. Recombinant adenovirus containing human SCL gene has a high transfection efficiency. |
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