Construction and identification of recombinant lentiviral vectors containing human stem cell leukemia gene

Objective To construct the recombinant lentiviral vectors containing human stem cell leukemia( SCL)gene( GV287-SCL),and to lay the foundation for gene therapy of diabetic cystopathy( DCP). Methods The genetic material from plasmid of SCL gene was amplified by PCR,and was connected to the shuttle plasmid to construct GV287-EGFP / SCL,then they were used to transfect 293 T cells. The recombinant lentivirus GV287-SCL was obtained by multiple infections,amplification and purification. 293 T cells were transfected by different concentrations of recombinant lentivirus. We used Western blotting to detect the target gene,and fluorescence labeling method to calculate the virus titer.Results The size of amplification product from human SCL gene was 1036 bp,which was the same as SCL-c DNA in the target gene associated fragment. After the extension of positive transformants,each formed 503 bp band,whose size was consistent with human SCL c DNA. The SCL target gene of recombinant plasmid GV287-EGFP / SCL positive clones were successfully inserted into the GV287-EGFP,the genetic sequence was the same as the human SCL gene mRNA in Genbank,and the titer of recombinant lentiviral was 5. 0 × 108 TU / m L. Conclusions The recombinant lentiviral vector GV287-SCL is successfully constructed,and the titer is 5. 0 × 108 TU / ml. This study provides a foundation for SCL gene transfection in vivo and gene therapy for DCP.