Construction And Identification Of Eukaryotic Expression Plasmid Carrying HTERT-P2A-GFP

ObjectiveTo construct the eukaryotic expression plasmid carrying h TERT-P2A-EGFP,and exploring its expression level and transfection rate in the HEK293 FT cells.And to lay the foundation for further study of h TERT gene function and construction of immortalized cell lines.MethodsThe recombinant plasmid was constructed by using p BABE-puro-h TERT and p RRLSIN-c PPT-MSCV-EGFP plasmids.The h TERT,P2 A,and EGFP genes were obtained by using plasmids as template by PCR.The PCR products were purified by kit.In addition,the target fragment h TERT-P2A-EGFP was obtained by overlapping PCR using the purified three fragments as template.Then the fragment h TERT-P2A-EGFP was inserted into p RRL-vector after digestion.So the recombinant plasmid containing h TERT-P2A-EGFP gene was obtained.The sequence of the recombinant plasmid was tested.The primers which containing the correct base was designed,and the site mutation was performed by using Fast Digest Dpn I.The recombinant plasmid containing h TERT-P2A-EGFP gene was identified.The HEK293 FT cells were transfected by recombinant plasmid.And the expression of green fluorescent protein was observed by fluorescence microscope.The transfection efficiency of recombinant plasmid in HEK293 FT cells was detected by flow cytometry.Four plasmids were packaged for virus and the required plasmids were transfected into the 293 FT cells line by using lipo2000.The collected virus was subjected to flow detection and thus learned the virus titer and infectivity.After the human embryonic hepatic cells were transfected by recombinant virus,extracted RNA,then detected the expression level of h TERT gene by RT-PCR.Results1.The PCR results showed that the fragments of target genes h TERT,P2 A,and EGFP were 3500,110 and 720 bp.2.The electrophoretic results showed that the fragments of h TERT-P2A-EGFP was about 4300 bp,which was consistent with the expected results.3.The results of sequencing showed that the 1547 site of the target gene was mutated.By using site-directed mutagenesis,the site was successfully mutated.And the target gene sequence was completely identical with the sequence published in Gen Bank.4.The recombinant plasmid p RRL-h TERT-P2A-EGFP was digested and electrophoresed to produce about 4300 bp target bands.Thus the plasmid was successfully constructed.5.The recombinant plasmid was transfected into the HEK293 FT cells,and the green fluorescent protein could be observed in the cells.6.The results of flow cytometry showed that the transfection efficiency of recombinant plasmid was 44.8%.7.The results of flow cytometry showed that the positive rate of GFP in 293 FT cells was 32.7%,and the titer of recombinant lentivirus was 1.6×107 after the h TERT virus infected cells.8.The expression of h TERT gene in the experimental group was significantly higher than that in the control group by RT-PCR.Conclusion1.The sequence of target gene h TERT-P2A-EGFP was consistent with the sequence published in Gen Bank.2.The recombinant plasmid p RRL-h TERT-P2A-EGFP was successfully constructed after restriction enzyme digestion.3.The recombinant plasmid was transfected into HEK293 FT cells and the target gene could be expressed in cells.4.The expression of h TERT gene was increased in the cells after virus infection.