Construction and identification of eukaryotic expression plasmid carrying hTERT-P2A-EGFP
10.13481/j.1671-587x.20170201
- VernacularTitle:携带hTERT-P2A-EGFP基因真核表达质粒的构建和鉴定
- Author:
Xiaona CHEN
;
Xiaodan WANG
;
Liguang SUN
;
Fang FANG
;
Weiwei CUI
;
Yongguang YANG
;
Ya LIU
- Keywords:
human telomerase reverse transcriptase;
green fluorescence protein;
plasmid construction
- From:
Journal of Jilin University(Medicine Edition)
2017;43(2):213-219,封2
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct the eukaryotic expression plasmid carrying hTERT-P2A-EGFP, and to explore its expression and transfection efficiency in the HEK293FT cells.Methods:The recombinant plasmid was constructed by using pBABE-puro-hTERT and pRRLSIN-cPPT-MSCV-EGFP plasmids.The hTERT,P2A,and EGFP genes were obtained using pBABE-puro-hTERT as template by PCR.And the correct hTERT was inserted into pRRLSIN-cPPT-MSCV-EGFP vector.Then the recombinant plasmid containing hTERT-P2A-EGFP gene was obtained and identified.The HEK293FT cells were transfected by the recombinant plasmid, and the expression of green fluorescence protein(GFP) was observed by fluorescence microscope.Results:The PCR results showed that the fragments of hTERT, P2A, and EGFP were 3 400, 110 and 720 bp.And the length of gene fragment(hTERT-P2A-EGFP)was 4 300 bp by enzyme digestion.The results of sequencing showed that the 1 547 site of the target gene was mutated.Using site-directed mutagenesis, the 1 547 site was successfully mutated.And the target gene sequence was completely identical with the sequence published in GenBank.The recombinant plasmid was transfected into the HEK293FT cells, and GFP was observed in the cells.The results of flow cytometry showed that the transfection efficiency of recombinant plasmid was 44.8%.Conclusion:The recombinant plasmid carrying hTERT-P2A-EGFP gene is successfully constructed, and it can be used for cell transfection.
