[Construction of a lentiviral vector containing human NESG1 gene and its expression in 293FT cells]
Zhen Liu1, Yan Zhen, Xiao-li Yu
1Department of Pathology, Guangzhou Medical University, Guangzhou 510182, China.
Objective:
To construct a lentiviral vector carrying human NESG1-EGFP gene and observe its expression in 293FT cells.
Methods:
The CDS region of NESG1 gene was amplified from a plasmid containing the full-length NESG1 sequence and cloned into the lentiviral vector pGC-FU-EGFP by restriction endonuclease AgeI digestion and T(4) DNA ligase ligation. After transformation into competent E. coli cells, the candidate clones were identified by PCR and sequencing. The recombinant plasmid and the two packaging plasmids were co-transfected into human embryonic kidney cell line 293FT cells by lipofectamine 2000 to produce the lentiviral particles, and the viral titer was determined. The 293FT cells were infected by the lentiviral particles obtained and the transfection efficiency was assessed under fluorescent microscope. Western blotting was used to detect the expression of NESG1 protein in the transfected cells.
Results:
The lentiviral vector pGC-FU-NESG1-EGFP for NESG1 gene was constructed successfully. Strong green fluorescence was observed in 293FT cells under fluorescent microscope after co-transfection of the cells with the 3 plasmids of lentiviral vector. The virus in the supernatant reached a titer of 2×10(7) TU/ml. The transfection efficiency of the collected virus exceeded 90% in 293FT cells with a multiplicity of infection of 1. Western blotting identified the presence of NESG1 expression in the transfected 293FT cells.
Conclusion:
The lentiviral vector for NESG1 has been successfully constructed with a high yield of lentivirus, which facilitate further investigation of the roles of NESG1 gene in the development and progression of nasopharyngeal carcinoma.
