Construction of the lentiviral expression system for small proteins

Objective For further study the function and mechanism of chemotactic protein,to establish a lentiviral expression system and to construct a human ovarian cancer cell line that can express green fluorescent protein(GFP) stably.Methods Chemotactic factor CCL18,IL-8 and CXCL1 gene plasmids were amplified by PCR,restricted by endonuclease digestion and connected with lentiviral vector PWPI.The recombinant lentiviral vector was then mixed with the envelope plasmid PCMV-dR8.74 and packaging plasmids PMD2.G in proportion.LipofectinTM2000 293T cells were co-transfection with packaging viral particles.The viruses harvested from the supernatant were used to transfect ovarian cancer SKOV3 cells.The cells that strongly expressed GFP were selected by FACS,which served as the successfully transfected cells.The target genes were measured by Real-time fluorescent quantitative PCR.Western blot was adopted to confirm the expression levels of the target proteins.Results The efficiency of infection of the cells that express target gene was more than 95%.Real-time fluorescent quantitative PCR tests showed that the expression level of target gene improved significantly comparing to the control cells and tissues(P0.05).The expression of chemotactic protein in the transfected cells was validated by Western blot.Conclusion A lentiviral expression system for small protein is successfully constructed,which is an experimental basis for further study the function and mechanism of small-molecule protein.