Construction of the lentiviral expression plasmid carrying human interleukin-1 receptor antagonist gene and its expression in NIH3T3 cells

AIM: To construct the recombinant lentiviral expression plasmid pLenti-IL-1ra-IRES-GFP carrying human interleukin-1 receptor antagonist gene and test its stable expression in NIH3T3 cells.METHODS: Human interleukin-1 receptor antagonist gene was amplified from the plasmid pEGFP-N1-IL-1ra with PCR and subcloned into the plasmid pLenti.PCR and DNA sequencing were used to identify the recombinant plasmid pLenti-IL-1ra.IRES-GFP gene which was cut from plasmid PMIG was inserted into the correct plasmid pLenti-IL-1ra to construct the lentiviral expression plasmid pLenti-IL-1ra-IRES-GFP.The recombinant lentivirus was generated by 293T cells co-transfected with the packaging plasmid Δ8.91,the envelope plasmid pVSVG and the expression plasmid pLenti-IL-1ra-IRES-GFP.After NIH3T3 cells were infected by the recombinant lentivirus,the expression of IL-1ra gene in NIH3T3 cells was detected by fluorescence microscope.After screened with Blasticidin for 15d,the expression of mRNA of IL-1ra in NIH3T3 cells was detected by RT-PCR,and the expression of IL-1ra protein was detected by ELISA.RESULTS: The lentiviral expression plasmid pLenti-IL-1ra-IRES-GFP carrying the correct IL-1ra gene sequence and IRES-GFP gene,and the recombinant lentivirus Lenti-IL-1ra-IRES-GFP was generated by the packaging 293T cells.After the infection,the efficient and stable expression of IL-1ra gene in NIH3T3 cells could be identified by fluorescence microscope,RT-PCR and ELISA.CONCLUSION: The recombinant lentiviral expression plasmid pLenti-IL-1ra-IRES-GFP was successfully constructed and NIH3T3 cells could express IL-1ra stably after being infected by the recombinant lentivirus.