Construction of eukaryotic expression vector for IL-37 and EGFP fusion protein and its expression in 293T cells / 实用医学杂志

ABSTRACT

Objective To construct the eukaryotic expression vector for IL-37 fused with the enhanced green fluorescent protein (EGFP) and express the fusion protein IL-37-EGFP in 293T cells. Methods The IL-37 and EGFP gene were amplified by PCR, respectively. Then the gene fragments of IL-37 and EGFP were in-serted into the pcDNA3.1 vector to construct the recombinant eukaryotic expression vector pcDNA3.1/IL-37-EGFP. The pcDNA3.1/IL-37-EGFP recombinant expression vector was identified by enzymatic digestion , DNA sequencing, PCR identification. Then the recombinant plasmid pcDNA3.1/IL-37-EGFP was used to transfect 293T cells , the expression of EGFP in 293T cells was detected by fluorescence microscope , the expression of IL-37 in 293T cells was detected by RT-PCR and Western blot. Results The results of enzymatic digestion and DNA sequencing showed that the fusion gene of IL-37 and EGFP linked with (G4S)3 was cloned correctly into pcDNA3.1 vector. The green fluorescence was observed in 293T cells transfected with pcDNA3.1/IL-37-EGFP by fluorescence microscope. RT-PCR and Western blot showed that the expression with high level of IL-37 mRNA and IL-37 protein in 293T cells transfected with pcDNA3.1/IL-37-EGFP , respectively. Conclusion The recom-binant eukaryotic expression vector for IL-37 fused with EGFP, pcDNA3.1/IL-37-EGFP, was constructed suc-cessfully and expressed effectively in 293T cells.