Construction of human interleukin-26 lentivirus expression plasmid and gene knockout plasmid and their activity verification in HEK293T cells
Objective To construct a lentivirus-based expression plasmid and gene knockout plasmid of human interleukin(IL)-26 so as to lay a foundation of studying the function of IL-26 gene in cell signaling pathway and autophagy.Methods IL-26 gene sequence was amplified from human peripheral blood mononuclear cells by RT-PCR and cloned into pCDH-CMV-MCS-EF1-copGFP eukaryotic expression vector to construct overexpression plasmid;Four knockout targets,Exon1sgRNA1,Exon1sgRNA2,Exon3sgRNA1 and Exon3sgRNA2,were designed based on the exon sequence of IL-26,and constructed into lentiCRISPRv2 vector by CRISPR/Cas9 technology to construct gene knockout plasmid.The overexpression plasmid and gene knockout plasmid were transiently transfected into HEK293T cells respectively,and the expression of IL-26 was verified by RT-qPCR and Western blot.In addition,amino acid sequence analysis,structure prediction and subcellular localization observation of IL-26 were performed.Results The results of restriction digestion,sequencing and bioinfor-matics analysis showed that IL-26 was 516 bp in length,encoding 171 amino acids.The IL-26 mRNA level and protein level of HEK293T cells transfected with IL-26 overexpression plasmid increased by 656.789 times and 1.978 times respectively with significant differences as compared with the normal control group(t=17.976 and 7.859,P＜0.000 1 and＜0.001,respectively).With the transfection of 4 knockout targets Exon1sgRNA1,Exon1sgRNA2,Exon3sgRNA1 and Exon3sg-RNA2 into HEK293T cells,the expression of IL-26 decreased by 0.930,0.980,0.523 3 and 0.316 9 times,respec-tively,among which Exon3sgRNA2 significantly down-regulated the expression of IL-26(t=7.440,P＜0.001).IL-26 protein showed signal peptide structure and certain transmembrane function in the first 22 amino acids,which existed in cytoplasm.Conclusion IL-26 overexpression and gene knockout plasmids were successfully constructed,which laid a foundation of the follow-up study of the function of IL-26.
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