人白细胞介素-26慢病毒表达质粒与基因敲除质粒的构建及其在HEK293T细胞中的活性验证

Construction of human interleukin⁃26 lentivirus expression plasmid and gene knockout plasmid and their activity verification in HEK293T cells

目的 构建人白细胞介素(interleukin,IL)-26慢病毒表达质粒与基因敲除质粒,为研究IL-26基因在细胞信号通路及细胞自噬中的功能奠定基础。方法 利用RT-PCR从人外周血单个核细胞中扩增IL-26基因序列,克隆至pCDH-CMV-MCS-EF1-copGFP真核表达载体中,构建过表达质粒;并根据IL-26外显子序列设计4个敲除靶点Exon1sgRNA1、Exon1sgRNA2、Exon3sgRNA1、Exon3sgRNA2,利用CRISPR/Cas9技术构建至lentiCRISPRv2载体中,构建基因敲除质粒。将过表达质粒和基因敲除质粒分别瞬时转染至HEK293T细胞,利用RT-qPCR和Western blot验证IL-26 mRNA和蛋白的表达情况。并对IL-26进行氨基酸序列分析、结构预测及亚细胞定位观察。结果 通过酶切、测序鉴定及生物信息学分析显示,IL-26全长516 bp,编码171个氨基酸。IL-26过表达质粒转染的HEK293T细胞与正常对照组相比,IL-26 mRNA水平升高了656.789倍,蛋白质水平升高了1.978倍,差异均有统计学意义(t分别为17.976和7.859,P分别<0.000 1和<0.001)。4个敲除靶点Exon1sgRNA1、Exon1sgRNA2、Exon3sgRNA1、Exon3sgRNA2质粒转染至HEK293T细胞中后,IL-26的表达水平分别下降了0.930、0.980、0.523 3和0.316 9倍,其中Exon3-sgRNA2质粒能够显著下调IL-26的表达(t=7.440,P <0.001)。IL-26蛋白的前22个氨基酸存在信号肽结构,且具有一定的跨膜功能,IL-26存在于细胞质中。结论 成功构建了IL-26过表达和基因敲除质粒,为后续IL-26功能的研究奠定了基础。

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-CMVMCS-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 bioinformatics 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<0.0001 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 0.930,0.980,0.5233 and 0.3169 times,respectively,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.