IFN-λ1在HEK293T细胞中表达纯化及活性检测

Expression,purification and activity of IFN-λ1 in HEK293T cells

[目的]构建IFN-λ1真核表达质粒,利用人胚胎肾HEK293T细胞表达系统,获得具有良好生物学活性的IFN-λ1重组蛋白。[方法]将IFN-λ1目的基因克隆到pcDNA3.1+载体NheⅠ与XhoⅠ多克隆位点构建pcDNA3.1-IFN-λ1分泌表达质粒,并将其转染到HEK293T细胞中;采用Ni-NTA亲和层析方法分离纯化重组蛋白,SDS-PAGE和蛋白免疫印迹(Western Blotting)检测IFN-λ1的表达与纯度;采用qPCR、WB结合显微镜观察检测IFN-λ1的生物学活性。[结果]IFN-λ1真核表达质粒构建正确,而且能够在HEK293T细胞中分泌表达重组蛋白,分离纯化的IFN-λ1能够有效地诱导ISG15、ISG54、ISG56、OAS1、TNFα、MX1和TRAIL等凋亡相关基因的表达,激活p38促凋亡信号通路,抑制水泡性口炎病毒对BHK-21细胞的感染。[结论]成功构建了pcDNA3.1-IFN-λ1真核表达质粒,能够在HEK293T细胞中分泌表达IFN-λ1重组蛋白;分离纯化的IFN-λ1重组蛋白具有潜在的抗肿瘤和抗病毒生物学活性,为进一步研究IFN-λ1的功能和临床应用奠定了基础。

[Objective]To construct the eukaryotic expression plasmid of IFN-λ1,and to obtain the recombinant IFN-λ1 protein with good biological activity by using the human embryonic kidney HEK293 T cell expression system.[Method]The target IFN-λ1 gene was cloned into pcDNA3.1+vector NheⅠand XhoⅠpolyclonal sites to construct pcDNA3.1-IFN-λ1 expression plasmid.The recombinant protein was expression HEK293 T cells and isolated by Ni-NTA affinity chromatography.The expression level and purity of IFN-λ1 were assessed by SDS-PAGE and Western Blotting.Finally,The biological activity of IFN-λ1 was detected by qPCR,WB and microscope.[Result]The IFN-λ1 eukaryotic expression plasmid was correctly constructed,and the recombinant IFN-λ1 could be expressed in HEK293 T cells and secreted into cell culture supernatant The purified IFN-λ1 could effectively induce the expression of numerous apoptosis-related genes such as ISG15,ISG54,ISG56,OAS1,TNFα,MX1 and TRAIL,and activate the p38 pro-apoptotic signaling pathway.In addition,IFN-λ1 recombinant protein can also inhibit the infection of Vesicular Stomatitis Virus to BHK-21 cells.[Conclusion]The eukaryotic expression plasmid pcDNA3.1-IFN-λ1 was successfully constructed,which could secrete and express the recombinant protein IFN-λ1 in HEK293 T cells.The IFN-λ1 can be easily purified by Ni-NTA affinity chromatography from the cell culture supernatant.The purified recombinant IFN-λ1 protein has the potential of antitumor and antiviral biological activities,which lays a foundation for further study on IFN-λ1 function and clinical application.