人IFN-λ1的克隆表达及其在不同种属细胞中的抗病毒活性分析

Cloning and expression of human IFN-λ1 and its antiviral activity on cells from different sources

目的测定IFN-λ1在CaCo2、Vero、BHK-21、MDCK等4种不同种属来源细胞中的抗病毒活性。方法利用RT-PCR技术从人结直肠腺癌细胞(CaCo2)中扩增获得人IFN-λ1 (HuIFN-λ1)基因,然后克隆至真核表达载体pCDNA3.1-Flag-HA中,通过质粒转化、阳性菌落筛选、质粒提取及测序,获得重组质粒pCDNA3.1-HuIFN-λ1;利用脂质体将其转染293细胞,48 h后通过Western blot检测HuIFN-λ1瞬时表达情况,取转染后上清分别作用于CaCo2、Vero、BHK-21、MDCK等四种不同种属来源细胞进行HuIFN-λ1抗病毒活性分析。结果从人结直肠腺癌细胞中扩增获得HuIFN-λ1基因,大小为600 bp,并在293细胞内成功表达HuIFN-λ1,表达产物能抑制VSVG在CaCo2、Vero、BHK-21、MDCK中的增殖,流式细胞仪检测经HuIFN-λ1处理的上述细胞荧光率分别为73.69%、80.12%、71.73%、69.47%。结论重组表达的人IFN-λ1蛋白对4种不同种属来源的细胞均存在一定的抗病毒活性,为研究人IFN-λ1在宿主抗病毒免疫和黏膜免疫中的作用机制及其应用奠定了基础。

Objective To determine the antiviral activity of IFN-?1 in four different cell lines-CaCo2, Vero, BHK-21 and MDCK. Methods The human IFN-?1(HuIFN-?1) gene was amplified from human colorectal cancer cells(CaCo2) using RT-PCR and cloned into the eukaryotic expression vector pCDNA3.1-Flag-HA. Positive colony screening, plasmid extraction, and sequencing were performed to obtain the recombinant plasmid pCDNA3.1-HuIFN-?1;liposome was used to transfect 293 cells, and 48 hours later, the transient expression of HuIFN-?1 was detected using Western blotting. Four different cell lines-CaCo2, Vero, BHK-21, and MDCK-were treated with the supernatant to analyze HuIFN-?1 antiviral activity. Results The HuIFN-?1 gene was amplified from human colorectal adenocarcinoma cells, and it was 600 bp in length. HuIFN-?1 was successfully expressed in 293 cells. The expression product inhibited VSVG in CaCo2, Vero, BHK-21 and MDCK cells. Proliferation and flow cytometry indicated that the aforementioned cells fluoresced at a rate of 73.69%, 80.12%, 71.73%, and 69.47% when treated with HuIFN-?1. Conclusion The recombinant human IFN-?1 protein has certain antiviral activity in four different cell lines. This finding has laid the foundation for study of the mechanism and use of human IFN- 1 in host antiviral immunity and mucosal immunity.