扬子鳄α型干扰素蛋白抗病毒活性分析

Analysis of antiviral activity of Chinese alligator IFN-α

为了研究扬子鳄IFN-α抗病毒活性,构建真核表达载体pcDNA3.1-caIFN及pEGFP-N1-caIFN,将pc DNA3.1-caIFN转染至293T细胞中,通过间接免疫荧光试验证明IFN-α成功表达,并显示定位于细胞质中,第48小时扬子鳄IFN-α的表达量要高于第24小时的。将pEGFP-N1-caIFN转染至Vero细胞中,过表达24 h后接种水疱性口炎病毒(VSV),观察细胞,并在VSV感染后第24及48小时收集细胞,通过荧光定量PCR测定病毒VSV-G基因的变化,同时收集上清测定VSV子代病毒滴度,结果表明,与对照组相比,第24及48小时过表达扬子鳄IFN-α的试验组VSV-G基因的表达分别下调4倍及7倍,VSV子代病毒滴度分别低2.3及1.9个数量级。本研究结果证明,真核表达扬子鳄IFN-α蛋白能够抑制VSV mRNA的转录,并能抑制病毒增殖。

In order to study the antiviral activity of Chinese alligator IFN-α,the eukaryotic expression vectors pc DNA3.1-ca IFN and p EGFP-N1-ca IFN were constructed.Transfection of pc DNA3.1-ca IFN into 293 T cells proved the successful expression of IFN-αthrough indirect immunofluorescence experiments,and showed that it was localized in the cytoplasm.The expression level of IFN-αin Chinese alligator at 48 h was higher than that at 24 h.Transfection p EGFP-N1-ca IFN into Vero cells for 24 hours and then inoculate VSV virus.Observe and collect the cells at 24 h and 48 h after VSV infection.The changes in the VSV-G gene of the virus are measured by fluorescence quantitative PCR,and the supernatant is also collected to determine the virus titer of the VSV progeny.The results showed that compared with the control group,the expression of VSV-G gene was down-regulated by 4 times and 7 times in the experimental group overexpressing Chinese alligator IFN-αat 24 h and 48 h,and the VSV progeny virus titer was lower by 2.3 and 1.9 orders of magnitude.Research results have shown that eukaryotic expression of Chinese alligator IFN-αprotein can inhibit the transcription of VSV virus m RNA and inhibit virus proliferation.