猪流感病毒NS1蛋白关键氨基酸位点突变对蛋白定位及细胞因子水平的影响

Effect of the key amino acid of SIV NS1 on cellular localization and cytokine production

为探析猪流感病毒NS1蛋白中影响细胞定位和拮抗干扰素表达的关键氨基酸位点,本研究利用点突变技术扩增A/swine/Shanghai/1/2007(H1N2)分离株NS1突变基因,并构建重组表达质粒pCAGGS-NS1及突变体pCAGGS-NS1_(S42P)、pCAGGS-NS1D_(92E)、pCAGGS-NS1_(S42P/D92E)。将重组真核表达质粒转染细胞,SDS-PAGE和Western-blot均证实NS1蛋白及其突变体蛋白在A549和293T细胞中成功表达。间接免疫荧光试验表明,NS1及其突变体蛋白均主要分布在A549细胞核中,仅有少量分布于细胞浆。此外,试验证明另一株A/swine/Shanghai/3/2014(H1N1)分离株的NS1及其突变体蛋白也主要分布于细胞核中,说明点突变不影响SIV NS1蛋白的细胞定位。进一步利用荧光定量PCR和ELISA方法检测A/swine/Shanghai/1/2007(H1N2)分离株NS1点突变蛋白拮抗细胞中干扰素表达的影响,结果表明,pCAGGS-NS1_(S42P)转染组能够明显增强IFN-βmRNA、IFN-αmRNA转录水平(P<0.01;0.01<P<0.05),但对TNF-αmRNA转录水平无明显影响(P>0.05)。而pCAGGS-NS1D_(92E)和pCAGGS-NS1S_(42P/D92E_转染组细胞IFN-α、IFN-β和TNF-αmRNA转录水平均无明显变化(P>0.05)。试验证实NS1蛋白第42位氨基酸对拮抗宿主细胞中IFN-α/β表达具有重要作用。

To explore the effects of key amino acids of SIV NSI protein on the cellular localiza- tion and cytokine production,NSl gene of A/swine/Shanghai/1/2007（HIN2） was amplified and cloned into the eukaryotic plasmids,named pCAGGS-NSI. The 42 and 92 amino acid mutants of NSI proteins were also constructed and were named pCAGGS-NSls4p, pCAGGS-NSIm2E, pCAGGS-NSIs42P/m2E. SDS-PAGE and West- ern-blot analysis confirmed that NSI and mutant proteins were expressed in A549 and 293T cells suc- cessfully. Indirect immunofluorescence test indicates that NSI mainly located in the nucleus and only a few in cytoplasm which was the same as NSI and its mutant proteins of A/swine/Shanghai/1/2014 （HINI）. Mutation of 42 and/a 92 amino acids didn＇t change the cellular localization of NSI protein.Fluorescence quantitative PCR and ELISA were used to detect the cytokine production in the cells treated with poly（I：C）.Results showed that NSls4p enhanced the mRNA transcriptional level of IFN-β （P〈0.01） and IFN-a（0.01〈P〈0.05） with no influence on TNF-a（P〈0.05）.Besides,both of NSIand NSIS2E had no obvious influence on IFN-a/β and TNF-a production. This study confirmed the impor- tant role of the 42 amino acid of NS1 in antagonistic action on IFN-a/fl.