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prME和NS1为乙型脑炎病毒两个主要的免疫保护蛋白,且均为N-糖蛋白。为研究N-糖基化对乙型脑炎病毒免疫保护的作用,本研究用PCR介导的定点突变方法,分别消除乙型脑炎病毒prME和NS1基因的不同N-糖基化位点,并构建了prME和NS1突变基因的真核表达质粒。将质粒免疫四周龄雌性小白鼠,经两次免疫后,采集血清检测体液免疫反应,最后对小鼠用强毒进行攻击,观察并记录免疫保护力。研究结果显示,与野生型prME基因免疫组相比,消除单个糖基化位点后prME基因诱导的ELISA抗体、中和抗体和免疫保护力均略有升高,而同时消除两个糖基化位点的则会降低。NS1基因消除单个糖基化位点后保护率高达到100%,但消除两个糖基化位点后则免疫保护率略有降低(75%)。通过本研究证明,N-糖基化在维系乙型脑炎病毒prME和NS1蛋白的免疫保护中具有重要的作用,单个糖基化的缺失可增强蛋白的免疫原性,而两个糖基都缺失后,则造成了免疫效率的降低。
prME and NS1 are the two major immunoprotective proteins of Japanese encephalitis virus, both of which are N-glycoproteins. In order to study the role of N-glycosylation in the immune protection of Japanese encephalitis virus, we used PCR-mediated site-directed mutagenesis to eliminate the N-glycosylation sites of the eME and NS1 genes, respectively, The eukaryotic expression plasmids of prME and NS1 mutants were constructed. The plasmid was immunized four-week-old female mice. After two immunizations, the serum was collected to detect the humoral immune response. Finally, the mice were challenged with virulent virus, and the immunoprotective power was observed and recorded. The results showed that compared with the wild type prME gene immunization group, the prME gene induced a slight increase of ELISA antibody, neutralizing antibody and immunoprotective power after eliminating a single glycosylation site, while eliminating two glycosylation The site will be reduced. The protection rate of NS1 gene was 100% after eliminating single glycosylation site, but the immunoprotection rate decreased slightly (75%) after eliminating two glycosylation sites. It is demonstrated by this study that N-glycosylation plays an important role in maintaining the immunoprotection of prME and NS1 proteins of Japanese encephalitis virus. The deletion of a single glycosylation enhances the immunogenicity of the protein, whereas both glycosylation After deletion, the immune efficiency is reduced.