N-糖基化对乙型脑炎病毒prME和NS1基因免疫效果影响的研究

Roles of N-Glycosylation in Immunity of prME and NS1 Gene of JEV

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 gene were the two main immuneprotect proteins of Japanese encephalitis virus （JEV）, and they were also N-linked glycosylation proteins, To clear the effect of N-glycosylation on JEV immunity, the N-glycosylation site of prME and NS1 gene were eliminated by site-directed mutant PCR, subtituting the N to Q. And the the mutant genes were subcloned into eukaryotic expression plasmid. Four-weeks female mice were immuned with the wildtype and mutant gene by twice. The antibodies a- gainst prME were detected by ELISA and the neutralization antibodies were tested by viral neutralizing as- say. The immunoprotection were determined by attack with JEV virulent strain. Compare with the wild- type gene immune＆groups, one N-glycan eliminated prME gene could induce a little higher ELISA anti- body, neutralization antibody and immunoprotection, but the immunity of gene with both N-glycan ab- sence was decreased. The similar status were observed in the wildtype and mutant NS1 groups. Thus these results show that the N-linked glycosylation in the prME and NS1 gene were correlated with the immuni- ty, one glycan absent would enhance the immunity but both two loss would impair it.