2020年至今，新型冠状病毒SARS-CoV-2已经在全球大流行。新冠疫苗被寄于厚望用以减轻防控压力，成为解决疫情危机的有效手段之一。SARS-CoV-2通过S蛋白受体结合区RBD与宿主细胞ACE2受体结合侵染机体。本文提供了一种利用新型糖基工程毕赤酵母表达系统，制备基于S蛋白RBD重组亚单位候选疫苗的方法。该糖基工程酵母的糖基化修饰途径经过人源化改造后，具有类似于哺乳动物细胞糖基化修饰特点。研究发现，RBD候选疫苗可以有效地诱导小鼠产生高滴度的抗RBD特异性抗体，含Al(OH)₃和CpG双佐剂的RBD免疫组小鼠产生的特异性抗体滴度和病毒中和抗体滴度显著地高于Al(OH)₃单佐剂组，且中和抗体可以在小鼠体内持续6个月以上。综上所述，本文利用糖基工程酵母制备了SARS-CoV-2 S蛋白RBD糖蛋白疫苗，该疫苗能够诱导小鼠产生高滴度中和抗体，同时提供了一种可制备具有无岩藻糖的复杂型N-糖基化修饰重组蛋白的方法。

In 2020 and 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus, caused a global pandemic. Vaccines are expected to reduce the pressure of prevention and control, and have become the most effective strategy to solve the pandemic crisis. SARS-CoV-2 infects the host by binding to the cellular receptor angiotensin converting enzyme 2 (ACE2) via the receptor-binding domain (RBD) of the surface spike (S) glycoprotein. In this study, a candidate vaccine based on a RBD recombinant subunit was prepared by means of a novel glycoengineered yeast Pichia pastoris expression system with characteristics of glycosylation modification similar to those of mammalian cells. The candidate vaccine effectively stimulated mice to produce high-titer anti-RBD specific antibody. Furthermore, the specific antibody titer and virus-neutralizing antibody (NAb) titer induced by the vaccine were increased significantly by the combination of the double adjuvants Al(OH)₃ and CpG. Our results showed that the virus-NAb lasted for more than six months in mice. To summarize, we have obtained a SARS-CoV-2 vaccine based on the RBD of the S glycoprotein expressed in glycoengineered Pichia pastoris, which stimulates neutralizing and protective antibody responses. A technical route for fucose-free complex-type N-glycosylation modified recombinant subunit vaccine preparation has been established.