在flashBAC杆状病毒表达系统中制备人GⅡ.4型诺如病毒样颗粒

Preparation of virus-like particles of human GⅡ.4 norovirus in the flashBAC baculovirus expression system

摘要:

【目的】人诺如病毒(human norovirus,HuNoV)是全球范围内引起人急性胃肠炎的主要病原之一，目前尚无有效的疫苗对该病毒进行预防。本研究旨在利用flashBAC杆状病毒表达系统，制备HuNoV病毒样颗粒(virus-like particles,VLP)，为研发HuNoV疫苗奠定基础。【方法】将GⅡ.4型HuNoV的VP1蛋白全长基因序列进行密码子优化后合成，克隆至杆状病毒pBacPAK9转移载体，获得pBacPAK9-8his-VP1重组质粒。酶切、测序鉴定正确后将重组质粒与线性baculovirus plasmid (Bacmid)共转染SF9细胞，获得含有VP1基因的重组杆状病毒。重组杆状病毒感染Hi-Five (HF)细胞进行表达，利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)和Western blotting分析VP1蛋白的表达。用镍柱亲和层析方法纯化VP1蛋白；对纯化后的VP1蛋白进行SDS-PAGE和Western blotting检测。使用高效液相色谱(high performance liquid chromatography,HPLC)对纯化的VP1蛋白进行纯度鉴定。利用透射电镜观察VLP。【结果】构建了pBacPAK9-8his-VP1重组质粒，VP1蛋白在HF细胞中主要在细胞质内表达，分子量约为58 kDa。HPLC结果显示VP1蛋白纯度大于99%，透射电镜可以观察到形状规则、大小均一、直径约为30−40 nm的VLP。【结论】利用杆状病毒表达系统制备了GⅡ.4型HuNoV的VLP，为HuNoV疫苗的研发奠定了基础。

Abstract:

[Objective] Human norovirus (HuNoV) is one of the most common pathogens causing acute gastroenteritis in humans worldwide.Currently,there are no approved vaccines to prevent this disease.This study aimed to prepare virus-like particles (VLPs) of HuNoV in the flashBAC baculovirus expression system,laying a foundation for the development of vaccines against HuNoV.[Methods]After codon optimization,the full-length gene sequence of the VP1 protein of HuNoV GⅡ.4 was synthesized and cloned into the baculovirus pBacPAK9 transfer vector to obtain the recombinant plasmid pBacPAK9-8his-VP1.After enzyme digestion and sequencing,the recombinant plasmid was co-transfected with the linear baculovirus plasmid (Bacmid) into SF9 cells to obtain a recombinant baculovirus carrying the VP1 gene.Hi-Five (HF) cells were infected by the recombinant baculovirus for protein expression,and the expression of VP1 was analyzed by SDS-PAGE and Western blotting.VP1 was purified by Ni-NTA affinity chromatography and identified by SDS-PAGE and Western blotting.The purity of VP1 was examined by high performance liquid chromatography (HPLC).The VLPs were observed by transmission electron microscopy.[Results] A transfer plasmid pBacPAK9-8his-VP1 was constructed,and the VP1 protein,with a molecular weight of approximately 58 kDa,was mainly expressed in the cytoplasm of HF cells.The HPLC results showed that the purity of VP1 was over 99%.The VLPs with a regular shape,uniform sizes,and diameters of 30–40 nm were observed by transmission electron microscopy.[Conclusion] The VLPs of HuNoV GⅡ.4 were prepared with a baculovirus expression system,laying a foundation for the development of HuNoV vaccines.