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

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.