Abstract: Human norovirus (norovirus,NoV) is difficult to be cultured in vitro, so it is not suitable to use the classical vaccine method for vaccine development. The recombinant live virus vaccine based on adenovirus provides an alternative route for the development of norovirus vaccine. The construction of recombinant adenovirus expressing VP1 protein of humanG Ⅱ .4norovirus may lay a foundation for the research and development of vaccine of this technical route. Firstly, the VP1 fragment of norovirus was amplified by PCR, and the pshuttle-CMV-VP1 shuttle plasmid containing VP1 gene was cloned by enzyme digestion, ligation and transformation, and then homologous recombination was performed with adenovirus skeleton plasmid. After correct identification, the recombinant plasmid was digested and linearized byPacⅠenzyme, and transfected into HEK293 cells for virus packaging. The intact adenovirus particles packaged into replication-deficient type were named rAd-VP1. The titer of the virus was determined after passage, and the recombinant adenovirus was infected with HEK293 cells. The expression of recombinant adenovirus VP1 was identified by Western Blot. The results showed that the concentrated titer of recombinant adenovirus after packaging and passage reached CCID50=5×109/mL, and VP1 protein could be successfully expressed in HEK293 cells. It provided a basis for the evaluation of rAd-VP1 in animal immunization and the development of norovirus vaccine.

Key words: norovirus, major structural protein VP1, adenoviral vector, homologous recombination, virus packaging