ABSTRACT
@#Objective To express and identify recombinant adenovirus type 5-Norovirus(NoV)GⅡ.4-VP1 virus-like particles(VLPs)in 293T cells. Methods The recombinant adenovirus plasmids pAd5-eGFP and pAd5-NoV-GⅡ.4-VP1 were transfected into 293T cells respectively,and the recombinant adenovirus rAd5-eGFP and rAd5-NoV-GⅡ.4-VP1 were rescued. The rAd5-eGFP was subcultured in 293T cells to verify the function of the vector. The rAd5-NoV-GⅡ.4-VP1 was subcultured in293T cells,expressed and purified,and then NoV-GⅡ.4-VLP was formed by self-assembly,which was detected by Western blot,ELISA and observed by transmission electron microscope. Results The green fluorescence of the recombinant adenovirus rAd5-eGFP of various generations was observed under microscope,and the brightness increased with the increase of generations. NoV-GⅡ.4-VP1 protein was expressed in the harvested solution of recombinant adenovirus rAd5-NoV-GⅡ.4-VP1 of various generations,with a relative molecular mass of about 58 900. NoV-GⅡ.4-VLP showed specific binding to the rabbit anti-NoV-GⅡ.4-VP1 serum;it had similar conformation to natural NoV virus particles and can effectively identify NoV receptors in volunteer saliva samples;microscopic observation showed that the morphology was complete and spherical,with a diameter of 43. 5 — 58. 3 nm,while there were a few protrusions on the surface of the particles,which might be the P domain exposedon the particle surface during self-assembly of VP1. Conclusion The expressed recombinant adenovirus NoV-GⅡ. 4-VLP has complete VLP structure and good specificity,and is expected to be used in the related research of NoV adenovirus vector vaccine.
ABSTRACT
@#In recent years,more and more coronaviruses(CoV)have crossed the species barriers and spread from animals to human,causing many serious public health events and a blow to the global economy. Vaccination is the most effective measure to prevent coronavirus pandemic. A safe,effective and time-save vaccine platform is a critical requirement of vaccine research and development. With the increasing maturity of reverse genetics technology,it also plays an increasingly important role in the development of CoV vaccines. This paper reviewed several reverse genetics techniques used in CoV research and their possible directions in CoV vaccine development.