Expression and identification of recombinant adenovirus type 5-Norovirus GⅡ.4-VP1 virus-like particles
10.13200/j.cnki.cjb.004159
- VernacularTitle:重组腺病毒5型-诺如病毒GⅡ.4型-VP1病毒样颗粒的表达及鉴定
- Author:
WANG Mengjun
- Publication Type:Journal Article
- Keywords:
Adenovirus;
Norovirus;
VP1;
Virus-like particle(VLP);
Adenovirus vestor vaccine
- From:
Chinese Journal of Biologicals
2024;37(2):138-142
- CountryChina
- Language:Chinese
-
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.
