| Human Norovirus(HuNoV)is one of the main causes of global acute gastroenteritis.The lack of mature in vitro culture systems or small animal infection models has greatly hindered the study of virus infection mechanisms,and there are still no vaccines or specific drugs on the market.Virus-like particles(VLPs)are very similar to natural virus particles in terms of size and antigenicity,so they are used as a good substitute for studying Hu No V vaccines and protein mechanisms.Minor capsid protein(VP2)plays an important role in the protein mechanism,but the specific mechanism of interaction with major capsid protein(VP1)has not been reported yet,thus strengthening the research on VP2 protein is beneficial to the analysis of the virus mechanism.Hu No V has rich genetic diversity,and it is necessary to systematically grasp the protein interaction between different genotypes.The thesis combined with the previous epidemiological monitoring of Guangzhou local Hu No V,the VLP antigen library was constructed,and Guangzhou local Hu No V important genotype VLP was selected as the research object,and its immunogenicity and receptor binding ability were systematically evaluated.The effect of the minor capsid protein VP2 on VLP stability was investigated.The interaction of capsid proteins among different Hu No V genotypes was studied by yeast two-hybrid technique.The specific results are as follows:(1)Preparation and evaluation of virus-like particles of important genotypes of human Norovirus.The strains(GZ2013-L10 and GZ2013-L20)isolated from patients with acute diarrhea in Guangzhou were selected as the research objects,the recombinant baculovirus vector Bacmid-ORF2 was successfully constructed,VLP was expressed in insect cells sf9,and the complete VLP was obtained by ultra-purification.Electron microscope demonstrated that the particle diameter was about 30 nm.SDSPAGE and Western Blot showed that the relative molecular weight of the protein was about 58 k Da.The salivary receptor binding test results showed that the two strains VLP could bind to secretory HBGA receptor and porcine gastric mucin(Porcine Gastric Mucin,PGM),which the strain GZ2013-L20 can also bind to the non-secretory HBGA receptor.Polyclonal antibodies with a titer of above 105 was detected in the immunized mice.The cross-reaction results of VLP polyclonal antibodies showed that only the strain GZ2013-L20 showed broad-spectrum binding to the P particle proteins of most genotypes(20/28)of Hu No V,but the cross-blocking test results showed that they only had neutralizing and blocking effects on VLP of the same genotype,and had no neutralizing effect on VLP of different genotypes(including GII.8,GII.17,GII.2 and GII.3/GII.4).(2)The effect of the minor capsid protein VP2 on virus-like particle stability.GII.4and GII.17 strains were selected as the research objects,baculovirus vectors BacmidORF2 and Bacmid-(ORF2+ORF3)were successfully constructed,and VLP without VP2 protein and VLPs with VP2 protein were prepared.The electron microscope results showed that the diameter of VLPs was about 30 nm.SDS-PAGE and Western Blot showed that the relative molecular weight of VP2 protein was about 29 k Da.The protein expression of VP1 protein and VP2 protein after co-transfection of sf9 cells was higher than that after single transfection(VP1/VP2)protein expression.The immunogenicity and receptor function of VLPs and VLPs were verified after repeated freezing and thawing.The results showed that GII.4 and GII.17 VLPs still had immunogenicity and receptor binding function after 17 and 19 freeze-thaw times,and VLPs containing VP2 protein still have immunogenicity and receptor binding function after 2 more freeze-thaw times.(3)Study on the interaction mechanism of major and minor capsid proteins between different genotypes of human norovirus.14 genotypes were selected(common genotypes: GI.1,GII.3,GII.4 and GII.17,uncommon genotypes: GI.5,GI.8,GI.9,GII.1,GII.2,GII.5,GII.8,GII.10,GII.20 and GII.21)to construct yeast expression vectors p GADT7-ORF2 and p GBKT7-ORF3.The self-activation and toxicity verification results show that the bait protein does not have self-activation phenomenon,and had no obvious inhibitory effect on yeast growth compared with empty vector.Phylogenetic analyses based on ORF2 and ORF3 regions yielded roughly the same results.The results of the cross-interaction test showed that the major and minor capsid proteins of the 14 strains interacted,and the interaction ability of proteins of the same strain was stronger than the cross-interaction ability of different strains(p=0.0022).All have interactions within the GI genogroup,and most of the GII genogroups have interactions,which the cross-interaction results between the GI and GII genogroups are significantly different,and the protein interaction ability within the same genogroup is stronger than the different genogroups Protein interaction ability(p=0.043).Among the common genotypes,when GII.17 was used as a bait protein,the interaction ability was weak(p<0.0001).The closely related strains showed roughly the same protein interactions.It is worth noting that the GII.4 genotype Guangzhou strain GZ2013-L10 interacted with each other except for individual genotypes,whether as a bait protein or a prey protein.In conclusion,this study prepared and systematically characterized the receptor function and immunogenicity characteristics of VLPs of important Hu No V genotypes,which provided necessary basic data for the strain selection during the development of multivalent VLP vaccines and the exploration of virus evolution rules.It was investigated that VP2 protein can increase the expression of capsid protein and improve the freeze-thaw resistance of VLPs,which provides a new way for mechanism research and epidemic prevention and control.With the help of yeast two-hybrid technology,the interaction between major and minor capsid proteins of different genotypes was verified,and the interaction relationship between proteins was mastered,which provided a theoretical basis for mining the key amino acid sites of protein interaction. |
PDF Full Text Request