Preparation The P Particles Of GⅡ-4 Norovirus And The Relation With Histo-blood Group Antigens

Objectives To obtain information on viral gene structure and genome,full-length genomic sequence analysis of the norovirus strain GZ20133135 isolated from Guangzhou was performed.Molecular biology methods were applied for the preparation of GII-4 type No V P particles,then the correlation between main genotypes(95/96 US,2006b,and Sydney strains)and histo-blood group antigens(HBGAs)was detected by saliva- and oligosaccharides-based enzyme immune assays(EIA) binding assays,so to discover the binding patterns of the No V epidemic strains with HBGAs in our country, further more,it would contribute to determine the host range of No V epidemic strains,and provide experimental fundamental and theoretical data for further study on the relationship between No V and host receptor, No V vaccine and medicinal treatment of No V.Methods 1 The complete genomic sequence analysis of the Guangzhou Norovirus strain SZ20133135.Primers were designed according to the Sydney2012 full sequence. The full genome of the strain GZ20133135 was amplified by RT-PCR.The whole genome sequence was analyzed after cloned and sequenced. 2 Preparation of P particle of the GII-4 norovirus strain.Sydney2012 3135 which was the representative strains was chosen in this study.The PCR production of P domain from Sydney2012 3135 was inserted into p GEX-4T1 vector to construct recombinant expression plasmid p GEX-4T1-P. The recombinant plasmid p GEX4T1-P was transformed into BL21(DE3) competent cell, then small amount of expression was detected to identify whether fusion protein was expressed based on SDS-PAGE. Then the target protein was expressed abundantly and purified using the Glutathione Sepharose 4B. The P protein was released from fusion protein by thrombin cleavage.3 Afinity analysis of virus from different GII-4 norovirus sub-cluster with HBGAs.Saliva-based binding assays were used to analyze the binding patterns of the three mainly types No V epidemic strains with HBGAs in saliva. 54 saliva samples whose HBGAs phenotypes had been identified to were used coat 96-well microtiter plates. The bound GII-4 No V P protein were detected using rabbit serum anti-95/96 US,2006 b,Sydney No V P, followed by addition of horseradish peroxidase(HRP)-conjugated goat anti-rabbit Ig G. The signal intensities were displayed by a TMB kit, and then the optical density(OD) at 450 nm was read under EIA spectra reader. The oligosaccharides-based binding assays were performed by EIA between the P protein of three No V epidemic strains and the synthetic oligosaccharides of lea、leb、lex、ley、A、B、H1 and H2. The binding patterns of three No V epidemic strains with HBGAs receptor were confirmed by saliva and oligosaccharides-based binding assays in this study.Results 1 The complete genomic sequence analysis of the Guangzhou Norovirus strain SZ20133135.The genome of GII-4 norovirus strain GZ20133135 consist of 7566 bp,which forms three ORFs(ORF1 is male up of 5100 bp, ORF2 is 1623 bp, and ORF3 is807bp), there is 19 nt overlap in the transition domain from ORF1 to ORF2.The total homology sequence is up to 99.07% by evolutionary comparative analysis,which indicates the homology of GZ20133135 and GII-4 Sydney2012 is the highest than that of genomic nucleotide sequences with reference strains of GII-4Sydney2012 strains the GZ20133135 and the other 2.Phylogenetic analyses showed GZ20133135 belonging to GII-4 Sydney2012 variant.Comparision of 541 amino acid from capsid demonstrated that the variant of Sydney2012 strains were as the followings, aa294 V or A or P�'T, aa296 S or T�'S,aa297 H or Q�'R, aa298 D or N or T�'N, aa368 T or N or S or A�'E,aa372 N orS�'D,aa393 N or D or S,aa394 T or G or S�'T,aa395 T or A�'T,aa407 N or D�'S,aa412 T or D�'N,aa413 G or I�'T. 2 Preparation of P particle of the GII-4norovirus strain.991 bp of 3135 P domain were amplified by PCR.Recombinant plasmids4T1-3135-P was successfully constructed, and the sequencing results proved the insert fragment were 972 bp and encoded 324 amino acids. The expression of the soluble recombinant fusion protein and P protein were shown by the SDS-PAGE, in which the fusion protein was 62 k Da and P protein was 36 k Da. 3 Afinity analysis of virus from different GII-4 norovirus sub-cluster with HBGAs.The binding patterns between 3135-P,9711-P and 55019-P No V and saliva HBGAs were discovered by saliva-based binding assays of free P protein. 9711 and 55019 No V could bind to positive saliva in A, B and O+, but not O-; 3135-P No V could bind to positive saliva in A, B, O+ and O- saliva.3135 could combined with lea,leb,A,B,and H2 antigens by oligosaccharides-based binding assays, 55019 could combined with lea,leb,A,B,and H2 antigens,9711 could combined with leb,ley,A,and B antigens.Conclusions 1 GZ20133135 strains belong to GII-4 Sydney2012 mutant strain. 2Norovirus 4T1-3135 expression plasmid P was constructed successful, and the target protein of P particles was prepared.3 3135,9711,and 55019 GII-4 No V bound HBGAs in a strain-specific manner.The binding patterns between 3135-P, 9711-P and 55019-P No V with saliva HBGAs by saliva-based binding assays of free P protein and oligosaccharides-based binding assays were as the followings,9711 and 55019 No V could bind to positive saliva in A, B and O+, but not O-; 3135-P No V could bind to positive saliva in A, B, O+ and O- saliva.