Selection and inhibitory effect analysis of siRNAs specific to ORF2-4 of porcine reproductive and respiratory syndrome virus / 生物工程学报

RNA interference (RNAi) is a powerful tool in gene function research. In order to investigate the role of GP2, GP3 and GP4 of porcine reproductive and respiratory syndrome virus (PRRSV) in the viral replication, small interference RNAs (siRNAs) directed to ORF2, ORF3 and ORF4 were designed and 12 short hairpin RNA (shRNA) expression vectors were constructed (designed as 21,22,23,24,31,32,33,34,41,42,43 and 44). Cells treated with shRNA expression vectors were infected by PRRSV. The effective shRNA expression vectors were selected by fluorescent quantatitive PCR (FQ-PCR). The virus titer of supernatant of the cells treated with effective shRNA expression vectors (23,24,31,34 and 41) were reduced by 184 to 4.65 folds compared with that of controls.

Identification of mimotope peptides which bind to the SARS-CoV spike protein specific monoclonal antibody 2C5 with phage-displayed peptides library / 生物工程学报

To identify the epitope of SARS-CoV spike protein specific neutralizing monoclonal antibody (MAb) 2C5. The antibody was used as target and three rounds of bio-panning were conducted with phage-display peptide library. After the third panning, 20 phage-plague clones were randomly picked and analyzed for the binding ability with the MAb 2C5 by ELISA. The display sequence analysis demonstrated that among the twenty phage clones, eight clones displayed the same seven-peptide TPEQQFT. All these eight phage-clones showed strongest binding activity with 2C5 in phage ELISA analysis. Furthermore, phages displaying peptide TPEQQFT could specifically inhibit the binding of MAb 2C5 with SARS-CoV spike protein. The results demonstrated that TPEQQFT is a mimic epitope peptide containing neutralizing MAb 2C5. This study may provide information for further structural and functional analysis of spike protein and development vaccine for severe acute respiratory syndrome.

Development of monoclonal antibodies against SARS-CoV and identification of antigenic epitopes / 生物工程学报

Based on the genomic sequence of SARS-CoV strain BJ101, antigenic immunodominant genes coding for the structure proteins of SARS-CoV were predicted by bio-informatics methods, and two chimeric genes A and B with multi-immunodominants lined up by Gly-Pro-Gly linker were synthesized. The chimeric genes were cloned into plasmid pGEX-6p-1 and expressed in E. coli with IPGT inducing. BALB/c mice were immunized with the purified recombinant fusion protein. The specificity of monoclonal antibodies were tested with a commercial ELISA kit for detecting antibody against SARS-CoV. The results showed that two peptides with molecular weights of 34kD and 35kD expressed by the two chimeric genes could be recognized by SARS patient convalescent serum in Western blot. Six positive hybridoma cell lines stably secreting monoclonal antibodies were selected. The subtype of monoclonal antibody D3C5 is IgG2a, and subtypes of all other five monoclonal antibodies are IgG1. Light chains of all monoclonal antibodies are kappa. With a commercial SARS-CoV antibodies detection ELISA kit, five out of six monoclonal antibodies were positively recognized. In western blot analysis with inactived virus cultures, D3D1 specifically recognized a band of about 180 kD. To further analyse the epitopes corresponding to the monoclonal antibodies, six oligoes (S1-S6) from S gene were synthesized and expressed. The results showed that the monoclonal antibodies D3D1 and D3C5 specifically recognized expression product of S2 and S5 oligoes, respectively. The S2 and S5 oligoes are corresponding to 447-458aa and 789-799aa of SARS-CoV S protein respectively.