RESUMO
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.
Assuntos
Vetores Genéticos , Fases de Leitura Aberta , Vírus da Síndrome Respiratória e Reprodutiva Suína , Genética , Interferência de RNA , RNA Interferente Pequeno , Genética , Metabolismo , RNA Viral , Química , Metabolismo , Transdução Genética , Proteínas Virais , Genética , Replicação Viral , GenéticaRESUMO
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.