RESUMEN
We have constructed 2 small interfering RNAs (siRNAs) specifically targeting homogenous 3D and 2B1 regions of 7 serotypes of the foot and mouth disease virus (FMDV) and tested the ability of siRNAs to inhibit virus replication in baby hamster kidney (BHK-21) cells and suckling mice. In this study, we generated transgenic mouse models integrating short hairpin RNA (shRNA) targeting microinfected FMDV. When examined at the 7th passage in transgenic mice, the target gene was still found by PCR to be integrated in the genome. Compared to the control mice, the transgenic mice showed only slightly abnormal pathology when they were infected with the FMDV serotype Asia 1. The number of viruses in the tissues of the transgenic mouse was very low and in some tissues no virus could be detected by immunohistochemistry.
Asunto(s)
Virus de la Fiebre Aftosa/genética , Virus de la Fiebre Aftosa/patogenicidad , Fiebre Aftosa/genética , Fiebre Aftosa/virología , Terapia Genética/métodos , ARN Interferente Pequeño/genética , Replicación Viral , Animales , Línea Celular , Cricetinae , Virus de la Fiebre Aftosa/aislamiento & purificación , Inmunohistoquímica , Ratones , Ratones Transgénicos , Carga ViralRESUMEN
By using bioinformatics computer programs, all foot-and-mouth disease virus (FMDV) genome sequences in public-domain databases were analyzed. Based on the results of homology analysis, 2 specific small interfering RNA (siRNA) targeting homogenous 3D and 2B1 regions of 7 serotypes of FMDV were prepared and 2 siRNA-expression vectors, pSi-FMD2 and pSi-FMD3, were constructed. The siRNA-expressing vectors were used to test the ability of siRNAs to inhibit virus replication in baby hamster kidney (BHK-21) cells and suckling mice, a commonly used small animal model. The results demonstrated that transfection of BHK-21 cells with siRNA-expressing plasmids significantly weakened the cytopathic effect (CPE). Moreover, BHK-21 cells transiently transfected with short hairpin RNA (shRNA)-expressing plasmids were specifically resistant to the infection of the FMDV serotypes A, O, and Asia I and this the antiviral effects persisted for almost 48 hours. We measured the viral titers, the 50% tissue culture infective dose (TCID50) in cells transfected with anti-FMDV siRNAs was found to be lower than that of the control cells. Furthermore, subcutaneous injection of siRNA-expressing plasmids in the neck of the suckling mice made them less susceptible to infection with O, and Asia I serotypes of FMDV.
Asunto(s)
Virus de la Fiebre Aftosa/fisiología , Fiebre Aftosa/virología , Interferencia de ARN , ARN Interferente Pequeño/genética , Replicación Viral , Animales , Línea Celular , Cricetinae , Efecto Citopatogénico Viral , Fiebre Aftosa/mortalidad , Fiebre Aftosa/terapia , Virus de la Fiebre Aftosa/clasificación , Virus de la Fiebre Aftosa/genética , Ratones , ARN Interferente Pequeño/farmacologíaRESUMEN
Objective:To highly express HIV-1p17 in E.coli and purify the protein.Methods:①Recombinant plasmid was constructed by inserting HIV-1p17 gene amplified by PCR into plasmid vector,pET28c;②The recombinant plasmid was expressed in BL21,BL21(DE3),BL21(DE3) plysS and HMS174(DE3) of E.coli separately;③The target protein were purified with Ni-NTA resin;④The purified protein was detected by western blot and ELISA.Results:The expression of the P17 protein in BL21(DE3) represented up to 32% of total protein in E.coli,which was the most amounts compared with other kinds of E.coli.The purity of the purified protein reached 95%.The purified protein was recognized by HIV-1P17McAb as well as by HIV-1 positive serum.Conclusion:The recombinant plasmid is highly expressed in BL21(DE3) of E.coli that can be proceeded to the immunocompetence and the bioactivity research.The method of Ni-NTA resin is simple with low protein losing and high purity.And the purified p17 can be employed in early detection of HIV-1 infection and prediction of the clinical progression.