RESUMEN
OBJECTIVE: To construct, express and purify human fusion proteins composed of a single-chain antibody fragment scFv that recognizes the prostate specific membrane antigen (PSMA) protein, Fdt, HA2 and tp, and to analyze the binding activity of the expressed fusion proteins. METHODS: The fusion protein genes scFv, scFv-tp, and scFv-Fdt-HA2-tp were amplified by PCR, and the genes obtained were then cloned into the expression vector pET28 and expressed in E. coli BL21. The expressed products were identified by SDS-PAGE and Western blot and purified with Ni(2+)-NTA chelating agarose. The antigen-binding activity of the fusion proteins was determined by ELISA. RESULTS: The human anti-PSMA fusion gene was successfully constructed and expressed in M15 as the inclusion body after induced with IPTG. All the target proteins expressed could bind the PSMA antigen. CONCLUSION: Fusion proteins can specifically bind the PSMA antigen. This finding contributes to the study of the targeted delivery of siRNA.
Asunto(s)
Antígenos de Superficie/inmunología , Glutamato Carboxipeptidasa II/inmunología , Anticuerpos de Cadena Única/genética , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/inmunología , Humanos , Masculino , Reacción en Cadena de la Polimerasa , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Anticuerpos de Cadena Única/inmunologíaRESUMEN
OBJECTIVE: To investigate the inhibitory effect of RNA interference (RNAi) on the expression of survivin mRNA and its inducibility of the apoptosis of PC-3 cells. METHODS: siRNA expression vectors were designed and constructed to be directed at survivin and transfected into PC-3 cells by Lipofectamine in 4 groups: plasmid A, plasmid B, negative sequence and control E. RT-PCR, Western blot and flow cytometry were used to detect the expression of survivin mRNA and the apoptosis of PC-3 cells. RESULTS: The expression rates of survivin protein were 18.94% +/- 0.63%, 16.35 +/- 0.23%, 46.41% +/- 0.76% and 46.20 +/- 1.47%, those of survivin mRNA were 27.94% +/- 1.43%, 24.51% +/- 1.37%, 49.46% +/- 0.71% and 48.49% +/- 1.32%, and the apoptosis rates of PC-3 cells were 12.80% +/- 1.33%, 16.48% +/- 1.00%, 3.03% +/- 0.62% and 2.96% +/- 0.41% respectively in different groups. CONCLUSION: RNAi can effectively inhibit the expression of survivin mRNA and induce the apoptosis of PC-3 cells.