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  • Title: [Targeted inhibition of rabies virus replication in vitro by single chain antibody domain mediated vector expression shRNA delivery].
    Author: Yang R, Cui Y, Yang S, Wang C, Shan H, Wang H, Xia X.
    Journal: Wei Sheng Wu Xue Bao; 2010 Feb; 50(2):256-62. PubMed ID: 20387470.
    Abstract:
    OBJECTIVE: Single chain antibody-mediated delivery is a novel approach for targeting shRNA to appropriate cells. In this report, we studied whether this shRNA delivery strategy would be effective against rabies virus. METHODS: Rabies virus scFv (G) gene and ETA-GAL4 gene were amplified by PCR from vector scFv (G)-T and PE40-GAL4-T respectively. Then, the chimeric gene scFv (G)-ETA-GAL4 was constructed by lapextension PCR and cloned into the prokaryotic expression vector pET28a( +). Recombinant expression plasmid of pET28a(+)-scFv(G)-ETA-GAL4 was constructed and then transformed into the competent E. coli BL21 (DE3) cells for expression under the induction of IPTG. scFv(G)-ETA-GAL4 protein was purified by Ni-NTA His Bind Resin affinity chromatograph and identified by SDS-PAGE gel and Western blot assay. Binding of the fusion protein scFv(G)-ETA-GAL4 to rabies virus was determined by ELISA. Complexes which formed spontaneously by the fusion protein scFv(G)-ETA-GAL4 with plasmid pRNATU6.3-shRNA were added to BHK-21 cells culture medium that infected with RV. Green fluorescent protein (GFP) was observed after 35 h and judged the transferring efficiency of the complexes. The inhibition of RV replication by shRNA was detected by direct immune fluorescence test. RESULTS: A 1557 bp DNA encoding scFv (G)-ETA-GAL4 protein gene was cloned and successfully expressed in inclusion body with approximate molecular weight of 57.0 kDa, which could be recognized by anti-His mAb. The scFv(G)-ETA-GAL4 proteins were purified by Ni-NTA column , and after renatured with the yield of 2.8 mg/mL. The ELISA results showed that when concentration of the scFv(G)-ETA-GAL4 protein ranging from 2.8 nmol/L to 1000 nmol/L, binding affinity is directly related with RV. The GFP expressed in BHK-21 cell after transfection with the complexes and effectively inhibited RV replication in BHK-21 cell. CONCLUSION: scFv(G)-ETA-GAL4 fusion protein could mediated plasmid pRNATU6.3-shRNA transferred into BHK-21 cell infected with RV, and then inhibited RV replication.
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