Targeted inhibition of Rabies Virus replication in vitro by single chain antibody domain mediated vector expression shRNA delivery

Abstract:

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 35h 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.