慢病毒介导siRNA抑制1型鸭肝炎病毒复制

doi:10.11843/j.issn.0366-6964.2017.03.016

Abstract

Abstract:

To screen siRNAs that can suppress the replication of duck hepatitis A virus 1 (DHAV-1) and explore the application of RNAi technology for the control of DHAV-1. The RNA-dependent RNA polymerase (RdRp) gene was amplified by PCR. pEGFP-RdRp was constructed for fusion expression of EGFP-RdRp protein. According to the sequence of RdRp, three gene-specific siRNAs were designed and corresponding shRNA was inserted into pmiRZipΔ to construct pRdRp-shRNA. pRdRp-shRNA and pEGFP-RdRp were co-transfected into HEK293T cells for effective siRNA screening with fluorescent microscopy, flow cytometry, and quantitative fluorescence PCR. More effective siRNAs were selected for preparation of recombinant lentivirus using lentivirus vector pmiRZip. The suppressing effect of entivirus-Mediated siRNA was determined by calculating the TCID₅₀ and RdRp gene expression of DHAV-1 in the duck embryo fibroblast (DEF) cells infected with recombinant lentivirus followed by DHAV-1. The results indicated that the three siRNAs screened all could suppress the RdRp gene expression, and the shRNA2 containing GDD motif had the highest efficiency. The recombinant lentivirus corresponding to shRNA2 reduced the TCID₅₀of DHAV-1 by 6.2 lg and RdRp gene transcription by 89.6%, with the suppressing effect continued for at least 120 h. This work provides a new idea for the clinical control of duck virus hepatitis.

CLC Number:

S852.659.6

WANG Yong-juan, DONG Ya-qing, ZHU Shan-yuan, WANG An-ping, HONG Wei-ming, WU Shuang, ZUO Wei-yong. Suppression of Duck Hepatitis A Virus 1 Replication by Lentivirus-Mediated siRNA[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(3): 522-529.

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Suppression of Duck Hepatitis A Virus 1 Replication by Lentivirus-Mediated siRNA

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