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

畜牧兽医学报 ›› 2017, Vol. 48 ›› Issue (3): 522-529.doi: 10.11843/j.issn.0366-6964.2017.03.016

慢病毒介导siRNA抑制1型鸭肝炎病毒复制

王永娟，董亚青，朱善元，王安平，洪伟鸣，吴双，左伟勇^*

（江苏农牧科技职业学院/江苏省兽用生物制药高技术研究重点实验室，泰州 225300）

收稿日期:2016-10-11 出版日期:2017-03-23 发布日期:2017-03-23
通讯作者: 左伟勇(1978-)，男，山东泰安人，博士，教授，主要从事生物工程技术研究，E-mail:979490023@qq.com
作者简介:王永娟(1980-)，女，江苏海门人，博士，副教授，主要从事生物工程技术研究，E-mail: 43088591@qq.com
基金资助:
2016年度江苏省高校自然科学研究面上资助项目（16KJB230004）；江苏省“六大人才高峰”第十二批培养对象资助项目（NY-023，10410015002）；江苏省大学生创新创业训练计划校企合作基金项目（201612806030H）

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

WANG Yong-juan, DONG Ya-qing, ZHU Shan-yuan, WANG An-ping, HONG Wei-ming, WU Shuang, ZUO Wei-yong^*

(Jiangsu Agri-Animal Husbandry Vocational College, Jiangsu Provincial Key Laboratory of Veterinary Bio-pharmaceutical High Tech Research,Taizhou 225300, China)

Received:2016-10-11 Online:2017-03-23 Published:2017-03-23

摘要/Abstract

摘要：

为筛选能抑制DHAV-1复制的siRNA，探索RNAi技术在鸭病毒性肝炎防治中的应用，采用PCR法扩增DHAV-1 RdRp基因，并构建pEGFP-RdRp融合表达EGFP-RdRp蛋白；根据RdRp的序列测定结果，设计3条RdRp特异siRNA，并合成相应shRNA分别插入pmiRZipΔ中构建pRdRp-shRNA；共转染pRdRp-shRNA和pEGFP-RdRp于HEK-293T细胞，通过荧光显微镜法、流式细胞术、相对荧光定量PCR法筛选抑制效率较高的siRNA；将高效siRNA用于病毒载体pmiRZip介导的慢病毒制备；通过计算重组慢病毒转导、DHAV-1感染的鸭胚胎成纤维细胞中病毒TCID₅₀和RdRp基因表达量确定siRNA对病毒及病毒基因的抑制作用。结果显示，3个siRNA均能抑制RdRp基因的表达，包含GDD模体的shRNA2的抑制率最高，对应重组病毒能使DHAV-1的TCID₅₀下降6.2 lg，RdRp基因转录量下降89.6%，抑制时间至少达120 h，为鸭病毒性肝炎临床防治提供了新思路。

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.

中图分类号:

S852.659.6

王永娟，董亚青，朱善元，王安平，洪伟鸣，吴双，左伟勇. 慢病毒介导siRNA抑制1型鸭肝炎病毒复制[J]. 畜牧兽医学报, 2017, 48(3): 522-529.

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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慢病毒介导siRNA抑制1型鸭肝炎病毒复制

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

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