摘要
以新城疫病毒(NDV)NP基因为标靶,构建3个细胞内表达发夹样结构小干扰RNA(shRNA)的质粒载体,在鸡胚成纤维细胞(CEF)和鸡胚上进行了RNAi试验,筛选出一个有效抑制病毒复制的小分子ndv1.用阳离子脂质体转染试剂Silent-fect将ndv1转染CEF,以不相关shRNA质粒载体HK为阴性对照,4h后接种NDV,与对照相比,干涉组在病毒感染后3hNP基因的表达量降低2.3倍,6h降低21.1倍,9h降低9.8倍;ndv1能在48h内完全阻断NDV在CEF中的增殖,延缓病变出现时间,减轻病变程度.将Silent-fect-ndv1混合物与NDV同时注入10日龄SPF鸡胚绒毛尿囊腔,能使105ELD50NDV感染后17h鸡胚尿囊液中病毒增殖量减少94.4%,使106ELD50 NDV感染后17h鸡胚尿囊液中病毒增殖量减少62.5%.实验结果证实,在CEF中存在RNAi机制,抑制ND VNP基因的表达能有效阻断该病毒增殖,说明NP基因在NDV复制过程中起重要作用.实验结果为进一步利用RNAi技术在CEF和鸡胚中研究病毒基因组功能及筛选抗病毒小分子奠定了基础.
Three plasmid vectors expressing short hairpin RNAs （shRNAs） targeted against new castle disease virus （NDV） NP gene were constructed in this study. One of them was provided inducing a block of viral replication in chicken embryo fibroblasts （CEF） and chicken embryo. Four hours prior infection with NDV, CEF was transfected with the plasmids by silent-fect. Unrelated shRNA sequence （HK） was used as mock transfection. The expression of a potent shRNA resulted in up to 2.3,21.1 and 9.8 folds decrease of NP gene expression 3,6 and 9 hours post infection in CEF respectively. The NDV replication complete inhibition in CEF can be induced by ndvl within 48 hours, furthermore, the pathological changes of CEF caused by NDV are later and the degree of pathological changes is lighter compare with mock in the presence of ndvl. When the complex of shRNA-silent-fect and NDV was co-injected into allantoic cavity of 10-day-old embryonated corresponding with the NDV 10^5 and 10^6 ELD50 attacked. Our studies demonstrate that RNAi chicken eggs, the NDV replication was dropped 94.4% and 62.5% in 17 hours respectively, mechanism was presence in CEF like in other type cells. The findings reveal that newly synthesized NP protein is critical for NDV transcription and replication and provide a basis for the identifying the function of ,virus genes and effective siRNAs against virus in CEF and chicken embryo by RNAi.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2007年第2期154-159,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家"十五"科技攻关项目(No2004BA519A58)
四川省应用基础项目(No05JY029-007-5)~~
关键词
RNA干涉
小发夹结构RNA
新城疫病毒
鸡胚成纤维细胞
鸡胚
RNAi
short hairpin RNAs
new castle disease virus
chicken embryo fibroblasts
embryonated chicken eggs
作者简介
联系人Tel：028-85528276，Fax：028-85522855 E-mail：yhua900@yahoo．com．cn
