新城疫病毒3′及5′末端非编码区序列的反向遗传研究  

Reverse genetic studies of Newcastle disease virus 3′ and 5′ end non-coding region sequence

作　　者：李想[1] 高超[1] 李芹[1] 王建忠[1] 杨闽楠[1] 王泽[1] 张晓东[1] 丁壮[1] 

机构地区：[1]吉林大学动物医学学院,吉林长春130062

出　　处：《中国兽医学报》2014年第5期751-755,760,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31272561);公益性行业(农业)科研专项基金资助项目(201303033)

摘　　要：以新城疫病毒(NDV)LaSota株感染性克隆PCI-La为平台进行反向遗传操作,并利用PCR技术将其3′端和5′端非编码区分别替换为鹅源NDV NA-1株3′端和5′端非编码区,以及3′端和5′端非编码区同时替换,通过生物学软件DNAStar对替换后的序列进行比对分析,所替换的序列未出现基因突变现象,证明非编码区替换成功,从而分别获得3株突变全长感染性克隆pCI-rL-NL、pCI-rL-NT、pCI-rL-NL-NT;将所获得的这3株突变感染性克隆分别与辅助质粒pCI-NP、pCI-P、pCI-L共转染BSR细胞,进行重组病毒的拯救。结果显示,pCI-rL-NL、pCI-rL-NT两种质粒表达系统均可拯救到有感染活性的病毒粒子,其HA效价为24,23,HI试验及间接免疫荧光试验结果均为阳性,并且通过电镜观察到具有典型副黏病毒科特征的、有囊膜的NDV颗粒。本试验成功构建了2株NDV LaSota突变株的反向遗传操作系统,为后期进行重组NDV致病性的研究奠定基础,也为进一步研究筛选新型分子标记疫苗、病毒活载体疫苗和抗病毒药物提供了可能。Reverse genetic operation was done with NDV LaSota strain infectious cloned PCI-La as platform,and replace non-coding regions of 3＇ end,5＇ end with goose derived NDV NA-1 strain, respectively and simultaneously. Alignment analysis of the replaced sequence by DNAStar showed that the replaced sequence did not display the gene mutation,indicating that the replacement of non--coding region is successful. Calcium phosphate was used to transfect BSR cells with pCI-rL- NL.pCI-rL-NT or pCI-rL-NL-NT together with helper plasmids to rescue recombinant NDV. Re- sults the expression plasmids pCI-rL-NL and pCI-rL-NT allowed successful recovery of NDV. Hemagglutination titers were 2^4 , 2^3 respectively, HI test and indirect immunofluorescence test re- sults were all positive. The typical characteristics of the paramyxovirus family and enveloped NDV particles were observed by electron microscopy. The reverse genetics system of two mutant strains of NDV LaSota lay a foundation for the further study of pathogenicity, screening of new molecular marker vaccine,live viral vector vaccines and antivirals drugs.

关 键 词：NDV 末端序列 替换 拯救 

分 类 号：S852.65[农业科学—基础兽医学]