西尼罗病毒NS1与E基因重组腺病毒的构建及免疫原性研究  

Construction and identification of recombinant adenovirus co-expressing the E and NS1 gene of West Nile virus

作　　者：刘二战[1,2] 赵国辉[2] 孙恩成[2] 崔焕忠[1] 杨涛[2] 徐青元[2] 孙晶[2] 孙亮[2] 席娜[1,2] 魏天 吴东来[2] 

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130000 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/农业部兽医公共卫生重点开放实验室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2014年第11期881-884,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31302065);中国博士后科学基金(2013M540172);公益性行业(农业)科研专项资助(201203056);新型重大动物疫苗与诊断试剂创制及生产工艺创新(2011AA10A213);兽医生物技术国家重点实验室基金(SKLVBP201316)

摘　　要：为构建串联表达西尼罗病毒(WNV)NS1和E基因的重组腺病毒,本研究利用口蹄疫病毒具有自我切割功能的2A蛋白的编码序列将NS1和E基因串联,构建穿梭载体p Shuttle-WNV-NS1-2A-E,通过细菌内同源重组获得重组腺病毒DNA,经脂质体转染AD293细胞,在细胞内完成病毒粒子的组装制备重组腺病毒。通过间接免疫荧光和western blot检测表明:重组腺病毒中的外源基因表达的融合蛋白NS1-2A-E可以自我裂解为NS1和E蛋白。将构建的重组腺病毒免疫小鼠,结果表明该重组病毒可以诱导机体产生良好的体液免疫应答。本实验为WNV重组腺病毒活载体疫苗的研制奠定了基础。In order to construct a recombinant adenovirus co-expressing NS1 and E protein of West Nile virus （WNV）,the sequence encoding 2A peptide of foot-and-mouth disease virus was introduced as a linker between NS1 and E gene to insert into the shuttle plasmid to construct pshuttle-WNV-NS1-2A-E,which was transformed into E.coli BJ5183 to produce the pAd-WNV-NS1-2A-E by homologous recombination.Then the recombinant adenovims of rAd-WNV-NS1-2A-E was generated by transfection of pAd-WNV-NS1-2A-E into AD293 cells.The indirect immunofluorescence and western blot assay showed that E-2A-NS1 protein was expressed and self-cleavaged into E and NS1 proteins in rAd-WNV-NS1-2A-E infected AD293 cells.In addition,the antibodies against both adenovirus and WNV were efficiently produced in mice immunized with the rAd-WNV-NS1-2A-E detected by indirect ELISA.This study provided the basis for the development of recombinant adenovims vaccine against WNV infection.

关 键 词：西尼罗病毒 NS1 E 重组腺病毒 体液免疫 

分 类 号：S852.65[农业科学—基础兽医学]