水貂阿留申病毒NS1基因在大肠杆菌中的分段表达  

Segmented Expression of Aleutian Disease Virus(ADV) NS1 Gene Fragment in E.coli

作　　者：刘红娜[1] 王文玉[1] 张云[1] 杨宇航[1] 郝俊伟[1] 时坤[2] 李健明[2] 杜锐[2] 

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130118 [2]吉林农业大学中药材学院,吉林长春130118

出　　处：《中国畜牧兽医》2014年第4期32-36,共5页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(31272565)

摘　　要：利用DNAStar Protean模块预测分析水貂阿留申病毒(aleutian disease virus,ADV)NS1基因后,设计3对特异性引物,以吉林农业大学经济动物实验室保存的NS1全长克隆质粒NS1-pMD18-T为模板扩增NS1基因的3个截短片段,分别命名为L1 N1、L2 N2、L3 N3基因,构建克隆质粒pMD18-T-L1N1、pGEM-T-L2N2及pMD18-T-L3N3。经测序鉴定正确后,将经EcoRⅠ、SalⅠ双酶切的3个NS1基因片段连接至经相同双酶切的pGEX-4T-1原核表达载体,并转化入表达宿主菌BL21(DE3)感受态细胞,利用IPTG进行诱导表达。采用SDS-PAGE和Western blotting检测重组菌在大肠杆菌中的表达情况。结果表明,成功构建了3段重组原核表达质粒pGEX-4T-L1N1、pGEX-4T-L2N2、pGEX-4T-L3N3,SDS-PAGE结果显示分别表达出大小约57、51、44ku的目的条带,与预期相符。Western blotting进一步证实获得的3种蛋白能被ADV阳性血清识别,具有反应原性。本试验首次对ADV NS1基因在大肠杆菌中进行分段表达,为后续研制ADV的亚单位疫苗奠定基础。We predicted the non-structural protein NS1 of aleutian disease virus （ADV） by DNAStar software Protean mod- ule. Then three fragments of NS1 gene named as L1N1, L2N2 and L3N3 were amplified using the laboratory saved NS1- pMD18-T plasmid as template. We constructed three cloning plasmids pMD18-T-L1N1, pGEM-T-L2N2 and pMD18-T-L3N3. After identification, they were linked to pGEX-4T 1 prokaryotie expression vector by two enzymes EcoR I and Sal I , respec tively, and transformed into E. coli BL21 （DE3） inducing with IPTG. The expressed recombinant proteins were analyzed by SDS-PAGE and Western blotting. The results showed that three recombination prokaryotic expression plasmids pGEX-4T- L1N1, pGEX-4T-L2N2 and pGEX-4T-L3N3 were successfully constructed and the SDS-PAGE results showed that three purpose proteins were expressed about 57, 55 and 44 ku as expected. Furthermore, the proteins were recognized by ADV positive sera and had the reaetionogenieity. In the study, the NS1 gene of ADV was expressed segmentedly in E. coli for the first time and laid a foundation for the further study of ADV subunit vaccines.

关 键 词：水貂阿留申病毒NS1基因 大肠杆菌 分段表达 

分 类 号：S858.92[农业科学—临床兽医学]