表达猪圆环病毒2型Cap蛋白重组腺病毒的构建  

Construction of Recombinant Adenovirus Expressing PCV-2 Cap Protein

作　　者：许晗[1] 李海敏[1] 王爱玲[1] 李春燕[1] 李河林[1] 吕其壮[1] 张彦明[1] 郭抗抗[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《动物医学进展》2015年第9期1-7,共7页Progress In Veterinary Medicine

基　　金：陕西省科技统筹创新工程计划项目(2014KTCQ02-02);陕西省重点科技创新团队计划项目(2013KCT-28)

摘　　要：为研制表达猪圆环病毒2型(PCV-2)Cap蛋白的重组腺病毒疫苗,根据PCV-2ORF2(Cap)基因序列和耶尔森菌侵袭素C端(InvC)序列,克隆获得Cap和InvC基因后,插入pAdShttle-CMV载体构建重组穿梭载体pAdShttle-Cap-InvC,经PmeI线性化后,转化入BJ5183感受态细菌与腺病毒骨架载体pAdEasy-1同源重组获得重组腺病毒质粒pAd-Cap-InvC。重组质粒经PacⅠ线性化后,转染HEK293细胞,连续传代后获得重组腺病毒。应用PCR、Western blot和间接免疫荧光(IFA)技术分别检测重组腺病毒中Cap和InvC基因及表达。结果表明,成功构建了表达PCV-2Cap蛋白和InvC的重组腺病毒rAd-CapInvC,经过噬斑纯化和连续传代,重组腺病毒TCID50可达10-9.32/mL,为PCV-2重组腺病毒活载体疫苗的研发提供了基础材料。To obtain the genetically engineering vaccine of recombinant adenovirus containing Cap gene of porcine circovirus type 2 （PCV-2）,Cap gene of PCV-2 and C-terminal domain of Yersinia enterocolitica in- vasion （InvC） gene were inserted into pAdShttle-CMV, linearized pAdShttle-Cap-InvC with Pine I was transformed into Escherichia coli BJ5183 containing pAdEasy-1 vector to constructe recombinant adenovi- rus plasmid pAd-Cap-InvC. AD293 cells were transfected with Pac I -linearized plasmid pAdShttle-Cap-In- vC to produce the recombinant adenovirus rAd-Cap-InvC. The expressions of targeted genes were verified by PCR,RT-PCR,Western blot and IFA. Results indicated that the recombinant adenovirus rAd-Cap-InvC with the objective genes can be expressed stably. After plaque purification and serial passages, the TCIDs0 of rAd-Cap-InvC was 10-9. 32/mL. Therefore,it laid a good foundation for developing PCV-2 genetically en- gineering vaccine.

关 键 词：猪圆环病毒2型 CAP蛋白 侵袭素蛋白C端 重组腺病毒 

分 类 号：S852.659.2[农业科学—基础兽医学]