【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。更多还原

【Abstract】 To obtain the genetically engineering vaccine of recombinant adenovirus containing Cap gene of porcine circovirus type 2(PCV-2),Cap gene of PCV-2and C-terminal domain of Yersinia enterocoliticainvasion(InvC)gene were inserted into pAdShttle-CMV,linearized pAdShttle-Cap-InvC with PmeⅠ was transformed into Escherichia coli BJ5183 containing pAdEasy-1vector to constructe recombinant adenovirus plasmid pAd-Cap-InvC.AD293 cells were transfected with PacⅠ-linearized plasmid pAdShttle-Cap-InvC 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 TCID50 of rAd-Cap-InvC was 10-9.32/mL.Therefore,it laid a good foundation for developing PCV-2genetically engineering vaccine.更多还原