摘要
为构建携带分子标记的PCV1-2嵌合病毒,本研究通过基因工程技术将V5标签引入PCV2的ORF2末端,以ORF2-V5替换PCV1的ORF2后克隆于pcDNA3.1(+)载体中,构建pcDNA-PCV1-2m-V5。为检验pcDNA-PCV1-2m-V5的体外感染性,将其转染PK-15细胞后,采用IPMA、RT-PCR等技术进行检测,结果显示:pcDNA-PCV1-2m-V5具有感染性。采集已注射pcDNA-PCV1-2m-V5质粒的小鼠血清接种于PK-15细胞,应用细胞试验、PCR技术、IFA、western blot及电镜等试验对嵌合病毒进行鉴定。结果显示:在小鼠体内拯救出PCV1-2m-V5嵌合病毒,且V5标签的引入能够很好地区分嵌合病毒和亲本病毒产生的Cap蛋白。本实验可为PCV2的感染性DNA (iDNA)疫苗研发提供素材和参考。
In order to construct the infectious cloned plasmid of PCV1 and PCV2 chimeric virus carrying molecular markers, the infectious clone of pcDNA-PCV1-2m-V5 was constructed by introducing the V5 tag encoding sequence into the ORF2 end of PCV2 and replacing ORF2 of PCV1 with ORF2-V5, then ligated it into pcDNA3.1 （＋）, and the infectious clone was transfected into PK-15 cells to investigate the infectivity of the rescued chimeric virus carrying molecular markers （PCV1-2m-V5） detected using IPMA and RT-PCR. The results showed that the PCV1-2m-V5 chimeric virus was rescued and replicated in the pcDNA-PCV1-2m-V5 transfected PK-15 cells. Moveover, the sera were collected from the mice injected with the pcDNA-PCVI- 2m-V5 and inoculated into PK-15 cells. Then the rescued chimeric virus was identified by cell test, PCR, IFA, western blot and electronic microscope. The results showed that PCV1-2m-V5 chimeric virus was also rescued in mice and the introduced V5 tag was well distinguished the capsid protein produced from the chimeric virus and the parental virus. This experiment provided the material and reference for PCV2 iDNA vaccine research and development.
作者
徐国
代振江
曾智勇
梁海英
汤德元
王彬
黄涛
叶百川
张爱琼
何小莉
咸文
XU Guo;DAI Zhen-jiang;ZENG Zhi-yong;LIANG Hai-ying;TANG De-yuan;WANG Bin;HUANG Tao;YE Bai-chuan;ZHANG Ai-qiong;HE Xiao-li;XIAN Wen(College of Animal Science,Guizhou University,Gui yang 550025,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2018年第9期779-784,共6页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金资助项目"猪圆环病毒iDNA标记疫苗及其免疫应答效果的研究"(31560688)
