摘要
试验旨在利用真核表达系统构建V5标记的猪圆环病毒2型(PCV2)Cap蛋白的表达载体。采用基因工程技术将V5标签引入到PCV2 ORF2基因C末端,并将标记基因定向克隆入pFastBacHTA载体,将pFastBacHTA-ORF2-V5重组转移载体转化大肠杆菌DH10Bac感受态细胞,使用脂质体介导法将鉴定后的重组杆状病毒质粒转染于Sf9细胞中;运用间接免疫荧光试验(IFA)、SDS-PAGE和Western blotting试验对Sf9细胞中V5标记Cap蛋白的表达进行验证。结果显示,本试验成功将V5标签引入到PCV2 ORF2基因末端,pFastBacHTAORF2-V5重组转移载体转化大肠杆菌DH10Bac感受态细胞后获得了Bacmid-ORF2-V5重组杆状病毒质粒;IFA、SDS-PAGE和Western blotting试验结果显示,在Sf9细胞中能检测到重组杆状病毒V5标记的PCV2Cap蛋白,具有良好的反应原性,说明本试验成功获得一株rAcMNPV Cap-V5重组杆状病毒。试验结果为PCV2标记亚单位疫苗的研制提供一定的理论依据。
The aim of the experiment was to construct the expression vector of V5-labeled porcine circovirus type 2(PCV2)Cap protein using eukaryotic expression system.The V5 tag was introduced into the end of the PCV2 ORF2 gene using genetic engineering,and the tagged gene was cloned into the pFastBacHTA vector.Recombinant transfer vector pFastBacHTA-ORF2-V5 was transformed into E.coli DH10 Bac.The identified recombinant bacmid was transfected into Sf9 cells using liposome-mediated method;Indirect immunofluorescence assay(IFA),SDS-PAGE and Western blotting were used to test the expression of V5-labeled Cap protein in Sf9 cells.The results showed that this experiment successfully introduced the V5 tag to the end of the PCV2 ORF2 gene.After the recombinant transfer vector pFastBacHTA-ORF2-V5 transformed into E.coli DH10 Bac,Bacmid-ORF2-V5 recombinant bacmid was obtained.The result of IFA,SDSPAGE and Western blotting showed that it could be expressed in Sf9 cells.The recombinant baculovirus V5-labeled PCV2 Cap protein was detected to have good reactogenicity,indicating that this experiment successfully obtained a rAcMNPV-Cap-V5 recombinant baculovirus.The test resultsprovided a theoretical basis for the development of PCV2 marker subunit vaccine.
作者
徐国
梁海英
曾智勇
王彬
黄涛
汤德元
代振江
叶百川
张爱琼
何小莉
咸文
XU Guo;LIANG Haiying;ZENG Zhiyong;WANG Bin;HUANG Tao;TANG Deyuan;DAI Zhenjiang;YE Baichuan;ZHANG Aiqiong;HE Xiaoli;XIAN Wen(College of Animal Science,Guizhou University,Guiyang 550025,China)
出处
《中国畜牧兽医》
CAS
北大核心
2018年第7期1775-1782,共8页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金"猪圆环病毒iDNA标记疫苗及其免疫应答效果的研究"(31560688)