表达猪圆环病毒2型Rep蛋白的重组杆状病毒的构建与鉴定

猪圆环病毒2型(PCV2)是近年来新发现的引起断奶仔猪多系统衰弱综合征的主要病原,含有两个主要的阅读框ORF1和ORF2,分别编码复制相关蛋白(Rep)和核衣壳蛋白(Cap)。为了探究ORF1基因结构及其与功能的关系,本研究利用Bac-to-Bac杆状病毒表达系统将PCV2的ORF1基因克隆到杆状病毒转移载体pFastBacTMDual中,获得重组转移载体pFBD-ORF1,再将其转化进含穿梭载体Bacmid的感受态细胞DH10Bac中,经蓝白菌落筛选获得含有ORF1基因的重组穿梭载体rBacmid-ORF1,经脂质体介导转染昆虫细胞sf9,获得重组杆状病毒rAc-Rep,SDS-PAGE和Western-blotting分析可见大小约为36ku的特异性条带,表明rAc-Rep在sf9中成功表达了PCV2Rep蛋白。

Construction and identification of recombinant insect baculovirus expressing Rep protein of Porcine Circovirus type 2

Porcine circovirus type 2(PCV2) has been implicated as the etiological agent of a new disease,named postweaning multisystemic wasting syndrome(PMWS).PCV2 contains two major open reading frames(ORFs):ORF1 and ORF2,encoding replication-associated(Rep) protein and capsid(Cap) protein,respectively.In order to find out the relationship between structure and function of Rep protein,the ORF1 gene of PCV2 was cloned into baculovirus transfer vector(pFastBacTMDual) using Bac-to-Bac baculovirus expression system.E.coli DH10Bac containing baculovirus shutter vector(bacmid) and helper vector was transformed with recombinant plasmid pFBD-ORF1.The colonies of E.coli containing recombinant bacmid(rBacmid-ORF1) were collected by blue/white selection.The rBacmid-ORF1 was transfected into sf9 cells to yield Ac.NPV carrying the PCV2 ORF1 gene,referred to as rAc-Rep.Expression of the ORF1 gene of PCV2 was confirmed by SDS-PAGE and Western-blotting.The Rep protein expressed by rAc-Rep had a molecular weight of 36 ku.