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

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

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。

Porcine circovirus type 2 （PCV2） has been implicated as the etiological agent of a new disease, named postweaning muhisystemic 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 sit） cells to yield Ac. NPV carrying the PCV20RF1 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.