【摘要】 利用BactoBac杆状病毒表达系统将圆环病毒2型的ORF2全基因克隆到杆状病毒转移载体pFastBacTM1中,获得重组转移载体pFastOFR2,再将其转化进含穿梭载体Bacmid的感受态细胞DH10Bac中,发生转座作用,经蓝白菌落筛选得到含ORF2基因的重组穿梭载体Bac.ORF2,以脂质体介导的方法将重组穿梭载体转染sf9细胞,获得重组病毒,命名为Ac.ORF2。间接免疫荧光分析表明,PCV2阳性血清能使Ac.ORF2感染的sf9昆虫细胞呈强的荧光着色;SDSPAGE与Westernblotting分析可见大小约为28kD的特异性带,表明Ac.ORF2在sf9细胞中成功表达了PCV2ORF2蛋白。将该表达蛋白纯化并经磷钨酸负染后,通过电镜观察可见形态与PCV2病毒粒子相似的病毒样颗粒(VLPs),其中某些颗粒由于中心浓染而似空衣壳,其直径也为17nm左右。更多还原

【Abstract】 To produce the recombinant baculovirus transfer plasmid pFast-ORF2, the ORF2 gene of Porcine Circovirus type 2 (PCV2) was subcloned into baculovirus transfer vector (pFastBac~ TM1) using Bac-to-Bac baculovirus expression system. E.coli DH10Bac (Gibco BRL) containing baculovirus shutter vector (bacmid) and helper vector was transformed with recombinant plasmid pFast-ORF2. Within E.coli DH10Bac, the ORF2 gene was transposed into the bacmid. The colonies of E.coli containing recombinant bacmid (Bac.ORF2) were collected by blue/white selection. The Bac.ORF2 was transfected into sf9 cells to yield AcNPV carrying the PCV2 ORF2 gene, referred to as Ac.ORF2. Expression of the ORF2 gene of PCV2 was confirmed by indirect immunofluorescent assay (IIFA), SDS-PAGE and Western-blotting. The expressed ORF2 gene product had a molecular mass of 28kD and could be recognized by the positive serum of PCV2.The results indicated the ORF2 gene was properly expressed in sf9 cell. It was noteworthy that many self-assembled virus-like particles (VLPs) were found in purified and phosphotungstic acid (PTA) stained PCV2 ORF2 protein by electron microscope. The particles were of similar morphology to the PCV2 virion and some self-assembled virus-like particles had darkly stained centers that made them appear to be empty capsids. Both PCV2 particles and self-assembled particles were approximately 17nm in diameter.更多还原