猪细小病毒VP_2蛋白在昆虫细胞中的表达及其特性

The Expression of Porcine Parvovirus VP_2 Protein in Insect Cells and It's Character

将猪细小病毒 (PorcineParvovirus,PPV)vp2 基因重组到杆状病毒Bac To Bac表达系统的pFastBacⅠ质粒中 ,构建了pFast vp2 质粒。在DH10 Bac大肠杆菌中 ,pFast vp2 与改造过的苜蓿夜蛾核型多角体病毒 (AcNPV)基因组 (Bacmid)发生同源重组 ,从而获得重组穿梭载体Bac mid vp2 ,转染Sf9细胞得到重组病毒AcNPV vp2 。SDS PAGE和Western blotting分析可见大小约为 6 4kD的特异性带 ,表明AcNPV vp2 在Sf9细胞中成功地表达了PPVVP2 蛋白。红细胞凝集试验和间接ELISA进一步证实 ,表达的VP2 蛋白具有与全病毒相同的血凝活性和相似的抗原性。电镜观察VP2 蛋白的粗提物 ,发现VP2 蛋白可自行装配成许多病毒样粒子 (VLPs)

vp 2 gene of PPV was cloned into pFastBacⅠ plasmid of Bac-To-Bac expression system. Bacmid-vp 2 was obtained, through homologous recombination between pFast-vp 2 and bacmid which is genome modified of Autogragha californica nuclear polyhedrosis virus(AcNPV). After transfection,recombined virus(AcNPV-vp 2) was developed. The specific 64kD band was showed by analysis of SDS-PAGE and Western-blotting, which indicated that PPV VP 2 protein had expressed in insect sf9 cells. Moreover, VP 2 protein had identical haemagglutinating activity and similar antigenic activity with full PPV by haemagglutination assay and ELISA. It wad noteworthy that many self-assembled virus-like particles(VLPs) were found in VP 2 crude by electron microscope.