鸡传染性法氏囊病毒病毒样颗粒的制备  

The preparation of virus-like particles of infectious brusal disease virus

作　　者：李芹[1] 王建忠[1] 黄楚荻 穆昱[1] 周玉龙[1] 高超[1] 杨闽楠[1] 赛度 丁壮[1] 

机构地区：[1]吉林大学动物医学学院,吉林长春130062 [2]兰州大学资源环境学院,甘肃兰州730001

出　　处：《中国兽医学报》2014年第11期1725-1731,共7页Chinese Journal of Veterinary Science

摘　　要：为了制备鸡传染性法氏囊病毒病毒样颗粒,本试验通过RT-PCR技术扩增出鸡传染性法氏囊病病毒超强毒株JL株的VP2全长基因,并将其插入杆状病毒表达系统的供体质粒pFastBacHTA中,构建含有IBDV VP2基因的重组供体质粒pFast-VP2,转化DH10Bac E.coli感受态细胞中,经蓝白斑筛选,获得重组杆粒rBacmid-VP2,将重组杆粒rBacmid-VP2转染sf9细胞后得到重组杆状病毒vBac-VP2。rBac-VP2感染sf9细胞,提取DNA,经PCR电泳分析,得到1条与预期大小相符的特异性条带;间接免疫荧光检测,可见特异性荧光;Western-blotting分析,在大约53 000处出现1条特异性的蛋白条带;电镜观察感染重组杆状病毒的细胞上清和沉淀,均可见重组VP2蛋白自行组装形成的病毒样颗粒。试验结果表明IBDV VP2基因在昆虫细胞中得到了表达,并自组装成了IBDV病毒样颗粒。本试验为研制鸡传染性法氏囊病病毒样颗粒疫苗奠定了基础。Toprepare IBDV virus-like particles, the VP2 gene of infectious bursal disease virus （IBDV） JL strain was amplified and cloned into pFastBacHAT donor plasmid. The recombinant plasmid pFast-VP2 was transformed into competent E. coli DH10Bac cells, then the recombinant bacmid DNA rBacmid-VP2 was obtained by blue-white screening and transfected into insect cell Sf9 to produce recombinant baculovirus vBac-VP2. DNA of sf9 cells infected with vBac-VP2 was extracted and followed by PCR, showed a specific band which was matched with the expected size. The Sf9 cells infected with vBac-VP2 were stained positive against IBDV antibody using the indirect immunofluorescence assay （IFA）. Western-blotting analysis displayed a specific protein band of approximate 53 000,and the self-assembly virus-like particles （VLP） could were observed by electron microgcopy. The above results indicated that the VP2 gene of IBDV was expressed in sf9 cells and self-assembled into virus-like particles. This study paved the way for developing the IBDV virus-like particle vaccine.

关 键 词：鸡传染性法氏囊病毒 VP2 真核表达 病毒样颗粒 SF9细胞 

分 类 号：S852.65[农业科学—基础兽医学]