Ⅰ型鸭甲型肝炎病毒VP3基因在昆虫细胞中的表达与鉴定

发布时间：2018-03-30 23:17

本文选题：I型鸭甲型肝炎病毒　切入点：VP基因　出处：《江苏农业学报》2017年03期

【摘要】：为在昆虫细胞中表达Ⅰ型鸭甲型肝炎病毒(Duck hepatitis A virus type-I,DHAV-I)SH株的主要结构蛋白VP3,首先根据DHAV-I SH株VP3基因序列设计1对引物,RT-PCR方法扩增出VP3基因,克隆至杆状病毒表达载体p Fast Bac1,获得重组杆状病毒转移载体p FB-VP3,将其转化到DH10Bac感受态细胞中,经抗性和蓝白斑筛选,获得重组穿梭质粒r Bacmid-VP3,在脂质体介导下转染昆虫细胞Sf9,获得重组杆状病毒r Bac-VP3。间接免疫荧光结果显示重组蛋白获得了正确表达,能与鸭抗全病毒阳性血清发生特异性反应。Western blot结果显示表达的重组蛋白分子量约为27 000。表明,DHAV-I SH株的主要结构蛋白VP3在昆虫细胞中获得了成功表达,为VP3结构蛋白的功能研究及相关亚单位疫苗的研制奠定了基础。
[Abstract]:In order to express the main structural protein VP3 of Duck hepatitis A virus type-I DHAV-ISH strain in insect cells, a pair of primers RT-PCR was designed to amplify the VP3 gene according to the VP3 gene sequence of DHAV-I SH strain.The recombinant baculovirus transfer vector, pFB-VP3, was cloned into baculovirus expression vector p Fast Bac1 and transformed into DH10Bac competent cells.The recombinant shuttle plasmid r Bacmid-VP3 was obtained and transfected into insect cell Sf9 by liposome, and the recombinant baculovirus rBac-VP3 was obtained.The results of indirect immunofluorescence showed that the recombinant protein was correctly expressed and could react specifically with duck anti-virus positive serum. Western blot showed that the molecular weight of the expressed recombinant protein was about 27000.The results showed that the main structural protein VP3 of DHAV-I SH strain was successfully expressed in insect cells, which laid a foundation for the functional study of VP3 structural protein and the development of related subunit vaccine.
【作者单位】：江苏农牧科技职业学院/江苏省兽用生物制药高技术研究重点实验室;
【基金】：国家自然科学基金项目(31302096) 江苏省农业科技支撑项目(BE2013415) 江苏省六大人才高峰项目(NY-009)
【分类号】：S852.65

【相似文献】