昆虫杆状病毒系统表达登革2型病毒prM/E蛋白  

Expression of dengue virus type Ⅱpremembrane and envelope proteins in baculovirus expression system

作　　者：刘晓宇[1] 姚立红[2] 陈爱珺[2] 郭建强[2] 张智清[2] 陈辉[3] 

机构地区：[1]中国疾病预防控制中心,北京102206 [2]中国疾病预防控制中心病毒病预防控制所,北京102206 [3]首都医科大学基础医学院病原生物学系,北京100069

出　　处：《中国医学装备》2014年第5期52-55,共4页China Medical Equipment

摘　　要：目的:构建表达登革2型病毒prM/E蛋白的真核细胞系。方法:应用聚合酶链反应(PCR)方法从含有登革2型病毒prM/E基因的质粒中扩增得到prM/E基因。将该片段亚克隆到pGEM-T Easy载体上,用XhoⅠ和NheⅠ双酶切将其与同样双酶切的pFastBac Dual质粒连接,构建转移载体pFBD-prM/E。将转移载体转化的同时,含有杆状病毒穿梭载体Bacmid和Helper质粒的感受态DH10 Bac得到重组Bacmid;用后者转染Sf 9细胞获得重组杆状病毒。双酶切鉴定构建的重组杆状病毒转移载体pFBD-prM/E,间接免疫荧光法检测目的蛋白的表达。结果:通过间接免疫荧光法可观察到特异性绿色荧光,即检测到prM/E蛋白的表达。结论:利用昆虫杆状病毒系统成功表达了登革2型病毒prM/E蛋白,为登革病毒prM和E蛋白的功能研究、登革病毒感染的诊断以及登革病毒样颗粒疫苗的研制奠定了基础。Objective： Co-expression of dengue virus premembrane and envelope（prM/E） proteins is required for production of virus-like-particles, and the dengue virus-like-particle has become one of the most important aspects in dengue virus vaccine research. Methods： To establish the baculovirus expression system that expresses prM/E proteins, the prM/E gene was obtained by PCR amplification from the plasmid containing the prM/E gene of dengue virustype Ⅱ. The PCR product was cloned into the Xho Ⅰ/ Nhe Ⅰ restriction site of pFastBac Dual vector to establish the transfer vector pFBD-prM/E. Then the pFBD-prM/E was transformed into the competent DH10 Bac containing Bacmid and Helper vector, which established the shuttle plasmid rBacmid-prM/E. The latter was transfected into Sf9 cells, and the recombinant baculovirus was obtained. Results： The expression of prM/E proteins was then confirmed by indirect immunofluorescence assay. Conclusion： The baculovirus expression system expressing prM/E will be useful for further functional studies of prM and E proteins, and development of dengue virus-like-particle vaccine.

关 键 词：登革2型病毒 prM蛋白 E蛋白 杆状病毒表达系统 

分 类 号：R512.8[医药卫生—内科学]