昆虫杆状病毒系统表达登革2型病毒prM/E蛋白

摘要

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 virus typeⅡ. The PCR product was cloned into the XhoⅠ/NheⅠrestriction 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.%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。