CAJ | 학술논문

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。
목적：구건표체등혁2형병독prM/E단백적진핵세포계。방법：응용취합매련반응(PCR)방법종함유등혁2형병독prM/E기인적질립중확증득도prM/E기인。장해편단아극륭도pGEM-T Easy재체상，용XhoⅠ화NheⅠ쌍매절장기여동양쌍매절적pFastBac Dual질립련접，구건전이재체pFBD-prM/E。장전이재체전화적동시，함유간상병독천사재체Bacmid화Helper질립적감수태DH10 Bac득도중조Bacmid；용후자전염Sf 9세포획득중조간상병독。쌍매절감정구건적중조간상병독전이재체pFBD-prM/E，간접면역형광법검측목적단백적표체。결과：통과간접면역형광법가관찰도특이성록색형광，즉검측도prM/E단백적표체。결론：이용곤충간상병독계통성공표체료등혁2형병독prM/E단백，위등혁병독prM화E단백적공능연구、등혁병독감염적진단이급등혁병독양과립역묘적연제전정료기출。
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.