Zhang Shuo, Gu Wen, Li Chuan, Miao Fang, Lu Peng, Qu Jing, Wei Yan, Zhang Quan-fu, Liu Qin-zhi, Li Jian-dong, Liang Mi-fang, Li De-xin
State Key Laboratory for Molecular Virology & Genetic Engineering, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China.
Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2011 Apr;25(2):85-8.
To secreted express envelope glycoprotein (E) of dengue virus type 2 extracellularly.
The entire prM/E gene was amplified by RT-PCR. An optimized signal sequence gene from Japanese encephalits virus (JEV, SA14-14-2 strain) was introduced using fusion PCR. The impact of E protein transmembrane and cytoplasmatic domains was compared by amplifying prM and E with full length of E gene, with 20% truncation of the E gene at 3' terminus and one chimeric gene, which was generated by replacing the 3' terminal 20% region of E gene with the corresponding sequence of JEV (SA14-14-2 strain). The PCR segments were inserted into the NheI and NotI sites of pcDNA5/FRT vector or into the NheI and XhoI sites of pAcUW51-M. Then they were transfected into 293T cells or Sf9 cells respectively. The expression and secretion of E protein were detected by immunofluorescence assay (IFA) and Western Blot.
After transected into 293T cells or Sf9 cells, all constructs expressed E protein intracellularly indentified by IFA while only two plasmids could secret detectable E protein into tissue culture using Western Blot analysis.
Signal peptide as well as the transmembrane and cytoplasmatic domains is crucial for the secretion of dengue E protein.
在细胞外分泌表达2型登革病毒包膜糖蛋白（E）。
通过逆转录聚合酶链反应（RT-PCR）扩增完整的prM/E基因。使用融合PCR引入来自日本脑炎病毒（JEV，SA14-14-2株）的优化信号序列基因。通过扩增具有全长E基因的prM和E、E基因3'末端截短20%的片段以及一个嵌合基因（通过用JEV（SA14-14-2株）的相应序列替换E基因3'末端20%区域产生）来比较E蛋白跨膜和细胞质结构域的影响。将PCR片段插入pcDNA5/FRT载体的NheI和NotI位点或pAcUW51-M的NheI和XhoI位点。然后分别将它们转染到293T细胞或Sf9细胞中。通过免疫荧光测定（IFA）和蛋白质免疫印迹法检测E蛋白的表达和分泌。
转染到293T细胞或Sf9细胞后，所有构建体在细胞内均表达E蛋白，通过IFA鉴定，而使用蛋白质免疫印迹分析仅两种质粒能将可检测到的E蛋白分泌到组织培养物中。
信号肽以及跨膜和细胞质结构域对登革病毒E蛋白的分泌至关重要。
