Miao Fang, Li Chuan, Zhang Shuo, Wang Xiao-fang, Li Jian-dong, Zhang Quan-fu, Liu Qin-zhi, Wei Yan, Hang Xiao-tong, Liang Mi-fang, Li De-xin
National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory for Molecular Virology & Genetic Engineering, Beijing 100052, China.
Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2009 Dec;23(6):415-7.
To expression prM/E gene of dengue virus type I in mammalia cells.
The full-length prM/E gene of dengue virus type I strain GZ01/95 was amplified by RT-PCR, the signal peptide preceding the prM gene was added or the carboxyl-terminal 20% of DEN-1 E was replaced with the corresponding JE sequence in the meanwhile, and three of the constructions were cloned into the pcDNA5/FRT.Then they were transfected into 293T cells by lipofectamine respectively. The expression of recombinant proteins were identified by indirect immuno-fluorescence assay(IFA) as well as Western blot.
In the cytoplasm of 293T cells transfected with all the recombinant plasmids DNA, the expressed products for gene of dengue virus type I were confirmed by IFA. The secreted expression products for gene of dengue virus type I specific protein bands were confirmed by Western blot only existing in the cell supernatants transfected with the modified recombinant plasmids DNA.
The prM/E protein of dengue virus type 1 were expressed in 293T cells transfected with all the three recombinant plasmids DNA. The prM/E protein was obtained secretion after transfecting the modified recombinant plasmids adding a signal peptide preceding the prM gene or replacing the carboxyl-terminal 20% of E with the corresponding JE sequence.
在哺乳动物细胞中表达Ⅰ型登革病毒的prM/E基因。
通过RT-PCR扩增Ⅰ型登革病毒GZ01/95株的全长prM/E基因，同时在prM基因前添加信号肽或用相应的乙型脑炎（JE）序列替换DEN-1 E羧基末端的20%，将三种构建体克隆到pcDNA5/FRT中。然后分别用脂质体将它们转染到293T细胞中。通过间接免疫荧光法（IFA）和蛋白质免疫印迹法鉴定重组蛋白的表达。
IFA证实，在转染了所有重组质粒DNA的293T细胞的细胞质中，有Ⅰ型登革病毒基因的表达产物。蛋白质免疫印迹法证实，仅在转染了修饰重组质粒DNA的细胞上清液中存在Ⅰ型登革病毒基因特异性蛋白条带的分泌表达产物。
转染三种重组质粒DNA的293T细胞均表达了1型登革病毒的prM/E蛋白。在prM基因前添加信号肽或用相应的JE序列替换E羧基末端的20%后转染修饰的重组质粒，可使prM/E蛋白获得分泌表达。
