摘要
目的分泌表达登革I型病毒prM／E基因，为研究该蛋白的免疫学功能和特性奠定基础。方法用RT-PCR法获得登革I型病毒广州分离株全长prM／E基因，在prM基因前添加乙型脑炎病毒的信号肽或同时替换E基因羧基末端的20％为乙脑病毒E基因相应的部分，分别将其克隆入真核表达载体pcDNA5／FRT中，获得三种重组质粒D1prME-pc5，D1JsprME．pc5，D1JsprM80E20JE-pc5。用脂质体法分别将重组质粒DNA转入293T细胞，通过免疫荧光、Western印迹检测外源基因在真核细胞中的分泌表达。结果用免疫荧光法检测到分别转染了三种重组质粒的293T细胞的胞质中均有登革I型病毒蛋白的表达。Western印迹检测转染了D1prME-pc5重组质粒的293T细胞上清中没有特异蛋白条带，转染了经基因改造的重组质粒D1JsprME-pc5和D1JsprM80E20JE-pc5的细胞上清中均存在登革I型病毒的特异蛋白条带。结论转染了三种重组质粒的293T细胞均可表达登革I型病毒prM／E蛋白，只有在prM基因前添加了信号肽的重组质粒转染后蛋白才获得分泌表达。
Objective To expression prM/E gene of dengue virus type I in mammalia cells. Methods The full-length prM/E gene of dengue virus type I strain GZO1/95 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. Results In the cytoplasm of 293T cells transfeeted 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. Conclusion 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 transfeeting 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.
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
北大核心
2009年第6期415-417,共3页
Chinese Journal of Experimental and Clinical Virology
基金
国家高技术研究发展计划（863计划）（2006AA02A223）.
