DEVELOPMENT OF VECTORS FOR EXPRESSION OF VIRAL ENVELOPE GLYCOPROTEIN ANTIGENS

登革病毒基因工程候选疫苗抗原的研制 工程学已经集中在病毒载体的修饰上， 蛋白表达和抗原分泌。 辛德毕斯病毒 复制子、SIN病毒双相病毒和T7牛痘重组体 系统被遗传工程化以表达DEN病毒前膜 (prM)和包膜（E）蛋白抗原。 DEN的高水平表达 在用这些载体感染的细胞中观察到病毒抗原 含有登革病毒prM和E基因。 不过大部分 蛋白质仍然在细胞内，这表明表达的蛋白质 被不适当地处理或折叠，防止细胞外分泌。 为了改善细胞内的加工和增强分泌， 从受感染的细胞中提取的蛋白质， DEN蛋白质基因盒，编码编码DEN蛋白质基因盒上最后15个氨基酸的基因。 除了碱性氨基酸之外，衣壳蛋白的羧基末端 在prM/E切割位点。 登革病毒基因编码的双顺反子 SIN病毒不稳定。 为了进一步增强基因的表达， SIN病毒复制子，该载体被基因工程改造以促进 通过添加RNA在体内转录亚基因组RNA 在RSV真核启动子控制下的转录起始位点，和 在病毒3 '-NTR的末端连接deltaribozyme，所述病毒3'-NTR编码 DEN插入物。 这些改变简化了转染过程， 改善基因表达。 重组牛痘载体pTM-1导入 克隆到的登革病毒prM和E基因在大肠杆菌中表达量较高， 瞬时系统和重组牛痘病毒中的病毒蛋白 携带T7聚合酶。 为了便于选择重组 编码病毒基因的牛痘病毒，新霉素抗性基因， 在7.5启动子的控制下工程化到牛痘载体中 在用于同源重组的TK区域之外。 研究来 增强DEN病毒prM和E蛋白产生的水平，并促进 目前正在进行抗原的分泌。

Development of dengue (DEN) virus candidate vaccine antigens by genetic engineering has focused on modification of viral vectors to enhance protein expression and antigen secreation. Sindbis (SIN) virus replicons, SIN virus biphasic viruses and the T7 vaccinia recombinant systems were genetically engineered to express DEN virus premembrane (prM) and envelope (E) protein antigens. High level expression of DEN virus antigens was observed in cells infected with these vectors containing the DEN virus prM and E genes. However, the majority of the proteins remained intracellular suggesting that the expressed proteins were improperly processed or folded preventing extracellular secreation. To improve intracellular processing and enhance secreation of the proteins from infected cells it was essential to include in the cloned DEN protein gene cassette, genes encoding the last 15 amino acids at the carboxyl end of the capsid protein in addition to the basic amino acids at the prM/E cleavage site. DEN virus genes encoded into the bicistronic SIN virus were unstable. To further enhance expression of genes by the SIN virus replicon, the vector was genetically engineered to facilitate in vivo transcription of the subgenomic RNA by the addition of a RNA transcription start site under control of a RSV eukaryotic promoter and attachment of a deltaribozyme at the end of the viral 3'-NTR encoding the DEN insert. These changes simplified the transfection process and improved gene expression. The recombinant vaccinia vector pTM-1 into which DEN virus prM and E gene were cloned expressed high levels of DEN virus protein in both the transient system and recombinant vaccinia virus carrying the T7 polymerase. To facilitate selection of recombinant vaccinia viruses encoding viral genes, the neomycin resistance gene was engineered into the vaccinia vector under control of the 7.5 promoter outside of the TK-region used for homologous recombinantion. Studies to enhance levels of DEN virus prM and E protein produced and facilitate secreation of the antigens are currently in progress.