DEVELOPMENT OF VECTORS FOR EXPRESSION OF VIRAL ENVELOPE GLYCOPROTEIN ANTIGENS

登革热(DEN)病毒候选疫苗抗原的研究 工程学已将重点放在病毒载体的修饰上，以增强 蛋白质表达和抗原分泌。辛德比斯(SIN)病毒 复制子、SIN病毒双相病毒和T7重组痘苗病毒 基因工程系统被用来表达登革热病毒前膜 (PRM)和包膜(E)蛋白抗原。DEN的高水平表达 在感染这些载体的细胞中观察到病毒抗原。 含有登革热病毒PrM和E基因。然而，大多数人 蛋白质仍然在细胞内，这表明表达的蛋白质 不适当的加工或折叠，阻止细胞外分泌。 改善细胞内的处理和增强细胞的分泌 来自受感染细胞的蛋白质必须包括在克隆的 DEN蛋白基因盒，编码最后15个氨基酸的基因 衣壳蛋白的羧基末端以及碱性氨基酸 在Prm/E裂解位点。编码到双顺反子中的DEN病毒基因 SIN病毒不稳定。为了进一步提高基因的表达， 在病毒复制子中，该载体经过基因工程改造，以促进 通过添加RNA在体内转录亚基因组RNA RSV真核启动子控制下的转录起始点 在病毒3‘-NTR末端附加一个Deltabozyme编码 书房插入物。这些变化简化了转导过程， 改善了基因表达。重组痘苗病毒载体PTM-1转化成 所克隆的哪些DEN病毒PrM和E基因表达高水平的DEN 瞬时系统和重组痘苗病毒中的病毒蛋白 携带T7聚合酶。为了方便选择重组人 痘苗病毒编码病毒基因，新霉素抗性基因 在7.5启动子的控制下进入痘苗载体 在用于同源重组的TK-区之外。研究到 提高DEN病毒PrM和E蛋白的水平，并促进 抗原的分泌目前正在进行中。

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.