INFECTIOUS SINDBIS VIRUS TRANSIENT EXPRESSION VECTORS FOR STUDYING ANTIGEN PROCESSING AND PRESENTATION
INFECTIOUS SINDBIS VIRUS TRANSIENT EXPRESSION VECTORS FOR STUDYING ANTIGEN PROCESSING AND PRESENTATION
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DOI：
10.1073/pnas.89.7.2679
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发表时间：
1992-04-01
影响因子：
11.1
通讯作者：
RICE, CM
中科院分区：
文献类型：
--
作者：
HAHN, CS;HAHN, YS;RICE, CM
辛德毕斯病毒（SIN）是一种小的正链包膜RNA病毒，感染范围广泛的脊椎动物和昆虫细胞。设计用于瞬时表达异源RNA和蛋白质的SIN载体（称为dsSIN）通过将第二亚基因组mRNA启动子序列插入SIN基因组的非必需区域来工程化。通过使用这种载体，dsSIN重组体已被构建，其表达细菌氯霉素乙酰转移酶、流感血凝素（HA）的截短形式或编码两种不同的免疫显性细胞毒性T淋巴细胞（CTL）HA表位的小基因。用这些重组体感染鼠细胞系，导致每个细胞几乎等于10（6）-10（7）个氯霉素乙酰转移酶多肽的表达，并在体外通过适当的主要组织相容性复合物限制的HA特异性CTL克隆有效地致敏靶细胞以裂解。此外，在用表达截短形式的HA或免疫显性流感CTL表位的dsSIN重组体免疫小鼠后，观察到流感特异性T细胞应答的引发。这种SIN表达系统允许在几天内产生高滴度的重组病毒原液，并应有助于通过抗原加工和呈递的内源性途径表达的I类主要组织相容性复合物限制性T细胞表位的作图和突变分析。
Sindbis virus (SIN) is a small positive-strand enveloped RNA virus that infects a broad range of vertebrate and insect cells. A SIN vector (called dsSIN), designed for transient expression of heterologous RNAs and proteins, was engineered by inserting a second subgenomic mRNA promoter sequence into a nonessential region of the SIN genome. By using this vector, dsSIN recombinants have been constructed that express either bacterial chloramphenicol acetyltransferase, a truncated form of the influenza hemagglutinin (HA), or mini-genes encoding two distinct immunodominant cytotoxic T lymphocyte (CTL) HA epitopes. Infection of murine cell lines with these recombinants resulted in the expression of almost-equal-to 10(6)-10(7) chloramphenicol acetyltransferase polypeptides per cell and efficient sensitization of target cells for lysis by appropriate major histocompatibility complex-restricted HA-specific CTL clones in vitro. In addition, priming of an influenza-specific T-cell response was observed after immunizing mice with dsSIN recombinants expressing either a truncated form of HA or the immunodominant influenza CTL epitopes. This SIN expression system allows the generation of high-titered recombinant virus stocks in a matter of days and should facilitate mapping and mutational analysis of class I major histocompatibility complex-restricted T-cell epitopes expressed via the endogenous pathway of antigen processing and presentation.