EXPRESSION OF FOREIGN GENES IN CULTURED HUMAN PRIMARY MACROPHAGES USING RECOMBINANT VACCINIA VIRUS VECTORS
EXPRESSION OF FOREIGN GENES IN CULTURED HUMAN PRIMARY MACROPHAGES USING RECOMBINANT VACCINIA VIRUS VECTORS
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DOI：
10.1016/0378-1119(94)90257-7
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发表时间：
1994-05-16
期刊：
影响因子：
3.5
通讯作者：
BERGER, EA
中科院分区：
文献类型：
--
作者：
BRODER, CC;KENNEDY, PE;BERGER, EA
重组牛痘病毒 (re-VV) 提供了一种极其通用的方法，可在不同谱系和物种的多种培养细胞类型中表达外源基因。在本报告中，我们研究了re-VV载体在通过外周血单核细胞体外分化获得的培养的人原代巨噬细胞中生产re蛋白的效用。原代巨噬细胞支持 VV 感染周期的早期阶段，包括形态细胞病变效应、宿主蛋白合成的关闭和早期病毒蛋白合成的激活；然而，感染的后期阶段被阻断，包括晚期病毒蛋白的合成、病毒DNA的复制以及感染性子代病毒颗粒的产生。在几个独立的 VV 菌株中观察到流产感染。使用含有大肠杆菌 lacZ 的 re-VV 作为报告基因，我们测定了不同类别的 VV 启动子的活性。与上述结果一致，人原代巨噬细胞支持由早期或中间 VV 启动子驱动的报告基因表达，但不由晚期启动子驱动；使用含有早期和/或中间成分的合成双功能启动子获得表达。原代巨噬细胞还支持 VV/噬菌体 T7 RNA 聚合酶杂合基因表达系统。使用编码人 CD4 和人免疫缺陷病毒 1 型包膜糖蛋白的 re-VV 证明了 re-VV 载体在人原代巨噬细胞中生产具有生物学意义的蛋白质的效用。
Recombinant vaccinia viruses (re-VVs) provide an extremely versatile method for the expression of foreign genes in a wide range of cultured cell types of different lineages and species. In the present report, we examine the utility of re-VV vectors for re-protein production in cultured human primary macrophages obtained through in vitro differentiation of peripheral blood monocytes. Primary macrophages supported early stages of the VV infection cycle, including morphologic cytopathic effect, shut-off of host protein synthesis and activation of early viral protein synthesis; however, late stages of infection were blocked, including synthesis of late viral proteins, replication of viral DNA, and production of infectious progeny virions. Abortive infection was observed with several independent VV strains. Using re-VVs containing Escherichia coli lacZ as a reporter gene, we assayed the activities of different classes of VV promoters. Consistent with the results noted above, human primary macrophages supported reporter gene expression driven by an early or intermediate VV promoter, but not by a late promoter; expression was obtained with synthetic bifunctional promoters containing early and/or intermediate components. Primary macrophages also supported the VV/bacteriophage T7 RNA polymerase hybrid gene expression system. The utility of re-VV vectors for production of proteins of biological interest in human primary macrophages was demonstrated using re-VVs encoding human CD4 and the human immunodeficiency virus type-1 envelope glycoprotein.