EXPRESSION OF HUMAN CLASS-II MAJOR HISTOCOMPATIBILITY COMPLEX ANTIGENS USING RETROVIRUS VECTORS
EXPRESSION OF HUMAN CLASS-II MAJOR HISTOCOMPATIBILITY COMPLEX ANTIGENS USING RETROVIRUS VECTORS
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DOI：
10.1073/pnas.84.8.2150
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发表时间：
1987-04-01
影响因子：
11.1
通讯作者：
MULLIGAN, RC
中科院分区：
文献类型：
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作者：
KORMAN, AJ;FRANTZ, JD;MULLIGAN, RC
已经构建了逆转录病毒载体[直接定向（DO）载体]，其允许同时表达插入的蛋白质编码序列和显性作用的选择标记。在这些载体中，内部猿猴病毒40或人金属硫蛋白启动子序列用于驱动细菌新霉素磷酸转移酶或鸟嘌呤-黄嘌呤磷酸核糖基转移酶基因的表达，而病毒长末端重复序列用于促进插入序列的表达。在一些载体中，通常用于亚基因组env编码mRNA的生物发生的病毒5“剪接位点已被消除。这些载体在转染病毒包装细胞系后产生高瞬时和稳定滴度的病毒，用各种插入物显示很少或没有抑制病毒滴度，并忠实地将重组前病毒序列传递给受体细胞。为了表征这些载体的表达潜力，使用编码α-葡聚糖酶的多种插入片段。和β已将人主要组织相容性复合体II类抗原HLA-DR的亚基引入这些载体中。用含有HLA-DR α的病毒感染的NIH 3 T3细胞。或βcDNA表达这些蛋白质，如代谢标记提取物的免疫沉淀所示。此外，通过用表达两种不同选择性标记的逆转录病毒结构连续感染细胞，I类抗原的两个亚基都被引入NIH 3 T3细胞。这种感染的细胞在细胞表面表达HLA-DR分子。
Retrovirus vectors [direct orientation (DO) vectors] that permit the simultaneous expression of an inserted protein-coding sequence and a dominant-acting selectable marker have been constructed. In these vectors, an internal simian virus 40 or human metallothionein promoter sequence serves to drive the expression of the bacterial neomycin phosphotransferase or guanine-xanthine phosphoribosyltransferase genes, whereas the viral long terminal repeat sequences are utilized to promote expression of inserted sequences. In some of the vectors, the viral 5'' splice site, normally used in the biogenesis of the subgenomic env-encoding mRNA has been eliminated. These vectors yield high transient and stable titers of virus after transfection of viral packaging cell lines, show little or no depression of virus titer with a variety of inserts, and faithfully transmit recombinant proviral sequences to recipient cells. To characterize the expression potential of these vectors, a variety of inserts encoding the .alpha. and .beta. subunits of the human major histocompatibility complex class II antigen HLA-DR have been introduced into these vectors. NIH 3T3 cells infected by viruses containing HLA-DR .alpha. or .beta. cDNAs express these proteins as shown by immunoprecipitation of metabolically labeled extracts. In addition, through the sequential infection of cells with retrovirus constructions expressing two different selectable markers, both subunits of the class I antigen have been introduced into NIH 3T3 cells. Such infected cells express HLA-DR molecules at the cell surface.