EFFICIENT ISOLATION OF GENES BY USING ANTIBODY PROBES
EFFICIENT ISOLATION OF GENES BY USING ANTIBODY PROBES
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DOI：
10.1073/pnas.80.5.1194
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发表时间：
1983-01-01
期刊：
影响因子：
--
通讯作者：
DAVIS, RW
中科院分区：
文献类型：
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作者：
YOUNG, RA;DAVIS, RW
设计了一种灵敏和通用的技术，用于通过使用抗体作为探针克隆基因和分离由克隆的DNA编码的未知蛋白质的双重目的。所述方法使用表达载体，Δ gt 11（lac 5 nin 5 c1857 S100），其允许将外源DNA插入β-半乳糖苷酶结构基因lacZ和促进杂合蛋白质的合成。在大肠杆菌的hflA（高频溶原性）突变细胞中通过噬菌体文库的溶原性实现了在大肠杆菌gt 11重组c[互补]DNA文库中有效筛选产生抗原的克隆;溶原性在诱导时产生可检测量的抗原，甚至当以高细胞密度铺板时。该载体还被设计为促进由先前克隆的基因序列指定的蛋白质的分离。由重组噬菌体编码的杂合蛋白以适于大规模纯化的量在蛋白质降解缺陷的菌株（lon突变体）中积累。针对由外源DNA编码的杂合体部分产生的抗体又可用于从真核细胞中分离天然多肽。
A sensitive and general technique was devised for the dual purposes of cloning genes by using antibodies as probes and isolating unknown proteins encoded by cloned DNA. The method uses an expression vector, .lambda.gt11 (lac5 nin5 c1857 S100), that permits insertion of foreign DNA into the .beta.-galactosidase structural gene lacZ and promotes synthesis of hybrid proteins. Efficient screening of antigen-producing clones in .lambda.gt11 recombinant c[complementary]DNA libraries is achieved through lysogeny of the phage library in hflA (high-frequency lysogeny) mutant cells of Escherichia coli; lysogens produce detectable quantities of antigen on induction, even when plated at high cell densities. The vector is also designed to facilitate the isolation of proteins specified by previously cloned gene sequences. Hybrid proteins encoded by recombinant phage accumulate in strains defective in protein degradation (lon mutants) in amounts amenable to large-scale purification. Antibodies produced against the portion of the hybrid encoded by foreign DNA could in turn be used to isolate the native polypeptide from eukaryotic cells.