HIGH-LEVEL EXPRESSION OF CHIMERIC ANTIBODIES USING ADAPTED CDNA VARIABLE REGION CASSETTES
HIGH-LEVEL EXPRESSION OF CHIMERIC ANTIBODIES USING ADAPTED CDNA VARIABLE REGION CASSETTES
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DOI：
10.1016/0022-1759(89)90093-8
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发表时间：
1989-12-20
影响因子：
2.2
通讯作者：
WESOLOWSKI, J
中科院分区：
文献类型：
--
作者：
GILLIES, SD;LO, KM;WESOLOWSKI, J
开发了一种用于修饰免疫球蛋白cDNA的快速且普遍适用的方法，使得可变（V）区可以与各种恒定区一起表达为盒。分离鼠cDNA，测序，并将V区连接到短寡核苷酸上，提供剪接供体位点和插入表达载体的独特限制性位点。使用这种策略，我们已经表达了几种鼠抗体的V区，以及人γ 1恒定区。虽然这些嵌合抗体中的大多数容易表达，但一个鼠轻链cDNA序列不能在转染的杂交瘤细胞中表达。重建实验表明，由鼠前导序列和可变区融合产生的序列在RNA积累水平上阻断表达。所描述的方法，以及表达的潜在问题，适用于传统的cDNA片段和通过体外扩增技术获得的那些。
A rapid and generally applicable method for the modification of immunoglobulin cDNAs was developed so that the variable (V) regions could be expressed as cassettes, together with a variety of constant regions. Murine cDNAs were isolated, sequenced and the V regions joined to short oligonucleotides providing both splice donor sites and unique restriction sites for insertion into an expression vector. Using this strategy we have expressed the V regions of several murine antibodies, together with the human gamma 1 constant region. Although most of these chimeric antibodies were readily expressed, one murine light-chain cDNA sequence could not be expressed in transfected hybridoma cells. Reconstruction experiments indicate that the sequence created by the fusion of the murine leader and variable region blocked expression at the level of RNA accumulation. The methods described, as well as the potential problems of expression, are applicable to both traditional cDNA fragments and those obtained by in vitro amplification techniques.