Gillies S D, Lo K M, Wesolowski J
Damon Biotech, Inc., Needham Heights, MA 02194.
J Immunol Methods. 1989 Dec 20;125(1-2):191-202. doi: 10.1016/0022-1759(89)90093-8.
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.
我们开发了一种快速且普遍适用的方法来修饰免疫球蛋白cDNA，以便可变（V）区能够作为盒式结构与多种恒定区一起表达。分离并测序了鼠源cDNA，将V区与短寡核苷酸连接，这些寡核苷酸提供了剪接供体位点和用于插入表达载体的独特限制性位点。利用这一策略，我们表达了几种鼠源抗体的V区以及人γ1恒定区。尽管大多数这些嵌合抗体很容易表达，但一个鼠源轻链cDNA序列在转染的杂交瘤细胞中无法表达。重建实验表明，由鼠源前导肽和可变区融合产生的序列在RNA积累水平上阻断了表达。所描述的方法以及表达中可能出现的问题，适用于传统的cDNA片段以及通过体外扩增技术获得的片段。
