Walls M A, Hsiao K C, Harris L J
Bristol-Myers Squibb Pharmaceutical Research Institute-Seattle, Department of Molecular Immunology, WA 98121.
Nucleic Acids Res. 1993 Jun 25;21(12):2921-9. doi: 10.1093/nar/21.12.2921.
Cassette vectors have been constructed for mammalian expression of complete immunoglobulin heavy and light chain genes whose variable regions are produced by the polymerase chain reaction (PCR). The light and heavy chain vectors have promoter, leader, partial intron, enhancer and constant region segments within modified pSV2-gpt and pSV2-neo plasmids, respectively. Variable (V) regions are obtained by PCR using a two step process: 1) the V gene is amplified from genomic or cDNA, cloned into an intermediate vector and sequenced; 2) the first PCR product serves as the template for a second amplification in which restriction enzyme recognition sites and limited flanking intron sequence are added. The second PCR product is inserted into the expression vector, which is then transfected into mouse myeloma cells. These vectors contain human constant regions and may be used to express chimeric, humanized or human Ig genes. This report describes the design of these vectors and their application for the expression of chimeric 60.3, an anti-CD18 antibody.
已构建用于完整免疫球蛋白重链和轻链基因哺乳动物表达的盒式载体，其可变区由聚合酶链反应（PCR）产生。轻链和重链载体分别在修饰的pSV2 - gpt和pSV2 - neo质粒中具有启动子、前导序列、部分内含子、增强子和恒定区片段。可变（V）区通过两步PCR过程获得：1）从基因组或cDNA扩增V基因，克隆到中间载体并测序；2）第一个PCR产物用作第二次扩增的模板，其中添加限制酶识别位点和有限的侧翼内含子序列。第二个PCR产物插入表达载体，然后转染到小鼠骨髓瘤细胞中。这些载体包含人恒定区，可用于表达嵌合、人源化或人Ig基因。本报告描述了这些载体的设计及其在嵌合60.3（一种抗CD18抗体）表达中的应用。
