Sarantopoulos S, Kao C Y, Den W, Sharon J
Department of Pathology, Boston University School of Medicine, MA 02118.
J Immunol. 1994 Jun 1;152(11):5344-51.
Libraries of Ab fragments have been produced by others from light and heavy chain cDNAs derived from populations of B lymphocytes and expressed in bacteria. However, such libraries have not been transferred to eukaryotic expression vectors to generate polyclonal libraries of intact glycosylated Abs that can mediate effector functions. We present a method for transferring pairs of linked VL-VH region genes between circular prokaryotic and eukaryotic vectors. The key feature of the transfer is that the VL and VH region genes are linked head to head (<-->) in opposite transcriptional orientations. To illustrate this method, a pair of VL and VH region cDNAs derived from an existing hybridoma cell line were linked head to head by PCR, transferred as a unit between vectors, and expressed as an IgG Ab with Ag binding activity. Although we tested the transfer of a single VL-VH region gene pair, this system is expected to allow the bulk transfer of physically linked VL-VH region gene combinations between different circular vectors and the expression of the same library as either Ab fragments or intact Abs.
其他人已利用源自B淋巴细胞群体的轻链和重链cDNA在细菌中表达，构建了抗体（Ab）片段文库。然而，此类文库尚未被转移至真核表达载体，以生成能够介导效应功能的完整糖基化抗体的多克隆文库。我们提出了一种在环状原核和真核载体之间转移相连的VL-VH区域基因对的方法。该转移方法的关键特征在于，VL和VH区域基因以头对头（<-->）的方式相连，转录方向相反。为说明此方法，通过PCR将源自现有杂交瘤细胞系的一对VL和VH区域cDNA头对头相连，作为一个单元在载体之间转移，并表达为具有抗原结合活性的IgG抗体。尽管我们测试了单个VL-VH区域基因对的转移，但该系统有望实现不同环状载体之间物理相连的VL-VH区域基因组合的批量转移，并将同一文库表达为抗体片段或完整抗体。
