Laboratory of Molecular and Cellular Biology, Faculty of Science and Engineering, Graduate School, University of Toyama, 3190 Gofuku, Toyama-shi, Toyama, 930-8555, Japan.
BMC Biotechnol. 2011 Apr 13;11:39. doi: 10.1186/1472-6750-11-39.
Molecular cloning of functional immunoglobulin genes from single plasma cells is one of the most promising technologies for the rapid development of monoclonal antibody drugs. However, the proper insertion of PCR-amplified immunoglobulin genes into expression vectors remains an obstacle to the high-throughput production of recombinant monoclonal antibodies.
We developed a single-step cloning method, target-selective homologous recombination (TS-HR), in which PCR-amplified immunoglobulin variable genes were selectively inserted into vectors, even in the presence of nonspecifically amplified DNA. TS-HR utilizes Red/ET-mediated homologous recombination with a target-selective vector (TS-vector) with unique homology arms on its termini. Using TS-HR, immunoglobulin variable genes were cloned directly into expression vectors by co-transforming unpurified PCR products and the TS-vector into E. coli. Furthermore, the high cloning specificity of TS-HR allowed plasmids to be extracted from pools of transformed bacteria without screening single colonies for correct clones. We present a one-week protocol for the production of recombinant mouse monoclonal antibodies from large numbers of single plasma cells.
The time requirements and limitations of traditional cloning procedures for the production of recombinant immunoglobulins have been significantly reduced with the development of the TS-HR cloning technique.
从单个浆细胞中克隆功能性免疫球蛋白基因是快速开发单克隆抗体药物最有前途的技术之一。然而，将 PCR 扩增的免疫球蛋白基因正确插入表达载体仍然是高通量生产重组单克隆抗体的障碍。
我们开发了一种一步法克隆方法，即靶向选择性同源重组（TS-HR），其中 PCR 扩增的免疫球蛋白可变基因可选择性地插入载体中，即使存在非特异性扩增的 DNA 也是如此。TS-HR 利用 Red/ET 介导的同源重组，其末端带有独特同源臂的靶向选择载体（TS-vector）。使用 TS-HR，可通过将未经纯化的 PCR 产物和 TS-vector 共转化入大肠杆菌，直接将免疫球蛋白可变基因克隆到表达载体中。此外，TS-HR 的高克隆特异性允许从转化细菌的混合物中提取质粒，而无需筛选单个菌落以获得正确的克隆。我们提出了一种从大量单个浆细胞生产重组鼠单克隆抗体的一周方案。
随着 TS-HR 克隆技术的发展，传统克隆程序生产重组免疫球蛋白的时间要求和局限性得到了显著降低。
