Cloning and expression of murine Ig genes from single B cells
Cloning and expression of murine Ig genes from single B cells
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DOI：
10.1016/j.jim.2009.08.009
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发表时间：
2009-10-31
影响因子：
2.2
通讯作者：
Wardemann, Hedda
中科院分区：
文献类型：
--
作者：
Tiller, Thomas;Busse, Christian E.;Wardemann, Hedda
我们已经建立了一种高效的基于96孔格式的策略，通过将免疫球蛋白(Ig)基因克隆与单细胞水平的抗体表达和反应性分析相结合来表征表达的小鼠抗体谱系。通过荧光激活细胞分选技术分离单个小鼠B系细胞，并通过RT-PCR法扩增相应的免疫球蛋白重链(H)和免疫球蛋白轻链可变区(V)基因转录产物。将扩增产物克隆到真核表达载体中，可以在体外产生具有与初始B细胞抗原受体相同的抗原特异性的单抗。IgH和IGL链基因序列信息是作为克隆过程的一部分获得的，并且可以直接与重组抗体的反应谱相关联。总之，我们基于RT-PCR的策略，从单个小鼠B细胞产生重组单抗，允许通过序列分析和平行抗体反应性测试对表达的小鼠抗体库进行高效和无偏见的表征。(C)2009爱思唯尔B.V.保留所有权利。
We have established a highly efficient 96-well format based strategy to characterize the expressed murine antibody repertoire by combining immunoglobulin (Ig) gene cloning with antibody expression and reactivity profiling at the single cell level. Individual mouse B lineage cells are isolated based on defined surface marker expression patterns by fluorescence-activated cell sorting (FACS) and corresponding full-length Ig heavy (H) and Ig light (L) chain variable (V) region gene transcripts are amplified by RT-PCR. Cloning of the amplified products into eukaryotic expression vectors enables the in vitro production of monoclonal antibodies with antigen specificities identical to the initial B cell antigen receptors. IgH and IgL chain gene sequence information is obtained as part of the cloning procedure and can be directly linked to reactivity profiles of the recombinant antibodies. In summary, our RT-PCR based strategy to generate recombinant monoclonal antibodies from single mouse B cells allows the highly efficient and unbiased characterization of the expressed murine antibody repertoire by sequence analysis and parallel antibody reactivity testing. (C) 2009 Elsevier B.V. All rights reserved.