Sequencing and cloning of antigen-specific antibodies from mouse memory B cells
Sequencing and cloning of antigen-specific antibodies from mouse memory B cells
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DOI：
10.1038/nprot.2016.102
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发表时间：
2016-10-01
期刊：
影响因子：
14.8
通讯作者：
Nussenzweig, Michel C.
中科院分区：
文献类型：
--
作者：
von Boehmer, Lotta;Liu, Cassie;Nussenzweig, Michel C.
从单个B淋巴细胞中鉴定编码免疫球蛋白重链和轻链的基因的方法在效率、错误率和实用性方面各不相同。在这里，我们描述了一个协议的序列和克隆的可变抗体区的单抗原特异性小鼠记忆B细胞的抗体生产。纯化后，首先通过荧光激活细胞分选（FACS）对抗原特异性小鼠记忆B细胞进行单细胞分选，并通过RT-PCR扩增V（D）J转录物。然后将片段与线性化表达载体组合，作为序列和连接非依赖性克隆（SLSLIC）反应的一部分在体外组装，然后转化到大肠杆菌中。纯化的载体然后可用于在HEK 293 E悬浮细胞中产生单克隆抗体。该方案提高了抗体可变区基因的扩增效率，加快了克隆工作流程。抗体序列将在3-4天内获得，微克至毫克量的抗体将在14天内产生。新的协议应该是有用的解决有关抗原特异性记忆B细胞反应的基本问题，以及表征抗原特异性抗体。
Methods to identify genes encoding immunoglobulin heavy and light chains from single B lymphocytes vary in efficiency, error rate and practicability. Here we describe a protocol to sequence and clone the variable antibody region of single antigen-specific mouse memory B cells for antibody production. After purification, antigen-specific mouse memory B cells are first single-cellsorted by fluorescence-activated cell sorting (FACS), and V(D)J transcripts are amplified by RT-PCR. Fragments are then combined with linearized expression vectors, assembled in vitro as part of a sequence-and ligation-independent cloning (SLSLIC) reaction and then transformed into Escherichia coli. Purified vectors can then be used to produce monoclonal antibodies in HEK293E suspension cells. This protocol improves the amplification efficiency of antibody variable genes and accelerates the cloning workflow. Antibody sequences will be available in 3-4 d, and microgram to milligram amounts of antibodies are produced within 14 d. The new protocol should be useful for addressing fundamental questions about antigen-specific memory B cell responses, as well as for characterizing antigen-specific antibodies.