OPTIMIZATION OF PRIMERS FOR CLONING LIBRARIES OF MOUSE IMMUNOGLOBULIN GENES USING THE POLYMERASE CHAIN-REACTION
OPTIMIZATION OF PRIMERS FOR CLONING LIBRARIES OF MOUSE IMMUNOGLOBULIN GENES USING THE POLYMERASE CHAIN-REACTION
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DOI：
10.1002/eji.1830230132
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发表时间：
1993-01-01
影响因子：
5.4
通讯作者：
BENDIG, MM
中科院分区：
文献类型：
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作者：
KETTLEBOROUGH, CA;SALDANHA, J;BENDIG, MM
我们利用聚合酶链式反应优化了克隆小鼠重链可变区和轻链可变区文库的引物。由于我们有兴趣克隆小鼠Fab片段以在细菌细胞中表达，因此设计了重链引物来克隆包含重链可变区和Ig G恒定区第一结构域的Fd片段。轻链引物被设计用来克隆整个鼠卡伯链。用10个简并5‘和1个简并3’引物扩增小鼠Fd，用7个简并5‘引物加3’引物扩增kappa链，从小鼠脾中克隆了一系列小鼠可变区。
We have optimized primers for cloning libraries of murine heavy and light chain variable regions using the polymerase chain reaction. Since we are interested in cloning murine Fab fragments for expression in bacterial cells, the heavy chain primers were designed to clone Fd fragments comprising the heavy chain variable domain and the first domain of the IgG constant region. The light chain primers were designed to clone the entire murine kappa chain. Using ten degenerate 5' primers and a degenerate 3' primer to amplify murine Fd and seven degenerate 5' primers with a single 3' primer to amplify kappa chains, a diverse repertoire of mouse variable regions was cloned from mouse spleens.