V-gene amplification revisited - An optimised procedure for amplification of rearranged human antibody genes of different isotypes
V-gene amplification revisited - An optimised procedure for amplification of rearranged human antibody genes of different isotypes
复制标题
DOI：
10.1016/j.nbt.2010.01.001
复制
发表时间：
2010-05-31
影响因子：
5.4
通讯作者：
Konthur, Zoltan
中科院分区：
文献类型：
--
作者：
Lim, Theam Soon;Mollova, Svetlana;Konthur, Zoltan
为了研究人抗体可变（V）基因在任何个体组中的使用或为了产生用于噬菌体展示的重组人抗体文库，扩增的V基因库的质量和产量是极其重要的。影响完整抗体库扩增的关键参数是V基因特异性引物设计、从外周血单核细胞（PBMC）的总RNA提取物合成互补DNA（cDNA）以及最终聚合酶链反应（PCR）。在这项工作中，我们分析了所有这些因素，我们进行了详细的生物信息学分析的V-基因特异性引物的基础上VBASE 2和不同的商业可用的逆转录酶的cDNA合成和聚合酶的PCR效率的影响进行了评估。所提供的引物覆盖了VBASE 2中几乎100%的所有功能性和纯功能性V基因，并且最终方案提供了用于cDNA合成的商业酶和反应添加剂的优化组合以及用于V基因扩增的PCR条件。最后，将该方案与不同的免疫球蛋白（IG）链特异性反向引物组合应用，我们能够扩增所研究的不同同种型的重排抗体基因。
For studying human antibody variable (V)-gene usage in any group of individuals or for the generation of recombinant human antibody libraries for phage display, quality and yield of the amplified V-gene repertoire is of utmost importance. Key parameters affecting the amplification of full antibody repertoires are V-gene specific primer design, complementary DNA (cDNA) synthesis from total RNA extracts of peripheral blood mononuclear cells (PBMCs) and ultimately the polymerase chain reaction (PCR). In this work we analysed all these factors; we performed a detailed bioinformatic analysis of V-gene specific primers based on VBASE2 and evaluated the influence of different commercially available reverse transcriptases on cDNA synthesis and polymerases on PCR efficiency. The primers presented cover near to 100% of all functional and putatively functional V-genes in VBASE2 and the final protocol presents an optimised combination of commercial enzymes and reaction additives for cDNA synthesis and PCR conditions for V-gene amplification. Finally, applying this protocol in combination with different immunoglobulin (Ig) chain specific reverse primers we were able to amplify rearranged antibody genes of different isotypes under investigation.