扩增抗体重链可变区的一个高效方法

A Novel Method of Amplifying the Heavy-chain Variable Regions from Immunoglobulin cDNAs

从杂交瘤细胞或脾细胞中扩增抗体可变区,是构建单链抗体(Single-chainFvfragment,ScFv)、克隆单克隆抗体和研究抗原与抗体相互作用的关键一步。而抗体可变区(Variableregions,Fv)高度变异,扩增相对困难,至今仍是一个难点,有待解决。我们在构建ScFv过程中,发现2株杂交瘤细胞用普通方法难于扩增出抗体重链可变区(Heavy-chainvariableregion,VH)抗体。于是提出了一种多序列比对和简并引物设计算法,并加以实现,设计出通用的鼠源性抗体VH引物;应用上述引物,采用反向多聚酶链反应(Reversepolymerasechainreaction,reversePCR)方法——3'RACE和5'RACE法,准确地扩增出2株杂交瘤细胞的VH基因。该算法很好地解决了引物特异性和最小化问题,具有简单、实现容易的特点,可用于基因家族中未知基因克隆和文库构建中的引物设计。与反向PCR方法结合,提高了难扩增的未知基因的成功率。

The amplification of variable regions （Fv） of immunoglobulins has become a crucial technique for studying antigen-antibody interactions and cloning the monoclonal antibodies （mAbs）. But this work becomes a major challenge in cloning antibody genes either from hybridoma cell lines or B cells. Isolation of the immunoglobulin heavy-chaln variable regions （VH） from two mouse hybridoma cell lines based on conventional protocols using both the specific consensus primers and the commercial primer set of VH regions, was unsuccessful. This prompted us to design our own primers for VH regions of immunoglobulins. A novel algorithms and reverse polymerase chain reaction （PCR） protocol 3＇ RACE and 5＇ RACE were established in order to recognize the VH genes. The most conserved region in the end of framework region （FR1） of the VH eDNA region was identified with the program and a degenerated primer were designed in the most conserved region followed by 3＇ RACE and 5＇ RACE, The new protocol rescued the failed PCR amplifications. Thereby, a improvement in the degenerate primers designing programs and a notable increase in amplification specificity were achieved. This approach can be useful in primer design and amplification of VH and light-chain variable regions （VL） gene libraries or cloning of the unknown genes in the large gene families and extend to other species or members of the immunoglobulin superfamily.