菌落PCR快速分析抗体库重组率时的假阳性现象

False positives resulted from colony PCR in rapid identification of recombinant clones from phage antibody library

目的 评价菌落PCR法鉴定噬菌体抗体库阳性重组率的可靠性。方法 鼠抗体基因Lc片段、Fd片段经酶切、纯化后 ,依次克隆入pComb3载体上相应的酶切位点 ,电转化XL1 blue菌后建立鼠源性Fab噬菌体抗体库。比较菌落PCR法、质粒PCR法及质粒酶切法鉴定转化菌阳性重组率的一致性。同时用空菌、空载体、空载体菌等作模板为对照PCR ,以排除相关组分的干扰。结果 建立的Lc库的库容为 1.175×10 6CFU ,Fab库的库容为 1.0 2×10 6CFU。 3种方法鉴定的阳性重组率不同 (Lc的重组率依次为 10 0 %、78%、78% ,Fd的重组率依次为 90 %、6 6 %、6 6 % ,Lc与Fd同时插入的重组率依次为 90 %、5 0 %、5 0 % )。菌落PCR法鉴定的重组率偏高 ,存在假阳性现象。对照PCR提示 ,XL1 blue菌本身可能是导致假阳性扩增的原因。结论 用菌落PCR法不能准确鉴定噬菌体抗体库的重组率 。

Objective To evaluate the reliability of colony PCR in identifying the recombinant clones selected from phage antibody libraries. Methods The digested pComb3 vector and Lc fragments, Lc library plasmid, and Fd fragments were ligated successively. The ligation product was transformed into Escherichia Coli strain XL 1-blue bacilli by electroporation and thus murine Fab phage antibody library was constructed.The transformed recombinant clones selected randomly from libraries were identified simultaneously by colony PCR, plasmid PCR and restriction enzyme digestion. The identification consistency was analyzed. The interference was excluded by control PCR using the relevant constituents as template. Results The educed library content of Lc library was 1.175×10 6 CFU and the content of Fab antibody library was 1.02×10 6 CFU. Different recombinant percentages were obtained through three different identification methods (the Lc positive recombinant percentages by colony PCR, plasmid PCR and enzyme digestion were 100%, 78% and 78%, respectively; the Fd positive recombinant percentages by three methods were 90%, 66% and 66%, respectively; the dual positive recombinant (Fd and Lc insert simultaneously) percentages by three methods were 90%, 50% and 50%, respectively). A high frequency of false-positives appeared in colony PCR identification. Nonspecific amplification of control PCR was presumably induced by some intracellular components in XL 1-blue bacilli. Conclusion The identification of the recombinant clones selected from phage antibody libraries by colony PCR remains ambiguous. So it is our assertion that the traditional identification methods such as plasmid PCR or enzyme digestion are more accurate and will less false positive results.