新式SOE PCR高效组装人单链抗体在高容量人天然噬菌体抗体库中的应用

Application of an improved efficient SOE PCR in assembling scFv genes during construction of large human antibody library

目的探讨新式SOEPCR高效组装人单链抗体在高容量人天然噬菌体抗体库中的应用。方法通过引物设计的改变,将6种人VH基因和11种人VL基因利用两步法直接互相两两连接为人scFv基因,并且与传统的SOE法进行对比。scFv基因用于构建噬菌体单链抗体库。结果新式SOEPCR法成功的将6种人VH基因和11种人VL基因连接成66种不同的人scFv基因,与传统的SOEPCR法相比,效率高且方法简便,经过约130次电转化后,抗体库的总库容为5·58×109。结论成功地改进了传统的SOEPCR方法,高效组装了66种不同的人scFv基因,提高了抗体库的多样性。

Objective To establish an improved efficient SOE PCR applied to assemble scFv genes during construction of a large human antibody library. Methods In this improved SOE PCR, 6 kinds of VH gene families and 11 kinds of VL gene families were amplified, then connected with linker genes respectively and finally assembled into scFv genes. The assembled scFv genes were used to construct a human phage antibody library. The scFv genes obtained by SOE PCR and this improved SOE PCR were compared. Results 66 different scFv gene families were successfully assembled by the improved SOE PCR. Comparing with SOE PCR, this new method was more efficient and easier. A naive human phage display antibody library, which contained 5.58 × 10^9 clones and exhibited high diversity, was constructed. Conclusion We successfully improve SOE PCR, assemble 66 different scFv gene families efficiently and optimize the diversity of antibody library.