噬菌体展示抗体微型文库的构建

Construction of scFv Antibody Mini-library to 3 Cellular Surface Molecules

用噬菌体展示技术构建抗神经钙粘连素(N-cadherin,N-cad)分子胞外结构域、CD34分子胞外结构域及AC133分子的噬菌体展示单链抗体库。将3种分子分别表达于噬菌体表面,再以此噬菌体免疫BALB/c小鼠,提取脾总RNA,RT-PCR扩增抗体重、轻链基因cDNA并插入噬菌体表达载体,构建分别抗N-cad分子胞外结构域、CD34分子胞外结构域及AC133分子的噬菌体表达文库。然后对文库的库容量、多样性、滴度及表达能力进行鉴定。结果显示,用噬菌体展示的3种分子免疫小鼠获得成功,所构建抗体文库的库容量、多样性、滴度及表达能力均能满足进一步筛选目的抗体基因的需要,为制备相应的特异性抗体奠定了基础。

To construct a scFv antibody mini-library by phage display technique from the spleen cells of BALB/c mice immunized with extracellular domain of N-cadherin（N-cad）, CD34 and AC133, the extracellular domain genes of N-cad, CD34 and AC133 were cloned into a phagemid pSEX81 respectively, and were then displayed on the phagemid in the form of fusion protein with p I protein. After being effectively immunized with the fusion protein, the spleen of the mice were harvested, and total RNA were extracted from the spleen. The eDNA of VH and VL genes were amplified by RT-PCR. and a scFv-phage display antibody library was constructed with the amplified V-genes, The content . multiplicity and expressing potential of the library were examined, As a result, we had produced a scFv library containing 1, 4 ×10^6 individual clones which showed different patterns after being digested with restriction endoneuclease Mua 1. The surface display expression of the library was also verified. It indicated that the capacity and diversity of the library was sufficient for screening specific scFv antibody against N-cad, CD34 and AC133, The library will be useful for isolating corresponding specific scFv antibodies.