抗D二聚体的人源性噬菌体抗体库的构建

目的：应用噬菌体展示技术构建人源性抗D二聚体噬菌体抗体抗体组合文库。 方法： 从不同人群外周血淋巴细胞中提取总RNA，经反转录后，以免疫球蛋白信号肽序列引物和家族特异性免疫球蛋白可变区基因引物,进行半套式PCR扩增人全套抗体基因片段，并克隆于pComb3H载体，电转化大肠杆菌XL1-Blu，在辅助噬菌体的超感染下，构建噬菌体抗体组合文库。 结果： 采用不同的引物进行重链、轻链Kappa和Lambda链的半套式PCR扩增，均能获得相应大小的PCR产物，其结果扩增率达到100％。经4次电转化构建了库容为2.8×108的抗体库,轻、重链基因的重组率为46％，经辅助噬菌体的超感染，得到噬菌体滴度为4.1×1017PFU /L的人源性噬菌体抗体库。 结论： 成功构建了含抗D二聚体的人源性噬菌体抗体库，为进一步筛选抗D二聚体的Fab噬菌体抗体奠定了基础。

Construction of human immunoglobulin combinatorial library containing D-Dimer on the surface of phage

AIM: To construct a human recombinant immunoglobulin library containing D-Dimer by using phage surface display technology. METHODS: Human immunoglobulin heavy chain and light chain genes were amplified respectively by RT-PCR from different human lymphocytes using family specific primers and signal sequences of immunoglobulin as half-nested PCR primers. The heavy chain and light chain PCR products were cloned into phagemid vector pComb3H and the human immunoglobulin recombination library was generated with helper phage VCSM13. RESULTS: A human combinatorial antibody library consisting of 2.8×108 in dependent clones was generated with a titer of 4.1×1017PFU/L. The recombinant frequency of Fab genes was 46%. CONCLUSION: A human combinatorial antibody library was generated. It will be beneficial for selecting Fab antibodies of D dimer from the library.