人源性抗核抗体Fab片段噬菌体抗体库的构建

Construction of human recombinant antinuclear antibody Fab library on the surface of phage

目的构建人源性抗核抗体(ANA)Fab片段噬菌体抗体库。方法从4名ANA滴度大于1∶10000的自身免疫病患者外周血淋巴细胞中抽提总RNA,用RT-PCR技术扩增免疫球蛋白分子轻链κ、λ基因及重链Fd基因。将轻链基因片段克隆入pComb3Hss载体以构建轻链文库,随后将重链基因载入κ/λ-pComb3Hss载体,形成κ/λ-Fd-pComb3Hss质粒,然后将重组质粒转化大肠杆菌XL1-Blue。用辅助噬菌体M13KO7感染XL1-Blue,随机的重组抗体文库即表达于丝状噬菌体表面。结果扩增了长度为660bp的轻链κ、λ基因及重链Fd基因,并成功构建了库容为2×104的轻链基因抗体库和库容为4×104的人Fab抗体库,获得了噬菌体滴度为2×109CFU/ml的富含人源性抗核抗体Fab片段的噬菌体抗体库。结论成功构建了人源性抗核抗体Fab片段噬菌体抗体库,为人源性抗核抗体Fab片段的制备以及进一步评价其在肿瘤免疫靶向治疗中的作用奠定了基础。

Objective To construct a phage antibody Fab library of human antinuclear antibody. Methods Total RNA was extracted from peripheral blood lymphoeytes of 4 patients suffering from autoallergic disease, with antinuclear IgG antibody titers in the serum higher than 1：10 000 as estimated by indirect immunofluorescence technique. Taking oligo-dT as the primers, human immunoglobulin light chains κ/λgenes as well as heavy chains Fd genes were reversely transcribed to cDNA by PCIL The κ/λ light chains were initially cloned into pComb3Hss vector to construct a human recombinant light chain library, and the heavy chain genes were subsequently inserted into the corresponding sites of κ/λ-pComb3Hss plasmid to generate a recombinant κ/λ,-Fd-pComb3Hss plasmid. The plasmid was then transformed into E. coli XL1-Blue, which was subsequently infected by the helper phage M13KO7. A random recombinant antibody library was expressed on the surface of filamentous phage. Results Human immunoglobulin κ/λ, light chain and heavy chain Fd genes （660bp） were amplified successfully. The light chain library with the capacity size of 2 ×10^4 , and the human recombinant Fab antibody library with the ca- pacity size of 4 ×10^1 were also successfully obtained. Finally, the phage antibody library of the Fab fragment of human antinuclear antibody, containing 2×10^9 CFU/ml phage, was constructed. Conclusion A phage antibody library of Fab fragment of human antinuclear antibody is constructed successfully. This research lays a foundation for the preparation for phage library of Fab fragment of human antinuclear antibody, and also paves the way for the advanced assessment for its effect on the immuno-targeting therapy of tumors.