抗血小板人源噬菌体抗体库的构建和初步筛选

Construction and Screening to Human Immunoglobulin Combinatorial Library of Phage Antibodies Against Platelet

目的运用噬菌体抗体库技术构建一个经血小板自身免疫的抗体组合文库。方法从3例慢性特发性血小板减少性紫癜(ITP)患者的外周血中分离淋巴细胞,提取总RNA,经逆转录合成cDNA,通过半巢式PCR扩增抗体轻链κ链基因和重链Fd段基因,并依次克隆到载体pComb3中构建人源噬菌体Fab抗体库。结果20次电转后得到库容约为2.3×106cfu的人源噬菌体Fab抗体库。用多聚甲醛固定的血小板细胞作为抗原,通过3轮亲和选择,ELISA鉴定结合活性较高者,选取3个经测序分析后显示抗体轻链和重链Fd段在核苷酸和氨基酸水平都符合人免疫球蛋白基因结构特征。结论成功构建了抗血小板人源噬菌体抗体库,可用于进一步筛选针对血小板特异性靶点如GPIIb/IIIa等的人源抗体,进行以GPIIb/IIIa受体等作为靶点的治疗性人源抗体的研究和开发。

Objective To construct a human immunoglobulin combinatorial library by using phage surface display expression system and to screen the autoantibodies against platelet. Methods The total RNA was extracted from three patients with idiopathic thrombocytopenic purpura（ITP）, and cDNA was synthesized by reverse transcription. Half-nested PCR was employed to amplify the light chain and heavy chain Fd which were cloned into plasmid pComb3. 3 rounds of biopanning was conducted using activated coated platelet as antigen and the antibodies with the highest binding activity were sequenced. Results After 20 electroporations ,we got a Fab-phage display library with the capacity of 2.3×10^6cfu. The recombination rate reached 40%. Conclusion The constructed antibody library may be useful to further screening of antibodies against the GPIIb/IIIa.