抗血小板人源噬菌体抗体库的构建和初步筛选

发布时间：2018-05-29 07:22

本文选题：血小板 + 噬菌体展示　；参考：《临床输血与检验》2006年01期

【摘要】：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。
[Abstract]:Objective to construct a platelet autoimmune antibody library using phage antibody library. Methods lymphocytes were isolated from peripheral blood of three patients with chronic idiopathic thrombocytopenic purpura (ITP). Total RNAs were extracted and synthesized by reverse transcription. The light chain 魏 chain gene and heavy chain FD gene were amplified by semi-nested PCR. The human phage Fab antibody library was constructed by cloning into the vector pComb3 in turn. Results the human phage Fab antibody library containing about 2.3 脳 106cfu was obtained after 20 times of electroporation. Platelet cells fixed with paraformaldehyde were used as antigens, and three rounds of affinity selection were used to identify those with high binding activity. The sequence analysis showed that the Fd fragment of light chain and heavy chain of antibody were in accordance with the structural characteristics of human immunoglobulin gene at the level of nucleotide and amino acid. Conclusion the anti-platelet human phage antibody library was successfully constructed, which can be used to screen human antibodies against platelet-specific targets such as GPIIb/IIIa, and to study and develop therapeutic human antibodies targeting GPIIb/IIIa receptors.
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【分类号】：Q789

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