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目的:构建抗体库并筛选Rh抗原的Fab抗体。方法:收集5份血清中抗Rh抗体效价在1∶256～512的人淋巴细胞,提取RNA,用多对引物PCR扩增VH、Vκ、Vλ基因,并分别从pComb3xTT和pComb3xλ噬粒中扩增CH1、Cκ、Cλ,通过重叠PCR,构建重链Fd基因及完整的kappa、Lamda轻链,并进一步重叠PCR,获得Fab抗体片段。Fab经酶切、连接并电转化到噬菌体抗体表达载体pComb3xSS中,构建获得抗Rh的Fab抗体库,经4轮Rh(-)/Rh(+)红细胞阴/阳淘筛,获得的阳性克隆分别进行血凝试验、Western blot分析及测序鉴定。结果:构建获得总库容为7.4×106的Fab抗体库,并从中筛选得到1株能特异结合Rh抗原的Fab抗体。结论:应用基因工程抗体技术,成功获得了1株能与Rh(+)红细胞特异凝集的Fab抗体,为制备能用于临床的特异性强、效价高,并具有自主知识产权的Rh抗体打下基础。
OBJECTIVE: To construct an antibody library and screen for Fab antibodies against Rh antigen. Methods: Five human lymphocytes with anti-Rh antibody titers ranging from 1: 256 to 512 were collected and RNA was extracted. VH, Vκ and Vλ genes were amplified by PCR with multiple pairs of primers and amplified from pComb3xTT and pComb3xλ phage By adding CH1, Cκ and Cλ, the heavy chain Fd gene and complete kappa and Lamda light chain were constructed by overlap PCR, and further overlapped PCR to obtain the Fab antibody fragment. Fab was digested, ligated and electrotransformed into the phage antibody expression vector pComb3xSS to construct a library of anti-Rh Fab antibody. The positive clones obtained after 4 rounds of Rh (-) / Rh (+) erythrocytes Yin / Yang panning Hemagglutination test, Western blot analysis and sequencing identification. Results: The Fab library with a total volume of 7.4 × 106 was constructed and a Fab antibody that specifically bound to Rh antigen was screened out. CONCLUSION: A Fab antibody that can specifically agglutinate Rh (+) erythrocytes was successfully obtained by using genetically engineered antibody technology and was prepared for Rh antibody with high specificity, high potency and independent intellectual property for clinical application basis.