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目的:构建超大容量天然噬菌体抗体库。方法:从正常人外周血和新生儿脐血中分离淋巴细胞(>180份),提取RNA,用RT-PCR分别扩增抗体可变区轻重链基因(VH和VL),通过重叠PCR技术将VH和VL连接为单链抗体ScFv形式,克隆插入到pDF噬菌粒载体,转化XL1-Blue细菌得到ScFv初级抗体库,并以高感染复数(MOI≥100)感染Cre+菌株BS1365,利用Cre/LoxP位点特异性重组原理,使VH和VL基因定向同源重组匹配,随后以低感染复数(MOI<1)感染XL1-Blue,获取次级工作库。分别用5种不同抗原进行筛选,所获阳性克隆送测序以获取抗体基因。结果:抗体V区基因得到有效扩增,初级库库容3.6×107,工作库容1.8×1011,5种不同抗原筛选均得到特异性结合噬菌体抗体;测序结果表明,所获取抗体涵盖了不同的基因亚群,进一步证明抗体库具有良好的多样性。结论:经Cre/Loxp定位重组系统成功构建了超大容量天然噬菌体抗体库,初步尝试对5种抗原进行筛选均获成功,提示该抗体库多样性较好,可用于制备人源抗体。
Objective: To construct a large capacity natural phage antibody library. Methods: Lymphocytes (> 180 copies) were isolated from normal human peripheral blood and neonatal umbilical blood and RNA was extracted. The heavy and light chain variable domains (VH and VL) of antibody variable regions were amplified by RT-PCR. VH and VL were ligated into single chain antibody ScFv, cloned into pDF phagemid vector and transformed into XL1-Blue bacterium to obtain ScFv primary antibody library, and infected Cre + strain BS1365 with high multiplicity of infection (MOI≥100), using Cre / LoxP Site-specific recombination principle, the VH and VL genes targeted homologous recombination matching, and then infected with a low multiplicity of infection (MOI <1) XL1-Blue, to obtain a sub-working library. Five different antigens were screened respectively, and the positive clones were sent for sequencing to obtain antibody genes. Results: The gene of V region of the antibody was effectively amplified. The primary library had a capacity of 3.6 × 107 and a working capacity of 1.8 × 1011. The results showed that the obtained antibodies contained different genes The group further proved that the antibody library has a good diversity. CONCLUSION: The large-capacity natural phage antibody library was constructed successfully by Cre / Loxp site-directed recombination system. The initial attempts to screen all five antigens were successful, suggesting that the antibody library has good diversity and can be used for the preparation of human antibodies.