大容量人乳腺癌单链抗体库的构建与初步鉴定

Construction of a large phage displayed scFv library of human breast carcinoma

目的利用细胞内重组技术构建大容量人乳腺癌单链抗体(scFv)库。方法收集30例乳腺癌外周转移淋巴结,依次提取总RNA、mRNA,RT-PCR扩增全套可变区基因(VL、VH),各自克隆到T载体,构建T载体库。然后从T载体上酶切VL、VH分步克隆到噬粒载体pDAN5,经电转化TG1得到初级库。辅助性噬菌体感染初级库,使噬菌体抗体表面呈现。提纯噬菌体抗体,滴定浓度后感染大肠杆菌BS1365进行细胞内重组。噬菌体挽救后再次提纯滴定噬菌体,感染大肠杆菌DH5αF',得到次级库,并测序验证。结果VH、VLT载体库分别为3.5×108和1.7×108,初级库库容量2.5×109。次级库库容量为2.5×1011。结论所采用的引物能够高效扩增目的片段,T载体的应用能够提高抗体基因的克隆效率,细胞内重组将进一步扩大库容量及增加其多样性,这种基于RT-PCR、T载体过渡、细胞内重组的路线能够构建大容量抗体库。

Objective To prepare a large phage displayed scFv library of human breast carcinoma by using in vivo recombination. Methods Peripheral lymphadens of 30 patients with breast carcinoma were collected, from which total RNA was extracted and mRNA was derived. Then the V gene was amplified by RT - PCR. The PCR products were cloned into T vector firstly in order to be efficiently cloned into expression vector pDANS. By rescuing with the helper phage, primary library was obtained. Then E. coliBS1365 was infected by the primary library at high multiplicity of infection （ MOI ） so the in - vivo recombination was carried oh , the VH and VL genes were exchanged between different phagemids, creating many new VH/VL combinations the secondary library was constructed. Results The primers adopted can efficiently recognize functional V genes. T clone libraries of VH and VL were 3.5×10^8and 1.7 ×10^8 respectively. The primary library was 2.5 ×10^9 , and the secondary, 2.5×10^11. Conclusion A large antibody library of human breast carcinoma was constructed successfully. The application of T vector can greatly improve the cloning efficiency. The in - vivo recombination can expand the diversity. By adopting RT - PCR,T vector and in -vivo recombination ,large antibody library can be constructed.