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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 construct large scFv human breast cancer scFv library by intracellular recombination technology. Methods Totally 30 cases of breast cancer peripheral lymph node metastasis were collected and the total RNA and mRNA were extracted. The complete set of variable region genes (VL and VH) were amplified by RT-PCR and cloned into T vector to construct T vector library. Then the VL vector was digested from the T vector, and the VH vector was cloned stepwise into the phagemid vector pDAN5. The primary library was obtained by electrotransformation of TG1. The helper phage infects the primary pool, rendering the phage antibody surface present. Phage antibody was purified and titrated to infect E. coli BS1365 for intracellular recombination. After the phage was rescued, the titer was purified again to infect E. coli DH5αF ’to obtain a secondary library and verified by sequencing. Results The VH and VLT vector libraries were 3.5 × 108 and 1.7 × 108, respectively. The initial library capacity was 2.5 × 109. Secondary library capacity of 2.5 × 1011. Conclusion The primers used can efficiently amplify the target fragment. The application of T vector can improve the cloning efficiency of antibody genes. The intracellular recombination will further expand the library capacity and increase its diversity. Based on RT-PCR, T vector transition, Internal reorganization of the route to build a large capacity antibody library.