抗人CD3单链抗体基因的构建及序列分析

本文在已克隆抗人ＣＤ３抗体ＶＨ和ＶＫ基因的基础上，设计并合成了ＰＣＲ引物。两个外侧引物分别含有ＥｃｏＲＩ和ＳａｌＩ酶切位点及起始码和终止码序列，４个内侧引物各含部分连肽基因序列，回收后混合退火。

Construction and Sequence Analysis of the Single-Chain Fv Gene of an Anti-CD_3 Monoclonal Antibody

We designed and synthesized six primers.The two outer primers(VH-For,Vk-Back)contained EcoRI and SaiI restriction sites for insertion into pUC19, and the initiation and termination codon for expression.The four inner primers (VH-Back1, VH-Back2,Vk-Forl, Vk-For2)contained gene sequences encoding the linker peptide. The VH and Vk genes amplified by addingon PCR,as well as a part of linker peptide gene sequence was added on VH 3' and Vk 5' terminus respectively. The PCR products were mixed and reamplified using only the outer primers and then the ScFv gene was spliced by overlap extension. The resulting product containing the entireScFv gene was digested with EcoRI and SalI and ligated to pUC19 predigested with the same restriction endonucleases. By screening and identification,several recombinants that had been inserted with the target fragment were obtained. The recombinants were sequenced using the T7sequencing kit.The sequences of VH,Vk and linker peptide were all correct. The size of linker peptide gene sequence was 45 bp and the predicted amino acid sequence of the linker peptide was (Gly4 Ser)3.