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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接肽基因序列。用加端ＰＣＲ分别在ＶＨ基因３＇端和Ｖｋ基因５＇端延伸部分连接肽基因序列，回收后混合运火；应用重叠延伸拼接法，将ＶＨ和Ｖｋ基因通过Ｌｉｎｋｅｒ序列串联为单链抗体基因；利用ＰＣＲ产物两端预先设计的ＳａｌＩ和ＥｃｏＲＩ酶切位点，将其克隆到ｐＵＣ１９质粒中，筛选到阳性克隆。经双脱氧终止法序列测定：ＶＨ和ＶＫ基因及Ｌｉｎｋｅｒ序列均正确，为用基因工程技术生产单链抗体奠定了基础。
In this paper, based on the cloned VH and Vk anti-human CD3 antibodies, PCR primers were designed and synthesized. The two outer primers contained the EcoRI and SalI restriction sites and the start code and stop code sequences, respectively. Each of the four inner primers contained part of the linker peptide sequences. Peptide was ligated to the 3 ’end of VH gene and 5’ end of Vk gene respectively by end-PCR, and then mixed for firefighting. The overlapped extension splicing method was used to link VH and Vk genes into single-chain antibody genes by Linker sequence The SalI and EcoRI sites pre-designed at both ends of the PCR product were cloned into the pUC19 plasmid, and the positive clones were screened out. After dideoxy termination assay, the VH and VK genes and Linker sequences were correct, which laid the foundation for the production of single-chain antibodies by genetic engineering.