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目的为降低鼠源抗体对人体的免疫原性 ,构建表达了 Vm D11的单链抗体。方法用 overlap PCR构建出单链抗体的基因 ,克隆入表达载体 p Kp L- 3a,在大肠杆菌 pop2 136中表达 ,采用抗原结合 EL ISA和竞争抑制 EL ISA测定活性。结果经SDS- PAGE检测 ,诱导后的细菌表达了 2 6 k D的蛋白 ;Western Blot证实该蛋白为表达的单链抗体。经变性、复性后 ,该单链抗体可特异结合人 VEGF16 5抗原 ,并与 Vm D11具有相同的抗原结合位点。结论成功构建和表达了抗人 VEGF16 5单链抗体 ,可望进一步应用于肿瘤复发转移的诊治研究。
Objective To reduce the immunogenicity of mouse antibodies against humans and construct single-chain antibodies expressing Vm D11. Methods The single-chain antibody gene was constructed by overlap PCR and cloned into the expression vector p Kp L-3a. It was expressed in E. coli pop2 136 and the activity was determined using the antigen-binding EL ISA and competitive inhibition EL ISA. Results The SDS-PAGE analysis showed that the induced bacteria expressed 2 6 kD protein; Western Blot confirmed that the protein was expressed as single-chain antibody. After denaturation and renaturation, the single-chain antibody specifically binds human VEGF16 5 antigen and has the same antigen binding site as Vm D11. Conclusion The successful construction and expression of anti-VEGF165 single chain antibody is expected to be further applied to the diagnosis and treatment of tumor recurrence and metastasis.