摘要
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。
ObjectiveIn order to reduce the immunogenicity of murine antibody,a ScFv against human VEGF165 was expressed in E.coli. MethodsThe variable region genes of heavy chain and light chain were amplified by PCR.The ScFv gene was produced by overlap PCR of VH and VL,then ligated into expression vector pKpL 3a,and expressed in E.coli pop2136.The antigen binding activity of the ScFv was assayed by antigen binding ELISA and competition ELISA with the parent McAb.ResultsWith the method of SDS PAGE,western blot,antigen binding ELISA and competition ELISA,it confirmed that E coli . expressed the correct 26kD of ScFv,which could specifically bind to the huaman VEGF165 and recognized the same epitope with the parent McAb.ConclusionAn anti human VEGF165 ScFV was successfully constructed expressed in E.coli. [
出处
《免疫学杂志》
CAS
CSCD
北大核心
2000年第2期92-95,共4页
Immunological Journal
基金
国家863计划资助!项目 (86 3- 10 2 - 0 9- 0 2 - 0 3)
