抗转铁蛋白受体双价抗体的构建、表达及鉴定

Construction,expression and characterization of a bivalent single chain variable fragment against transferrin receptor

目的:抗转铁蛋白受体(TfR)双价抗体(bsFv)构建、表达及其与细胞结合的活性鉴定。方法:以抗TfR的单链抗体(scFv)为模板,设计引物引入中间连接肽(G4S)及酶切位点AscI,通过PCR方法扩增两条scFv片段,将其连接并克隆至原核表达载体pAB1,转化大肠杆菌TG1,阳性菌经IPTG诱导表达,FCM检测bsFv与K562,HepG2肿瘤细胞结合的活性及特异性。结果:酶切鉴定及DNA测序证明该bsFv构建成功;SDS-PAGE、Western blot鉴定表明表达蛋白分子量与bsFv理论值一致;FCM结果证明bsFv可与K562,HepG2肿瘤细胞特异性结合,且结合的阳性率较scFv有所提高。结论:成功构建了抗转TfR的bsFv,且能与K562,HepG2肿瘤细胞特异性结合。

Objective： To construct and express bsFv against transferrin receptor, and identify its cell conjugation activity. Methods：Designed PCR primers from the parent scFv to introduce interlinker G4 S and the restriction endonuclease AscI. Two scFv fragments were produced by PCR. The two scFv fragments gene were connected into the secretory expression vector pAB1 and transformed into E. coli. TG1. The positive clones were induced by IPTG. The protein was identified by SDS-PAGE and Western blot. Cell conjugation activity of bsFv was characterized by FCM. Results ：The digesting and sequencing results of bsFv were demonstrated that the constructing was successful. Protein was analyzed by Western blot, which molecular weight was identical with the protein size of bsFv. The results of FCM proved that bsFv had the specificity of conjugating with TfR on the cell surface. The avidity of bsFv was higher than that of the parent scFv. Conclusion： BsFv against transferrin receptor have been successfully constructed and expressed. Our study demonstrated expression products could specifically bind to TfR on the cell surface of K562.