Liu Jing, Xiao Daiwen, Zhou Xiaoou, Wen Xue, Dai Hong, Wang Zhihua, Shen Xin, Dai Wei, Yang Daofeng, Shen Guanxin
Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
J Huazhong Univ Sci Technolog Med Sci. 2008 Dec;28(6):621-5. doi: 10.1007/s11596-008-0601-z. Epub 2008 Dec 24.
To obtain single chain variable fragment (scFv) and bivalent single chain variable fragment (bsFv) against transferrin receptor, up-stream and down-stream primers were designed according to the complementary sequences of FR1 region of variable heavy (VH) and FR4 of variable light (VL), respectively, which contained inter-linker G4S and the restriction endonuclease SfiI, AscI and NotI. Two pieces of scFv fragments were first amplified through PCR and then inserted into plasmid pAB1, which could express scFv protein once induced by IPTG in the host bacteria. To express scFv and bsFv, E. coli TG1 was cultured in LB broth and was induced by IPTG. The restriction enzyme digestion map and DNA sequencing demonstrated that scFv and bsFv genes were successfully inserted into the expression plasmid. SDS-PAGE and Western blotting revealed the protein band at 35kD and 60kD, which were consistent with the molecular weight of scFv and bsFv respectively. Flow cytometry showed that scFv and bsFv harbored the specific binding activity with TfR expressed in various tumor cells, and the avidity of bsFv was higher than that of the parent scFv.
为了获得抗转铁蛋白受体的单链可变片段（scFv）和二价单链可变片段（bsFv），分别根据可变重链（VH）的FR1区域和可变轻链（VL）的FR4的互补序列设计上下游引物，其中包含连接子G4S以及限制性内切酶SfiI、AscI和NotI。首先通过PCR扩增两条scFv片段，然后将其插入质粒pAB1中，该质粒在宿主细菌中经IPTG诱导后可表达scFv蛋白。为了表达scFv和bsFv，将大肠杆菌TG1在LB肉汤中培养并经IPTG诱导。限制性酶切图谱和DNA测序表明scFv和bsFv基因已成功插入表达质粒。SDS-PAGE和蛋白质印迹显示在35kD和60kD处有蛋白条带，分别与scFv和bsFv的分子量一致。流式细胞术表明scFv和bsFv与各种肿瘤细胞中表达的TfR具有特异性结合活性，且bsFv的亲和力高于亲本scFv。
