抗转铁蛋白受体抗体的筛选和鉴定

Screening and identification of antibodies against transferrin receptor

目的:从全人源噬菌体抗体库中筛选抗转铁蛋白受体(TfR)的抗体。方法:从HeLa细胞中提取总RNA,分两段反转录TfR胞外区cDNA,经两轮PCR后,获得胞外区基因。测序正确后,将其插入表达载体pPROEXTMHTa中,并在E.coliBL21(DE3)中表达。目的蛋白通过NiNTA金属螯合层析柱进行纯化、复性。用复性的TfR包被免疫管,从库容为1013cfu/L的全合成人源噬菌体单链抗体库中,筛选抗TfR的抗体。结果:得到约1.9kb的TfR胞外区基因,该基因以包涵体的形式在大肠杆菌中表达;利用纯化、复性的TfR从抗体库中筛选到了8个可与人TfR特异性结合的单链抗体。结论:利用TfR在大肠杆菌表达的复性产物,可以筛选到与天然TfR特异性结合的单链抗体。

AIM： To screen the antibodies against transferrin receptor（TfR） from a full humanized phage antibody library. METHODS： Total RNA was extracted from HeLa cells and then the extracellular domain of TfR gene was amplified by RT-PCR in two parts. A full-length gene of extracellular domain was obtained by two rounds of PCR. The gene was inserted into the plasmid pPROEXTM HTa and transformed into E. coil BL21 （ DE3 ）. Transformant was expressed under 1mmol/L IPTG induction. The target protein was purified and refolded by one-step affinity chromatography with Ni＋-NTA agarose. The antibodies against TfR were screened by proteins coupled to immunotubes from a whole-hog humanized phage single chain antibody library with sink size of 10^13 cfu/L. RESULTS： The extracellular domain of TfR gene with about l. 9 kb was obtained. DNA sequencing of TfR gene proved to be identical to the published sequence. Eight single chain antibodies specifically binding to human TfR have been screened. CONCLUSION： Antibodies specifically binding to natural TfR can be obtained by screening the library with refolded protein, which lays the foundation for treatment of tumor and central nervous system diseases.