Construction, Expression And Characterization Of A Tetravalent Miniantibody Specifically Binding To Transferrin Receptor (TfR)

Objectives: In order to improve the binding affinity of single chain antibody (scFv) to its target, a prokaryotic plasmid encoding the fusion gene of anti-transferrin scFv and tetramerization domain of p53 was constructed to express a tetravalent miniantibody specifically binding to transferrin receptor(TfR-TeAb).Methods: A pair of primers were designed with introduction of restriction endonuclease sites of NcoⅠand EcoRⅠ. The anti-TfR-scFv gene was amplified from pET28(a)-TfR-scFv-D4S×5 by PCR. Then the fragment was cloned into the plasmid pET28a–HSA-p53 to construct the prokaryotic expression vector pET28(a)-TfR-TeAb. After the recombinant plasmid was transformed into E.coil BL21, positive clones were selected and identified by restriction enzyme digestion and sequencing. Expression conditions were optimized and the expression style in E.coli was analyzed. After large scale expression, the inclusion body were collected and purified by IMAC under denaturing condition. The purity was analyzed by SDS-PAGE and western-blot. For refolding the protein, the purity was subjected to a stepwise urea-removal dialysis. The nature protein weight was analyzed by nondenaturing polyacrylamidegel electrophoresis (native-PAGE) and the cell-binding activity of the protein was analyzed by FCM and immunoflourescence microscopy.Results: 1. DNA sequencing showed that anti-TfR-scFv gene was successfully subcloned into pET28a–HSA-p53 and was in frame with the downstream ORF of tetramerization domain of p53. 2. After four hour expression, the protein was accumulated as inclusion body. A special protein band with 36kD was found in SDS-PAGE and western blot, which was in conformity with the theoretical value. 3. Native PAGE showed the molecular weight of nature protein was about 144KD, which meant that the protein formed tetrameric structure. 4. Results from FCM and immunoflourescence microscopy showed TfR-TeAb binding to TfR positive cells more specifically than TfR-scFv.Conclusions: The prokaryotic expression vector pET28(a)-TfR-TeAb was successfully constructed. It has the advantage in specificity and avidity binding to transferrin receptor comparing to TfR-scFv. The construction and expression of TfR-TeAb paved our way for further research on tumor diagnosis and therapy.