Construction And Expression Of Multivalent Anti-CD3×anti-prostate-cancer Bispecific Single-Chain Antibody

Objective To construct anti-CDs X anti-prostate-cancer bispecific single-chain antibody(BsAb) and the fusion gene of human IgG3 upper hinge region and p53 tetramerization domain respectively. Then the genes mentioned above were sequentially cloned into pUCIS vector to construct multivalent BsAb which can self-assemble tetramerization. It will exhibite an enormous gain in affinity.Methods The fusion gene of human IgG3 upper hinge region and p53 tetramerization domain was obtained by recursive polymerase chain reaction (R-PCR), and was cloned into pUC 18 vector. The positive clone was selected by restriction endonuclease digestion and sequenced with PE310 auto-sequencer, which was named Ip-pUC18. The genes of anti-CDs single-chain antibody(scFv) and anti-prostate-cancer scFv were obtained by PCR respectively. The sequence of linker was included in the reverse primer of anti-prostate-cancer scFv. The PCR products were sequentially inserted into pUCIS and Ip-pUC18 respectively, which then underwent endonuclease-6-digestion and sequencing and were named BsAb-pUC18 and MAb- pUC18. Followingly, PCR products were subcloned into the pSectag2-B plasmid from the pUCIS vector by digestion with EcoR I and Hind HI restriction endonucleases, whose sites exist in both the vectors. Then the pSectag2-B plasmids concluding PCR products were transfected Hela cell line. The expression products were analyzed by both SDS-PAGE and Western blot, then were purified with Ni~^-NTAsuperflow affinity chromatography.Results (1) After R-PCR reaction, a fragment of 160 bp or so was amplified. Restriction endonuclease digestion confirmed that the fusion gene has been inserted correctly into the pUCIS vector. The result of sequencing showed that the fusion gene was identical with that designed. (2) Both PCR products of anti-CDa scFv and anti-prostate-cancer scFv concluded a fragment of 750 bp or so. After restriction endonuclease digestion, two fragments of 1.5kp and 1.7 kb or so were seen in the digestion products of BsAb-pUC18 and MAb- pUCIS respectively. The results of sequencing showed that the fusion genes of anti-CDs X anti-prostate-cancer BsAb and multivalent BsAb had been constructed successfully. (3) The results of restriction endonuclease digestion showed that the eukaryotic expression vectors of anti-CDs X anti-prostate-cancer BsAb and multivalent BsAb had been constructed successfully. (4) The expression products of anti-CD3 X anti-prostate-cancer BsAb and multivalent BsAb were confirmed by SDS-PAGE and Western blot, which molecular weight were 61 KD and 67 KD or so respectively. After purified with Ni2+-NTAsuperflow affinity chromatography, the purifity of both proteins reached 90%.Conclution Successful construction of the fusion gene of human IgG3-7-upper hinge region and p53 tetramerization domain lays the foundation for further preparation of multivalent gene engineering antibody and break a new path to the improvement of antibody's affinity. Anti-CDs X anti-prostate-cancer BsAb and multivalent BsAb were prepared successfully, which could be used for the study on the biological activities of both antibodies. Furthermore, our experiment is helpful to the exploration of the treatment of prostate cancer.