title: Construction and Expression of a Recombinant Antibody-targeted Plasminogen Activator Composed of a Humanized Monoclonal Antibody against Activated Human Platelets and a Single-chain Urokinase.
abstract: To obtain a thromblytic agent with high efficacy and specifity, we have engineered a recombinant chimeric plasminogen activator SZ51Hu-scuPA, which was composed of a humanized monoclonal antibody (SZ51Hu) directed against activated human platelets and a single-chain urokinase (scuPA). The cDNA sequence encoding scuPA was fused through 5' end to 3' end of the CH(3) of SZ51Hu heavy-chain sequence in the expression vector alphalys30(alphalys30-SZ51VH/Hu-scuPA) by PCR. This construct was transfected into a murine myeloma line secreting SZ51Hu light chain with Lipofectin reagent and three lines which showed stable integration and high level expression with concentration of 5 mg/L in supernatant, were selected in the end. Purified SZ51Hu-scuPA migrated as a 160 kD band on non-reduced SDS/PAGE and had the same characteristic of binding to the activated platelets compared with its parent murine antibody molecule SZ51 by Western-blot analysis. The fibrinolytic activity of purified products was 39 000 IU/mg. Thus, by recombinant techniques, an expressed fusion protein was capable of specific binding to activated platelets and plasminogen activation. These observations laid a solid foundation for further study of targeting of thrombolysis in vitro and in vivo.
authors.affiliation: Jiangsu Institute of Haematology, Suzhou Medical College, Suzhou 215007, China. smcklnmb@public1.sz.js.cn
authors.forename: Zheng-Hui
authors.initials: ZH
authors.lastname: Liu
authors.forename: Hai-Ying
authors.initials: HY
authors.lastname: Wan
authors.forename: Bin
authors.initials: B
authors.lastname: Wang
authors.forename: Pei-Xia
authors.initials: PX
authors.lastname: Li
authors.forename: Bing-Gen
authors.initials: BG
authors.lastname: Ru
authors.forename: Mei-Hao
authors.initials: MH
authors.lastname: Hu
authors.forename: Chang-Geng
authors.initials: CG
authors.lastname: Ruan