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.
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.
Liu
Zheng-Hui
ZH
Jiangsu Institute of Haematology, Suzhou Medical College, Suzhou 215007, China. smcklnmb@public1.sz.js.cn
Wan
Hai-Ying
HY
Wang
Bin
B
Li
Pei-Xia
PX
Ru
Bing-Gen
BG
Hu
Mei-Hao
MH
Ruan
Chang-Geng
CG