Expression, purification of a synthetic fuse-protein-TSF from PF4 and TSP1 fragments and its effect on angio-genesis and tumor growth  

作　　者：SHEN Xianrong JI Dongmei HU Youqing JIA Fuxing DENG Xinxian WANG Ling CHU Zhiyong JIANG Dingwen LEI Chengxiang HUANG Jiansheng HU Xinran REN Darning 

机构地区：[1]School of Life Science,Institute of Genetics,State Key Lab of Genetic Engineering,Fudan University,Shanghai 200433,China [2]Naval Medical Research Institute,Shanghai 200433.China

出　　处：《Chinese Science Bulletin》2001年第24期2037-2042,共6页

基　　金：This work was supported in part by the National Natural Science Foundation of China (Grant No. 39970814).

摘　　要：Sequences encoding PF4 (58-70) and TSP1 (429-459) were linked to yield a single gene TSF which encodes the fuse-protein of TSF. The gene was cloned into a pGEX-2T expression vector to generate a protein GST-TSF, which was strongly expressed in E. coli. The purified GST-TSF was degraded with thrombin to generate the protein TSF. With the methods of MTT and wound repair assay, the effects of TSF on the proliferation and migration of EC were detected, respectively. The results showed that TSF significantly suppressed BAEC proliferation and migration in a dose-dependent manner. The fuse protein GST-TSF, and the peptides PF4 (58-70) and TSP1 (429-459) also inhibited BAEC proliferation and migration, respectively, but their inhibition rates were not as high as TSF. Using the CAM assay, it was shown that TSF, GST-TSF, PF4 (58-70) and TSP1 (429-459) inhibited angiogenesis in chick CAM potentially, the effect of TSF was the highest. In vivo, the growth of Lewis lung carcinoma was potently inhibited by TSFSequences encoding PF4 (58-70) and TSP1 (429-459) were linked to yield a single gene TSF which encodes the fuse-protein of TSF. The gene was cloned into a pGEX-2T expression vector to generate a protein GST-TSF, which was strongly expressed in E. coli. The purified GST-TSF was degraded with thrombin to generate the protein TSF. With the methods of MTT and wound repair assay, the effects of TSF on the proliferation and migration of EC were detected, respectively. The results showed that TSF significantly suppressed BAEC proliferation and migration in a dose-dependent manner. The fuse protein GST-TSF, and the peptides PF4 (58-70) and TSP1 (429-459) also inhibited BAEC proliferation and migration, respectively, but their inhibition rates were not as high as TSF. Using the CAM assay, it was shown that TSF, GST-TSF, PF4 (58?0) and TSP1 (429-459) inhibited angiogenesis in chick CAM potentially, the effect of TSF was the highest. In vivo, the growth of Lewis lung carcinoma was potently inhibited by TSF treatment, and the inhibition rate was 68.75% at a dose of 1.00 μmol/kg ·d. These findings suggest that the design on TSF gene was successful, and TSF with its anti-angiogenic and anti-tumor activity, should be a useful source of the inhibitor.

关 键 词：fusion protein angiogenesis tumor therapy PF4 thrombodspondin-1 anti-angiogenesis. 

分 类 号：R73-36[医药卫生—肿瘤]