1.Construction Of Fusion Gene Coding For RGD Peptide And Urokinase B Chain And Its Expression In Escherichia Coli And Characterization 2.The Study On Suppression Of Metastatic Potential Of Cancer Cells With Urokinase Amino-terminal Fragment Gene Tran

一 Construction of Fusion Gene Coding for RGD Peptide and Urokinase B Chain and Its Expression in Escherichia coli and CharacterizationA RGD peptide-coding cDNA was chemically synthesized and fused with urokinase B-chain cDNA and then cloned into the prokaryotic expression vector pBV220. The fused gene was expressed in E. coli DH5α under the control of PRPL promoter by 42 ℃ heat induction. The expression level of the fused gene was over 9.2% of the total bacterial proteins as an inactive form of inclusion bodies. The isolated products were obtained by denaturation and renaturation. It was demonstrated that the fusion proteins have the similar antigenicity, fibrinolysis as entire urokinase and displayed anti-platelets aggregation activity in vitro. 二 The Study on Suppression of Metastatic Potential of Cancer Cells with Urokinase Amino-terminal Fragment Gene TransferTo explore the suppressive effects of gene transfer of urokinase amino-terminal fragment (ATF) on metastatic potential of cancer cell, the eukaryotic expression vector containing ATF cDNA controlled under CMV promotor/enhancer was constructed and transfected into a highly metastatic human lung cancer PG and breast cancer MCF-7 cells line, which expressed uPA and uPAR. Then the expression of ATF was analyzed by means of reverse transcription polymerase chain reaction (RT-PCR). Latter, we observed the effects of ATF gene transfer on invasive potential of cancer cells in vitro. To determine the effcts of ATF gene transfer on tumorigenesis and spontaneous metastasis, the gene-transfected cancer cells were inoculated subcutaneously into nude mice. After ATF gene transfection, the ATF mRNA was obviously expressed. The invasive potential through matrigel in vitro of these cancer cells was decreased significantly. Its tumorigenesis was not affected but its spontaneous metastasis potential remarkably inhibited. Therefore, ATF gene transfer results in the suppression of spontaneous metastasis through competitive binding of exogenous ATF to urokinase receptor with urokinase.