Studies On Preparation And Biological Activity Of Chimeric Proteins Possessing Anti-tumor And Thrombolytic Activity

Thrombosis is one of the most common complications of the patients with tumor. As a tight relationship exists between tumor and thrombosis, thrombosis plays an important role on the mechanism of tumor metastasis and tumor angiogenesis.The thrombus incidence in patients with malignant tumor is about 10%～30%. Clinical studies have shown that thrombosis is the second cause of death in patients with cancer at present. So thrombosis is the stealth killer of tumor patients. Therefore, thrombosis in cancer is a problem that can not be neglected.Staphylococcal enterotoxin C2 (SEC2) is a bacterial superantigen secreted by Staphylococcus aureus. In clinic, SEC2 has a positive effect in curing malignant tumor such as lung cancer, gastric cancer, ovarian cancer, bladder cancer. Staphylokinase (Sak) is another important protein secreted by Staphylococcus aureus. Sak has many advantages such as strong fibrinolytic activity, specific thrombolysis, fewer allergic reactions, and less bleeding.In our previous study, the natural SEC2 had been truncated 17 amino acids in the N terminal and 132 amino acids in the C terminal. In this way, we obtained the mutant protein ASEC2 with retained superantigen activity and reduced side effects. At the same time, the mutant protein △Sak was also obtained by truncating staphylokinase 10 amino acids in the N terminal, but it had no effect on thrombolytic activity by genetic engineering technology. Then we used these mutant proteins to create the chimeric proteins ASak-ASEC2 and △SEC2-ASak.Based on our study, the researchs on optimum cultivating conditions and the optimum induction conditions of preparation of chimeric proteins possessing anti-tumor and thrombolytic were conducted. Superantigen activity of the chimeric proteins was conducted and thrombolytic was examined by the major histocompatibility complex Ⅱ (MHCⅡ) affinity experiment, mouse spleen T-cells proliferation assay, tumor inhibition experiment and mouse tumor inhibition experiment. The results were as follows.1) Optimize on preparation of chimeric proteinsThe optimum cultivating conditions were determined through orthogonal test of culture volume, seed dose and medium original pH value. The optimum induction conditions were determined through orthogonal test of inductive time, inductive temperature and inductive concentration. Results showed that the optimum cultivating conditions for preparation of chimeric proteins possessing anti-tumor and thrombolytic were summarized as follows, Culture volume is 100ml, seed dose is 2%, medium original pH value is 7.5.The optimum induction conditions were summarized as follows, inductive time is 4h, inductive temperature is 33℃, inductive concentration is 0.6mM.2) Tumor cell affinity experimentOn the surface of the cancer cells, chimeric protein and MHC class Ⅱ molecules were combined with each other. Results showed that chimeric proteins may have the ability of cross-linking to MHC Ⅱ. Affinity ability were increased with the increase of protein concentration, and they have superantigen activity same as SEC2. Affinity ability has nothing to do with the time.3) Bioactivities assay of chimeric proteins in vitroBioactivities assay of chimeric proteins in vitro were verified by mouse spleen T-cells proliferation assay and tumor inhibition experiment. The results showed that chimeric proteins stimulated lymphocyte proliferation and inhibited the growth of 823, HT-29, k562 cells in vitro. The activity of mouse spleen T-cells proliferation and tumor inhibition were increased with the increase of protein concentration, and they have superantigen activity same as SEC2.4) Activity analysis of mouse tumor inhibitionTo investigate the anti-tumor effect of chimeric proteins possessing anti-tumor and thrombolytic using mouse model ofxenograft and further understand the role of chimeric proteins possessing anti-tumor and thrombolytic in tumor therapy. Mouse models of xenograft S180 sarcoma were established. The model mice were injected with chimeric proteins at high(100 μg/ml),moderate(50 μg/ml)and low(10 μg/ml)dosages respectively for 8 d starting from day 1 after inoculation of xengraft tumor cells, using SEC2 as positive control and PBS as negative control.The mice were killed on day 9 after injection, and the tumor tissues were isolated and weighed. Results showed that the tumor weight of S180 bearing tumor mice administrated chimeric proteins was lower than that of control group. The life extension rate of S180 bearing tumor mice administrated with chimeric proteins was higher than that of control group. Spleen index and liver index were significantly increased in the chimeric proteins group. In summary, our studies can improve the expression of chimeric protein, and it was proved that chimeric proteins possessing anti-tumor and thrombolytic had good superantigen activity both in vitro and in vivo.Moreover, our studies would provide positive effects on the research of thrombosis treatment,which might make the chimeric proteins to be a new biological preparation. And out studies also contributed to the further chimeric proteins research.