| [Background]One of the key links between the tumor growth and metastasis is angiogenesis, thus blocking or inhibiting angiogenesis is an effective anti-tumor therapeutic strategy. In recent years, the formation of the vascular mechanism has been extensively and deeply researched, and it has been proved by a variety of angiogenesis-related factor of adjustment. People have found a variety of angiogenic factors and angiogenesis inhibitor factors. Angiogenesis as a target in the treatment of tumors has become a research point in cancer therapy. There are numbers of angiogenesis inhibitors that have entered clinical trials.Human Plasminogen Kringle5 (referred to as K5) is a newly discovered angiogenesis inhibitor. It can act on the proliferation of vascular endothelial cells, inhibit cell proliferation, migration, and induction of apoptosis, and it is the most powerful endogenous inhibitor of angiogenesis. Effect of K5 as an inhibiting endothelial cell proliferation and anti-migration is strong, the applicationμperspective of K5 is prominent. At.present, K5 was gained mainly through genetic engineering of recombinant technology. Its purification is complex, outcome is low, and the relatively molecular weight of K5 is large.[Objective] The purpose of this experiment is to find a quick and efficient way to gain the K5 protein, laying foundation for the development of anticancer drugs.[Methods] We observed the expression and optimized the expression conditions with different competent cell BL-21, DH-5α, Top 10 and different induction conditions K5/pGEX-5X-1. In order to explore the optimum conditions, we analyzed the dissolubility of protein, obtained high purity protein by dissolving, washing, dissolution, renaturation, etc.. We used glutathione agarose 4B affinity column with GST tag to purify interest protein, obtain high purity K5 fusion protein, identify the product, analyze the purity and determine the concentration. After cutting off the tag with thrombin, we planted ECV304 tumor cells to nude mice, analyzed its biological activity and detect its anti-tumor effect in vitro.[Results] We selected strains with efficient expression, and obtained the best expression conditions. Competent cell, induced concentration, induction time, induction temperature and hydrogen ion exponent were BL21,1 mmol/L,6 hours,37 degrees and 7.3, respectively.Using the method of ultrasonic lysis buffer plus, we broken body of bacteria. Then we made it resuspension in PBS solution (1 mmol/L), and wash it with inclusion body of 2m urea,4m urea and 6m urea.8m urea was used to dissolve inclusion bodies. Glutathione agarose 4B affinity column with GST tag was used to refold diluted. The concentration was 727μg/ml.After cutting off the tag with thrombin, interest protein was purified. We measured the activity of it with ECV304 cells. The results showed it can inhibit the growth of cells. When the concentration at 5μg/ml had the highest inhibition rate.We made a construction of nude mice xenograft model to determin the inhibition of tumor in nude mice. The results indicated that the treatment group can inhibit tumor growth.[Conclusion] High expression strain BL21 was obtained after experimental screening. Through repeated experiments, we mastered the best expression conditions which could increase expression of target protein. Using the advantages of its convenience to purify GST tag, we obtained high purity protein by dissolving, washing, dissolution, renaturation, etc. Cutting off the tag with thrombin, which indicated that GST tag is feasibility and had advantages of the purification of K.5 protein. This experiment had a preliminary determination that inhibitory activity of ECV304 cells and transplanted bladder cancer of nude mice in vitro, can inhibit the growth of endothelial cells and tumor proliferation. This experiment lay the foundation of drug development in future.
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