| Malignant tumors is one of the most serious diseases threaten to human health and has become the focus of all governments, medical scientists, life scientists and pharmaceutical companies. Currently, chemotherapy is still a very important tool to inhibit the development of tumor. There are some serious problems of chemical anti-tumor drugs, such as drug resistances and poor securities. So high anti-tumor activities, strong targeting, high security and less toxic has became the hot fields in the processes of anti-tumor drug discovery and development.Survivin gene can be expressed in embryonic stem cells and tumor cells but not in normal cells. And the protein expressed by surviving gene can inhibit the apoptosis of tumor cells, regulate the cell division and promote angiogenesis of tumors. So it became a hot target to cure diseases and develop new anti-tumor drugs. For example, the mutant of Survivin(T34A) can promote the apoptosis of tumor cells.With the characteristic that can help to transport protein into cells, TAT sequence was designed to fuse with Survivin(T34A) in writer's laboratory. Furthermore, preparation technologies and anti-tumor activities of fusion protein TATm-survivin (T34A), that can also be named TmSm in this paper, had also been studied in this laboratory. To develop a candidate anti-tumor drug, many works necessary to drug development has been succeeded in this paper, such as freeze-dried powder preparation, quality evaluation and activity evaluation etc.At first, the preparation process of TmSm fusion protein and the optimized technology of freezed-dried TmSm were studied in this paper. And an optimized fomula for TmSm lyophilizates is Mannitol 20mg/ml, Glycine 5mg/ml, Trehalose 30mg/ml, Tween 80 0.8mg/ml, TmSm protein 5mg/ml and 50mM pH6.5 Sodium phosphate buffers. The best freeze-drying process for TmSm is 3 hours at -80℃,2 hours annealing at -8℃and 3 hours freezing at -45℃for freezing process that is the first period of freeze-drying process,20 hours sublimation at -20℃that is the temperature of shelf, and 8 hours at 20℃for the second and third period of the freeze-drying process. In addition, the anti-tumor activity of TmSm is first time defined in this paper. And a standard activity detection method is also constructed here. Furthermore, appearances of freeze-dried TmSm, residual waters, reconstitution characteristics, purity, activity, special activity, UV scanning, endotoxin level, haemolysis and the stability of TmSm freeze-dried powder are also detected in this paper. And fortunately, results of these all items are stratified with normal antitumor drugs. And so the freeze-dried form of TmSm prepared in this paper can be used to deeper development.Anti-tumor activity in vitro of freeze-dried TmSm is found in this paper with four types of tumor cells such as B-Cap-37, MCF-7/s, SW1990 and A549. Furthermore studies found that A549 cell is the most sensitive one to TmSm in four tumor cells and the MCF-7/s is the worst one.Finally, the combination with doxorubicin, a market chemical antitumor drug, is also studied in this paper. And a strong chemicalsensitivity effect has been observed here, especially to MCF-7/ADR cell, a type of drug-resistant breast tumor cell. |
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