Preparation, Detection And Functional Analysis Of Targeting P53 Fusion Protein

Biological medicine, prepared by modern biological technology, is a kind of drug for prevention, treatment and diagnosis. Biological medicine has become the most promising new drugs in the 21st century because of its good properties of high pharmacological activities, little toxic side-effects, high nutritional value and strong therapeutical ditections. So medical and pharmaceutical fields at home and abroad pay high attention to the preparation and research of biological drugs, and its analysis, detection and identification are the foundation for further clinical applicationp53 gene is an important tumor suppressor gene, and its encoding product p53 protein play a critical role in recognition and repair of DNA damage, inhibition of cell malignant proliferation, induction of cell apoptosis and regulation of cell cycle arrest. Deficiency of wild type p53 could lead to the occurrence of tumors due to the genetic mutation or deletion, and then the cells appear significant disorder in cell growth, apoptosis and DNA repair. It was reported that 25%～35% mutation rate of p53 in cancerous neural cells and this rate in glioma cells line reaches 70%～76%, so recovery p53 function in cancer cells is considered as a method of antitumor therapy. The preparation, detection and functional analysis of p53 protein are the foundation for the treatment of nerve tumors. The conventional detection method of p53 protein such as ELISA, Western blot, is only used for qualitative and quantitative analysis, which couldn’t ensure the biological activity of p53. So methods that not only guarantee the accuracy of p53 protein but also examine its function need to detect p53 protein.The application of p53 protein is limited due to its low cell-permeability. Thus, combination of p53 protein and specific membrane penetrating peptide may provide an approach of improving the cell uptake efficiency of p53 protein. Our group previously found a novel derived peptide, named RDP (RVG-derived peptides), whose partial sequence comes from the key segment of rabies virus glycoprotein (RVG). Experiments show that it can rapidly and specifically carry BDNF protein and gene into the brain and exert its function. Therefore we consider conjunction RDP and p53 protein by chemical bond, determination its analysis method and detection the intra-cellular biological activity of p53 protein in order to provide a new way for inhibiting the proliferation of nervous system tumor such as malignant gliomas.The paper is divided into three parts, the first part:the pET28a-p53 recombinant plasmid was constructed by genetic engineering technology. The preparation condition of p53 protein was optimized; the accuracy of p53 protein was analyzed by SDS-PAGE and its content was determined by Folin-phenol method. Then targeting p53 fusion protein and p53 protein were prepared and purified, respectively. The second part: targeting p53 fusion protein and p53 protein which were labeled by FITC and rhodamine B isothiocyanate (Rho B) were synthesized, respectively. The fluorescence intensity of proteins were analyzed by the fluorescence spectrophotometry, and then the cellular selectivity and cerebullar feasibility of targeting p53 fusion protein were examined through the way of cell experiment and in vivo imaging. The third part:we analyzed the function of targeting p53 fusion protein. The biological activity of targeting p53 fusion protein was detected by MTT assay and its mechanism was analyzed by apoptosis and necrosis assay and flow cytometry. The results showed that targeting p53 fusion protein had higher selectivity of nerve tumor cells and held biological activity of p53 protein. It demonstrated that targeting p53 fusion protein, prepared by biotechnology, neither interfere the biological function of RDP nor impair the activity of p53 protein, which could provide a simple, effective and new approach for inhibiting the growth on nerve tumor cells.