| Aurora kinase A is a member of the Aurora kinase family, a mitosis - serine / threonine kinase family which is very conservative during the evolution. The amount of the kinase changes with the process of the cell cycle. Aurora kinase A is mainly localized in the centrosome and spindle, involved in the mature, separation of the centrosome and the formation of spindle. It is highly expressed in a variety of tumor tissues, and its over-expression may lead to genetic instability and tumorigenesis. Aurora kinase A is widely participated in the different stages of cell cycle, and plays a vital role in the occurring and development of various human malignant tumors. As a result, this kinase is likely to be a target for the molecular therapy of malignant tumor in the future.The final purpose in this study is to transforme the aurora kinase A expression vector into eukaryotic cell line MCF-7, and detected the influence on cell proliferation and apoptosis after this transformation. Then, we use this cell line as a platform to select several drugs which can play an inhibiting role to this kinase from Chinese and Tibetan medicine, or look for some small molecule kinase inhibitors, including naturally existing macromolecular substances, chemical modified small molecules, as well as chemically synthesized SiRNA fragments. We hope that our work can do some help in tumor treatment.One starting point of this study is to construct human Aurora kinase A gene onto the yeast expression vector pPICZαA. Then we use the electroporation method to transfer the recombinant plasmid pPICZαA-Aurora A into Pichia pastoris X33, and pick up positive clones by Zeocin screening. After that, we use the yeast expression system to express the human Aurora kinase A protein. In this way, we can determine its kinase activity. In order to test its activity, we use this kinase to phosphorate its optimal substrate histone 3 (H3); Also, we have another substrate [γ-32P] ATP which can provide the phosphate for the phosphorylation. By using the method of autoradiography, we detect the phosphorylation of Histone 3, therefore the activity of Aurora kinase A can be determined in qualitative and quantitative. After the determination of the activity of Aurora kinase A, we construct the fragment onto the eukaryotic expression vector pcDNA3.1 (+), transfer the recombinant plasmid pcDNA3.1 (+)-Aurora A into the MCF-7 by using electroporation, select G418 positive clones, and then use the method of RT-PCR to detect the gene transcription in MCF-7, and detect the protein expression in MCF-7 by using western blot method. We ultimately establish a MCF-7 cell line which can stably express Aurora kinase A, a platform for the screening of the inhibitor of Aurora A kinase.The results of this study:①Construction of the Aurora-A gene in yeast expression vector pPICZαA-Aurora-A, and electric shocks the recombinant vector into Pichia pastoris X33, methanol-induced expression of Aurora kinase A protein.②The successful expression of the Aurora A kinase substrate histone 3, determined the IPTG-induced expression of the protein in the best conditions: 37℃, 200rpm, IPTG final concentration of 1mmol/L, induced by 4 hours. Through the Ni column affinity chromatography purification of the Histone 3 protein.③By means of autoradiography measured the activity of Aurora kinase A, proved in Pichia pastoris expression of a kinase activity of Aurora A protein.④Construction of the Aurora-A gene eukaryotic expression vector pcDNA3.1 (+)-Aurora-A, and the recombinant vector of the electroporation of eukaryotic cells MCF-7, G418 positive clones were screened out from 96-well plates.
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