Establishment Of Platelet Membrane Chromatograph And Expression And Purification Of P2Y₁₂ Receptor

Recently, the antithrombotic drugs develop for anti-platelete aggregation especially antagonist of P2Y₁₂ receptor (P2Y₁₂R) have been studied extensively. In contrast to the high toxic and side effects of antithrombotic chemical drugs, traditional Chinese medicine （TCM） has its unique advantages in the treatment of thrombotic disease. But due to the composition of TCM is complex and efficacy of material basis is unknown, that restricts its further development and modernization. Receptor affinity chromatography has good biological activity, and has been widely used in studying the interaction between protein and drug and its mechanism of action, and active components of TCM.P2Y₁₂R is the most ideal target of antithrombotic drugs, and the acquisition of the receptor is the core of this study. Due to the P2Y₁₂R is GPCR, the traditional prokaryotic expression system is difficult to achieve P2Y₁₂R, so we are trying to construct the P2Y₁₂R eukaryotic expression system. At the same time, we make the whole platelet as the breakthrough point, in view of the platelet membrane surface exist multiple receptor, enzyme and protein, that can mediate platelet aggregation and thrombosis, so we want to construct the model of platelet cell membrane chromatography, aims to study the interaction of biological membrane protein and effective ingredients of TCM. Thus, in this research, a platelet cell membrane chromatography （CMC） was established by using the physical adsorption method, and the chromatographic behavior was investigated. The platelet cell membrane chromatography stationary phase （CMSP） was examined by electron microscopy scanning and surface energy spectrum analysis. The protein content of the CMSP and biological activity were determined by BCA protein assay kit and Na+/K+-ATPase enzyme assay kit. The retention characteristics of model drugs were investigated to characterization of the CMC model. Once the model was constructed, it was used to screen the active ingredients of Salvia miltiorrhiza, the active substances were then isolated and identified by HPLC and mass spectrometry （HPLC-Q-TOF-MS/MS） analysis method. The fast, efficient, high-throughput drug screening separation model is able to be applied in the complex system of TCM, it provides a new method for the development of the main compound of TCM.The baculovirus mediated Sf 9 expression system was carried out to express the P2Y₁₂R in this study. Then separated by HP HisTrap affinity chromatography column and detected by SDS-PAGE, Western-Blot experiments, the purity of the P2Yi2R is about 80%. P2Y₁₂R with high purity can be further used to construct the model of drug screening and to explore the mechanism of the anti-thrombus drugs. Hereon, we express and purify the P2Y₁₂R by using biological engineering technology, which provides a reliable source for the construction of the P2Y₁₂R affinity chromatographic model in the later stage.The combination of platelet CMC-HPLC and HPLC-Q-TOF-MS/MS identification, which provids a modle that could be used for active ingredient recognition, online enrichment, isolation, and identification in simultaneously, obviously improves the drug screening process accuracy and success rate. The baculovirus expression system to obtain the P2Y₁₂R and binds nickel ion affinity chromatography to purify P2Y₁₂R, provide a strong guarantee for successfully built P2Y₁₂R affinity chromatographic model. Our studies provide a methodological support for the detection ofanti platelet active components and the foundation of the active substance of the traditional Chinese medicine. It has important theoretical and practical significance for the modernization of Chinese medicine.