b族g蛋白偶联受体pac1四环素诱导可控表达系统构建及应用word格式论文.docxVIP

The Construction and application of tetracycline-inducible expression system for Class B G-protein coupled receptor PAC1 AbstractObjective: PAC1 is the neuropeptide pituitary adenylate cyclase activatingpolypeptide (PACAP) preferring receptor, which belongs to class B G protein-coupled receptors (GPCRs) family. PAC1 mediates the most effects of PACAP as neurotransmitter, neuroregulator and neuroprotectant, while its high expression has close relationship with some physiological and pathological processes such as nerve-injury and tumor. Tet (tetracycline)-on gene expression systems is developed quickly in recent years. It becomes the most widely used system of regulating gene expression in eukaryotes for its efficiency and strict switch function. For further understanding the functional role of PAC1, a cell line that expressed inducible PAC1 was constructed to achieve the Doxycycline (Dox) dependent expression of PAC1 in Chinese hamster ovary (CHO) cell using the improved Tet-on Advanced System in this research to investigate the correlation between the receptor expression level and its constitutive activity.Method: Firstly, the PAC1-EYFP fusion gene composed of PAC1 gene and gene encoding EYFP (enhanced yellow fluorescent protein) was sub-cloned to the tetracycline response element pTRE-Tight vector to construct the recombinant vector pEYFP-PAC1-EYFP by double enzyme digestion. Secondly, the tetracycline regulation components including pTet-On advanced vector and the response element pTRE-PAC1-EYFP vector were both introduced into CHO cells successively. The positive clones were screened with G418 and hygromycin respectively. Thirdly, the controlled expression of PAC1-EYFP in CHO was induced by tetracycline analogues Dox in different concentrations and the corresponding levels of receptor PAC1-EYFP were detected. Meanwhile the proliferative activities and the anti-apoptotic abilities against serum-withdraw induced apoptosis of the cells with differen