B族G蛋白偶联受体PAC1可调控表达体系的建立

Construction of controlled expression system of Class B G-protein coupled receptor PAC1

摘要:

PAC1是神经肽垂体腺苷酸环化酶激活多肽(Pituitary adenylate cyclase activating polypeptide，PACAP)的特异受体，属于B族G蛋白偶联受体，介导PACAP的神经递质、神经调质、神经保护、抗神经损伤及调控神经再生等功能，PAC1高表达和神经损伤、肿瘤等生理病理过程密切相关。为了深入了解PAC1的功能，构建PAC1可调控表达的细胞系，通过优化的四环素控制表达系统实现PAC1在中国仓鼠卵巢(Chinese hamster ovary，CHO)细胞的强力霉素(doxycycline，Dox)依赖的可控表达。首先通过双酶切将编码PAC1和增强型黄色荧光蛋白(EYFP, enhanced yellow fluorescent protein)的融和基因PAC1-EYFP克隆到pTRE-Tight载体上，获得重组载体pTRE-PAC1-EYFP；基因测序鉴定正确后将新型的四环素调节元件载体pTet-on advanced和反应元件载体pTRE-PAC1-EYFP分别转入CHO细胞中，G418和潮霉素(Hygromycin)双抗筛选阳性克隆PAC1-Tet-CHO，使用梯度浓度四环素类似物强力霉素Dox 诱导PAC1-EYFP表达，48 h后检测受体表达水平，并通过MTT法检测不同PAC1表达水平的细胞增殖活性。荧光检测和Western印迹结果显示，成功获得了具有良好诱导性的Dox依赖的PAC1可控表达的细胞系，这些细胞株在传10代后仍能稳定地可控表达PAC1。MTT结果显示PAC1表达水平越高，细胞增殖活性越强。成功所构建的Dox依赖的PAC1可控表达细胞系，为PAC1的生物学功能的深入研究奠定了基础。

Abstract:

PAC1 is the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) preferring receptor, which belongs to class B G protein-coupled receptors (GPCR) 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. To further understand the function of PAC1, a cell line that expressed inducible PAC1 was constructed to achieve Doxycycline (Dox) dependent expression of PAC1 in CHO (Chinese hamster ovary) cell using the improved Tet (tetracycline)-on Advanced System. First, 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. Second, the tetracycline regulation components pTet-On advanced vector and the response element pTRE-PAC1-EYFP vector were both introduced into CHO cells successively and the positive clones were screened with G418 and hygromycin respectively. Third, the controlled expression of PAC1-EYFP in CHO was induced by tetracycline analogues Dox in different concentrations and the different levels of receptor PAC1-EYFP were detected. The results of fluorescence analysis and western blotting show that the cell strain with Dox dependent expression of PAC1-EYFP named PAC1-Tet-CHO was obtained. Moreover, in PAC1-Tet-CHO cells the expression of PAC1-EYFP was induced by Dox in a dose-dependent manner. The inducible expression of PAC1 still was stable after sub-culturing for more than 10 passages. It was also found by MTT assay that the higher expression level of PAC1 endowed the cells with higher proliferative viabilities. The construction of controlled expression system of PAC1 will lay a foundation for the further research on PAC1 profiles.