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.
Animals ; Blotting, Western ; CHO Cells ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Genetic Vectors ; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I ; biosynthesis

Objective This report aims to assess the exposure risk of aflatoxin B1 in edible vegetable oil in Guangxi.Methods By using margin of exposure (MOE),the report analyzes the dietary exposure of aflatoxin B1 in edible vegetable oil with the data from contamination survey and dietary intake survey.Results For the vegetable oil sample,the content of aflatoxin B1 was between 0.50-320.00 μg/kg.The detection rate of peanut oil was 78.08％ (114/146) which was higher than other vegetable oil,and the exceeding rate was 31.51％ (46/146).For peanut oil,the average content was 30.80 μg/kg,the dietary exposure of the population was 17.30 ng/kg BW,and the MOE was 18.For the prepackaged peanut oil samples,the average content of aflatoxin B1 was 6.33 μg/kg,which was below the limit.While for the bulk peanut oil,the average content of AFB1 was 41.50 μg/kg,which was more than 1.08 times of the limit,and the dietary exposure was 25.59 ng/kg BW.The MOE of bulk peanut oil was 12,1/8 of the prepackaged peanut oil.Conclusion Food safety regulators should pay more attention to bulk peanut oil products,the priority in the risk management measures.At the same time,related department should also promote healthy education for the residents.