猪PACAP基因真核表达载体的构建及其在3T3-L1前体脂肪细胞上的表达

发布时间：2018-09-14 18:06

【摘要】：垂体腺苷酸环化酶激活肽(pituitary adenylate cyclase-activating polypeptide,PACAP)是由下丘脑合成并分泌,存在于人(Homo sapiens)和动物体内的生物活性物质,能够刺激垂体生长激素(growth hormone,GH)的合成与分泌,从而促进动物体的生长发育。本研究首先克隆了猪(Sus scrofa)PACAP基因(p PACAP)的全长c DNA,利用油红O染色技术和q RT-PCR技术研究PACAP基因在3T3-L1前体脂肪细胞成脂分化过程中的表达规律;继而构建了真核表达载体p EGFP-p PACAP,将其转染至小鼠(Mus musculus)3T3-L1前体脂肪细胞,48 h内采用CCK-8法检测细胞生长情况并于48 h后检测3T3-L1前体脂肪细胞中的p PACAP、脂肪酸合成酶(fatty acid synthase,FAS)和乙酰辅酶A羧化酶(acetyl-Co A carboxylase,ACC)m RNA的表达及pacap-EGFP融合蛋白的表达。成脂分化结果表明,PACAP m RNA随着脂肪细胞的成熟,其表达量逐渐上升,在第4天时,表达量最高;双酶切及测序结果表明,成功构建了p PACAP基因的真核表达载体p EGFP-p PACAP;真核表达载体转入培养的3T3-L1前体脂肪细胞中后,在荧光显微镜下,重组质粒组和空载体组均发出绿色荧光,48 h时转染效率最高,且PACAP、FAS和ACC m RNA相对表达量均有极显著的提高(P0.01),成功检测到pacap融合蛋白。本研究结果表明,PACAP参与到脂肪形成的机制中。该结果为体外研究猪PACAP基因对脂肪合成代谢及其机体代谢的调节机制提供了理论依据。
[Abstract]:Pituitary adenylate cyclase-activating polypeptide (PACAP) is synthesized and secreted by hypothalamus. It is a bioactive substance in human (Homo sapiens) and animals. It can stimulate the synthesis and secretion of pituitary growth hormone (GH) and promote the growth and development of animals. * we first cloned the full-length C DNA of the Sus scrofa PACAP gene (P PACAP), and studied the expression pattern of the PACAP gene in the adipocyte differentiation process of the pre adipocytes by using oil red O staining and Q RT-PCR technology. Then we constructed the eukaryotic expression vector, which was transfected into the mouse adipocytes, 48 CCK-8 method was used to detect the growth of cells within hours and the expression of p-PACAP, fatty acid synthase (FAS), acetyl-Co A carboxylase (ACC) m RNA and pacap-EGFP fusion protein in 3T3-L1 preadipocytes were detected 48 hours later. The expression level of P PACAP gene was the highest on the 4th day after maturation. The results of double enzyme digestion and sequencing showed that the eukaryotic expression vector p EGFP-p PACAP was successfully constructed. After the eukaryotic expression vector was transfected into the cultured 3T3-L1 preadipocyte, the recombinant plasmid and the empty vector both emitted green fluorescence under fluorescence microscope. At 8 h, the transfection efficiency was the highest, and the relative expression of PACAP, FAS and ACC m RNA increased significantly (P0.01). PACAP fusion protein was successfully detected. The results showed that PACAP was involved in the mechanism of adipose formation. * the results provide a theoretical basis for studying the regulation mechanism of Porcine PACAP gene on fat metabolism and metabolism in vitro. Basis.
【作者单位】：浙江大学动物科学学院;
【基金】：国家转基因生物新品种培育科技重大专项(No.2014ZX08006-003)
【分类号】：S828

【参考文献】