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  

Construction of Eukaryotic Expression Vector for Pig(Sus scrofa) PACAP Gene and Its Expression in 3T3-L1 Cell Line

作　　者：高甜甜[1] 毛海光 沈一飞[1] 徐宁迎[1] 

机构地区：[1]浙江大学动物科学学院,杭州310058

出　　处：《农业生物技术学报》2017年第3期415-424,共10页Journal of Agricultural Biotechnology

基　　金：国家转基因生物新品种培育科技重大专项(No.2014ZX08006-003)

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(P<0.01),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。Pituitary adenylate cyclase-activating polypeptide （PACAP） is synthesized and secreted by the hypothalamus. As a bioactivator, it can stimulate the synthesis and secretion of growth hormone （GH） to promote the growth of animals. The objective of this study was to obtain full-length cDNA of pig （Sus scrofa） PACAP gene （pPACAP）, and to analyse the relative expression level of PACAP gene during the differentiation of 3T3-L1 preadipocytes into adipocytes and then analyze the relative expression levels of PACAP, FAS, ACC mRNA and detect the target protein pacap-EGFP after the recombinant plasmid pEGFP-PACAP was transfected into 3T3-L1 cells in 48 h. Besides, cell counting kit-8 （CCK-8） was applied to see the growth curve of 3T3-L1 cells transfected. Firstly lipid droplet and the expression levels of PACAP gene were detected respectively by Oil red O and qRT-PCR after XDI was induced to differentiate the 3T3-L1 preadipocytes into adipocytes. And then primers with EcoRⅠand BglⅡ restriction enzymes were designed and amplified by PCR in accordance with the deposited sequence of pPACAP in GenBank. Further, the digested PCR product was ligated into eukaryotic vector pEGFP-C1 and the recombinant plasmid pEGFP-PACAP was successfully constructed. During the transfection in 48 h, the cell growth was observed with CCK-8 under in vitro transfection with XfectTM transfection reagent. Besides, the expected mRNA including PACAP, FAS and ACC and target protein pacap were successfully detected in 3T3-L1 cells, respectively by using qRT-PCR and Western blot. The adipogenic differentiation results showed that the expression level of PACAP of mRNA is gradually increasing and is the highest at 4 day after 3T3-L1 cells were induced; Double restriction enzymes digestion and sequence results indicated that the eukaryotic expression vector of pPACAP gene is successfully cloned, named as pEGFP-PACAP; By fluorescence microscopy, cells of the empty plasmid pEGFP-C1 transfection group and the recombinant plasmid pEGFP-PACA

关 键 词：垂体腺苷酸环化酶激活肽基因(PACAP) 真核表达 转染 3T3-L1前体脂肪细胞 

分 类 号：S828[农业科学—畜牧学]