Abstract 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-PACAP transfection group both emit green fluorescence with high transfectection efficiency after 48 h post transfection. The growth curve showed that the cells grew well. Both mRNA and protein can expectedly express in 3T3-L1 cells including PACAP, FAS, ACC mRNA and pacap-EGFP protein. Especially the relative expression levels of PACAP, FAS, ACC mRNA in the pEGFP-PACAP group are significantly higher than the pEGFP-C1 group. All the results indicated PACAP may participate in adipogenesis. This study can be the theory basis for further study on adipogenesis and metabolism for pig in vitro.
GAO Tian-Tian,MAO Hai-Guang,CHEN Yi-Fei, et al. Construction of Eukaryotic Expression Vector for Pig (Sus scrofa) PACAP Gene and Its Expression in 3T3-L1 Cell Line[J]. , 2017, 25(3): 415-424.
[1]何君贤.2010. 梅山、大白猪FIT1基因真核表达载体的构建及其转染研究[D] . 硕士学位论文，华中农业大学, 导师：熊远著, pp. 50-56. ( He J X. 2010. Construction of Eukaryotic-Expressing Vectors for Meishan and Large White FITI gene and Transfeetion Research[D]. Thesis for M. S., Huazhong Agricultural University, Supervisor: Xiong Y Z, pp. 50-56) [2]刘松财, 戴建威, 任晓慧, 等.2009. 共表达生长激素释放激素 (GHRH) 与垂体腺苷酸环化酶 (PACAP) 对动物生长的影响[J]. 生物化学与生物物理进展, (6): 761-769. (Liu S C, Dai J W, Ren X H, et al. 2009. Ectopic Co-expression of Growth Hormone Releasing Hormone and Pituitary Adenylate Activating Peptide in Skeletal Muscle Enhance Animal Growth[J]. Progress in Biochemistry and Biophysics, (6):761-769.) [3]潘登科, 万荣, 刘吉, 等.2007. 猪前脂肪细胞为核供体生产表达绿色荧光蛋白的转基因克隆胚胎[J].畜牧兽医学报, 38(12): 1389-1393. (Pan D K, Wang R, Liu J, et al. 2007. Production of Transgenic Embryos Expressing Green Fluorescent Protein by Nuclear Transfer of Preadipocytes in Pig [J]. Animal Husbandry and Veterinary Medicine, 38(12) : 1389-1393. ) [4]王丽静.2007. 地塞米松对3T3-L1脂肪细胞葡萄糖转运及其信号分子的影响[D] . 硕士学位论文， 福建医科大学，导师：刘礼斌，pp. 3-8. (Wang L J. 2007. The Effect of Dexamethasone on Glucose Transport and the Signaling Proteins in 3T3-L1 Adipocyte [D]. Thesis for M. S., Fujian Medical University, Supervisor: Liu L B, pp. 3-8. ) [5]徐昊, 王明荣, 颜光涛 .2000. 脂质体转染技术进展及其应用[J]. 国外医学.预防. 诊断. 治疗用生物制品分册, 23(6): 263-265. [6]Arsenijevic T, Gregoire F, Chiadak J, et al.2013. Pituitary adenylate cyclase activating peptide (PACAP) participates in adipogenesis by activating ERK signaling pathway[J]. PloS ONE, 8(9): e72607. [7]CHARTREL N, TONON M C, VAUDRY H, et al.1991. Primary Structure of Frog Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and Effects of Ovine PACAP on Frog Pituitary*[J]. Endocrinology, 129(6): 3367-3371. [8]Green H, Meuth M.1974. An established pre-adipose cell line and its differentiation in culture[J]. Cell, 3(2): 127-133. [9]Harmar A J, Fahrenkrug J, Gozes I, et al.2012. Pharmacology and functions of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide: IUPHAR Review 1[J]. British journal of pharmacology, 166(1): 4-17. [10]Hugo H, Ackland M L, Blick T, et al.2007. Epithelial—mesenchymal and mesenchymal—epithelial transitions in carcinoma progression[J]. Journal of cellular physiology, 213(2): 374-383. [11]Kimura C, Ohkubo S, Ogi K, et al.1990. A novel peptide which stimulates adenylate cyclase: molecular cloning and characterization of the ovine and human cDNAs[J]. Biochemical and biophysical research communications, 166(1): 81-89. [12]Lambe N R, McLean K A, Gordon J, et al.2017. Prediction of intramuscular fat content using CT scanning of packaged lamb cuts and relationships with meat eating quality[J]. Meat Science, 123: 112-119. [13]Lee S H, Choi Y M, Choe J H, et al.2010. Association between polymorphisms of the heart fatty acid binding protein gene and intramuscular fat content, fatty acid composition, and meat quality in Berkshire breed[J]. Meat science, 86(3): 794-800. [14]Lefaucheur L.2010. A second look into fibre typing–Relation to meat quality[J]. Meat Science, 84(2): 257-270. [15]Miyata A, Arimura A, Dahl R R, et al.1989. Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells[J]. Biochemical and biophysical research communications, 164(1): 567-574. [16]Miyata A, Jiang L, Dahl R D, et al.1990. Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38)[J]. Biochemical and biophysical research communications, 170(2): 643-648. [17]Monaghan T K, Mackenzie C J, Plevin R, et al.2008. PACAP-38 induces neuronal differentiation of human SH-SY5Y neuroblastoma cells via cAMP‐mediated activation of ERK and p38 MAP kinases1[J]. Journal of neurochemistry, 104(1): 74-88. [18]Ogi K, Kimura C, Onda H, et al.1990. Molecular cloning and characterization of cDNA for the precursor of rat pituitary adenylate cyclase activating polypeptide (PACAP)[J]. Biochemical and biophysical research communications, 173(3): 1271-1279. [19]Webb E C, O’neill H A.2008. The animal fat paradox and meat quality[J]. Meat Science, 80(1): 28-36.
