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

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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.

Key words： Pituitary adenylate cyclase-activating polypeptide gene (PACAP) Eukaryotic expression Transfection 3T3-L1 cells

Received: 10 October 2016 Published: 02 March 2017

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	GAO Tian-Tian
	MAO Hai-Guang
	CHEN Yi-Fei
	XU Ning-Ying

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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.

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http://journal05.magtech.org.cn/Jwk_ny/EN/ OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2017/V25/I3/415

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