鸡Visfatin蛋白的原核表达、纯化及其活性研究

doi:10.11843/j.issn.0366-6964.2016.09.006

Abstract

Abstract:

The objective of the present study was to obtain recombinant chicken Visfatin protein with biological activity.The total RNA was extracted from livers of AA broilers.The Visfatin cDNA was first amplified by qRT-PCR and cloned into pGEM-T vector.The cDNA was then cut out from pGEM-T vector by digestion with endonuclease NcoⅠ and XhoⅠand inserted into corresponding cloning sites of the plasmid pET-30a.The positive recombinant plasmid designated as pET-30a-Visfatin was transformed into E.coli BL21 (DE3) competent cells.The cells containing pET-30a-Visfatin were identified.The conditions of IPTG induction were optimized by examining the expression level of recombinant protein using SDS-PAGE analysis.The recombinant protein was purified by Nickel ion affinity chromatography.The identification of the purified recombinant protein was checked by Western blot and the bioactivity was examined by looking into differentiation status of 3T3-L1 cells.A fragment which was the same size to prediction was observed when the recombinant plasmid pET-30a-Visfatin was digested with endonuclease NcoⅠ and XhoⅠ.It indicated that the recombinant expression vector was constructed successfully.The SDS-PAGE analysis revealed that the optimum conditions of induction were 0.4 mmol•L^-1 IPTG，pH 8.0，at 30 ℃ for 10-12 h.The molecular weight of the recombinant protein was about 60 ku，which was the same to prediction.The Western blot analysis indicated that the recombinant protein could be recognized by anti-His antibody.Additionally，the Oil Red O stain assay showed that there were more lipid droplets formation in 3T3-L1 cells induced by Visfatin than that in the controls.Meanwhile，the results of qRT-PCR revealed that the expression levels of genes involved in cell differentiation such as PPARγ， aP2，FAS and C/EBPα increased significantly (P<0.05) when 3T3-L1 cells induced by Visfatin.This study successfully established a standardized expression program for recombinant chicken Visfatin protein production，and obtained the purified recombinant protein with biological activity，which laid a foundation for further research in the field of poultry.

CLC Number:

S831.2

ZHANG Pan-pan，SHANG Peng-fei，TIAN Fang-yuan，HAN Rui-li，JIANG Rui-rui，SUN Gui-rong，KANG Xiang-tao，LIU Xiao-jun，TIAN Ya-dong. Prokaryotic Expression，Purification and Bioactivity Identification of Recombinant Chicken Visfatin Protein[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(9): 1785-1794.

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References

［1］FUKUHARA A，MATSUDA M，NISHIZAWA M，et al.Visfatin：a protein secreted by visceral fat that mimics the effects of insulin［J］.Science，2005，307(5708)：426-430.
［2］REVOLLO J R，KÖMER A，MILLS K F，et al.Nampt/PBEF/visfatin regulates insulin secretion in β cells as a systemic NAD biosynthetic enzyme［J］.Cell Metab,2007，6(5)：363-375.
［3］REVOLLO J R，GRIMM A A，IMAI S.The regulation of nicotinamide adenine dinucleotide biosynthesis by Nampt/PBEF/visfatin in mammals［J］.Curr Opin Gastroenterol，2007，23(2)：164-170.
［4］ONS E，GERTLER A，BUYSE J，et al.Visfatin gene expression in chickens is sex and tissue dependent［J］.Domest Anim Endocrinol，2010，38(2)：63-74.
［5］韩瑞丽.鸡 visfatin 和 PRDMl6 基因遗传变异及其效应与表达研究［D］.杨凌：西北农林科技大学，2010.
HAN R L.Study on genetic variation and its effects and expression of visfatin and PRDM16 gene in chickens［D］.Yangling：North West Agriculture and Forestry University，2010.(in Chinese)
［6］SAMAL B，SUN Y，STEARNS G，et al.Cloning and characterization of the cDNA encoding a novel human pre-B-cell colony-enhancing factor［J］. Mol Cell Biol，1994，14(2)：1431-1437.
［7］KLÖTING N，KLÖTING I.Visfatin：gene expression in isolated adipocytes and sequence analysis in obese WOKW rats compared with lean control rats［J］.Biochem Biophys Res Commun，2005，332(4)：1070-1072.
［8］CHEN H，XIA T，ZHOU L，et al.Gene organization，alternate splicing and expression pattern of porcine visfatin gene［J］.Domest Anim Endocrinol，2007，32(3)：235-245.
［9］CHEN M P，CHUNG F M，CHANG D M，et al.Elevated plasma level of visfatin/pre-B cell colony-enhancing factor in patients with type 2 diabetes mellitus［J］.J Clin Endocrinol Metab，2006，91(1)：295-299.
［10］OGNJANOVIC S，BRYANT-GREENWOOD G D.Pre-B-cell colony-enhancing factor，a novel cytokine of human fetal membranes［J］.Am J Obstet Gynecol，2002，187(4)：1051-1058.
［11］YE S Q，SIMON B A，MALONEY J P，et al.Pre-B-cell colony-enhancing factor as a potential novel biomarker in acute lung injury［J］.Am J Respir Crit Care Med，2005，171(4)：361-370.
［12］CLINE M A，NANDAR W，PRALL B C，et al.Central visfatin causes orexigenic effects in chicks［J］.Behav Brain Res，2008，186(2)：293-297.
［13］BRUNETTI L，RECINELLA L，DI NISIO C，et al.Effects of visfatin/PBEF/NAMPT on feeding behaviour and hypothalamic neuromodulators in the rat［J］.J Biol Regul Homeost Agents，2011，26(2)：295-302.
［14］韩瑞丽，康相涛，魏杨，等.鸡 visfatin 基因第 9 内含子 4 个 SNPs 的关联分析［C］.南京：第十二次全国畜禽遗传标记研讨会，2010.
HAN R L，KANG X T，WEI Y，et al.Association analysis of 4 SNPs in the ninth intron of visfatin gene in chicken［C］.Nanjing：Conference Proceedings of the 12th National Symposium on Genetic Markers of Livestock and Poultry，2010.(in Chinese)
［15］LI J，MENG F，SONG C，et al.Characterization of chicken visfatin gene：cDNA cloning，tissue distribution，and promoter analysis［J］.Poult Sci，2012，91(11)：2885-2894.
［16］SETHI J K，VIDAL-PUIG A.Visfatin：the missing link between intra-abdominal obesity and diabetes［J］.Trends Mol Med，2005，11(8)：344-347.
［17］范翠英，冯利兴，樊金玲，等.重组蛋白表达系统的研究进展［J］.生物技术，2012，22(2)：76-80.
FAN C Y，FENG L X，FAN J L，et al.Recent advances on the expression systems for recombinant protein production［J］.Biotechnology，2012，22(2)：76-80.(in Chinese)
［18］RIBAS A V，HO P L，TANIZAKI M M，et al.High-level expression of tetanus toxin fragment C-thioredoxin fusion protein in Escherichia coli［J］.Biotechnol Appl Biochem，2000，31(2)：91-94.
［19］ZHAO J H，XU Z，HUA Z C.Expression of human cardiac-specific homeobox protein in Escherichia coli［J］. Protein Expr Purif，2000，18(3)：316-319.
［20］KANE J F，HARTLEY D L.Formation of recombinant protein inclusion bodies in Escherichia coli［J］.Trends Biotechnol，1988，6(5)：95-101.
［21］叶姣，陈长华，夏杰，等.温度对重组大肠杆菌生长及外源蛋白表达的影响［J］.华东理工大学学报：自然科学版，2002，28(4)：364-367.
YE J，CHEN C H，XIA J，et al.Effect of temperature on the growth of recombinant E.coli and on the expression of recombiant protein［J］. Journal of East China University of Science and Technology，2002，28(4)：364-367.(in Chinese )
［22］PORATH J.Immobilized metal ion affinity chromatography［J］.Protein Expr Purif，1992，3(4)：263-281.
［23］LIN F T，LANE M D.CCAAT/enhancer binding protein alpha is sufficient to initiate the 3T3-L1 adipocyte differentiation program［J］. Proc Natl Acad Sci，1994，91(19)：8757-8761.
［24］初明，胡志强，魏兰兰，等.白黎芦醇对垂体腺瘤GH3细胞生长及其分子机制的研究［J］.中华医学杂志，2003，83(22)：2004-2006.
CHU M，HU Z Q，WEI L L，et al.Studies on the effect of resveratrol on growth of GH3 cells and its molecular mechanism in pituitary adenomas［J］.National Medical Journal of China，2003，83(22)：2004-2006.(in Chinese )
［25］WU Z，ROSEN E D，BRUN R，et al.Cross-regulation of C/EBPα and PPARγ controls the transcriptional pathway of adipogenesis and insulin sensitivity［J］.Mol Cell，1999，3(2)：151-158.

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Prokaryotic Expression，Purification and Bioactivity Identification of Recombinant Chicken Visfatin Protein

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