小鼠FGF21基因克隆及his-mFGF21融合蛋白的诱导表达
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摘要
目的:对小鼠FGF21基因进行克隆并诱导表达FGF21融合蛋白。方法:以带有小鼠FGF21的T载体pMD19-T-mFGF21为模板特异性扩增FGF21基因片段,将扩增产物克隆至pET-28a(+),得到pET-28a(+)-mFGF21的重组子,并转化于大肠杆菌BL21(DE3),使用IPTG诱导融合蛋白的表达,纯化后采用Western Blot进行鉴定。结果:pET-28(+)-mFGF21重组质粒构建成功,表达出可溶性的his-mFGF21融合蛋白,经纯化后该蛋白his-mFGF21可与FGF21抗体特异性结合。结论:成功构建pET-28(+)-mFGF21重组子,并表达出his-mFGF21蛋白。
Objective: To clone the gene of mice FGF21 to the expression vector pET-28a(+), express the pET-28a(+)-mFGF21 in E.coli, and obtain the fusion protein of mice FGF21. Methods: To specifically amplificate the gene fragment of mFGF21,which was preserved as pMD19-T-mFGF21 in the early stage of the laboratory, and then clone it into pET-28a(+) to get the recombinant plasmids of pET-28a(+)-mFGF21.The pET-28a(+)-mFGF21 was transformed into E.coli BL21(DE3). The fusion protein induced with IPTG and purified in affinity chromatography, was identified by Western blotting. Results: The recombinant plasmid of pET-28a(+)-mFGF21 was successfully constructed and expressed in a soluble form, and the his-mFGF21 protein had specific interaction with FGF21 antibody after being purified. Conclusion: The pET-28a(+)-mFGF21 recombinant can be successfully constructed and the fusion protein of his-mFGF21 can be expressed.
Objective: To clone the gene of mice FGF21 to the expression vector pET-28a(+), express the pET-28a(+)-mFGF21 in E.coli, and obtain the fusion protein of mice FGF21. Methods: To specifically amplificate the gene fragment of mFGF21,which was preserved as pMD19-T-mFGF21 in the early stage of the laboratory, and then clone it into pET-28a(+) to get the recombinant plasmids of pET-28a(+)-mFGF21.The pET-28a(+)-mFGF21 was transformed into E.coli BL21(DE3). The fusion protein induced with IPTG and purified in affinity chromatography, was identified by Western blotting. Results: The recombinant plasmid of pET-28a(+)-mFGF21 was successfully constructed and expressed in a soluble form, and the his-mFGF21 protein had specific interaction with FGF21 antibody after being purified. Conclusion: The pET-28a(+)-mFGF21 recombinant can be successfully constructed and the fusion protein of his-mFGF21 can be expressed.
引文
[1] JIN L,LIN Z,XU A.Fibroblast growth factor 21 protects against atherosclerosis via fine-tuning the multiorgan crosstalk[J].Diabetes Metab J,2016,40(2):22-31.
[2] 肖卫晋,李国华,屈顺林,等.成纤维细胞生长因子21 心肌保护作用的研究进展[J].中国动脉硬化杂志,2017,25(5):527-530.
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[11]石维.2型糖尿病合并动脉粥样硬化的发病机制、临床诊断与治疗[J].中国老年学杂志,2016,36(7):1777-1781.
[12]刘丽,张腾,陈瑜.免疫系统与动脉粥样硬化发病机制的研究进展[J].中西医结合心脑血管病杂志,2015,13(8):1000-1003.
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[14]吕欣,任毅,杨静.FGF21生物学特征及对脂肪组织糖代谢的调节[J].中国现代医药杂志,2017,19(1):101-104.
[15]LI H,ZHANG J,JIA W.L.Fibroblast Growth factor 21:a novel metabolic regulator from pharmacology to physiology[J].Front Med,2013,7(1):25-30.
[16]FISHER FM,KLEINER S,DOURIS N,et al.FGF21 regulates PGC-1α and browning of white adipose tissues in adaptive thermogenesis[J].Genes Dev,2012,26(3):271-281.
[17]MURIELLE M,VENIANT,GLENN S,et al.Pharmacologic effects of FGF21 are independent of the“browning”of white adipose tissue[J].Cell Metabolism,2015,21(5):731-738.
[18]TANAJAK P,CHATTIPAKORN S C,CHATTIPAKORN N,et al.Effects of fibroblast growth factor 21 on the heart[J].J Endocrinol,2015,227(2):13-30.
[19]KOKKINOS J,TANG S,RYE K A,et al.The role of fibroblast growth factor 21 in atherosclerosis[J].Atherosclerosis,2017,257:259-265.
[20]ZHU S,MA L,WU Y,et al.FGF21 treatment ameliorates alcoholic fatty liver through activation of AMPK-SIRT1 pathway[J].Acta Biochim Biophys Sin,2014,46(12):1041-1048.
[21]SINGHAL G,FISHER F M,CHEE M J,et al.Fibroblast growth factor 21(FGF21) protects against high fat diet induced inflammation and islet hyperplasia in pancreas[J].PLoS One,2016,11(2):e0148252.
[22]WANG W F,LI S M,REN G P,et al.Recombinant murine fibroblast growth factor 21 ameliorates obesity-related inflammation in monosodium glutamate-induced obesity rats[J].Endocrine,2015,49(1):119-129.
[23]YU Y,HE J,LI S,et al.Fibroblast growth factor 21(FGF21) inhibits macrophage-mediated inflammation by activating Nrf2 and suppressing the NF-kappa B signaling pathway[J].Int Immunopharmacol,2016,38:144-152.
[24]王大秀,韩继武.成纤维细胞生长因子21对小鼠肝纤维化的作用及其机制[J].临床肝胆病杂志,2018,34(6):1215-1219.
[25]VIDELA LA,FERNANDEZ V,VARGAS R,et al.Upregulation of rat liver PPARalphaFGF21 signaling by a docosahexaenoic acid and thyroid hormone combined protocol[J].Biofactors,2016,42(6):638-646.
[26]XIAO Y,LIU L,XU A,et al.Serum fibroblast growth factor 21 levels are related to subclinical atherosclerosis in patients with type 2 diabetes[J].Cardiovasc Diabetol,2015,14:72.
[2] 肖卫晋,李国华,屈顺林,等.成纤维细胞生长因子21 心肌保护作用的研究进展[J].中国动脉硬化杂志,2017,25(5):527-530.
[3] LUO F,GUO Y,RUAN G,et al.Metformin promotes cholesterol efflux in macrophages by up-regulating FGF21 expression:a novel anti-atherosclerotic mechanism[J].Lipids Health Dis,2016,15(1):1-2.
[4] CHENG P,ZHANG F,YU L,et al.Physiological and pharmacological roles of FGF21 in cardiovascular diseases[J].J Diabetes Res,2016(2):1-8.
[5] PLANAVILA A,REDONDO-ANGULO I,RIBAS F,et al.Fibroblast growth factor 21 protects the heart from oxidative stress[J].Cardiovasc Res,2015,106(1):19-31.
[6] 伍熙,赵东晖,范谦,等.成纤维细胞生长因子21 对载脂蛋白E 基因敲除小鼠动脉粥样硬化病变中内质网应激诱导的凋亡的影响[J].中国动脉硬化杂志,2014,22(4):325-328.
[7] LEE C H,WOO Y C,CHOW W S,et al.Role of circulating fibroblast growth factor 21 measurement in primary prevention of coronary heart disease among Chinese patients with type 2 diabetes mellitus[J].Journal of the American Heart Association,2017,6(6):30-35.
[8] 唐青蓝,李红梅,王剑青,等.FGF21与原发性高血压的相关性初探[J].贵阳医学院学报,2013,38(4):359-361.
[9] 刘俊田.动脉粥样硬化发病的炎症机制的研究进展[J].西安交通大学学报(医学版),2015,36(2):141-152.
[10]RASMUSSEN-TORVIK L J,LI M,KAO W H,et al.Association of a fasting glucose genetic risk score with subclinical Atherosclerosis [J].Diabetes,2011,60(1):33l-335.
[11]石维.2型糖尿病合并动脉粥样硬化的发病机制、临床诊断与治疗[J].中国老年学杂志,2016,36(7):1777-1781.
[12]刘丽,张腾,陈瑜.免疫系统与动脉粥样硬化发病机制的研究进展[J].中西医结合心脑血管病杂志,2015,13(8):1000-1003.
[13]NISHIMURA T,NAKATAKE Y,KONISHI M,et al.Identification of a novel FGF,FGF-21,preferentially expressed in the liver[J].Biochim Biophys Acta,2000,1492(1):203-206.
[14]吕欣,任毅,杨静.FGF21生物学特征及对脂肪组织糖代谢的调节[J].中国现代医药杂志,2017,19(1):101-104.
[15]LI H,ZHANG J,JIA W.L.Fibroblast Growth factor 21:a novel metabolic regulator from pharmacology to physiology[J].Front Med,2013,7(1):25-30.
[16]FISHER FM,KLEINER S,DOURIS N,et al.FGF21 regulates PGC-1α and browning of white adipose tissues in adaptive thermogenesis[J].Genes Dev,2012,26(3):271-281.
[17]MURIELLE M,VENIANT,GLENN S,et al.Pharmacologic effects of FGF21 are independent of the“browning”of white adipose tissue[J].Cell Metabolism,2015,21(5):731-738.
[18]TANAJAK P,CHATTIPAKORN S C,CHATTIPAKORN N,et al.Effects of fibroblast growth factor 21 on the heart[J].J Endocrinol,2015,227(2):13-30.
[19]KOKKINOS J,TANG S,RYE K A,et al.The role of fibroblast growth factor 21 in atherosclerosis[J].Atherosclerosis,2017,257:259-265.
[20]ZHU S,MA L,WU Y,et al.FGF21 treatment ameliorates alcoholic fatty liver through activation of AMPK-SIRT1 pathway[J].Acta Biochim Biophys Sin,2014,46(12):1041-1048.
[21]SINGHAL G,FISHER F M,CHEE M J,et al.Fibroblast growth factor 21(FGF21) protects against high fat diet induced inflammation and islet hyperplasia in pancreas[J].PLoS One,2016,11(2):e0148252.
[22]WANG W F,LI S M,REN G P,et al.Recombinant murine fibroblast growth factor 21 ameliorates obesity-related inflammation in monosodium glutamate-induced obesity rats[J].Endocrine,2015,49(1):119-129.
[23]YU Y,HE J,LI S,et al.Fibroblast growth factor 21(FGF21) inhibits macrophage-mediated inflammation by activating Nrf2 and suppressing the NF-kappa B signaling pathway[J].Int Immunopharmacol,2016,38:144-152.
[24]王大秀,韩继武.成纤维细胞生长因子21对小鼠肝纤维化的作用及其机制[J].临床肝胆病杂志,2018,34(6):1215-1219.
[25]VIDELA LA,FERNANDEZ V,VARGAS R,et al.Upregulation of rat liver PPARalphaFGF21 signaling by a docosahexaenoic acid and thyroid hormone combined protocol[J].Biofactors,2016,42(6):638-646.
[26]XIAO Y,LIU L,XU A,et al.Serum fibroblast growth factor 21 levels are related to subclinical atherosclerosis in patients with type 2 diabetes[J].Cardiovasc Diabetol,2015,14:72.