人脂联素的原核表达及生物活性检测
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摘要
近年来的研究发现脂肪组织除能量储存外,尚具有活跃的内分泌功能。在代谢状态改变或接受外界刺激时,脂肪组织可分泌多种具有生理活性的蛋白质,即脂肪细胞因子,包括瘦素、肿瘤坏死因子-α(TNF-α)、抵抗素、白细胞介素6(IL-6)、脂联素(adiponectin,ADPN)等。这些激素参与了神经、内分泌与免疫网络的调节,尤其对能量代谢调节具有重要的作用。
脂联素作为一种脂肪细胞因子,具有重要的生理功能:改善糖脂代谢及胰岛素抵抗;通过抑制人类主动脉内皮细胞(HAECs)表面的血管细胞粘附分子及细胞间粘附分子等的表达,起到抗动脉粥样硬化的作用;可使肥胖者个体质量减轻;通过抑制巨噬细胞前体细胞的生成和成熟巨噬细胞的功能调节炎症反应。因此,脂联素与胰岛素抵抗、2型糖尿病、肥胖以及心血管疾病等具有密切联系。近年来的研究还表明,脂联素可直接或间接影响肿瘤的发生发展。
在对脂联素多年的研究中发现,低脂联素水平不仅与胰岛素抵抗及2型糖尿病相关,低脂联素血症还可出现在心血管疾病、高血压、血脂异常中。血清脂联素水平的检测可以作为代谢性疾病诊断及治疗评价的指标,而补充重组脂联素或增加内源性脂联素的分泌则为胰岛素抵抗相关疾病的预防和治疗提供了一种新的思路与手段。
目前,国内外已有一些研究应用多种蛋白表达系统构建重组脂联素,包括原核及真核表达系统。其中原核表达体系因其成本低,操作简便以及产量高等众多优点而被广泛应用。本研究所采用的表达载体pQE-30Xa是Qiagen公司提供的一种高效表达和纯化外源蛋白的原核表达系统,相对于其他载体具有很多优势,目前国内尚未见使用该载体获得脂联素蛋白的相关报道。
研究目的:
1.克隆人脂联素目的基因,构建pQE-30Xa/ADPN原核表达载体。
2.转化入大肠杆菌E.coli M15[pREP4]进行重组蛋白的表达,鉴定表达产物。
3.优化表达条件,以期建立高效表达的技术流程。
实验方法:
1.从人的脂肪组织中提取总RNA,利用RT-PCR的方法得到人脂联素基因cDNA序列,构建模板质粒;自行设计引物一对,经PCR扩增人脂联素基因完全编码序列并使两端加上双酶切位点,构建pMD18-T/ADPN重组克隆质粒;pMD18-T/ADPN与pQE-30Xa质粒经限制性核酸内切酶BamHI与HindIII双酶切后,用T_4DNA连接酶进行连接,构建重组表达载体pQE-30Xa/ADPN。
2.将重组质粒转化入M15[pREP4]表达菌株中,IPTG诱导表达;提取包涵体,SDS-PAGE及Western blot鉴定表达产物;从诱导时间、温度及诱导剂浓度三方面摸索最适表达条件。
3.按照优化条件进行蛋白质的大量表达;在变性条件下利用His Band Kit纯化目的蛋白;PAPIB法梯度透析复性,SDS-PAGE鉴定蛋白纯度;Bradford法进行蛋白定量分析;MTS细胞增殖实验检测获得目的蛋白的生物学活性。
实验结果:
1.成功构建重组表达载体pQE-30Xa/ADPN,经测序证实插入的目的片段无误。
2.脂联素目的蛋白在E.coli.M15菌株中获得了表达,其中诱导剂IPTG终浓度为0.6mM、温度为37℃、诱导表达6小时后所获得的蛋白含量相对较高。
3.经His Band Kit纯化后,可获得单一的脂联素目的蛋白条带,蛋白纯化效果较好;蛋白表达效率约为24.8%,产量约为330μg/100ml LB培基;经MTS实验证实获得的重组蛋白具有生物活性。
实验结论:
成功构建了人脂联素原核表达载体pQE-30Xa/ADPN,并在E.coli.M15菌株中获得了表达,且获得的重组蛋白具有生物学活性。pQE-30Xa原核表达系统是一个高效、快速构建重组脂联素蛋白的良好选择。
In recent years,adipose tissue is considered not only as an energy storage but also as a very important endocrine organ.Adipose tissue secretes a number of biologically active adipokines such as leptin,tumor necrosis factor-a(TNF-a),resistin, interleukin-6(IL-6),adiponectin(ADPN) and so on.These hormones participate in the nervous,endocrine and immune regulation,and especially play an important role in the energy metabolism.
Adiponectin as one such adipokine has significantly physiological functions.It can improve glucose metabolism,lipid metabolism and insulin resistance. Adiponectin has been reported to have direct effects of antiatherosclerosis through inhibiting the expression of adhesion molecules,including intracellular adhesion molecule and vascular cellular adhesion molecule.Adiponectin could induce weight loss too.Recent studies suggest that adiponectin may play a role in the modulation of inflammatory vascular response by suppressing macrophage function.Therefore adiponectin has a close relation with insulin resistance,type 2 diabetes,obesity and cardiovascular disease.Lately it has been reported that adiponectin may directly or indirectly influence the occurrence and development of tumor.
The hypoadiponectinemia was correlated with insulin resistance and type 2 diabetes.We can find the plasma adiponectin level reduced in the condition of CVD, hypertension,and dyslipidemia.So the determination of plasma adiponectin will be a good marker for diagnosis and prognosis of diabetes mellitus,and addition of recombinant adiponectin or increasion of endogenous adiponectin secretion provides a new way for prevention and treatment of disease related to insulin resistance.
Till now researchers have applied many kinds of protein expression systems such as prokaryotic and eukaryotic systems to get recombinant adiponectin.
Prokaryotic expression system is widely used as the advantages of low cost,simple operation and high output.In our study,the expression vector of pQE-30 Xa we used is one kind of prokaryotic expression system from Qiagen company which has high efficiency in expressing and convenience for purifying.At present there is no domestic reporting about using this vector to get adiponectin protein.
Objectives:
1.To clone human adiponectin gene,and construct the prokaryotic expression vector-pQE-30 Xa/ADPN.
2.To transfect E.coli Ml 5 competent cells,obtain the expression of recombinant adiponection protein,and identify the expression product.
3.To optimize the expression conditions,and establish an efficient technological process.
Methods:
1.To extract the total RNA from human adipose tissue.We got the adiponectin cDNA with RT-PCR method.Design a pair of primers to amplify the encoding fragment of human ADPN gene by PCR.Construct recombinant clone vector pMD18-T/ADPN. Using BamHI、HindⅢenzymes to digest plasmids,the encoding fragment of human ADPN gene was ligated into expression vector pQE-30 Xa,and then recombinant vector pQE-30 Xa/ADPN was extracted.
2.The expression vector then was transformed into the M15 competent cells,and protein was expressed induced with IPTG.The protein in the inclusion body was extracted and identified by the methods of SDS-PAGE and Western Blot.The optimum of expression including induce time,temperature and concentration of induce reagent was tested.
3.The recombinant protein was largely expressed and purified by His Band Kit in the denatured condition.Protein was refolded and identified by SDS-PAGE.The concentration and bioactivity of the protein were determined.
Results:
1.Recombinant expression vector pQE-30 Xa/ADPN was successfully constructed. ADPN gene was inserted in correct location.
2.Recombinant protein was obtained by the expression of E.coli M15.The optimum of induce time was 6 hours,the temperature was 37℃and concentration IPTG was 0.6mM.
3.The protein was fairly purified by His Band Kit.The yield was 330μg/100ml LB and accounted for 24.8%of total protein in E.coli M15[pREP4].The result of MTS experiment showed that the protein had certain bioactivity.
Conclusions:
Recombinant prokaryotic expression vector pQE-30 Xa/ADPN was successfully constructed to express adiponectin protein with certain bioactivity in E.coli M15.The pQE-30 Xa expression system is efficient and suitable for adiponectin.
脂联素作为一种脂肪细胞因子,具有重要的生理功能:改善糖脂代谢及胰岛素抵抗;通过抑制人类主动脉内皮细胞(HAECs)表面的血管细胞粘附分子及细胞间粘附分子等的表达,起到抗动脉粥样硬化的作用;可使肥胖者个体质量减轻;通过抑制巨噬细胞前体细胞的生成和成熟巨噬细胞的功能调节炎症反应。因此,脂联素与胰岛素抵抗、2型糖尿病、肥胖以及心血管疾病等具有密切联系。近年来的研究还表明,脂联素可直接或间接影响肿瘤的发生发展。
在对脂联素多年的研究中发现,低脂联素水平不仅与胰岛素抵抗及2型糖尿病相关,低脂联素血症还可出现在心血管疾病、高血压、血脂异常中。血清脂联素水平的检测可以作为代谢性疾病诊断及治疗评价的指标,而补充重组脂联素或增加内源性脂联素的分泌则为胰岛素抵抗相关疾病的预防和治疗提供了一种新的思路与手段。
目前,国内外已有一些研究应用多种蛋白表达系统构建重组脂联素,包括原核及真核表达系统。其中原核表达体系因其成本低,操作简便以及产量高等众多优点而被广泛应用。本研究所采用的表达载体pQE-30Xa是Qiagen公司提供的一种高效表达和纯化外源蛋白的原核表达系统,相对于其他载体具有很多优势,目前国内尚未见使用该载体获得脂联素蛋白的相关报道。
研究目的:
1.克隆人脂联素目的基因,构建pQE-30Xa/ADPN原核表达载体。
2.转化入大肠杆菌E.coli M15[pREP4]进行重组蛋白的表达,鉴定表达产物。
3.优化表达条件,以期建立高效表达的技术流程。
实验方法:
1.从人的脂肪组织中提取总RNA,利用RT-PCR的方法得到人脂联素基因cDNA序列,构建模板质粒;自行设计引物一对,经PCR扩增人脂联素基因完全编码序列并使两端加上双酶切位点,构建pMD18-T/ADPN重组克隆质粒;pMD18-T/ADPN与pQE-30Xa质粒经限制性核酸内切酶BamHI与HindIII双酶切后,用T_4DNA连接酶进行连接,构建重组表达载体pQE-30Xa/ADPN。
2.将重组质粒转化入M15[pREP4]表达菌株中,IPTG诱导表达;提取包涵体,SDS-PAGE及Western blot鉴定表达产物;从诱导时间、温度及诱导剂浓度三方面摸索最适表达条件。
3.按照优化条件进行蛋白质的大量表达;在变性条件下利用His Band Kit纯化目的蛋白;PAPIB法梯度透析复性,SDS-PAGE鉴定蛋白纯度;Bradford法进行蛋白定量分析;MTS细胞增殖实验检测获得目的蛋白的生物学活性。
实验结果:
1.成功构建重组表达载体pQE-30Xa/ADPN,经测序证实插入的目的片段无误。
2.脂联素目的蛋白在E.coli.M15菌株中获得了表达,其中诱导剂IPTG终浓度为0.6mM、温度为37℃、诱导表达6小时后所获得的蛋白含量相对较高。
3.经His Band Kit纯化后,可获得单一的脂联素目的蛋白条带,蛋白纯化效果较好;蛋白表达效率约为24.8%,产量约为330μg/100ml LB培基;经MTS实验证实获得的重组蛋白具有生物活性。
实验结论:
成功构建了人脂联素原核表达载体pQE-30Xa/ADPN,并在E.coli.M15菌株中获得了表达,且获得的重组蛋白具有生物学活性。pQE-30Xa原核表达系统是一个高效、快速构建重组脂联素蛋白的良好选择。
In recent years,adipose tissue is considered not only as an energy storage but also as a very important endocrine organ.Adipose tissue secretes a number of biologically active adipokines such as leptin,tumor necrosis factor-a(TNF-a),resistin, interleukin-6(IL-6),adiponectin(ADPN) and so on.These hormones participate in the nervous,endocrine and immune regulation,and especially play an important role in the energy metabolism.
Adiponectin as one such adipokine has significantly physiological functions.It can improve glucose metabolism,lipid metabolism and insulin resistance. Adiponectin has been reported to have direct effects of antiatherosclerosis through inhibiting the expression of adhesion molecules,including intracellular adhesion molecule and vascular cellular adhesion molecule.Adiponectin could induce weight loss too.Recent studies suggest that adiponectin may play a role in the modulation of inflammatory vascular response by suppressing macrophage function.Therefore adiponectin has a close relation with insulin resistance,type 2 diabetes,obesity and cardiovascular disease.Lately it has been reported that adiponectin may directly or indirectly influence the occurrence and development of tumor.
The hypoadiponectinemia was correlated with insulin resistance and type 2 diabetes.We can find the plasma adiponectin level reduced in the condition of CVD, hypertension,and dyslipidemia.So the determination of plasma adiponectin will be a good marker for diagnosis and prognosis of diabetes mellitus,and addition of recombinant adiponectin or increasion of endogenous adiponectin secretion provides a new way for prevention and treatment of disease related to insulin resistance.
Till now researchers have applied many kinds of protein expression systems such as prokaryotic and eukaryotic systems to get recombinant adiponectin.
Prokaryotic expression system is widely used as the advantages of low cost,simple operation and high output.In our study,the expression vector of pQE-30 Xa we used is one kind of prokaryotic expression system from Qiagen company which has high efficiency in expressing and convenience for purifying.At present there is no domestic reporting about using this vector to get adiponectin protein.
Objectives:
1.To clone human adiponectin gene,and construct the prokaryotic expression vector-pQE-30 Xa/ADPN.
2.To transfect E.coli Ml 5 competent cells,obtain the expression of recombinant adiponection protein,and identify the expression product.
3.To optimize the expression conditions,and establish an efficient technological process.
Methods:
1.To extract the total RNA from human adipose tissue.We got the adiponectin cDNA with RT-PCR method.Design a pair of primers to amplify the encoding fragment of human ADPN gene by PCR.Construct recombinant clone vector pMD18-T/ADPN. Using BamHI、HindⅢenzymes to digest plasmids,the encoding fragment of human ADPN gene was ligated into expression vector pQE-30 Xa,and then recombinant vector pQE-30 Xa/ADPN was extracted.
2.The expression vector then was transformed into the M15 competent cells,and protein was expressed induced with IPTG.The protein in the inclusion body was extracted and identified by the methods of SDS-PAGE and Western Blot.The optimum of expression including induce time,temperature and concentration of induce reagent was tested.
3.The recombinant protein was largely expressed and purified by His Band Kit in the denatured condition.Protein was refolded and identified by SDS-PAGE.The concentration and bioactivity of the protein were determined.
Results:
1.Recombinant expression vector pQE-30 Xa/ADPN was successfully constructed. ADPN gene was inserted in correct location.
2.Recombinant protein was obtained by the expression of E.coli M15.The optimum of induce time was 6 hours,the temperature was 37℃and concentration IPTG was 0.6mM.
3.The protein was fairly purified by His Band Kit.The yield was 330μg/100ml LB and accounted for 24.8%of total protein in E.coli M15[pREP4].The result of MTS experiment showed that the protein had certain bioactivity.
Conclusions:
Recombinant prokaryotic expression vector pQE-30 Xa/ADPN was successfully constructed to express adiponectin protein with certain bioactivity in E.coli M15.The pQE-30 Xa expression system is efficient and suitable for adiponectin.
引文
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[53]Luo Z,Saha AK,Xiang X,et al.AMPK,the metabolic syndrome and cancer[J].Trends Pharmacol Sci,2005,26(2):69-76.
[54]Angulo P,Lindor KD.Insulin resistance and mitochondrial abnormalities in NASH:a cool look into a burning issue[J].Gastroenterology,2001,120:1281-1284.
[55]Jeong WI,Jeong DH,Do SH,et al.Mild hepatic fibrosis in cholesterol and sodium cholate diet-fed rats[J]J Vet Med Sci,2005,67(3):235-242.
[56]Lieber CS,Leo MA,Mak KM,et al.Model of nonalcoholic steato-hepatitis[J]Am J Clin Nutr,2004,79:502-509.
[57]Louthan M Vos,Barve S,McClain CJ,et al.Decrease Serum Adiponectin:An Early Event in Pediatric Nonalcoholic Fatty Liver Disease[J].J Pediat,2005,147(6):835-838.
[1]Biesalski HK,Nohr D.Importance of vitamin A for lung function and development[J].Mol Aspect Med,2003,24:431-440.
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[3]Vanitallie TB.Frailty in the elderly:Contributions of sarcopenia andvisceral protein depletion[J].Metabolism,2003,52:22-26.
[4]Yang Q,Graham TE,Mody N,et al.Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes[J].Nature,2005,436:356-362.
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[7]Reaven GM.The insulin resistance syndrome:definition and dietary approaches to treatment[J].Annu Rev Nutr,2005,25:391-406.
[8]Timothy E.Graham,M.D,Qin Yahg,M.D,Ph.D.,Matthias Bluher,et al.Retinol-Binding Protein 4 and Insulin Resistance in Lean,Obese,and Diabetic Subjects[J].The new England journal of medicine,2006,354:2552-2563.
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[1]Biesalski HK,Nohr D.Importance of vitamin A for lung function and development[J].Mol Aspect Med,2003,24:431-440.
[2]Noy N.Retinoid-binding proteins:mediators of retinoid action[J].Biochem J,2000,348(3):481-495.
[3]Vanitallie TB.Frailty in the elderly:Contributions of sarcopenia andvisceral protein depletion[J].Metabolism,2003,52:22-26.
[4]Yang Q,Graham TE,Mody N,et al.Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes[J].Nature,2005,436:356-362.
[5]Abel,E.D,et al.Adipose-selective targeting of the GLUT4 gene impairs insulin action in muscle and liver[J].Nature,2001,409:729-773.
[6]Shin,D.J,Odom,D.P,et al.Retinoid regulation of the phosphoenolpyruvate carboxykinase gene in liver[J].Mol.Cell Endocrinol,2002,195:39-54.
[7]Reaven GM.The insulin resistance syndrome:definition and dietary approaches to treatment[J].Annu Rev Nutr,2005,25:391-406.
[8]Timothy E.Graham,M.D,Qin Yahg,M.D,Ph.D.,Matthias Bluher,et al.Retinol-Binding Protein 4 and Insulin Resistance in Lean,Obese,and Diabetic Subjects[J].The new England journal of medicine,2006,354:2552-2563.
[9]Rexrode KM,Buring JE,Manson JE.Abdominal and total adiposity and risk of coronary heart disease in men[J].Int J Obes Relat Metab Disord,2001,25:1047-1056.
[10]Engelgau,M,M.et al.The evolving diabetes burden in the United States[J].Ann.Intern.Med.,2004,140:945-50.
[11]Miyazaki Y,Glass L,Triplitt C,Wajcberg E,Mandarino LJ,DeFronzo RA.Abdominal fat distribution and peripheral and hepatic insulin resistance in type 2diabetes mellitus[J].Am J Physiol Endocrinol Metab,2002,283:1135-1143.
[12]Stefan Engelil,Michael Boschmannl,et al.Retinol-Binding Protein 4 in Human Obesity[J].Diabetes,2006,55:2805-2810.
[13]Bjornholm M,Al Khalili L,Dicker A,et al.Insulin signal transduction and glucose transport in human adipocytes:effects of obesity and low calorie diet[J].Diabetologia,2002,45:1128-1135.
[14]吴海娅,贾伟平等.肥胖及2型糖尿病患者血清视黄醇结合蛋白4水平的变化及其临床意义[J].中华内分泌代谢杂志,2006,22(3):290-293.
[15]Newcomer ME,Ong DE.Plasma retinol binding protein:structure and function of the prototypic lipocalin[J].Biochim Biophys Acta,2000,1482:57-64.
[16]Um,S.J.et al.Antiproliferative mechanism of retinoid derivatives in ovarian cancer cells[J].Cancer Lett,2001,174:127-134.
[17]Shen,Q,Cline,G.W,et al.Effects of retinoid on glucose transport and insulin-mediated signaling in skeletal museles of diabetic(db/db)mice[J].J.Biol.Chem.,2004,279:19721-19731.
[18]Kershaw EE,Flier JS.Adipose tissue as an endocrine organ[J].J Clin Endocrinol Metab,2004,89:2548-2556.
[19]Spranger J,Kroke A,Bergmann MM,et al.Adiponectin and protection against type 2 diabetes mellitus[J].Lancet,2003,361:226-228.
[20]Rahmouni K,Morgan DA,Morgan GM,Mark AL,Haylies WG.Role of selective leptin resistance in diet-induced obesity hypertension[J].Diabetes,2005,54:2012-2018.
[21]Lee,C.H,Olson.P,et al.Lipid metabolism,metabolic diseases and peroxisome proliferators-activated receptors[J].Endocrinology,2003,144:2201-2207.
[22]Kotani,K,Kim,Y.B.,Peroni,O.,Mundt,A & Kahn,B.B.Rosiglitazone treatment normalizes glucose tolerance in adipose-specific GLUT4 knockout mice but renders muscle-specific GLUT4 knockout more diabetic[J].Diabetes,2001,50:A271.