人脂联素基因克隆、融合表达及纯化的研究
摘要
目的构建重组人脂联素(adiponectin,ADPN)原核表达载体,获取同源性、可溶性、高纯度的人重组脂联素融合蛋白,为进一步脂联素的深入研究及Ⅱ型糖尿病的早期诊断奠定基础。
方法从人脂肪组织中提取RNA,经RT-PCR扩增,克隆出脂联素基因;用EcoRⅠ和HIndⅢ对人脂联素基因和载体pET32进行双酶切,构建pET32—ADPN重组质粒;将重组质粒转化BL21感受态细胞,通过氨苄青霉素抗性、PCR和重组质粒双酶切筛选重组子转化菌落,经DNA测序及BLAST比对确认脂联素基因序列正确性以及插入pET32质粒的位点和方向;用IPTG诱导表达、Western blotting鉴定,用镍离子金属螯合层析柱纯化重组融合蛋白。
结果(1)通过RT-PCR,经1%的琼脂糖凝胶电泳观察,扩增的脂联素DNA片段与预期大小一致。(2)经双酶切及DNA测序鉴定脂联素DNA定向插入了pET32质粒。(3)用重组质粒转化大肠杆菌BL21、诱导表达和纯化目的蛋白。经Western—Blotting鉴定所诱导的蛋白为重组脂联素融合蛋白。
结论成功构建了人重组脂联素蛋白原核表达载体、表达和纯化了脂联素融合蛋白,为进一步研究其生学物学功能和在Ⅱ型糖尿病中的作用机制奠定了基础。
Objective: To construct a prokaryotic expression vector in which the maximum level of homogeneous, soluble, and fully bioactive of recombinant human adiponectin fusion protein was obtained from E.coli.
Methods: Total RNA were extracted from human adipose tissue and the adiponectin gene were fished by reverse transcription PCR; the adiponectin gene were amplified by a pair of special primer including endonucleases sites of EcoR I and Hind III by PCR and inserted into the prokaryotic expression vector pET 32a(+) . After being selected by Ampicillin resistance, identified by PCR and double digestion of endonucleases, the sequenced DNA of adiponectin was analyzed by BLAST. The Adiponectin/Trx fusion protein expressed in E.coli. BL21 (DE3) was induced with IPTG, identified by western blot and purified with Ni—NTA agarose respectively.
Results: Through RT-PCR and 1% agarose gel electrophoresis, the products of 1000bp were obtained as expected . After double digestion with Endonucleases, the Adiponectin gene has been inserted into the prokaryotic expression vector PET 32a (+). After detected the DNA sequence, the constructed expression plasmid was transformed into competent E.coli. BL21(DE3). The recombinant Adiponectin/Trx fusion protein was specific against adiponectin antibody identified by western blot .The purity of the protein was over 80% after been purified by Ni—NTA agarose.
Conclusion:
The prokaryotic expression plasmid PET 32a (+)/Adipoenctin issuccessfully constructed and high purified Adiponectin/Trx fusion proteinis obtained ether.
方法从人脂肪组织中提取RNA,经RT-PCR扩增,克隆出脂联素基因;用EcoRⅠ和HIndⅢ对人脂联素基因和载体pET32进行双酶切,构建pET32—ADPN重组质粒;将重组质粒转化BL21感受态细胞,通过氨苄青霉素抗性、PCR和重组质粒双酶切筛选重组子转化菌落,经DNA测序及BLAST比对确认脂联素基因序列正确性以及插入pET32质粒的位点和方向;用IPTG诱导表达、Western blotting鉴定,用镍离子金属螯合层析柱纯化重组融合蛋白。
结果(1)通过RT-PCR,经1%的琼脂糖凝胶电泳观察,扩增的脂联素DNA片段与预期大小一致。(2)经双酶切及DNA测序鉴定脂联素DNA定向插入了pET32质粒。(3)用重组质粒转化大肠杆菌BL21、诱导表达和纯化目的蛋白。经Western—Blotting鉴定所诱导的蛋白为重组脂联素融合蛋白。
结论成功构建了人重组脂联素蛋白原核表达载体、表达和纯化了脂联素融合蛋白,为进一步研究其生学物学功能和在Ⅱ型糖尿病中的作用机制奠定了基础。
Objective: To construct a prokaryotic expression vector in which the maximum level of homogeneous, soluble, and fully bioactive of recombinant human adiponectin fusion protein was obtained from E.coli.
Methods: Total RNA were extracted from human adipose tissue and the adiponectin gene were fished by reverse transcription PCR; the adiponectin gene were amplified by a pair of special primer including endonucleases sites of EcoR I and Hind III by PCR and inserted into the prokaryotic expression vector pET 32a(+) . After being selected by Ampicillin resistance, identified by PCR and double digestion of endonucleases, the sequenced DNA of adiponectin was analyzed by BLAST. The Adiponectin/Trx fusion protein expressed in E.coli. BL21 (DE3) was induced with IPTG, identified by western blot and purified with Ni—NTA agarose respectively.
Results: Through RT-PCR and 1% agarose gel electrophoresis, the products of 1000bp were obtained as expected . After double digestion with Endonucleases, the Adiponectin gene has been inserted into the prokaryotic expression vector PET 32a (+). After detected the DNA sequence, the constructed expression plasmid was transformed into competent E.coli. BL21(DE3). The recombinant Adiponectin/Trx fusion protein was specific against adiponectin antibody identified by western blot .The purity of the protein was over 80% after been purified by Ni—NTA agarose.
Conclusion:
The prokaryotic expression plasmid PET 32a (+)/Adipoenctin issuccessfully constructed and high purified Adiponectin/Trx fusion proteinis obtained ether.
引文
1.Scheen AJ.Pathophysiology of type 2 diabetes.Acta Clin Belg.2003;58(6):335-341.
2.Nakano Y,Tobe T,Choi-Miura N,et al.Isolation and characterization of GBP,a novel gelatin-binding protein purified from human plasma.J Biochem.1996/120:803-812.
3.Maeda K,Okubo k,Shimomura I:cDNA cloning and expression of a novel adipose specific collagen-like factor,aPM1.Biochem Biophys Res Commun.1996;221:286-289
4.Yamauchi T,Kamon J,Waki H,et al.The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity.Nat Med.2001;7:941-946
5.Schaffler A,Langmann T,Palitzsch KD,et al.Identification and characterization of the human adipocyte aPM1 promoter.Biochim Biophys Acta,1998;1339:187-197
6.Saito K,Tobe T,Minoshima S,et al.Organization of the gene for gelatin-binding protein(GBP28).Gene 1999;229:67-73.
7.Maeda K,Okubo K,Shimomura I et al.cKNA cloning and expression of a novel adipose soecific collagen-like factor,apM1(Adipose Most abundant Gene transcript 1).Biochem Biophys Res Commun.1996;221(2):286-289.
8.Weyer C,Funahashi T Tanaka S,et al.Hypoadiponectinemia in obesity and type 2diabetes:close association with insulin resistance and hyperinsulinemia.J Clin Endocrinol Metad.2001;86:1930-1935.
9.Okamoto Y,Ariat Y,Nishida M,et al.An adipocyte-derived plasma protein,adiponectin,adheres to injured vascular walls.Horm Metab Res.2000;32:47-50
10.Nakata A,Nakagawa Y,Nishida M,et al.CD6,a novel receptor for oxidized low-density lipoproteins is highly expressed on lipid daden marophages in human athero sclerotic aorta.Arterioscler Thromb Vasc Biol.1999;19:1333-1339.
11.时照明 王长江 王佑民等.人脂联基因全长cDNA克隆.安微医科大学学报.2004;39(2):116-118
12.Devis LG,Kuehl WM,Battey JF.Basic methods in molecular biology.Norwalk Appleton & Lange.1994:147-148
13.Takahashi M,Arita Y,Yamagata K.Genomic structure and mutations in adipose-specific gene,adiponectin.int J Obse.2000;24:861-868
14.Heilbronn LK,Meka CS,Medina KL,et al.The insulin-sensitizing role of the fat derived hormone adiponectin.Curr Pharm Des.2003;9(17):1411-1418
15.Nemet D,Wang p,Funahashi T,et al.Adipocytokines,body composition and fitness in children.Pediatr Res.2003;5(1)148-152
16.Yokota T, Oritani K, Takahashi I, et al.Adiponectin,a new member of the family of soluble defense collagens,negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages.Blood.200;96(5): 1723-1732
17.Lavallie ER, Diblasio EA, Kovacic S.A thioredoxingene fusion expression system that circumvents inclusion body formation in the Ecolicytoplasm. Biotechnology. 1993 ,11(2): 187-193.Bacteriol 1988 1 70:1245.1253.
18. LaVaillie E.R., Lu Z.J , DiBlasio E.A, et al. Thioredoxin gene fusion partner for production of soluble recombinant Proteins in Escherichia coli[J]. Methods in Enmology.2000,326:322-340
19.Pace CN, Vagdos L, Gray T, et al. how to measure and predict the molar absorption coefficient of a protein[J].Protien Science. 1995, 4:2411-2413.
20. AmannE, BrosiusJ, Ptashne M. Vectors bearing a hybrid trp--lac Promoter usdful for regulated expression of cloned genes in Escherichia coli[J] .Gene.1983, 25: 167-178.
21. Ford CF, Suominenl, Glatz CE. Fusion tails for the recovery and purification of recombinant proteins[J]..Protein ExPr Purif. 1991, 2:95-107.
22.Porath J, et al. Immobilized metal ion affinity chlomato-Graphy [J]. Protein ExPr Purif. 1992, 3:206-281.
2.Nakano Y,Tobe T,Choi-Miura N,et al.Isolation and characterization of GBP,a novel gelatin-binding protein purified from human plasma.J Biochem.1996/120:803-812.
3.Maeda K,Okubo k,Shimomura I:cDNA cloning and expression of a novel adipose specific collagen-like factor,aPM1.Biochem Biophys Res Commun.1996;221:286-289
4.Yamauchi T,Kamon J,Waki H,et al.The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity.Nat Med.2001;7:941-946
5.Schaffler A,Langmann T,Palitzsch KD,et al.Identification and characterization of the human adipocyte aPM1 promoter.Biochim Biophys Acta,1998;1339:187-197
6.Saito K,Tobe T,Minoshima S,et al.Organization of the gene for gelatin-binding protein(GBP28).Gene 1999;229:67-73.
7.Maeda K,Okubo K,Shimomura I et al.cKNA cloning and expression of a novel adipose soecific collagen-like factor,apM1(Adipose Most abundant Gene transcript 1).Biochem Biophys Res Commun.1996;221(2):286-289.
8.Weyer C,Funahashi T Tanaka S,et al.Hypoadiponectinemia in obesity and type 2diabetes:close association with insulin resistance and hyperinsulinemia.J Clin Endocrinol Metad.2001;86:1930-1935.
9.Okamoto Y,Ariat Y,Nishida M,et al.An adipocyte-derived plasma protein,adiponectin,adheres to injured vascular walls.Horm Metab Res.2000;32:47-50
10.Nakata A,Nakagawa Y,Nishida M,et al.CD6,a novel receptor for oxidized low-density lipoproteins is highly expressed on lipid daden marophages in human athero sclerotic aorta.Arterioscler Thromb Vasc Biol.1999;19:1333-1339.
11.时照明 王长江 王佑民等.人脂联基因全长cDNA克隆.安微医科大学学报.2004;39(2):116-118
12.Devis LG,Kuehl WM,Battey JF.Basic methods in molecular biology.Norwalk Appleton & Lange.1994:147-148
13.Takahashi M,Arita Y,Yamagata K.Genomic structure and mutations in adipose-specific gene,adiponectin.int J Obse.2000;24:861-868
14.Heilbronn LK,Meka CS,Medina KL,et al.The insulin-sensitizing role of the fat derived hormone adiponectin.Curr Pharm Des.2003;9(17):1411-1418
15.Nemet D,Wang p,Funahashi T,et al.Adipocytokines,body composition and fitness in children.Pediatr Res.2003;5(1)148-152
16.Yokota T, Oritani K, Takahashi I, et al.Adiponectin,a new member of the family of soluble defense collagens,negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages.Blood.200;96(5): 1723-1732
17.Lavallie ER, Diblasio EA, Kovacic S.A thioredoxingene fusion expression system that circumvents inclusion body formation in the Ecolicytoplasm. Biotechnology. 1993 ,11(2): 187-193.Bacteriol 1988 1 70:1245.1253.
18. LaVaillie E.R., Lu Z.J , DiBlasio E.A, et al. Thioredoxin gene fusion partner for production of soluble recombinant Proteins in Escherichia coli[J]. Methods in Enmology.2000,326:322-340
19.Pace CN, Vagdos L, Gray T, et al. how to measure and predict the molar absorption coefficient of a protein[J].Protien Science. 1995, 4:2411-2413.
20. AmannE, BrosiusJ, Ptashne M. Vectors bearing a hybrid trp--lac Promoter usdful for regulated expression of cloned genes in Escherichia coli[J] .Gene.1983, 25: 167-178.
21. Ford CF, Suominenl, Glatz CE. Fusion tails for the recovery and purification of recombinant proteins[J]..Protein ExPr Purif. 1991, 2:95-107.
22.Porath J, et al. Immobilized metal ion affinity chlomato-Graphy [J]. Protein ExPr Purif. 1992, 3:206-281.