尼克酰胺N-甲基化酶表达载体的构建、表达产物的分离纯化、及多克隆抗体的制备
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摘要
尼克酰胺N-甲基化酶(NNMT,EC 2.1.1.1)是S—腺苷—L蛋氨酸(SAM)依赖的胞质酶,它以SAM为甲基供体,催化尼克酰胺和其他吡啶类物质形成吡啶盐类物质。据文献报道,NMMT酶蛋白含量和mRNA水平在多种疾病中的明显增高,例如肾癌、帕金森病、甲状腺癌等。在肾透明细胞癌中,NNMT基因表达是正常肾组织和其他亚型肾癌表达的三倍以上。在甲状腺癌中,几乎所有的乳头状甲状腺癌的NNMT蛋白及其mRNA水平都明显增高,但在滤泡状、未分化、髓样甲状腺癌中却不明显。在帕金森病人的脑脊液中,NNMT蛋白含量明显高于正常对照组。目前检测NNMT的方法主要有放射性同位素酶化学法,免疫印迹,PROTEOMEX分析,高密度寡核苷酸微阵法等。这些方法存在易污染或费用昂贵或同位素废物处理等缺点不适应用于临床诊断。本实验的目的在于构建NNMT的表达载体,表达正确的人NNMT蛋白,并制备其多克隆抗体,为下一步制备单克隆抗体及建立检测NNMT的ELISA法奠定基础。
目的 利用pGEX-4T-2系统构建重组尼克酰胺N-甲基化酶(NNMT)表达载体,诱导表达并对表达产物进行分离纯化,制备多克隆抗体及纯化抗体。
方法 根据NCBI核苷酸数据库编码为gi:12652950人NNMT cDNA序列,设计合成编码NNMT的DNA引物,并分别在5'端和3'端设计BamHI和XhoI位点。从人肝脏组织中提取总RNA,进行RT-PCR获得NNMT基因。经T—A克隆至pGEM-T-Easy载体,经过BamHI/XhoI双酶切与同样经过双酶切的pGEX-4T-2连接。重组质粒pGEX-4T-2/NNMT转化至大肠杆菌BL-21-STAR(DE3),重组菌经异丙基—β—D—硫代乳糖苷(IPTG)诱导表达,用Glutathione Sepharose 4B亲和层析制备融合蛋白GST-NNMT。通过DNA测序来证实质粒pGEX-4T-2/NNMT的正确性,SDS-PAGE电泳以及Western—blot判断GST-NNMT蛋白的准确性。纯化后的融合蛋白作为抗原按常规方法免疫家兔,十周后收集兔血清,SAS沉淀法纯化IgG,用双向琼脂扩散和ELISA法测定多抗效价。
Nicotinamide N-Methyltransferase (NNMT, EC 2.1.1.1) is an S-adenosyl-L-methionine cytosolic enzyme that catalyzes the N-methylation of nicotinamide and other pyridines to form pyridinium ions. It is reported that NNMT and its mRNA were upregulate in some diseases such as renal cell carcinoma 、 Parkinson's disease and papillary thyroid carcinoma. In the clear-cell renal cell carcinoma, the mean expression of NNMT gene is more than three times higher than the chromophobe renal cell caricinoma and normal kidney tissue. In the papillary thyroid carcinoma cell, NNMT was identified for being highly expressed only in the papillary cell lines. Also NNMT is higher in the lumbar cerebrospinal fluid of patients with Parkinson's disease than the control. But now the method to measure the NNMT is too expensive, unsafety to apply for the clinic laboratory. We are want to establish the expression plasmid and get the human NNMT protein and its antibody.Object To prepare Nicotinamide N-methyltransferase with fusion protein of GST by pGEX-4T-2 expression system, induce its expression and purify the protein, establish the method of ELISA.Methods Acording to the nucleotide bank of NCBI(gi: 12652950), DNA primers with BamHI/XhoI site were designed. Total RNA was isolated from the human liver tissue using TRIzol reagent and was reverse transcripted into the c DNA. The polymerase chain reaction (PCR) was used to amplify the NNMT sequence with its cDNA as template and primers designed on the basis of the nnmt DNA sequence. The PCR product was ligated into the vector p GEM-T-Easy after T-A clone. Plasmid p GEM-T-Easy/NNMT and the expression vector p GEX-4T-2 were digested respectively with the restriction enzyme BamHI/XhoI,and then ligated to construct the recombinant expression plasmid pGEX-4T-2/NNMT. The correct plasmid pGEX-4T-2/NNMT were obtain after the restriction analysis and DNA sequencing. The recombined plasmid was transferred into E.coliBL-21-STAR(DE3) and the GST-NNMT fusion protein was expressed by inducing
with IPTG. The fusion protein was purified by Glutathione Sepharose 4B affinity chromatography. SDS-PAGE and Western-blot were used to verify the the fusion protein. In order to get the antibody, the rabbit were immunized with the fusion protein GST-NNMT according to the routine method and collected the serum after ten weeks. The titer of the rabbit anti-GST-NNMT antibody was measured by the agar diffusion assay and ELISA assayResults Restriction analysis and DNA sequencing confirmed the correct sequence and insertion site of the recombinant pGEM-T-Easy/NNMT and pGEX-4T-2/NNMT. E coli BL-21-STAR(DE3) with the pGEX-4T-2/NNMT successfully express the recombinant fusion protein GST-NNMT with the molecular weight of 55 Kda, which is consistent with the predicted putative calculating molecular weight. Expressed fusion protein was mainly in soluble supernatant and amount to about 20% of the total protein of the bacterium. SDS-PAGE and Western-blot result indicated that the fusion protein was GST-NNMT. The agar diffusion assay show that the anti-serum titer of immune rabbit was 1:16. After the purification of the Saturated ammonium sulphate (SAS),the titer of the antibody is about 103. In conclusion, the experiment laid a good foundation for research of the protein NNMT, the preparation of the monoclonal antibody against NNMT and the development ELISA antigen diagnosis kit.Conclusion 1. The plasmid pGEX-4T-2/NNMT is constructed successfully2.the plasmid can express the fusion protein GST-NNMT in vitro and the protein are mainly in soluble supernatant3. the GST-NNMT polyclonal antibody are prepared
目的 利用pGEX-4T-2系统构建重组尼克酰胺N-甲基化酶(NNMT)表达载体,诱导表达并对表达产物进行分离纯化,制备多克隆抗体及纯化抗体。
方法 根据NCBI核苷酸数据库编码为gi:12652950人NNMT cDNA序列,设计合成编码NNMT的DNA引物,并分别在5'端和3'端设计BamHI和XhoI位点。从人肝脏组织中提取总RNA,进行RT-PCR获得NNMT基因。经T—A克隆至pGEM-T-Easy载体,经过BamHI/XhoI双酶切与同样经过双酶切的pGEX-4T-2连接。重组质粒pGEX-4T-2/NNMT转化至大肠杆菌BL-21-STAR(DE3),重组菌经异丙基—β—D—硫代乳糖苷(IPTG)诱导表达,用Glutathione Sepharose 4B亲和层析制备融合蛋白GST-NNMT。通过DNA测序来证实质粒pGEX-4T-2/NNMT的正确性,SDS-PAGE电泳以及Western—blot判断GST-NNMT蛋白的准确性。纯化后的融合蛋白作为抗原按常规方法免疫家兔,十周后收集兔血清,SAS沉淀法纯化IgG,用双向琼脂扩散和ELISA法测定多抗效价。
Nicotinamide N-Methyltransferase (NNMT, EC 2.1.1.1) is an S-adenosyl-L-methionine cytosolic enzyme that catalyzes the N-methylation of nicotinamide and other pyridines to form pyridinium ions. It is reported that NNMT and its mRNA were upregulate in some diseases such as renal cell carcinoma 、 Parkinson's disease and papillary thyroid carcinoma. In the clear-cell renal cell carcinoma, the mean expression of NNMT gene is more than three times higher than the chromophobe renal cell caricinoma and normal kidney tissue. In the papillary thyroid carcinoma cell, NNMT was identified for being highly expressed only in the papillary cell lines. Also NNMT is higher in the lumbar cerebrospinal fluid of patients with Parkinson's disease than the control. But now the method to measure the NNMT is too expensive, unsafety to apply for the clinic laboratory. We are want to establish the expression plasmid and get the human NNMT protein and its antibody.Object To prepare Nicotinamide N-methyltransferase with fusion protein of GST by pGEX-4T-2 expression system, induce its expression and purify the protein, establish the method of ELISA.Methods Acording to the nucleotide bank of NCBI(gi: 12652950), DNA primers with BamHI/XhoI site were designed. Total RNA was isolated from the human liver tissue using TRIzol reagent and was reverse transcripted into the c DNA. The polymerase chain reaction (PCR) was used to amplify the NNMT sequence with its cDNA as template and primers designed on the basis of the nnmt DNA sequence. The PCR product was ligated into the vector p GEM-T-Easy after T-A clone. Plasmid p GEM-T-Easy/NNMT and the expression vector p GEX-4T-2 were digested respectively with the restriction enzyme BamHI/XhoI,and then ligated to construct the recombinant expression plasmid pGEX-4T-2/NNMT. The correct plasmid pGEX-4T-2/NNMT were obtain after the restriction analysis and DNA sequencing. The recombined plasmid was transferred into E.coliBL-21-STAR(DE3) and the GST-NNMT fusion protein was expressed by inducing
with IPTG. The fusion protein was purified by Glutathione Sepharose 4B affinity chromatography. SDS-PAGE and Western-blot were used to verify the the fusion protein. In order to get the antibody, the rabbit were immunized with the fusion protein GST-NNMT according to the routine method and collected the serum after ten weeks. The titer of the rabbit anti-GST-NNMT antibody was measured by the agar diffusion assay and ELISA assayResults Restriction analysis and DNA sequencing confirmed the correct sequence and insertion site of the recombinant pGEM-T-Easy/NNMT and pGEX-4T-2/NNMT. E coli BL-21-STAR(DE3) with the pGEX-4T-2/NNMT successfully express the recombinant fusion protein GST-NNMT with the molecular weight of 55 Kda, which is consistent with the predicted putative calculating molecular weight. Expressed fusion protein was mainly in soluble supernatant and amount to about 20% of the total protein of the bacterium. SDS-PAGE and Western-blot result indicated that the fusion protein was GST-NNMT. The agar diffusion assay show that the anti-serum titer of immune rabbit was 1:16. After the purification of the Saturated ammonium sulphate (SAS),the titer of the antibody is about 103. In conclusion, the experiment laid a good foundation for research of the protein NNMT, the preparation of the monoclonal antibody against NNMT and the development ELISA antigen diagnosis kit.Conclusion 1. The plasmid pGEX-4T-2/NNMT is constructed successfully2.the plasmid can express the fusion protein GST-NNMT in vitro and the protein are mainly in soluble supernatant3. the GST-NNMT polyclonal antibody are prepared
引文
1. Aksoy S, Szumlanski CL, Weinshiboum RM Human liver nicotinamide N-methyltransferase. cDNA cloning, expression, and biochemical characterization. J Biol Chem. 1994 May 20;269(20): 14835-40.
2. Rini J, Szumlanski C, Guerciolini R, Weinshiboum RM Human liver nicotinamide N-methyltransferase: ion-pairing radiochemical assay, biochemical properties and individual variation. Clin Chim Acta. 1990 Jan 31;186(3): 359-74.
3. Aksoy S, Brandriff BF, Ward A, et al Human nicotinamide N-methyltransferase gene: molecular cloning, structural characterization and chromosomal localization. Genomics. 1995 Oct 10;29(3): 555-61.
4. Smith ML, Bumett D, Bennett P A direct correlation between nicotinamide N-methyltransferase activity and protein levels in human liver cytosol. Biochim Biophys Acta. 1998 Nov 8;1442(2-3): 238-44.
5. Yan L, Otterness DM, Weinshiboum RM et al Human nicotinamide N-methyltransferase pharmacogenetics: gene sequence analysis and promoter characterization. Pharmacogenetics. 1999 Jun;9(3): 307-16.
6. Ohkubo M, Sakigama S, Fujimura S et al Increase of nicotinamide methyltransferase and N1-methyl-nicotinamide oxidase activities in the livers of the rats administered alkylating agents Cancer Lett. 1983 Dec 21(2): 175-81
7. Okamura A, Ohmura Y, Islam MM et al Increased hepatic nicotinamide N-methyltransferase activity as a marker of cancer cachexia in mice bearing colon 26 adenocarcinoma. Jpn J Cancer Res. 1998 Jun;89(6): 649-56.
8. Seifert R, Hoshino J, Kroger H et al Nicotinamide methylation. Tissue distribution, developmental and neoplastic changes. Biochim Biophys Acta. 1984 Sep 28;801(2): 259-64.
9. Yao M, Tabuchi H, Nagashima Y, et al Gene expression analysis of renal carcinoma: adipose differentiation-related protein as a potential diagnostic and prognostic biomarker for clear-cell renal carcinoma. J Pathol. 2005 Feb;205(3):377-87.
10. Akalin E,Hendrix RC,Polavarapu RG et al Gene expression analysis in human renal allograft biopsy samples using high-density oligoarray technology. Transplantation. 2001 Sep 15;72(5):948-53.
11. Xu J,Moatamed F,Caldwell JS et al Enhanced expression of nicotinamide N-methyltransferase in human papillary thyroid carcinoma cells. J Clin Endocrinol Metab. 2003 Oct;88(10):4990-6.
12. Xu J.Moatamed F,Caldwell JS et al Activation of nicotinamide N-methyltransferase gene promoter by hepatocyte nuclear factor-1 beta in human papillary thyroid cancer cells. Mol Endocrinol. 2005 Feb;19(2):527-39. Epub 2004 Oct 14.
13. Parsons RB,Smith ML, Williams AC, et al Expression of nicotinamide N-methyltransferase (E.C. 2.1.1.1) in the Parkinsonian brain. J Neuropathol Exp Neurol. 2002 Feb;61(2):111-24.
14. Ramsden DB,Parsons RB, Ho SL et al Further studies in xenobiotic metabolism and Parkinson's disease. Adv Neurol. 2001;86:105-13.
15. Aoyama K,Matsubara K,Kondo M et al Nicotinamide-N-methyltransferase is higher in the lumbar cerebrospinal fluid of patients with Parkinson's disease. Neurosci Lett. 2001 Jan 26;298(1):78-80.
16. Fukushima T,Kaetsu A,Lim H et al Possible role of 1-methylnicotinamide in the pathogenesis of Parkinson's disease. Exp Toxicol Pathol. 2002 Feb;53(6):469-73.
17. Matsubara K,Aoyama K,Suno M,et al N-methylation underlying Parkinson's disease. Neurotoxicol Teratol. 2002 Sep-Oct;24(5):593-8. Review.
18. Parsons RB,Smith SW,Waring RH et al High expression of nicotinamide N-methyltransferase in patients with idiopathic Parkinson's
disease. Neurosci Lett. 2003 May 15;342(1-2): 13-6.
19. Williams AC, Ramsden DB et al Autotoxicity, methylation and a road to the prevention of Parkinson's disease. J Clin Neurosci. 2005 Jan;12(1): 6-11
20. Williams AC, Cartwright LS, Ramsden DB et al Parkinson's disease: the first common neurological disease due to auto-intoxication? QJM. 2005 Mar;98(3): 215-26.
21. Jang JS, Cho HY, LeeYJ et al The differential proteome profile of stomach cancer: identification of the biomarker candidates Oncol Res 2004;14(10): 491-9
22. Roessler M, Rollinger W, Palme S et al Identification of nicotinamide N-methyltransferase as a novel serum tumor marker for colorectal cancer. Clin Cancer Res 2005 Sep 15;11(18): 6550-7
23.J 萨姆布鲁克、EF 弗里奇、T曼尼阿蒂斯著《分子克隆实验指南》第三版 科学出版社 27—30,87—93
24.T.A布朗 著《基因组》科学出版社 2002:22-47
25.胡福泉、杨恬主编《现代基因操作技术》第一版 人民军医出版社 2000:135-190
26.朱立平、陈学清主编《免疫学常用实验方法》第一版 北京:人民军医出版社 2000:1-19,78-86
27.鲍建芳、沈建根《医学免疫学实验技术》第二版 浙江:浙江大学医学院免疫教研室 2000:8-10,25-27
2. Rini J, Szumlanski C, Guerciolini R, Weinshiboum RM Human liver nicotinamide N-methyltransferase: ion-pairing radiochemical assay, biochemical properties and individual variation. Clin Chim Acta. 1990 Jan 31;186(3): 359-74.
3. Aksoy S, Brandriff BF, Ward A, et al Human nicotinamide N-methyltransferase gene: molecular cloning, structural characterization and chromosomal localization. Genomics. 1995 Oct 10;29(3): 555-61.
4. Smith ML, Bumett D, Bennett P A direct correlation between nicotinamide N-methyltransferase activity and protein levels in human liver cytosol. Biochim Biophys Acta. 1998 Nov 8;1442(2-3): 238-44.
5. Yan L, Otterness DM, Weinshiboum RM et al Human nicotinamide N-methyltransferase pharmacogenetics: gene sequence analysis and promoter characterization. Pharmacogenetics. 1999 Jun;9(3): 307-16.
6. Ohkubo M, Sakigama S, Fujimura S et al Increase of nicotinamide methyltransferase and N1-methyl-nicotinamide oxidase activities in the livers of the rats administered alkylating agents Cancer Lett. 1983 Dec 21(2): 175-81
7. Okamura A, Ohmura Y, Islam MM et al Increased hepatic nicotinamide N-methyltransferase activity as a marker of cancer cachexia in mice bearing colon 26 adenocarcinoma. Jpn J Cancer Res. 1998 Jun;89(6): 649-56.
8. Seifert R, Hoshino J, Kroger H et al Nicotinamide methylation. Tissue distribution, developmental and neoplastic changes. Biochim Biophys Acta. 1984 Sep 28;801(2): 259-64.
9. Yao M, Tabuchi H, Nagashima Y, et al Gene expression analysis of renal carcinoma: adipose differentiation-related protein as a potential diagnostic and prognostic biomarker for clear-cell renal carcinoma. J Pathol. 2005 Feb;205(3):377-87.
10. Akalin E,Hendrix RC,Polavarapu RG et al Gene expression analysis in human renal allograft biopsy samples using high-density oligoarray technology. Transplantation. 2001 Sep 15;72(5):948-53.
11. Xu J,Moatamed F,Caldwell JS et al Enhanced expression of nicotinamide N-methyltransferase in human papillary thyroid carcinoma cells. J Clin Endocrinol Metab. 2003 Oct;88(10):4990-6.
12. Xu J.Moatamed F,Caldwell JS et al Activation of nicotinamide N-methyltransferase gene promoter by hepatocyte nuclear factor-1 beta in human papillary thyroid cancer cells. Mol Endocrinol. 2005 Feb;19(2):527-39. Epub 2004 Oct 14.
13. Parsons RB,Smith ML, Williams AC, et al Expression of nicotinamide N-methyltransferase (E.C. 2.1.1.1) in the Parkinsonian brain. J Neuropathol Exp Neurol. 2002 Feb;61(2):111-24.
14. Ramsden DB,Parsons RB, Ho SL et al Further studies in xenobiotic metabolism and Parkinson's disease. Adv Neurol. 2001;86:105-13.
15. Aoyama K,Matsubara K,Kondo M et al Nicotinamide-N-methyltransferase is higher in the lumbar cerebrospinal fluid of patients with Parkinson's disease. Neurosci Lett. 2001 Jan 26;298(1):78-80.
16. Fukushima T,Kaetsu A,Lim H et al Possible role of 1-methylnicotinamide in the pathogenesis of Parkinson's disease. Exp Toxicol Pathol. 2002 Feb;53(6):469-73.
17. Matsubara K,Aoyama K,Suno M,et al N-methylation underlying Parkinson's disease. Neurotoxicol Teratol. 2002 Sep-Oct;24(5):593-8. Review.
18. Parsons RB,Smith SW,Waring RH et al High expression of nicotinamide N-methyltransferase in patients with idiopathic Parkinson's
disease. Neurosci Lett. 2003 May 15;342(1-2): 13-6.
19. Williams AC, Ramsden DB et al Autotoxicity, methylation and a road to the prevention of Parkinson's disease. J Clin Neurosci. 2005 Jan;12(1): 6-11
20. Williams AC, Cartwright LS, Ramsden DB et al Parkinson's disease: the first common neurological disease due to auto-intoxication? QJM. 2005 Mar;98(3): 215-26.
21. Jang JS, Cho HY, LeeYJ et al The differential proteome profile of stomach cancer: identification of the biomarker candidates Oncol Res 2004;14(10): 491-9
22. Roessler M, Rollinger W, Palme S et al Identification of nicotinamide N-methyltransferase as a novel serum tumor marker for colorectal cancer. Clin Cancer Res 2005 Sep 15;11(18): 6550-7
23.J 萨姆布鲁克、EF 弗里奇、T曼尼阿蒂斯著《分子克隆实验指南》第三版 科学出版社 27—30,87—93
24.T.A布朗 著《基因组》科学出版社 2002:22-47
25.胡福泉、杨恬主编《现代基因操作技术》第一版 人民军医出版社 2000:135-190
26.朱立平、陈学清主编《免疫学常用实验方法》第一版 北京:人民军医出版社 2000:1-19,78-86
27.鲍建芳、沈建根《医学免疫学实验技术》第二版 浙江:浙江大学医学院免疫教研室 2000:8-10,25-27