人NGAL克隆表达及抗体的制备与鉴定
|
摘要
目的:高效表达人中性粒细胞明胶酶相关脂质运载蛋白(neutrophil gelatinase-associated lipocalin,NGAL)重组蛋白并制备其多克隆及单克隆抗体。方法:人工合成经密码子优化的NGAL基因,构建原核表达重组质粒pW28-NGAL,IPTG诱导表达后分离纯化并分析在溶液中的聚集状态;获得的rhNGAL抗原免疫兔子制备多克隆抗体,免疫小鼠筛选高效价的特异性单克隆抗体并鉴定抗体亚型;Protein G亲合柱纯化,等温滴定量热法及非竞争ELISA法检测抗体的亲和力,SDS-PAGE分析纯度;最后运用Western blot、免疫荧光和免疫组化对抗体进行鉴定。结果:高效获得高纯度NGAL重组蛋白,主要以单体形式存在;成功制备兔多抗,同时筛选获得6株高效价单克隆抗体;其中,25号单抗为IgG1亚型,余者均属IgG2a亚型,且19、35号单抗的亲和常数分别为3.06×10~9、2.14×10~9。SDS-PAGE分析表明纯度均大于90%。进一步的鉴定结果表明制备的抗体皆具有良好免疫反应性及特异性,且单抗的特异性明显高于多抗。结论:本研究利用密码子优化技术高效表达制备了NGAL重组蛋白,获得了其高效价多克隆抗体和高特异性单克隆抗体,为NGAL的病理生理作用等功能研究提供了良好的实验材料,为NGAL临床免疫检测诊断试剂的国产化奠定了有力的基础。
Objective:To obtain human neutrophil gelatinase-associated lipocalin(NGAL) recombinant protein and its polyclonal and monoclonal antibodies,and to provide a powerful experimental basis for further functional research and clinical application of NGAL.Methods:The coding region of NGAL mature peptide gene was codon-optimized,chemically synthesized,and then inserted into the prokaryotic expression vector pW28. Transfected E.coli B834 cells were induced by IPTG to produce rhNGAL. The recombinant protein was was purified by Ni~(2+)-NTA affinity chromatography column. The aggregation in solution was analyzed by Hiload Superdex 75 gel flittration column. Rabbits and BALB/c mice were immunized with the rhNGAL to produce polyclonalant antibodies and high specificity monoclonal antibodies by hybridoma technology to identify antibody subtypes. After being purified by Protein G affinity column,the antibodies was detected by Isothermal Titration Calorimetry,non-competitive ELISA and SDS-PAGE analysis. Finally,Western blot,immunofluorescence and immunohistochemistry were used for antibody identification. Results:NGAL recombinant protein was successfully expressed with high purity and mainly existed in monomer form. Recombinant procalcitonin was successfully expressed. Rabbit polyclonal antibody was also produced. Six hybridoma cell lines stably secreting mAbs were screened,hereinto the mAb25 was IgG1 subtype,and the others belonged to IgG2 a subtype. The affinity constant of mAb19 and 35 was3.06×10~9 and 2.14×10~9,respectively. The SDS-PAGE analysis showed that the purity was higher than 90%. Excellent immunore activity and specificity of the antibodies was showed by Western blot immunofluorescence and immunohistochemistry analysis.Conclusion:Highly purified rhNGAL protein,anti-NGAL polyclonal antibodies and specific monoclonal antibodies are obtained in this study. The findings lay the foundation for the further study on the function of NGAL in pathophysiology and other regions,and promotes the development of domestic NGAL clinical diagnostic reagents.
Objective:To obtain human neutrophil gelatinase-associated lipocalin(NGAL) recombinant protein and its polyclonal and monoclonal antibodies,and to provide a powerful experimental basis for further functional research and clinical application of NGAL.Methods:The coding region of NGAL mature peptide gene was codon-optimized,chemically synthesized,and then inserted into the prokaryotic expression vector pW28. Transfected E.coli B834 cells were induced by IPTG to produce rhNGAL. The recombinant protein was was purified by Ni~(2+)-NTA affinity chromatography column. The aggregation in solution was analyzed by Hiload Superdex 75 gel flittration column. Rabbits and BALB/c mice were immunized with the rhNGAL to produce polyclonalant antibodies and high specificity monoclonal antibodies by hybridoma technology to identify antibody subtypes. After being purified by Protein G affinity column,the antibodies was detected by Isothermal Titration Calorimetry,non-competitive ELISA and SDS-PAGE analysis. Finally,Western blot,immunofluorescence and immunohistochemistry were used for antibody identification. Results:NGAL recombinant protein was successfully expressed with high purity and mainly existed in monomer form. Recombinant procalcitonin was successfully expressed. Rabbit polyclonal antibody was also produced. Six hybridoma cell lines stably secreting mAbs were screened,hereinto the mAb25 was IgG1 subtype,and the others belonged to IgG2 a subtype. The affinity constant of mAb19 and 35 was3.06×10~9 and 2.14×10~9,respectively. The SDS-PAGE analysis showed that the purity was higher than 90%. Excellent immunore activity and specificity of the antibodies was showed by Western blot immunofluorescence and immunohistochemistry analysis.Conclusion:Highly purified rhNGAL protein,anti-NGAL polyclonal antibodies and specific monoclonal antibodies are obtained in this study. The findings lay the foundation for the further study on the function of NGAL in pathophysiology and other regions,and promotes the development of domestic NGAL clinical diagnostic reagents.
引文
[1] Singer E,MarkóL,Paragas N,et al. Neutrophil gelatinase-associated lipocalin:pathophysiology and clinical applications[J]. Acta Physiol(Oxf),2013,207(4):663-672.
[2] Chakraborty S,Kaur S,Guha S,et al. The multifaceted roles of neutrophil gelatinase associated lipocalin(NGAL)in inflammation and cancer[J]. Biochim Biophys Acta,2012,1826(1):129-169.
[3] Paragas N,Qiu A,Zhang Q,et al. The Ngal reporter mouse detects the response of the kidney to injury in real time[J]. Nature Medicine,2011,17(2):216-222.
[4] Gaspari F,Cravedi P,MandalàM,et al. Predicting cisplatin-induced acute kidney injury by urinary neutrophil gelatinase-associated lipocalin excretion:a pilot prospective case-control study[J]. Nephron Clinical Practice,2010,115(2):c154-160.
[5] Haase M,Bellomo R,Devarajan P,et al. Accuracy of neutrophil gelatinase-associated lipocalin(NGAL)in diagnosis and prognosis in acute kidney injury:a systematic review and meta-analysis[J]. Am J Kidney Dis,2009,54(6):1012-1024.
[6] Nuntagowat C,Leelawat K,Tohtong R. NGAL knockdown by siRNA in human cholangiocarcinoma cells suppressed invasion by reducing NGAL/MMP-9 complex formation[J]. Clinical&Experimental Metastasis,2010,27(5):295-305.
[7]马玉国,赵红丽,任秀华,等.急性肾损伤的检测指标NGAL的研究进展[J].国际检验医学杂志,2013,34(14):1842-1844.
[8] Barasch J,Hollmen M,Deng R,et al. Disposal of iron by a mutant form of lipocalin 2[J]. Nature Communications,2016,7:12973.
[9] Bolignano D,Donato V,Lacquaniti A,et al. Neutrophil gelatinaseassociated lipocalin(NGAL)in human neoplasias:a new protein enters the scene[J]. Cancer Lett,2010,288(1):10-16.
[10] Makris K,Rizos D,Kafkas N,et al. Neurophil gelatinase-associated lipocalin as a new biomarker in laboratory medicine[J]. Clin Chem Lab Med,2012,50(9):1519-1532.
[11] Zhang Y,Fan Y,Mei Z. NGAL and NGALR overexpression in human hepatocellular carcinoma toward a molecular prognostic classification[J]. Cancer Epidemiology,2012,36(5):e294-299.
[12] Chakraborty S,Kaur S,Guha S,et al. The multifaceted roles of neutrophil gelatinase associated lipocalin(NGAL)in inflammation and cancer[J]. Biochim Biophys Acta,2012,1826(1):129-169.
[13] Heise D,Rentsch K,Braeuer A,et al. Comparison of urinary neutrophil glucosaminidase-associated lipocalin,cystatin C,andα1-microglobulin for early detection of acute renal injury after cardiac surgery[J]. Eur J Cardiothorac Surg,2011,39(1):38-43.
[14] Nielsen SE,Schjoedt KJ,Astrup AS,et al. Neutrophil gelatinaseassociated lipocalin(NGAL)and kidney injury molecule 1(KIM1)in patients with diabetic nephropathy:a cross-sectional study and the effects of lisinopril[J]. Diabet Med,2010,27(10):1144-1150.
[15] Fu WJ,Xiong SL,Fang YG,et al. Urinary tubular biomarkers in short-term type 2 diabetes mellitus patients:a cross-sectional study[J].Endocrine,2012,41(1):82-88.
[16] Prabhu A,Sujatha DI,Ninan B,et al. Neutrophil gelatinase associated lipocalin as a biomarker for acute kidney injury in patients undergoing coronary artery bypass grafting with cardiopulmonary bypass[J]. Ann Vasc Surg,2010,24(4):525-531.
[17] Zhang H,Xu L,Xiao D,et al. Upregulation of neutrophil gelatinase-associated lipocalin in oesophageal squamous cell carcinoma:significant correlation with cell differentiation and tumour invasion[J].Journal of Clinical Pathology,2007,60(5):555-561.
[18] Bauer M,Eickhoff JC,Gould MN,et al. Neutrophil gelatinaseassociated lipocalin(NGAL)is a predictor of poor prognosis in human primary breast cancer[J]. Breast Cancer Research&Treatment,2008,108(3):389-397.
[19] Roudkenar MH,Halabian R,Ghasemipour Z,et al. Neutrophil gelatinase-associated lipocalin acts as a protective factor against H2O2toxicity[J]. Archives of Medical Research,2008,39(6):560-566.
[20] Flo TH,Smith KD,Sato S,et al. Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron[J]. Nature,2004,432(7019):917-921.
[21]郑怡麟,陈安,易维京,等.抗人NGAL单克隆抗体的制备及其定量检测方法的建立[J].免疫学杂志,2013,29(1):50-54.
[2] Chakraborty S,Kaur S,Guha S,et al. The multifaceted roles of neutrophil gelatinase associated lipocalin(NGAL)in inflammation and cancer[J]. Biochim Biophys Acta,2012,1826(1):129-169.
[3] Paragas N,Qiu A,Zhang Q,et al. The Ngal reporter mouse detects the response of the kidney to injury in real time[J]. Nature Medicine,2011,17(2):216-222.
[4] Gaspari F,Cravedi P,MandalàM,et al. Predicting cisplatin-induced acute kidney injury by urinary neutrophil gelatinase-associated lipocalin excretion:a pilot prospective case-control study[J]. Nephron Clinical Practice,2010,115(2):c154-160.
[5] Haase M,Bellomo R,Devarajan P,et al. Accuracy of neutrophil gelatinase-associated lipocalin(NGAL)in diagnosis and prognosis in acute kidney injury:a systematic review and meta-analysis[J]. Am J Kidney Dis,2009,54(6):1012-1024.
[6] Nuntagowat C,Leelawat K,Tohtong R. NGAL knockdown by siRNA in human cholangiocarcinoma cells suppressed invasion by reducing NGAL/MMP-9 complex formation[J]. Clinical&Experimental Metastasis,2010,27(5):295-305.
[7]马玉国,赵红丽,任秀华,等.急性肾损伤的检测指标NGAL的研究进展[J].国际检验医学杂志,2013,34(14):1842-1844.
[8] Barasch J,Hollmen M,Deng R,et al. Disposal of iron by a mutant form of lipocalin 2[J]. Nature Communications,2016,7:12973.
[9] Bolignano D,Donato V,Lacquaniti A,et al. Neutrophil gelatinaseassociated lipocalin(NGAL)in human neoplasias:a new protein enters the scene[J]. Cancer Lett,2010,288(1):10-16.
[10] Makris K,Rizos D,Kafkas N,et al. Neurophil gelatinase-associated lipocalin as a new biomarker in laboratory medicine[J]. Clin Chem Lab Med,2012,50(9):1519-1532.
[11] Zhang Y,Fan Y,Mei Z. NGAL and NGALR overexpression in human hepatocellular carcinoma toward a molecular prognostic classification[J]. Cancer Epidemiology,2012,36(5):e294-299.
[12] Chakraborty S,Kaur S,Guha S,et al. The multifaceted roles of neutrophil gelatinase associated lipocalin(NGAL)in inflammation and cancer[J]. Biochim Biophys Acta,2012,1826(1):129-169.
[13] Heise D,Rentsch K,Braeuer A,et al. Comparison of urinary neutrophil glucosaminidase-associated lipocalin,cystatin C,andα1-microglobulin for early detection of acute renal injury after cardiac surgery[J]. Eur J Cardiothorac Surg,2011,39(1):38-43.
[14] Nielsen SE,Schjoedt KJ,Astrup AS,et al. Neutrophil gelatinaseassociated lipocalin(NGAL)and kidney injury molecule 1(KIM1)in patients with diabetic nephropathy:a cross-sectional study and the effects of lisinopril[J]. Diabet Med,2010,27(10):1144-1150.
[15] Fu WJ,Xiong SL,Fang YG,et al. Urinary tubular biomarkers in short-term type 2 diabetes mellitus patients:a cross-sectional study[J].Endocrine,2012,41(1):82-88.
[16] Prabhu A,Sujatha DI,Ninan B,et al. Neutrophil gelatinase associated lipocalin as a biomarker for acute kidney injury in patients undergoing coronary artery bypass grafting with cardiopulmonary bypass[J]. Ann Vasc Surg,2010,24(4):525-531.
[17] Zhang H,Xu L,Xiao D,et al. Upregulation of neutrophil gelatinase-associated lipocalin in oesophageal squamous cell carcinoma:significant correlation with cell differentiation and tumour invasion[J].Journal of Clinical Pathology,2007,60(5):555-561.
[18] Bauer M,Eickhoff JC,Gould MN,et al. Neutrophil gelatinaseassociated lipocalin(NGAL)is a predictor of poor prognosis in human primary breast cancer[J]. Breast Cancer Research&Treatment,2008,108(3):389-397.
[19] Roudkenar MH,Halabian R,Ghasemipour Z,et al. Neutrophil gelatinase-associated lipocalin acts as a protective factor against H2O2toxicity[J]. Archives of Medical Research,2008,39(6):560-566.
[20] Flo TH,Smith KD,Sato S,et al. Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron[J]. Nature,2004,432(7019):917-921.
[21]郑怡麟,陈安,易维京,等.抗人NGAL单克隆抗体的制备及其定量检测方法的建立[J].免疫学杂志,2013,29(1):50-54.