MMP-9蛋白的抗原表位分析及单克隆抗体制备
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摘要
利用生物信息学方法分析并预测MMP-9蛋白的抗原表位区,命名为Δmmp9。通过对Δmmp9的核苷酸序列进行分析,设计引物扩增目的片段,构建重组表达载体pET28α(+)-Δmmp9,并将其转化到BL21(DE3)中,诱导表达重组蛋白Δmmp9。纯化重组蛋白后,免疫BALB/c小鼠,筛选获得7株稳定分泌单克隆抗体的杂交瘤细胞株,检测抗体效价均达1:240 000。利用生物信息学筛选抗原表位区,成功制备单克隆抗体,通过抗体配对实验,证明其有良好特异性,并筛选出3株捕获抗体及对应标记抗体。
In this study,we used bioinformatics analysis to predict antigenic epitope region of MMP-9,which was designated asΔmmp9. Analyzing the nucleotide sequence of the Δmmp9,the primers were designed to amplify the target fragment,and the recombinant pET28α(+)-Δmmp9 expression vector was constructed,and transferred into Escherichia coli competent cell BL21(DE3)for inducing the recombinant protein Δmmp9. Then recombinant protein Δmmp9 was purified,and then was used to immunize BALB/c mice as antigen.Furthermore,7 hybridoma cell lines steadily secreting monoclonal antibodies(MAbs)were screened,and titers of seven MAbs all reached1 :240 000. In summary,MMP-9 antigenic epitope region is screened by bioinformatics methods and its MAbs are prepared successfully,its promising specificity is confirmed by antibody pairing test,and 3 captured antibodies and corresponding labeled antibodies are screened out.
In this study,we used bioinformatics analysis to predict antigenic epitope region of MMP-9,which was designated asΔmmp9. Analyzing the nucleotide sequence of the Δmmp9,the primers were designed to amplify the target fragment,and the recombinant pET28α(+)-Δmmp9 expression vector was constructed,and transferred into Escherichia coli competent cell BL21(DE3)for inducing the recombinant protein Δmmp9. Then recombinant protein Δmmp9 was purified,and then was used to immunize BALB/c mice as antigen.Furthermore,7 hybridoma cell lines steadily secreting monoclonal antibodies(MAbs)were screened,and titers of seven MAbs all reached1 :240 000. In summary,MMP-9 antigenic epitope region is screened by bioinformatics methods and its MAbs are prepared successfully,its promising specificity is confirmed by antibody pairing test,and 3 captured antibodies and corresponding labeled antibodies are screened out.
引文
[1]Chien MH, Lin CW, Cheng CW, et al. Matrix metalloproteinase-2 as a target for head and neck cancer therapy[J]. Expert Opinion on Therapeutic Targets, 2013, 17(2):203-216.
[2]Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases structure, function, and biochemistry[J].Circulation Research 2003, 92(8):827-839.
[3]韩寒,刘克良.基质金属蛋白酶抑制剂研究进展[J].国际药学研究杂志, 2008, 35(3):173-177.
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[5]Illemann M, Bird N, Majeed A. MMP-9 is differentially expressed in primary human colorectal adenocarcinomas and their metastases[J]. Molecular Cancer Research, 2006, 4(5):293-302.
[6]唐利娜.基质金属蛋白酶9及其在心血管疾病中的研究进展[J].医学综述, 2011, 17(6):801-804.
[7]Liang YF, Yang NL, Pan GQ, et al. Elevated IL-33 promotes expression of MMP2 and MMP9 via activating STAT3 in alveolar macrophages during LPS-induced acute lung injury[J]. Cellular&Molecular Biology Letters, 2018, 23(1):52-64.
[8]Royer PJ, Henrio K, et al. TLR3 promotes MMP-9 production in primary human airway epithelial cells through Wnt/β-catenin signaling[J]. Respiratory Research, 2017, 18(1):208-217.
[9]Huang QJ, Lan FH, et al. IL-1β-induced activation of p38 promotes metastasis in gastric adenocarcinoma via upregulation of AP-1/c-fos,MMP2 and MMP9[J]. Molecular Cancer, 2014, 13(1):18-32.
[10]Yushchenko M, et al. Interferon-β-1b decreased matrix metalloproteinase-9 serum levels in primary progressive multiple sclerosis[J]. Journal of Neurology, 2003, 250(10):1224-1228.
[11]Pei Jun, Lou YQ, et al. MMP9 activation triggered by epidermal growth factor induced FoxO1 nuclear exclusion in non-small cell lung cancer[J]. Tumor Biology, 2014, 35(7):6673-6678.
[12]Kao TH, Peng YJ, et al. Nerve growth factor increases MMP9 activity in annulus fibrosus cells by upregulating lipocalin 2 expression[J]. European Spine Journal, 2014, 24(9):1959-1968.
[13]Hu Shuai, Li Lei, Yeh SY, et al. Infiltrating T cells promote prostate cancer metastasis via modulation of FGF11→miRNA-541→androgen receptor(AR)→MMP9 signaling[J].Molecular Oncology, 2015, 9(1):44-57.
[14]Gupta A, Zhou CQ, Chellaia MA. Osteopontin and MMP9:associations with VEGF expression/secretion and angiogenesis in PC3 prostate cancer cells[J]. Cancers, 2013, 5(2):617-638.
[15]聂玉红,瞿雯,邢怡桥,等. PDGF对人RPE细胞中MMP-2、MMP-9和TIMP-1表达的影响[J].中华实验眼科杂志,2014, 32(1):6-11.
[16]Hsieh HL, Wang HH, et al. Transforming growth factor-b1 induces matrix metalloproteinase-9 and cell migration in astrocytes:roles of ROS-dependent ERK-and JNKNF-B pathways[J]. Journal of Neuroinflammation, 2010, 7(1):88-104.
[17]Ogawa K, Funaba M, et al. The effects of TGF-b1 on the expression of type IV collagenases in mouse peritoneal macrophages[J].Molecular Biology Reports, 2011, 38(2):1451-1456.
[18]Chen WL, Zhong X, et al. TGF-βregulates survivin to affect cell cycle and the expression of EGFR and MMP9 in glioblastoma[J].Molecular Neurobiology, 2016, 53(3):1648-1653.
[19]Malla N, Berg E, et al. Interaction of pro-matrix metalloproteinase-9/proteoglycan heteromer with gelatin and collagen[J]. J Biol Chem, 2008, 283(20):13652-13665.
[20]B?ck M, Ketelhuth DF, Agewall S. Matrix metalloproteinases in Atherothrombosis[J]. Progress in Cardiovascular Diseases,2010, 52(2):410-428.
[21]Li Xun, Wu JF. Recent developments in patent anti-cancer agents targeting the matrix metalloproteinases(MMPs)[J]. Recent Patents on Anticancer Drug Discovery, 2010, 5(2):109-141.
[22]王璐,张丽红,李玉林,等.基质金属蛋白酶-9及其m RNA在胃癌中的表达与血管新生的关系[J].中华医学杂志,2003, 83(9):782-786.
[23]王小侠,冯一中,周剑影,等. MMP-9在胃癌中的表达及临床意义[J].苏州大学学报:医学版, 2006, 26(1):83-85.
[24]高晨,李锋,周伟,等.血清MMP9蛋白ELISA检测方法的建立及在肝癌患者血清检测中的初步应用[J].中华检验医学杂志, 2007, 30(3):288-291.
[25]亓慧琴,张连莲,邢利平,等.慢性阻塞性肺疾病患者应用异丙托溴铵联合乙酰半胱氨酸治疗前后血清中TGF-β和MMP-9水平的变化及其临床意义[J].吉林大学学报:医学版,2016, 42(4):777-784.
[26]马婕,邱涵,张倩,等. TIMP-1、MMP-9在乳腺癌患者术前血清中的表达与临床病理特征的关系[J].湖南师范大学学报:医学版, 2016, 13(5):17-21.
[27]程欣. MMP-9和TIMP-1在结直肠癌组织和血清中的表达及其临床意义[D].长春:吉林大学, 2017.
[28]单体臻,王新,王海峰,等.血清MMP-2、MMP-9水平与阿尔茨海默病及轻度认知障碍关系的临床研究[D].青岛:青岛大学, 2016.
[29]庞岚,郑彦强,时志民,等. UPA和MMP-9在子宫内膜癌组织中的表达情况及临床意义[J].现代中西医结合杂志,2016, 25(32):3577-3579.
[30]白春侠. OPN和MMP-9在膀胱移行细胞癌中的表达及临床意义[J].当代医学, 2016, 22(1):23-24.
[31]金永梅,陈莫军,刘笑笑,等.抗鳞翅目害虫基因cry1C抗原表位分析及多克隆抗体制备[J].生物技术通报, 2018, 34(9):224-229.
[32]孙静娟,邱景璇,等.单增李斯特菌CdaA的抗原表位分析及抗体的制备[J].生物技术通报, 2018, 34(5):163-171.
[2]Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases structure, function, and biochemistry[J].Circulation Research 2003, 92(8):827-839.
[3]韩寒,刘克良.基质金属蛋白酶抑制剂研究进展[J].国际药学研究杂志, 2008, 35(3):173-177.
[4]CsehS,HajdúI,BarnaL,etal.Matrixmetalloproteinase inhibitors[J]. Drugs, 2010, 70(8):949-964.
[5]Illemann M, Bird N, Majeed A. MMP-9 is differentially expressed in primary human colorectal adenocarcinomas and their metastases[J]. Molecular Cancer Research, 2006, 4(5):293-302.
[6]唐利娜.基质金属蛋白酶9及其在心血管疾病中的研究进展[J].医学综述, 2011, 17(6):801-804.
[7]Liang YF, Yang NL, Pan GQ, et al. Elevated IL-33 promotes expression of MMP2 and MMP9 via activating STAT3 in alveolar macrophages during LPS-induced acute lung injury[J]. Cellular&Molecular Biology Letters, 2018, 23(1):52-64.
[8]Royer PJ, Henrio K, et al. TLR3 promotes MMP-9 production in primary human airway epithelial cells through Wnt/β-catenin signaling[J]. Respiratory Research, 2017, 18(1):208-217.
[9]Huang QJ, Lan FH, et al. IL-1β-induced activation of p38 promotes metastasis in gastric adenocarcinoma via upregulation of AP-1/c-fos,MMP2 and MMP9[J]. Molecular Cancer, 2014, 13(1):18-32.
[10]Yushchenko M, et al. Interferon-β-1b decreased matrix metalloproteinase-9 serum levels in primary progressive multiple sclerosis[J]. Journal of Neurology, 2003, 250(10):1224-1228.
[11]Pei Jun, Lou YQ, et al. MMP9 activation triggered by epidermal growth factor induced FoxO1 nuclear exclusion in non-small cell lung cancer[J]. Tumor Biology, 2014, 35(7):6673-6678.
[12]Kao TH, Peng YJ, et al. Nerve growth factor increases MMP9 activity in annulus fibrosus cells by upregulating lipocalin 2 expression[J]. European Spine Journal, 2014, 24(9):1959-1968.
[13]Hu Shuai, Li Lei, Yeh SY, et al. Infiltrating T cells promote prostate cancer metastasis via modulation of FGF11→miRNA-541→androgen receptor(AR)→MMP9 signaling[J].Molecular Oncology, 2015, 9(1):44-57.
[14]Gupta A, Zhou CQ, Chellaia MA. Osteopontin and MMP9:associations with VEGF expression/secretion and angiogenesis in PC3 prostate cancer cells[J]. Cancers, 2013, 5(2):617-638.
[15]聂玉红,瞿雯,邢怡桥,等. PDGF对人RPE细胞中MMP-2、MMP-9和TIMP-1表达的影响[J].中华实验眼科杂志,2014, 32(1):6-11.
[16]Hsieh HL, Wang HH, et al. Transforming growth factor-b1 induces matrix metalloproteinase-9 and cell migration in astrocytes:roles of ROS-dependent ERK-and JNKNF-B pathways[J]. Journal of Neuroinflammation, 2010, 7(1):88-104.
[17]Ogawa K, Funaba M, et al. The effects of TGF-b1 on the expression of type IV collagenases in mouse peritoneal macrophages[J].Molecular Biology Reports, 2011, 38(2):1451-1456.
[18]Chen WL, Zhong X, et al. TGF-βregulates survivin to affect cell cycle and the expression of EGFR and MMP9 in glioblastoma[J].Molecular Neurobiology, 2016, 53(3):1648-1653.
[19]Malla N, Berg E, et al. Interaction of pro-matrix metalloproteinase-9/proteoglycan heteromer with gelatin and collagen[J]. J Biol Chem, 2008, 283(20):13652-13665.
[20]B?ck M, Ketelhuth DF, Agewall S. Matrix metalloproteinases in Atherothrombosis[J]. Progress in Cardiovascular Diseases,2010, 52(2):410-428.
[21]Li Xun, Wu JF. Recent developments in patent anti-cancer agents targeting the matrix metalloproteinases(MMPs)[J]. Recent Patents on Anticancer Drug Discovery, 2010, 5(2):109-141.
[22]王璐,张丽红,李玉林,等.基质金属蛋白酶-9及其m RNA在胃癌中的表达与血管新生的关系[J].中华医学杂志,2003, 83(9):782-786.
[23]王小侠,冯一中,周剑影,等. MMP-9在胃癌中的表达及临床意义[J].苏州大学学报:医学版, 2006, 26(1):83-85.
[24]高晨,李锋,周伟,等.血清MMP9蛋白ELISA检测方法的建立及在肝癌患者血清检测中的初步应用[J].中华检验医学杂志, 2007, 30(3):288-291.
[25]亓慧琴,张连莲,邢利平,等.慢性阻塞性肺疾病患者应用异丙托溴铵联合乙酰半胱氨酸治疗前后血清中TGF-β和MMP-9水平的变化及其临床意义[J].吉林大学学报:医学版,2016, 42(4):777-784.
[26]马婕,邱涵,张倩,等. TIMP-1、MMP-9在乳腺癌患者术前血清中的表达与临床病理特征的关系[J].湖南师范大学学报:医学版, 2016, 13(5):17-21.
[27]程欣. MMP-9和TIMP-1在结直肠癌组织和血清中的表达及其临床意义[D].长春:吉林大学, 2017.
[28]单体臻,王新,王海峰,等.血清MMP-2、MMP-9水平与阿尔茨海默病及轻度认知障碍关系的临床研究[D].青岛:青岛大学, 2016.
[29]庞岚,郑彦强,时志民,等. UPA和MMP-9在子宫内膜癌组织中的表达情况及临床意义[J].现代中西医结合杂志,2016, 25(32):3577-3579.
[30]白春侠. OPN和MMP-9在膀胱移行细胞癌中的表达及临床意义[J].当代医学, 2016, 22(1):23-24.
[31]金永梅,陈莫军,刘笑笑,等.抗鳞翅目害虫基因cry1C抗原表位分析及多克隆抗体制备[J].生物技术通报, 2018, 34(9):224-229.
[32]孙静娟,邱景璇,等.单增李斯特菌CdaA的抗原表位分析及抗体的制备[J].生物技术通报, 2018, 34(5):163-171.