ATP5B原核表达和单克隆抗体的制备及其鉴定
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摘要
目的:制备高纯度ATP合酶β亚基(ATP5B)单克隆抗体并进行鉴定,为其后续研究奠定基础。方法:应用PCR法扩增ATP5B基因,克隆入pET28a载体中并转入大肠杆菌BL21 (DE3)。异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导蛋白表达并用镍亲和层析柱纯化融合蛋白,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测蛋白纯度。用纯化的融合蛋白免疫3只雌性Balb/C小鼠,取小鼠尾静脉血,间接酶联免疫吸附法(ELISA法)检测血清中ATP5B抗体效价。取血清效价最高的免疫小鼠脾细胞与小鼠骨髓瘤SP2/0细胞混合培养建立杂交瘤细胞,采用间接ELISA法筛选融合细胞并行单克隆化培养,对阳性细胞进行核型分析。取12周龄Balb/C小鼠,腹腔注射杂交瘤细胞制备腹水模型,收集腹水,间接ELISA法检测效价,SDS-PAGE检测抗体纯度,应用ELISA法检测抗体亚型。结果:PCR扩增后得到1 455bp的特异性条带,连接pET28a空载体获得重组pET28a/ATP5B载体。IPTG诱导转化的菌液表达目的蛋白,SDS-PAGE检测,在相对分子质量为51 000处出现明显的诱导蛋白条带。间接ELISA法检测免疫小鼠静脉血,血清效价最高达1∶64 000。杂交瘤细胞染色体核型分析,染色体总数约为骨髓瘤细胞与正常小鼠脾细胞之和。采用杂交瘤细胞株制备小鼠腹水,抗体最高效价为1∶240 000,杂交瘤细胞产生的单克隆抗体的亚型为IgG1。结论:通过克隆、表达和纯化重组蛋白,成功制备出抗ATP5B蛋白单克隆抗体。
Objective:To construct and identify the monoclonal antibody of ATP synthase beta subunit(ATP5 B)with high purity,and to lay foundation for further study.Methods:The ATP5 Bgene was amplified by PCR and cloned into the pET28 avector and transformed into E.coli BL21(DE3).The protein expression was induced by IPTG and then the fusion protein was purified by nickel affinity chromatography column.The protein purity was detected by SDS-polyacrylamide gel electrophoresis(SDS-PAGE).Three female Balb/C mice were immunized with purified fusion protein and the tail vein blood was taken to detect the titer of ATP5 Bantibody by indirect enzyme-linked immunosorbent assay(ELISA).The spleen cells from the immunized mice with the highest serum titer were mixed with the SP2/0 cells to establish the hybridoma cells and the fused cells were screened by indirect ELISA and monoclonally cultured.Karyotype analysis were performed in the positive cells.The hybridoma cells were intraperitoneally injected into 12 weeks old BALB/C mice to estabilish the ascites models.The titer of ascites was detected by indirect ELISA.The purity of the antibody was detected by SDS-PAGE.The antibody subtype was detected by ELISA.Results:After PCR amplification,a specific band of 1 455 bp was obtained,and the pET28 aempty vector was ligated to obtain a recombinant pET28 a/ATP5 Bvector.The target protein was expressed in the IPTG-induced bacteria solution;the SDS-PAGE results showed that the protein band was found at51 000.The indirect ELISA results showed that the serum titer of the venous blood of immunized mice was up to1∶64 000.In karyotype analysis,the total number of chromosomes in hybridoma cells was about the sum of myeloma cells and normal mouse spleen cells.The mouse ascites was prepared with the hybridoma cell line,and the highest titer of the antibody was 1∶240 000.The subtype of the monoclonal antibody produced by the hybridoma cells was IgG1.Conclusion:The monoclonal antibody against ATP5 Bprotein is successfully prepared by cloning,expressing and purifying the recombinant protein.
Objective:To construct and identify the monoclonal antibody of ATP synthase beta subunit(ATP5 B)with high purity,and to lay foundation for further study.Methods:The ATP5 Bgene was amplified by PCR and cloned into the pET28 avector and transformed into E.coli BL21(DE3).The protein expression was induced by IPTG and then the fusion protein was purified by nickel affinity chromatography column.The protein purity was detected by SDS-polyacrylamide gel electrophoresis(SDS-PAGE).Three female Balb/C mice were immunized with purified fusion protein and the tail vein blood was taken to detect the titer of ATP5 Bantibody by indirect enzyme-linked immunosorbent assay(ELISA).The spleen cells from the immunized mice with the highest serum titer were mixed with the SP2/0 cells to establish the hybridoma cells and the fused cells were screened by indirect ELISA and monoclonally cultured.Karyotype analysis were performed in the positive cells.The hybridoma cells were intraperitoneally injected into 12 weeks old BALB/C mice to estabilish the ascites models.The titer of ascites was detected by indirect ELISA.The purity of the antibody was detected by SDS-PAGE.The antibody subtype was detected by ELISA.Results:After PCR amplification,a specific band of 1 455 bp was obtained,and the pET28 aempty vector was ligated to obtain a recombinant pET28 a/ATP5 Bvector.The target protein was expressed in the IPTG-induced bacteria solution;the SDS-PAGE results showed that the protein band was found at51 000.The indirect ELISA results showed that the serum titer of the venous blood of immunized mice was up to1∶64 000.In karyotype analysis,the total number of chromosomes in hybridoma cells was about the sum of myeloma cells and normal mouse spleen cells.The mouse ascites was prepared with the hybridoma cell line,and the highest titer of the antibody was 1∶240 000.The subtype of the monoclonal antibody produced by the hybridoma cells was IgG1.Conclusion:The monoclonal antibody against ATP5 Bprotein is successfully prepared by cloning,expressing and purifying the recombinant protein.
引文
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[22]XIAO X,YANG J,LI R,et al.Deregulation of mitochondrial ATPsyn-βin acute myeloid leukemia cells and with increased drug resistance[J].PLoS ONE,2013,8(12):e83610.
[23]蒋灵旭,罗颖婉,佟红艳.急性髓系白血病靶向治疗新进展[J].中国实用内科杂志,2018,38(12):1198-1202.
[2]TAN A S,BATY J W,DONG L F,et al.Mitochondrial genome acquisition restores respiratory function and tumorigenic potential of cancer cells without mitochondrial DNA[J].Cell Metab,2015,21(1):81-94.
[3]FORMENTINI L,MARTNEZ-REYES I,CUEZVA J M.The mitochondrial bioenergetic capacity of carcinomas[J].IUBMB Life,2010,62(7):554-560.
[4]SENIOR A E.ATP synthase:motoring to the finish line[J].Cell,2007,130(2):220-221.
[5]GEYIK E,IGCI Y Z,PALA E,et al.Investigation of the association between ATP2B4and ATP5Bgenes with colorectal cancer[J].Gene,2014,540(2):178-182.
[6]XU G,LI J Y.ATP5A1and ATP5Bare highly expressed in glioblastoma tumor cells and endothelial cells of microvascular proliferation[J].J Neurooncol,2016,126(3):405-413.
[7]SUN J,YANG Z L,MIAO X,et al.ATP5b andβ2-microglobulin are predictive markers for the prognosis of patients with gallbladder cancer[J].J Mol Histol,2015,46(1):57-65.
[8]LU Z,SONG Q,SONG Q,et al.Preparation of monoclonal antibody against lung cancer and identification of its targeting antigen[J].J Biomed Engineering,2010,27(1):147-151.
[9]刘素霞.重组单克隆抗体生产的宿主细胞系的研究进展[J].科技风,2018,9(25):36.
[10]SALTZ L.Systemic therapy for metastatic colorectal cancer[J].J Natl Compr Canc Netw,2013,11(Suppl 5):649-652.
[11]SALTZ L B,LENZ H J,KINDLER H L,et al.Randomized phaseⅡtrial of cetuximab,bevacizumab and irinotecan compared with cetuximab and bevacizumab alone in irinotecanrefractory colorectal cancer:the BOND-2study[J].J Clin Oncol,2007,25(29):4557-4561.
[12]邓云秋.EGFR单克隆抗体靶向药物治疗脑胶质瘤的研究进展[J].中国处方药,2018,16(8):22-23.
[13]朱慧,李春蕊.多发性骨髓瘤的免疫治疗进展[J].医药导报,2018,37(10):1160-1165.
[14]赵源浩.靶向抗肿瘤单克隆抗体类药物联合常规化疗方案治疗转移性结直肠癌的临床进展[J].中国药房,2018,29(14):2012-2016.
[15]刘志,赵春清,张彬,等.VEGF、Bmi-1在结肠癌组织中的表达及意义[J].河北医药,2016,38(20):3104-3106.
[16]GREENING D W,LEE S T,JI H,et al.Molecular profiling of cetuximab and bevacizumab treatment of colorectal tumoursreveals perturbations in metabolic and hypoxic response pathways[J].Oncotarget,2015,6(35):38166-38180.
[17]SNCHEZ-ARAGM,CHAMORRO M,CUEZVA JM.Selection of cancer cells with repressed mitochondria triggers colon cancer progression[J].Carcinogenesis,2010,31(4):567-576.
[18]REN L R,ZHANG L P,HUANG S Y,et al.Effects of sialidase NEU1 siRNA on proliferation,apoptosis,and invasion in human ovarian cancer[J].Mol Cell Biochem,2016,411(1/2):213-219.
[19]FLIEDNER S M,YANG C,THOMPSON E,et al.Potential therapeutic target for malignant paragangliomas:ATP synthase on the surface of paraganglioma cells[J].Am JCancer Res,2015,5(4):1558-1570.
[20]LI W,LI Y,LI G,et al.Ectopic expression of the ATPsynthaseβsubunit on the membrane of PC-3Mcells supports its potential role in prostate cancer metastasis[J].Int JOncol,2017,50(4):1312-1320.
[21]LYNAM-LENNON N,Maher SG,Maguire A,et al.Altered mitochondrial function and energy metabolism is associated with a radioresistant phenotype in oesophageal adenocarcinoma[J].PLoS ONE,2014,9(6):e100738.
[22]XIAO X,YANG J,LI R,et al.Deregulation of mitochondrial ATPsyn-βin acute myeloid leukemia cells and with increased drug resistance[J].PLoS ONE,2013,8(12):e83610.
[23]蒋灵旭,罗颖婉,佟红艳.急性髓系白血病靶向治疗新进展[J].中国实用内科杂志,2018,38(12):1198-1202.