抗树鼩CD4单克隆抗体的制备及其生物学特性的鉴定
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摘要
目的制备鼠抗树鼩CD4单克隆抗体,并对其生物学特性进行鉴定。方法将重组质粒p GEX-5X-1-CD4和pET30a(+)-CD4分别转化感受态E. coli BL21和BL21(DE3),经IPTG诱导表达,并通过Glutathione Sepharose 4 Fast Flow亲和层析柱纯化重组蛋白GST-CD4和His-CD4。以His-CD4蛋白为免疫原免疫BALB/c小鼠,经杂交瘤技术筛选出能分泌抗树鼩CD4的杂交瘤细胞株,制备小鼠腹水单克隆抗体,间接ELISA法检测效价,并通过Protein A亲和层析柱纯化获得鼠抗树鼩CD4单克隆抗体,Western blot及FACS法分析其生物学特性。结果筛选出3株能稳定分泌抗树鼩CD4单克隆抗体的杂交瘤细胞株,分别命名为37D2、38E1、39F2,腹水单克隆抗体效价分别为1∶204 800、1∶204 800、1∶3 200。经HRP标记的38E1单克隆抗体可与树鼩PBMC总蛋白发生特异性结合,经PECy5. 5标记的38E1单克隆抗体能特异性识别树鼩PBMC。结论成功制备了鼠抗树鼩CD4的单克隆抗体,为进一步对树鼩作为动物模型的研究及应用奠定了基础。
Objective To prepare the monoclonal antibodies(McAbs) against tree shrew CD4,and identify their biological characteristics. Methods Recombinant plasmids p GEX-5 X-1-CD4 and p ET30 a(+)-CD4 were transformed to E. coli BL21 and BL21(DE3) respectively and induced with IPTG. The expressed recombinant proteins His-CD4 and GST-CD4 were purified by Glutathione Sepharose 4 Fast Flow affinity chromatography. BALB/c mice were immunized with His-CD4 protein,and hybridoma cell lines secreting McAb against CD4 of tree shrew were screened by hybridoma technique. The prepared McAb was prepared and determined for titer by indirect ELISA,then purified by Protein A affinity chromatography and analyzed for biological characteristics by Western blot and fluorescence activated cell sorting(FACS). Results Three hybridoma cell lines stably secreting McAbs against tree shrew CD4 were screened and named as 37 D2,38 E1,39 F2,by which the titers of secreted McAbs were 1 ∶ 204 800,1 ∶ 204 800 and 1 ∶ 3 200,respectively. The McAb 38 E1 labeled with HRP showed specific binding to the total protein of PBMCs of tree shrew,while that labeled with PE-Cy5. 5 recognized the PMBCs specifically. Conclusion McAbs against CD4 of tree shrew were successfully prepared,which laid a foundation of further research and application of tree shrews as animal models.
Objective To prepare the monoclonal antibodies(McAbs) against tree shrew CD4,and identify their biological characteristics. Methods Recombinant plasmids p GEX-5 X-1-CD4 and p ET30 a(+)-CD4 were transformed to E. coli BL21 and BL21(DE3) respectively and induced with IPTG. The expressed recombinant proteins His-CD4 and GST-CD4 were purified by Glutathione Sepharose 4 Fast Flow affinity chromatography. BALB/c mice were immunized with His-CD4 protein,and hybridoma cell lines secreting McAb against CD4 of tree shrew were screened by hybridoma technique. The prepared McAb was prepared and determined for titer by indirect ELISA,then purified by Protein A affinity chromatography and analyzed for biological characteristics by Western blot and fluorescence activated cell sorting(FACS). Results Three hybridoma cell lines stably secreting McAbs against tree shrew CD4 were screened and named as 37 D2,38 E1,39 F2,by which the titers of secreted McAbs were 1 ∶ 204 800,1 ∶ 204 800 and 1 ∶ 3 200,respectively. The McAb 38 E1 labeled with HRP showed specific binding to the total protein of PBMCs of tree shrew,while that labeled with PE-Cy5. 5 recognized the PMBCs specifically. Conclusion McAbs against CD4 of tree shrew were successfully prepared,which laid a foundation of further research and application of tree shrews as animal models.
引文
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[17] LI Y,LING W,XU H,et al. The activation and dynamics of cytokine expression by CD4+T cells and AIDS progression in HIV-1-infected Chinese individuals[J]. Microb Pathog,2012,53(5-6):189-197.
[18] TIAN W W,GAO Y D,GUO Y,et al. Cloning and molecular characterization of full-length coding sequence of CD4[J]. Zool Res,2012,33(1):60-66.(in Chinese)田巍威,高跃东,郭彦,等.树鼩CD4全长编码序列的克隆及分子特征分析[J].动物学研究,2012,33(1):60-66.
[19] LU K J,XU J W,ZHANG X M,et al. Preparation and detection of polyclonal antibody against tree shrew CD4 protein[J]. Lab Animals Comparative Med,2016,36(4):243-249.(in Chinese)卢孔杰,徐婧雯,张雪梅,等.树鼩CD4蛋白多克隆抗体的制备及检测[J].实验动物与比较医学,2016,36(4):243-249.
[20] XIAO J,LIU R,CHEN C S. Tree shrew(Tupaia belangeri)as a novel laboratory disease animal model[J]. Zool Res,2017,38(3):127.
[2] TANG Y P,CAO J,YANG X D,et al. Comparison of the genetic diversity between Guangxi and Yunnan tree shrew populations[J]. Acta Lab Anim Sci Sin,2016,24(6):572-578.(in Chinese)唐艳萍,曹骥,杨香娣,等.广西和云南树鼩群体遗传多样性的比较分析[J].中国实验动物学报,2016,24(6):572-578.
[3] LI Q,NI X. An early oligocene fossil demonstrates treeshrews are slowly evolving“living fossils”[J]. Sci Rep,2016,6(1):18627.
[4] KOUWAKI T,FUKUSHIMA Y,DAITO T,et al. Extracellular vesicles including exosomes regulate innate immune responses to hepatitis B virus infection[J]. Frontiers Immunol,2016,7:355.
[5] ZHOU L L,YANG C,CAO J. Research progress in tupaia animal model of hepatitis C[J]. Med Recapitulate,2015,21(11):1930-1932.(in Chinese)周玲丽,杨春,曹骥.树鼩丙型肝炎动物模型研究进展[J].医学综述,2015,21(11):1930-1932.
[6] YANG M,HU X X,WANG W G,et al. Establishment of a EV71 virus infection model of tree shrew primary renal cells[J].Acta Lab Anim Sci Sin,2017,25(2):117-122.(in Chinese)杨铭,胡晓星,王文广,等. EV71病毒对树鼩原代肾上皮细胞感染模型的建立[J].中国实验动物学报,2017,25(2):117-122.
[7] LI J P. The experiment infection of Tupia belangeri chinensis with Coxsackievirus A16[D]. Peking Union Medical College,2014.(in Chinese)李建平.柯萨奇病毒A组16型感染幼龄树鼩的研究[D].北京:北京协和医学院,2014.
[8] WANG Z,YI X,DU L,et al. A study of Epstein-Barr virus infection in the Chinese tree shrew(Tupaia belangeri chinensis)[J]. Virol J,2017,14(1):193.
[9] YU W,YANG C,BI Y,et al. Characterization of hepatitis E virus infection in tree shrew(Tupaia belangeri chinensis)[J].Bmc Infect Dis,2016,16(1):1-7.
[10] ZHOU F M. Study on the immune response of tree mites to influenza virus by various cytokines[D]. Kunming:Kunming University of Science and Technology,2018.(in Chinese)周风梅.多种细胞因子对树鼩感染流感病毒免疫反应的研究[D].云南昆明:昆明理工大学,2018.
[11] CHEN B,QIN M C,HUANG J L,et al. Preliminary establishment of integration of Alzheimer′s disease andblood stasis syndrome tree shrew model and evaluation of intervention of Panax notoginseng saponins[J]. Chin J Chin Materia Med,2017,42(6):1175-1182.(in Chinese)陈奔,覃梅春,黄金兰,等.老年性痴呆-血瘀证病证结合树鼩模型的初步建立及三七总皂苷干预的评价[J].中国中药杂志,2017,42(6):1175-1182.
[12] TONG Y,HAO J,TU Q,et al. A tree shrew glioblastoma model recapitulates features of human glioblastoma[J]. Oncotarget,2017,8(11):17897-17907.
[13] ZHAO L,LI S Q. Mechanism of metabolic disorders aggravated ischemic brain injury in tree shrews of type 2-diabetes mellitus[J].Acta Anatomica Sinica,2018,49(3):273-280.(in Chinese)赵玲,李树清.树鼩2型糖尿病加剧缺血性脑损伤的可能机制[J].解剖学报,2018,49(3):273-280.
[14] SAMUELS B C,et al. A novel tree shrew(Tupaia belangeri)model of glaucoma[J]. 2018, 59(7):3136.
[15] CAO X T. Medical immunology[M]. Beijing:People's Medical Publishing House,2015:200-201.(in Chinese)曹雪涛.医学免疫学[M].北京:人民卫生出版社,2015:200-201.
[16] WANG J,YAN Y,GARRETT T P J,et al. Atomic structure of a fragment of human CD4 containing two immunoglob?Lin-like domains[J]. Nature,1990,348(6300):411-418.
[17] LI Y,LING W,XU H,et al. The activation and dynamics of cytokine expression by CD4+T cells and AIDS progression in HIV-1-infected Chinese individuals[J]. Microb Pathog,2012,53(5-6):189-197.
[18] TIAN W W,GAO Y D,GUO Y,et al. Cloning and molecular characterization of full-length coding sequence of CD4[J]. Zool Res,2012,33(1):60-66.(in Chinese)田巍威,高跃东,郭彦,等.树鼩CD4全长编码序列的克隆及分子特征分析[J].动物学研究,2012,33(1):60-66.
[19] LU K J,XU J W,ZHANG X M,et al. Preparation and detection of polyclonal antibody against tree shrew CD4 protein[J]. Lab Animals Comparative Med,2016,36(4):243-249.(in Chinese)卢孔杰,徐婧雯,张雪梅,等.树鼩CD4蛋白多克隆抗体的制备及检测[J].实验动物与比较医学,2016,36(4):243-249.
[20] XIAO J,LIU R,CHEN C S. Tree shrew(Tupaia belangeri)as a novel laboratory disease animal model[J]. Zool Res,2017,38(3):127.
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