人巨细胞病毒US31蛋白特异性抗体的制备及鉴定
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摘要
目的:探讨人巨细胞病毒(HCMV)US31蛋白及其多克隆抗体的制备及鉴定方法。方法:HCMV US31基因经原核密码子优化后进行全基因合成,克隆至pET21a(+)原核表达载体,构建pET21a(+)/HCMV US31重组质粒,测序鉴定;将重组质粒转化大肠杆菌BL21(DE3),IPTG诱导目的蛋白表达,Ni-NTA亲和层析法纯化US31蛋白,免疫新西兰兔制备多克隆抗体,ELISA、Western blot及免疫荧光方法检测抗体效价和特异性。结果:在大肠杆菌中诱导出高表达的HCMV US31融合蛋白,经Ni-NTA亲和纯化后获得高纯度的目的蛋白;免疫新西兰兔诱导产生特异性的IgG抗体,效价为1:49 600;经ELISA、Western blot以及免疫荧光均证实制备的特异性抗体可识别细胞中表达的US31蛋白。结论:成功制备了HCMV US31蛋白及特异性抗体,为进一步研究US31蛋白的生物学功能奠定了基础。
Objective: To explore the preparation and identification of human cytomegalovirus(HCMV)US31 protein and its polyclonal antibody. Methods: After HCMV US31 gene was optimized by prokaryotic codon for whole gene synthesis and cloned to pET21 a(+) prokaryotic expression vector, pET21 a(+)/HCMV US31 recombinant plasmid was constructed, with identified sequence. Recombinant plasmid was transformed into Escherichia coli BL21(DE3), target protein expression induced by IPTG, and US31 protein purified by NiNTA affinity chromatography. Immunized New Zealand rabbits were used to prepare polyclonal antibodies, and antibody titer and specificity was detected by ELISA, Western blot and immunofluorescence method. Results:High expression of HCMV US31 fusion protein was induced in Escherichia coli, and high purity target protein was obtained by nickel column affinity purification, and the specific IgG antibody was induced by immune New Zealand rabbits, with the valence being 1:49 600. ELISA, Western blot and immunofluorescence have confirmed that the prepared specific antibodies were able to identify US31 proteins expressed in cells. Conclusion: HCMV US31 protein and specific antibodies were successfully prepared, which laid foundations for further study of the biological function of US31 protein.
Objective: To explore the preparation and identification of human cytomegalovirus(HCMV)US31 protein and its polyclonal antibody. Methods: After HCMV US31 gene was optimized by prokaryotic codon for whole gene synthesis and cloned to pET21 a(+) prokaryotic expression vector, pET21 a(+)/HCMV US31 recombinant plasmid was constructed, with identified sequence. Recombinant plasmid was transformed into Escherichia coli BL21(DE3), target protein expression induced by IPTG, and US31 protein purified by NiNTA affinity chromatography. Immunized New Zealand rabbits were used to prepare polyclonal antibodies, and antibody titer and specificity was detected by ELISA, Western blot and immunofluorescence method. Results:High expression of HCMV US31 fusion protein was induced in Escherichia coli, and high purity target protein was obtained by nickel column affinity purification, and the specific IgG antibody was induced by immune New Zealand rabbits, with the valence being 1:49 600. ELISA, Western blot and immunofluorescence have confirmed that the prepared specific antibodies were able to identify US31 proteins expressed in cells. Conclusion: HCMV US31 protein and specific antibodies were successfully prepared, which laid foundations for further study of the biological function of US31 protein.
引文
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[11]余晓霞,刘华峰,陈孝文,等.EBV LMP1和CMVpp65在肾脏疾病肾组织中的表达[J].临床与实验病理学杂志,2011,27(3):257-260.
[2]GUO G,YE S,XIE S,et al.The cytomegalovirus protein US31 induces inflammation through mono-macrophages in systemic lupus erythematosus by promoting NF-κB2 activation[J].Cell Death Dis,2018,9(2):104.
[3]李建蓉,黄汉菊.HCMV基因及其产物在感染中的作用[J].国外医学(病毒学分册),2005,12(6):175-178.
[4]BOECKH M,LJUNGMAN P.How we treat cytomegalovirus in hematopoietic cell transplant recipients[J].Blood,2009,113(23):5711-5719.
[5]PIGNATELLI S,DAL MONTE P,ROSSINI G,et al.Genetic polymorphisms among human cytomegalovirus(HCMV)wild-type strains[J].Rev Med Virol,2004,14(6):383-410.
[6]魏中南,胡洪波,彭巧英.人巨细胞病毒AD169株UL115基因编码gL蛋白序列分析及B细胞表位预测[J].临床血液学杂志(输血与检验版),2012,25(8):505-507.
[7]何雨.高危人群HCMV和BKV感染情况的调查及其与肾病关系的研究[D].南宁:广西医科大学,2010.
[8]BATE S L,DOLLARD S C,CONNON M J.Cytomegalovirus seroprevalence in the United States:the national health and nutrition examination surveys[J].Clin Infect Dis,2010,50(11):1439-1447.
[9]ENGSTRAND M,TOURNAY C,PEYRAT M A,et al.Characterization of CMVpp65-specific CD8+T lymphocytes using MHC tetramers in kidney transplant patients and healthy participants[J].Transplantation,2000,69(11):2243-2250.
[10]王羽,廖翔,岳俊杰,等.福氏志贺菌5a型M90T株GAPDH蛋白多克隆抗体的制备和鉴定[J].细胞与分子免疫学杂志,2014,30(4):407-410.
[11]余晓霞,刘华峰,陈孝文,等.EBV LMP1和CMVpp65在肾脏疾病肾组织中的表达[J].临床与实验病理学杂志,2011,27(3):257-260.