抗鸭乙肝病毒core蛋白单克隆抗体的制备与鉴定

发布时间：2018-04-16 02:24

本文选题：鸭乙肝病毒 + 核心蛋白　；参考：《西安交通大学学报(医学版)》2013年06期

【摘要】：目的制备抗鸭乙肝病毒core蛋白的单克隆抗体并进行鉴定。方法 PCR扩增鸭乙肝病毒(DHBV)core基因片段,构建表达载体pET28a(+)/DHBV core。转化诱导表达融合蛋白,用Ni 2+亲和柱纯化目的蛋白。免疫Balb/c小鼠,取脾细胞与SP2/0骨髓瘤细胞融合,ELISA筛选,采用有限稀释法筛选单克隆杂交瘤细胞并进行细胞克隆。体内诱生法大量制备单克隆抗体,进行抗体的特异性、效价和亚型的鉴定。结果成功构建表达载体pET28a(+)/DHBV core,并表达DHBV core蛋白。获得2株稳定分泌抗DHBV core抗体的杂交瘤细胞株,分别为2D7和5G10,细胞培养液抗体效价为1∶400和1∶800。选择5G10细胞株制备单克隆抗体,小鼠腹水抗体效价可达1∶320 000。鸭肝组织免疫组化结果显示,DHBV病毒载量1010copies/mg的肝组织中DHBV core蛋白的表达明显高于病毒载量105copies/mg的肝组织,而未感染DHBV的鸭肝细胞内不表达DHBV core蛋白。亚型鉴定结果为IgG2aκ链。结论制备并获得了抗DHBV core蛋白的单克隆抗体,为DHBV core蛋白的功能研究、诊断试剂的研制与抗病毒治疗研究奠定了基础。
[Abstract]:Objective to prepare and identify monoclonal antibodies against duck hepatitis B virus (core) protein.Methods DHBV core gene fragment was amplified by PCR and the expression vector pET28a was constructed.The fusion protein was expressed and purified by Ni 2 affinity column.Balb/c mice were immunized. Spleen cells were fused with SP2/0 myeloma cells to be screened by Elisa. Monoclonal hybridoma cells were screened by limited dilution method and cloned.Monoclonal antibodies were prepared in vivo to identify their specificity, titer and subtype.Results the expression vector pET28a was successfully constructed and the DHBV core protein was expressed.Two hybridoma cell lines stably secreting anti- core antibody were obtained, which were 2D7 and 5G10, respectively. The titers of antibody in cell culture medium were 1: 400 and 1: 800, respectively.5G10 cell line was selected to prepare monoclonal antibody. The titer of mouse ascites antibody was 1: 320 000.The results of immunohistochemistry showed that the expression of DHBV core protein was significantly higher in the liver tissue with 1010copies/mg viral load than in the liver tissue with the viral load of 105copies/mg, but the DHBV core protein was not expressed in the duck liver cells without DHBV infection.The subtype was identified as IgG2a 魏 chain.Conclusion Monoclonal antibodies against DHBV core protein were prepared and obtained, which laid a foundation for the study of the function of DHBV core protein, the development of diagnostic reagent and antiviral therapy.
【作者单位】：西安交通大学医学院第一附属医院检验科;西安交通大学医学院第一附属医院传染科;西安华广生物工程有限公司;
【基金】：国家自然科学基金资助项目(No.81101260)~~
【分类号】：Q78

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