人博卡病毒VP2蛋白的纯化及其单克隆抗体的制备

发布时间：2019-04-24 02:05

【摘要】：病毒感染所引起的呼吸道疾病已经成为世界性的常见疾病之一。人博卡病毒(HBoV)作为引起呼吸道感染的常见病毒之一,虽然发现相对较晚,但受到科研工作者的广泛关注。而有效的诊断和治疗需要具有高效价的抗体,才能建立系统的方法,单克隆抗体是病原检测和诊断的主要工具。目前,有关HBoV单克隆抗体制备的研究很少,因此,HBoV单克隆抗体的制备对于HBoV的诊断和所引起的疾病的治疗有重要意义。本研究利用强大的原核表达载体pET-30a构建重组质粒,通过大肠杆菌表达系统表达重组VP2蛋白,通过柱上变复性纯化目的蛋白。利用血清检查抗原。以纯化蛋白作为抗原,免疫小鼠,利用杂交瘤技术及间接ELISA方法筛选阳性杂交瘤细胞,小鼠体内诱导产生单克隆抗体,并对单克隆抗体进行鉴定。结果表明,构建出HBoVVP2基因的重组质粒,表达并纯化得到了重组HBoVVP2蛋白,利用呼吸道感染患者血清检测抗原,阳性率为77.7%,与国内外检出率基本一致,说明抗原较好。利用杂交瘤技术筛选得到了稳定细胞株,该细胞株可以分泌单克隆抗体。利用该株细胞在小鼠腹部诱导产生具有单克隆抗体的腹水,根据检测和鉴定,所产生的单克隆抗体为IgG抗体,具有较好抗原抗体结合能力,效价达到1：4×105。本实验得到的单克隆抗体,有益于对HBoV的深入研究。该研究结果对HBoV诊断、治疗和进一步研究奠定基础。
[Abstract]:Respiratory diseases caused by viral infection have become one of the most common diseases in the world. Human Boca virus (HBoV), one of the common viruses causing respiratory tract infection, has been paid more and more attention by researchers although it was found relatively late. Effective diagnosis and treatment requires high titer antibodies to establish a systematic method. Monoclonal antibodies are the main tools for the detection and diagnosis of pathogens. At present, there are few studies on the preparation of monoclonal antibodies against HBoV. Therefore, the preparation of monoclonal antibodies against HBoV is of great significance for the diagnosis of HBoV and the treatment of diseases caused by it. In this study, a powerful prokaryotic expression vector pET-30a was used to construct the recombinant plasmid, and the recombinant VP2 protein was expressed by E. coli expression system. The target protein was purified by renaturation on the column. Use the serum to test the antigen. The purified protein was used as antigen to immunize mice. The positive hybridoma cells were screened by hybridoma technique and indirect ELISA method. The monoclonal antibodies were induced to produce monoclonal antibodies in mice and the monoclonal antibodies were identified. The results showed that the recombinant plasmid of HBoVVP2 gene was constructed, and the recombinant HBoVVP2 protein was expressed and purified. The positive rate of the recombinant HBoVVP2 protein was 77. 7% by using the sera of patients with respiratory tract infection, which was basically consistent with the detection rate at home and abroad, indicating that the antigen was better. Stable cell lines were obtained by hybridoma technique, which could secrete monoclonal antibodies. The cell strain was used to induce ascites with monoclonal antibody in the abdomen of mice. According to the detection and identification, the monoclonal antibody produced was IgG antibody, which had good antigen-antibody binding ability, and the titer was up to 1:4 脳 10 ~ 5. The monoclonal antibodies obtained in this experiment are beneficial to the in-depth study of HBoV. The results of this study lay the foundation for the diagnosis, treatment and further study of HBoV.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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