人巨细胞病毒糖蛋白B单克隆抗体制备、鉴定及初步应用
发布时间：2018-12-29 13:30
【摘要】：研究背景人巨细胞病毒(HCMV)在人群中普遍感染,对于免疫系统发育不全和免疫抑制患者可引起严重并发症。包膜糖蛋白B(gB)是参与感染过程的关键蛋白,与病毒粘附、穿入宿主细胞及细胞间融合、传播密切相关；是HCMV中和抗体的主要靶蛋白,亦是疫苗研究的主要候选蛋白。研究HCMV-gB及其抗体对阐明HCMV致病机制及疫苗的研制有重要价值。本课题通过筛选和合成gB中具优势免疫原性的氨基酸短序列,并制备单克隆抗体,为HCMV-gB功能学研究,阐明HCMV致病机制和疫苗的研制打下基础。 实验目的设计含优势免疫原性表位的HCMV gB多肽片段；获得特异的gB单克隆抗体；将单克隆抗体初步应用于gB抗原检测。 实验方法以GenBank公布的Towne株、AD169株序列为基础,筛选并合成两条gB多肽(STRGTSATHSHHSS和SHATSSTHNGSHTS);免疫Balb/c小鼠,应用杂交瘤融合技术制备出gB单克隆抗体；采用ELISA、免疫细胞化学染色法、免疫印迹、免疫沉淀等方法鉴定抗体性质。将制备的单克隆抗体应用于外周血白细胞样本的gB抗原检测。 实验结果制备得到单克隆抗体ZJU-FH6STRGTSATHSHHSS和ZJU-FE6SHATSSTHNGSHTS,分别为IgGl-K和IgG2a-κ抗体亚型。经间接ELISA法鉴定,ZJU-FH6效价为1：60000(初始浓度4mg/mL); ZJU-FE6效价为1：240000(初始浓度1.2mg/mL)。免疫印迹和免疫沉淀显示ZJU-FH6和ZJU-FE6,均可识别变性的和天然的HCMVgB抗原。HCMV感染MRC-5的免疫细胞化学染色鉴定,ZJU-FH6和ZJU-FE6可检测Towne株和AD169株感染细胞的gB抗原。以制备的单克隆抗体检测Allo-HSCT受者PBLs中的gB抗原,阳性率分别为79.1%和84.6%,平均阳性细胞数分别为6.20/5×104WBC和6.88/5×104WBC,均无明显的统计学差异(P0.05)。单克隆抗体ZJU-FH6和ZJU-FE6检测gB抗原阳性的Allo-HSCT受者,随后3个月的HCMV IE、pp65、IgG平均值均较之前3个月平均值有统计学意义下降(P0.05)。 结论本研究成功制备了gB单克隆抗体ZJU-FH6和ZJU-FE6;可应用于Allo-HSCT受者的gB抗原检测,本课题制备的单克隆抗体所针对的抗原肽可能与机体产生相关抗体有一定关系。
[Abstract]:Background Human cytomegalovirus (HCMV) (HCMV) infection is common in the population and can cause serious complications in patients with hypoplasia and immunosuppression. Envelope glycoprotein (B (gB) is a key protein involved in the process of infection. It is closely related to virus adhesion, penetration into host cells and intercellular fusion and transmission. It is the main target protein of HCMV neutralizing antibody and the main candidate protein of vaccine research. The study of HCMV-gB and its antibodies is of great value in elucidating the pathogenesis of HCMV and the development of vaccine. By screening and synthesizing short amino acid sequences with dominant immunogenicity in gB and preparing monoclonal antibodies, this study will lay a foundation for the study of HCMV-gB function and the development of HCMV vaccine. Objective to design a HCMV gB polypeptide fragment containing dominant immunogenicity epitopes, to obtain a specific monoclonal antibody against gB, and to apply it to the detection of gB antigen. Methods based on the sequence of Towne strain and AD169 strain published by GenBank, two gB polypeptides (STRGTSATHSHHSS and SHATSSTHNGSHTS);) were selected and synthesized to immunize Balb/c mice. Monoclonal antibodies to gB were prepared by hybridoma fusion technique. ELISA, immunocytochemical staining, immunoblotting and immunoprecipitation were used to identify the antibody properties. The prepared monoclonal antibody was applied to the detection of gB antigen in peripheral blood leukocyte samples. The results showed that the monoclonal antibodies ZJU-FH6STRGTSATHSHHSS and ZJU-FE6SHATSSTHNGSHTS, were IgGl-K and IgG2a- 魏 antibody subtypes, respectively. The titer of ZJU-FH6 was 1: 60000 (initial concentration 4mg/mL) and that of ZJU-FE6 was 1: 240000 (initial concentration 1.2mg/mL) by indirect ELISA method. Immunoblotting and immunoprecipitation showed that both ZJU-FH6 and ZJU-FE6, could recognize the denatured and natural HCMVgB antigens. ZJU-FH6 and ZJU-FE6 could be used to detect gB antigens of Towne and AD169 strains infected with MRC-5 by immunocytochemical staining. The positive rates of gB antigen in PBLs of Allo-HSCT recipients were 79.1% and 84.6%, respectively. The average number of positive cells was 6.20 / 5 脳 104WBC and 6.88 / 5 脳 104WBCrespectively (P0.05). The mean value of HCMV IE,pp65,IgG in Allo-HSCT recipients with gB antigen positive detected by monoclonal antibody ZJU-FH6 and ZJU-FE6 was significantly lower than that in the previous 3 months (P0.05). Conclusion the gB monoclonal antibodies ZJU-FH6 and ZJU-FE6; can be used in the detection of gB antigens in Allo-HSCT recipients. The antigenic peptides targeted by the monoclonal antibodies in this study may be related to the production of related antibodies.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
本文编号：2394890
[Abstract]:Background Human cytomegalovirus (HCMV) (HCMV) infection is common in the population and can cause serious complications in patients with hypoplasia and immunosuppression. Envelope glycoprotein (B (gB) is a key protein involved in the process of infection. It is closely related to virus adhesion, penetration into host cells and intercellular fusion and transmission. It is the main target protein of HCMV neutralizing antibody and the main candidate protein of vaccine research. The study of HCMV-gB and its antibodies is of great value in elucidating the pathogenesis of HCMV and the development of vaccine. By screening and synthesizing short amino acid sequences with dominant immunogenicity in gB and preparing monoclonal antibodies, this study will lay a foundation for the study of HCMV-gB function and the development of HCMV vaccine. Objective to design a HCMV gB polypeptide fragment containing dominant immunogenicity epitopes, to obtain a specific monoclonal antibody against gB, and to apply it to the detection of gB antigen. Methods based on the sequence of Towne strain and AD169 strain published by GenBank, two gB polypeptides (STRGTSATHSHHSS and SHATSSTHNGSHTS);) were selected and synthesized to immunize Balb/c mice. Monoclonal antibodies to gB were prepared by hybridoma fusion technique. ELISA, immunocytochemical staining, immunoblotting and immunoprecipitation were used to identify the antibody properties. The prepared monoclonal antibody was applied to the detection of gB antigen in peripheral blood leukocyte samples. The results showed that the monoclonal antibodies ZJU-FH6STRGTSATHSHHSS and ZJU-FE6SHATSSTHNGSHTS, were IgGl-K and IgG2a- 魏 antibody subtypes, respectively. The titer of ZJU-FH6 was 1: 60000 (initial concentration 4mg/mL) and that of ZJU-FE6 was 1: 240000 (initial concentration 1.2mg/mL) by indirect ELISA method. Immunoblotting and immunoprecipitation showed that both ZJU-FH6 and ZJU-FE6, could recognize the denatured and natural HCMVgB antigens. ZJU-FH6 and ZJU-FE6 could be used to detect gB antigens of Towne and AD169 strains infected with MRC-5 by immunocytochemical staining. The positive rates of gB antigen in PBLs of Allo-HSCT recipients were 79.1% and 84.6%, respectively. The average number of positive cells was 6.20 / 5 脳 104WBC and 6.88 / 5 脳 104WBCrespectively (P0.05). The mean value of HCMV IE,pp65,IgG in Allo-HSCT recipients with gB antigen positive detected by monoclonal antibody ZJU-FH6 and ZJU-FE6 was significantly lower than that in the previous 3 months (P0.05). Conclusion the gB monoclonal antibodies ZJU-FH6 and ZJU-FE6; can be used in the detection of gB antigens in Allo-HSCT recipients. The antigenic peptides targeted by the monoclonal antibodies in this study may be related to the production of related antibodies.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
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