人巨細(xì)胞病毒糖蛋白B單克隆抗體制備、鑒定及初步應(yīng)用
發(fā)布時(shí)間：2018-12-29 13:30
【摘要】：研究背景人巨細(xì)胞病毒(HCMV)在人群中普遍感染,對(duì)于免疫系統(tǒng)發(fā)育不全和免疫抑制患者可引起嚴(yán)重并發(fā)癥。包膜糖蛋白B(gB)是參與感染過程的關(guān)鍵蛋白,與病毒粘附、穿入宿主細(xì)胞及細(xì)胞間融合、傳播密切相關(guān)；是HCMV中和抗體的主要靶蛋白,亦是疫苗研究的主要候選蛋白。研究HCMV-gB及其抗體對(duì)闡明HCMV致病機(jī)制及疫苗的研制有重要價(jià)值。本課題通過篩選和合成gB中具優(yōu)勢(shì)免疫原性的氨基酸短序列,并制備單克隆抗體,為HCMV-gB功能學(xué)研究,闡明HCMV致病機(jī)制和疫苗的研制打下基礎(chǔ)。 實(shí)驗(yàn)?zāi)康脑O(shè)計(jì)含優(yōu)勢(shì)免疫原性表位的HCMV gB多肽片段；獲得特異的gB單克隆抗體；將單克隆抗體初步應(yīng)用于gB抗原檢測(cè)。 實(shí)驗(yàn)方法以GenBank公布的Towne株、AD169株序列為基礎(chǔ),篩選并合成兩條gB多肽(STRGTSATHSHHSS和SHATSSTHNGSHTS);免疫Balb/c小鼠,應(yīng)用雜交瘤融合技術(shù)制備出gB單克隆抗體；采用ELISA、免疫細(xì)胞化學(xué)染色法、免疫印跡、免疫沉淀等方法鑒定抗體性質(zhì)。將制備的單克隆抗體應(yīng)用于外周血白細(xì)胞樣本的gB抗原檢測(cè)。 實(shí)驗(yàn)結(jié)果制備得到單克隆抗體ZJU-FH6STRGTSATHSHHSS和ZJU-FE6SHATSSTHNGSHTS,分別為IgGl-K和IgG2a-κ抗體亞型。經(jīng)間接ELISA法鑒定,ZJU-FH6效價(jià)為1：60000(初始濃度4mg/mL); ZJU-FE6效價(jià)為1：240000(初始濃度1.2mg/mL)。免疫印跡和免疫沉淀顯示ZJU-FH6和ZJU-FE6,均可識(shí)別變性的和天然的HCMVgB抗原。HCMV感染MRC-5的免疫細(xì)胞化學(xué)染色鑒定,ZJU-FH6和ZJU-FE6可檢測(cè)Towne株和AD169株感染細(xì)胞的gB抗原。以制備的單克隆抗體檢測(cè)Allo-HSCT受者PBLs中的gB抗原,陽(yáng)性率分別為79.1%和84.6%,平均陽(yáng)性細(xì)胞數(shù)分別為6.20/5×104WBC和6.88/5×104WBC,均無明顯的統(tǒng)計(jì)學(xué)差異(P0.05)。單克隆抗體ZJU-FH6和ZJU-FE6檢測(cè)gB抗原陽(yáng)性的Allo-HSCT受者,隨后3個(gè)月的HCMV IE、pp65、IgG平均值均較之前3個(gè)月平均值有統(tǒng)計(jì)學(xué)意義下降(P0.05)。 結(jié)論本研究成功制備了gB單克隆抗體ZJU-FH6和ZJU-FE6;可應(yīng)用于Allo-HSCT受者的gB抗原檢測(cè),本課題制備的單克隆抗體所針對(duì)的抗原肽可能與機(jī)體產(chǎn)生相關(guān)抗體有一定關(guān)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392
本文編號(hào)：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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392
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