H1N1型流感病毒小鼠杂交瘤单克隆抗体的制备和鉴定
发布时间：2019-06-20 21:48
【摘要】：目的纯化H1N1流感病毒作为抗原免疫小鼠制备单克隆抗体(mAb),并以mAb为工具分析病毒对宿主细胞的感染情况。方法鸡胚培养A/PR/8(H1N1),收获尿囊液,蔗糖密度梯度离心纯化病毒,透射电镜鉴定病毒颗粒,甲醛灭活病毒后免疫小鼠,评价抗原免疫效果,取鼠脾细胞与Sp2/0细胞融合制备mAb。用ELISA、免疫荧光细胞化学染色、Western blot法、血凝抑制试验和微量中和试验对mAb特性进行鉴定。依据流式细胞术用血凝素mAb分析流感病毒感染MDCK细胞后血凝素蛋白在宿主细胞膜上展示特征和病毒感染对细胞凋亡的影响。利用血凝素mAb建立基于细胞的ELISA(Cell-based ELISA)并对病毒增殖和感染动态进行分析。结果纯化出的A/PR/8病毒在电镜下呈圆形、椭圆形和棒状,免疫小鼠血清中流感病毒IgG滴度动态增长,免疫6周后IgG滴度达106。免疫小鼠脾细胞与Sp2/0细胞融合制备出6株流感病毒mAb,其中PR8-10mAb血凝抑制活性和中和活性均最高,分别为1∶2048和1∶640。免疫荧光细胞化学染色和Western blot结果表明该mAb可与血凝素结合。流式细胞术显示PR8-10也可识别细胞膜上的血凝素,同时发现病毒感染细胞后会引起细胞凋亡。依据PR8-10可识别细胞膜上的血凝素原理建立的Cell-based ELISA可分析病毒增殖情况。结论纯化出完整病毒颗粒免疫小鼠可刺激产生抗病毒IgG,并制备出高亲和活性和中和活性的H1N1病毒mAb,该mAb可分析病毒感染特征以及病毒对细胞的影响。
[Abstract]:Objective to purify H1N1 influenza virus as antigen to immunize mice to prepare monoclonal antibody (mAb), and to analyze the infection of influenza virus to host cells by mAb. Methods A/PR/8 (H1N1) was cultured in chicken embryo, allantoic fluid was collected, virus was purified by sucrose density gradient centrifugation, virus particles were identified by transmission electron microscope, mice were immunized with formaldehyde inactivated virus, antigen immunization effect was evaluated, and mouse spleen cells were fused with Sp2/0 cells to prepare mAb.. The characteristics of mAb were identified by ELISA, immunofluorescence cytochemical staining, Western blot assay, hemagglutination inhibition test and microneutralization test. According to flow cytometry, hemagglutinin mAb was used to analyze the characteristics of hemagglutinin protein on host cell membrane and the effect of virus infection on apoptosis of MDCK cells infected with influenza virus. Cell-based ELISA (Cell-based ELISA) was established by hemagglutinin mAb and the dynamics of virus proliferation and infection were analyzed. Results the purified A/PR/8 virus was round, oval and rod-shaped under electron microscope. The IgG titer of influenza virus in serum of immunized mice increased dynamically, and the IgG titer reached 106 after 6 weeks of immunization. Six strains of influenza virus mAb, were prepared by fusion of spleen cells and Sp2/0 cells in immunized mice. The hemagglutination inhibitory activity and neutralizing activity of PR8-10mAb were the highest, which were 1 鈮，
本文编号：2503532
[Abstract]:Objective to purify H1N1 influenza virus as antigen to immunize mice to prepare monoclonal antibody (mAb), and to analyze the infection of influenza virus to host cells by mAb. Methods A/PR/8 (H1N1) was cultured in chicken embryo, allantoic fluid was collected, virus was purified by sucrose density gradient centrifugation, virus particles were identified by transmission electron microscope, mice were immunized with formaldehyde inactivated virus, antigen immunization effect was evaluated, and mouse spleen cells were fused with Sp2/0 cells to prepare mAb.. The characteristics of mAb were identified by ELISA, immunofluorescence cytochemical staining, Western blot assay, hemagglutination inhibition test and microneutralization test. According to flow cytometry, hemagglutinin mAb was used to analyze the characteristics of hemagglutinin protein on host cell membrane and the effect of virus infection on apoptosis of MDCK cells infected with influenza virus. Cell-based ELISA (Cell-based ELISA) was established by hemagglutinin mAb and the dynamics of virus proliferation and infection were analyzed. Results the purified A/PR/8 virus was round, oval and rod-shaped under electron microscope. The IgG titer of influenza virus in serum of immunized mice increased dynamically, and the IgG titer reached 106 after 6 weeks of immunization. Six strains of influenza virus mAb, were prepared by fusion of spleen cells and Sp2/0 cells in immunized mice. The hemagglutination inhibitory activity and neutralizing activity of PR8-10mAb were the highest, which were 1 鈮，
本文编号：2503532
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