【摘要】：目的純化H1N1流感病毒作為抗原免疫小鼠制備單克隆抗體(mAb),并以mAb為工具分析病毒對(duì)宿主細(xì)胞的感染情況。方法雞胚培養(yǎng)A/PR/8(H1N1),收獲尿囊液,蔗糖密度梯度離心純化病毒,透射電鏡鑒定病毒顆粒,甲醛滅活病毒后免疫小鼠,評(píng)價(jià)抗原免疫效果,取鼠脾細(xì)胞與Sp2/0細(xì)胞融合制備mAb。用ELISA、免疫熒光細(xì)胞化學(xué)染色、Western blot法、血凝抑制試驗(yàn)和微量中和試驗(yàn)對(duì)mAb特性進(jìn)行鑒定。依據(jù)流式細(xì)胞術(shù)用血凝素mAb分析流感病毒感染MDCK細(xì)胞后血凝素蛋白在宿主細(xì)胞膜上展示特征和病毒感染對(duì)細(xì)胞凋亡的影響。利用血凝素mAb建立基于細(xì)胞的ELISA(Cell-based ELISA)并對(duì)病毒增殖和感染動(dòng)態(tài)進(jìn)行分析。結(jié)果純化出的A/PR/8病毒在電鏡下呈圓形、橢圓形和棒狀,免疫小鼠血清中流感病毒IgG滴度動(dòng)態(tài)增長(zhǎng),免疫6周后IgG滴度達(dá)106。免疫小鼠脾細(xì)胞與Sp2/0細(xì)胞融合制備出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,分別為1∶2048和1∶640。免疫熒光細(xì)胞化學(xué)染色和Western blot結(jié)果表明該mAb可與血凝素結(jié)合。流式細(xì)胞術(shù)顯示PR8-10也可識(shí)別細(xì)胞膜上的血凝素,同時(shí)發(fā)現(xiàn)病毒感染細(xì)胞后會(huì)引起細(xì)胞凋亡。依據(jù)PR8-10可識(shí)別細(xì)胞膜上的血凝素原理建立的Cell-based ELISA可分析病毒增殖情況。結(jié)論純化出完整病毒顆粒免疫小鼠可刺激產(chǎn)生抗病毒IgG,并制備出高親和活性和中和活性的H1N1病毒mAb,該mAb可分析病毒感染特征以及病毒對(duì)細(xì)胞的影響。
[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，

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