ev病毒c亚型重组病毒样.ppt

七、电子显微镜 VLP 样本用0.5%水样乙酸双氧铀负染, 用Philips CM-12S 放射电子显微镜观察。 Figure 4. Electron microscopy of EV71 VLPs. Partially purified VLPs were negatively stained with 0.5% aqueous uranyl acetate and visualized by transmission electron microscopy. Bar = 50 nm. 八、VLP 免疫小鼠诱导出抗EV71特异的抗体反应 免疫之前, EV71 VLPs 与同样制备的阴性对照 (Sf9) 抗原与明矾(Pierce, Rockford, IL, USA)根据厂商说明，以体积比1:1混合，混合物含有VLPs (相当于 5 mgVP0) 或者 Sf9阴性溶解产物. 每组5只雌性 Balb/c 小鼠(6–8 weeks old) 以 0, 2 和5周次腹腔注射。每次免疫之前，收集血清样本， 在第七周老鼠被处死。血清保存在-80 ℃备用。 九、抗体测定： 用间接ELISA测定血清样本中抗-EV71抗体的反应性。 包被：灭活的EV71 (10 ng/孔)包被96-孔ELISA反应板 37℃3 h. 封闭：用 5% 奶粉 PBST 孵育 37 ℃ 1h. 加血清：用1%奶粉PBST 稀释血清样本37℃ 2h。 二抗：HRP连接抗小鼠IgG 抗体 37 ℃ 1 h。 显色： 显色，450 nm测量OD值 。 Figure 5. Antibody responses following VLP immunization.Groups of five mice were injected i.p. at weeks 0, 2 and 5, with alumabsorbed antigens: EV71-VLP equivalent to 5 mg VP0, or the control lysate similarly prepared from uninfected Sf9 cells. The immunized mice were sacrificed at week 7 (2 weeks after the last immunization), and the serum, bone marrow and spleen samples were collected and assayed.Results are representative of two independent experiments. (A) EV71-specific serum IgG titers of the Sf9 and the VLP groups. Each symbol represents a mouse, and the line indicates the geometric mean value ofthe group. 十、斑点ELISA分析 在最后免疫之后的第三周，收集三只免疫小鼠的脾脏和骨髓，做B细胞ELISPOT。 分离脾细胞和骨髓细胞, 浓缩、计数。96孔PVDF板 (Millipore, Billerica, MA, USA)用纯化灭活的EV71以100 ng/孔 预先包被，4℃孵育过夜。 PVDF 板用完全RPMI 1640培养基37℃封闭2h 。 新鲜分离的脾细胞和骨髓细胞(1×105/孔) 在5% CO2中 37℃孵育40h 。用1% FBS 和 0.05%Tween-20的 PBS缓冲液稀释生物素化的山羊抗-小鼠IgG，以0.1 ug/孔加入到 PVDF板中，37℃ 2h。 然后用PBS以1:1000稀释碱性磷酸酶链接的链霉亲和素 37℃ 1h。 用水冲洗6次, 加NBT/BCIP酶底物100 ul/孔孵育20min 显色。 用水冲洗终止显色，然后晾干。 抗体分泌细胞的斑点用CTL免疫斑点器成像计数 Figure 5. Antibody responses following VLP immunization. (B) ELISPOT analysis of EV71-specific antibody-secreting cells in mouse bone marrow. Samples from na?¨ve mice were used as the control. Results of triplicate wells are shown. (C) ELISPOT analysis of EV71-specific antibody-secreting cells in mouse