课件：病毒亚型重组病毒样.ppt

十二、肽ELISA 用含有VP1整个区的58个合成肽镶嵌板做ELISA检测抗VLP抗血清的反应性。 抗血清显著地与43#肽 (FGEHKQEKDLEYGAC)反应,与以前鉴定的SP70 表位(YPTFGEHKQEKDLEY)重叠。43 # 肽定位于 VP1 蛋白的GH环。 Figure 7. Neutralizing antibodies in the anti-VLP sera predominantly target an epitope located in the GH loop of VP1 protein. (A) Mapping of immunodominant B-cell linear epitopes within the VP1 protein. The pooled anti-VLP sera were analyzed by indirect ELISA for reactivity with a panel of 58 synthetic peptides covering the entire sequences of VP1. 为了鉴定这个表位在产生中和抗体中的作用。43#肽以不同浓度与中和抗体混合做中和实验，43#肽以剂量依赖的方式降低了中和抗体的中和作用，而对照HIV肽没有干扰中和作用。证明中和抗体的目标表位在43#肽序列。 (B) Neutralization-inhibition by #43 peptide.The anti-VLP sera were incubated with #43 peptide or a negative control peptide (from HIV) at different concentrations for 1 hour at 37℃. The peptide/antiserum mixtures were subjected to neutralization testing. Neutralization-inhibition by the peptides was determined using an MTT method. Data are means+D of the OD490 readings of triplicate wells. 十四、吸附前试验 抗血清用含有2%FBS的DMEM培养基系列稀释, 8ul稀释的血清与对照培养基加入到400ul含有9×106TCID50的EV71基因亚型C4中，37孵育1h。 混合物加入到3.5×105/孔的RD细胞和Vero细胞的12孔板中。4孵育1h。 细胞用冷PBS冲洗三次。 收集细胞，用抗-EV71和抗-actin的多克隆抗体做western blotting. Figure 8. Inhibitory effect of the antisera on EV71 attachment to cells. Antisera at different dilutions (1:50, 1:500, and 1:5,000) were mixed with 400 ml of EV71 containing 9×106 TCID50, and incubated for 1 hour at 37 ℃. The mixtures were added to (A) RD or (B) Vero cells for incubation for 1 hour at 4 ℃ to allow virus attachment to the cells. After incubation, the cells were washed with cold PBS three times, collected, and subjected to Western blotting with a polyclonal antibody against EV71 VP1. For loading control, an antibody against actin was also used in Western blotting. 十五、吸附后试验 EV71病毒用含有2%DMEM的培养基稀释成2TCID50/ul, 稀释的病毒与对照培养基以50ul/孔加入到含有1.5×106/孔RD细胞的96孔板中，4℃孵育1h。 细胞用DMEM冲洗，洗去未结合的病毒。 加入用50ul/孔系列稀释的血清，37℃培育1h，细胞用DMEM冲洗3次，然后加入100ulDMEM。三天后观察CPE. Figure 9. Postattachment neutralization by the anti-VLP sera.