ev病毒c亚型重组病毒样.ppt

EV71病毒C4亚型重组病毒样颗粒诱导的中和抗体抑制吸附前及吸附后的感染。,研究生：王新刚 导 师：孟继鸿教授,前言：,EV71是小RNA病毒家族中的肠道病毒属，拥有大约7.4kb的单正链RNA，正二十面体衣壳。 病毒基因组 非结构基因P2和P3区 结构基因P1区 VP1、VP0、VP3。 VP2和VP4。,病毒蛋白酶3CD,空病毒衣壳,EV71是手足口病的病原体，手足口病发生于5岁以内的儿童，过去几年里，在远东地区如，中国、越南、韩国呈暴发流行，引起严重的发病率和死亡率。 感染EV71的个体常常显示轻微的症状，如在手、足及背臀部的水泡和发烧。一部分EV71感染者发展成严重的神经系统并发症，包括小儿麻痹症样瘫痪、脑干脑炎和肺水肿，最后导致死亡。,没有特异的疫苗能够用于阻止EV71的感染。 EV71分为三个基因型(A, B and C)，进一步分为11个基因亚型(A,B1B5, and C1C5)。 因此，理想的EV71疫苗能够阻止大部分基因型感染。 在所有研究EV71的疫苗中，病毒样颗粒(VLP)是最有前途的疫苗。在产量和安全方面都优于灭活疫苗。,Chung YC, Ho MS等用重组EV71病毒C2亚型病毒样颗粒能够在小鼠和猕猴中诱导产生高滴度的抗体。 用EV71VLP免疫的雌鼠能够通过被动免疫给予新生小鼠免受EV71致死性感染。证明中和抗体在免疫防御中起了重要作用。 然而，抗VLP抗血清在体外如何中和EV71病毒还不清楚。,现在的研究是： 用杆状病毒/昆虫细胞表达系统生产EV71病毒C4亚型的重组VLP。 评价VLP在小鼠体内诱导中和抗体的能力。 阐明抗VLP抗血清中和EV71的机制。,材料与方法：,一、细胞与病毒： 实验中用的RD 与 Vero 细胞（Ku Z, Shi J, Liu Q, Huang Z (2012) Development of murine monoclonal antibodies with potent neutralization effects on enterovirus 71.）， EV71 病毒C4亚型在RD细胞中增殖。根据ReedMuench 的方法以TCID50在RD细胞上滴定其浓度。纯化，灭活的病毒有hualan（中国河南）获得。,二、衣壳亚单位蛋白特异性多克隆抗体 抗-VP0豚鼠多抗 抗-VP3多抗用与EV71有交叉反应的重组CAV16的VP3蛋白免疫豚鼠获得，用于检测EV71的VP3蛋白。 抗-VP1多抗用大肠杆菌表达的重组EV71的VP1蛋白免疫兔子产生。,三、载体构建 从感染EV71病毒C4亚型的RD细胞抽提病毒RNA，随后用脱氧核酸引物和M-MLV反转录酶进行反转录。用cDNA作模板，扩增编码P1与3CD基因片段。 P1片段的扩增 引物 (forward 5,AATCCATGGGTTCGCAGGTGTCT-3, and reverse 5,- GACAAGCTTTCAAAGAGTAGTGATCGC-3,),用NcoI and HindIII酶切，然后插入到质粒pIExBac-1，成为pIExBac-P1,3CD 片段的扩增引物(forward 5,-AATCCATGGGCCCGAGCCTTGATTTT-3, and reverse 5,-GACAAGCTTTCAAAATAACTCGAGCC-3,), 用NcoI and HindIII酶切, 然后在同样位点插入到质粒pIExBac-1 成为pIExBac-3CD.,Figure 1. Diagrams of the constructs used in this study. lef2, the left sequence for homologous recombination; hr5-Enh, AcNPV hr5 enhancer;IE1-P, IE1 immediate early promoter; p10-P, p10 promoter; IE1-T, IE1 terminator; 1629, the right sequence for homologous recombination.,四、重组杆状病毒的产生。 重组载体pIExBac-P1 和 pIExBac-3CD，经过转染，产生，选择和重组病毒的增殖。分别产生相应的重组病毒，也就是IExBac-P1 和 IExBac-3CD。Sf9细胞用重组杆状病毒感染，感染后72小时，收集感染的细胞和上清，用于分析。,五、用ELISA 和 Western Blot分析表达的蛋白 杆状病毒感染的Sf9 cells 用 TrisNaCl buffer 12,000 rpm 离心5 min获得蛋白溶解产物. （一）间接ELISA： 包被：5 ul 蛋白产物用50 ul PBS 稀释,包被96孔酶标板4 12 h;用PBST (1PBS ，0.05%Tween 20)洗涤三次. 封闭：用5%牛奶PBST 200 ul/孔 37 1 h 一抗：第一抗体(豚鼠 anti-VP0抗血清,兔anti-VP1 抗血清 或者 豚鼠 anti-VP3 抗血清) 用1% 奶粉的 PBST 以1:1000 稀释，以50 ul/孔37 2 h。,二抗：标记的第二抗体 (1% 奶粉PBST 以 1:3000 稀释) 以 50 ul/孔 37 1h. 显色： TMB 混合物以 50ul/孔 510 min; 然后 50ul/ 1 N H3PO4终止. 450 nm测吸光度。 （二）Western blotting, 以12%聚丙烯酰胺凝胶分离蛋白然后转移至PVDF膜. 用抗原特异一抗结合抗原，HRP-连接的第二抗体.用BeyoECL Plus kit (Beyotime,Shanghai, China) 的化学发光试剂显示膜上的阳性标本，用 LAS-4000 冷光成像分析器记录。,Figure 2. Coexpression of P1 and 3CD of EV71 in insect cells. Lysates from the baculovirus-infected Sf9 cells were analyzed by ELISA with (A) anti-VP0, (B) anti-VP1, or (C) anti-VP3 polyclonal antibodies,(D) by Western blotting with the anti-VP0 polyclonal antibody. Sf9,mock-infected Sf9 cell lysate; P1, lysate from the IExBac-P1 infected cells; 3CD, lysate from the IExBac-3CD infected cells; P1+3CD, lysate from cells infected with both IExBac-P1 and IExBac-3CD. Error bars represent SE.,六、VLPs与对照抗原的制备 To evaluate VLP assembly, the P1/3CD co-infected cell lysate was subjected to sucrose gradient sedimentation 杆状病毒感染 Sf9 的溶解产物在20%的蔗糖缓冲液中以 25,000 rpm超速离心 6 h。 合成的球状物用PBS重悬，然后分层于1050% 之上39,000rpm超速离心3h。 从顶到底的片段用SDS-PAGE 户Western blotting分析，富含VLP的层用PBS浓缩、透析，然后在20%蔗糖缓冲液中超速离心。VLPs用PBS重悬，然后用于动物免疫。用Western blotting 检测VP0含量作为参考标准定量VLPs。 未感染的Sf9溶解物如同纯化VLP一样处理，作为阴性对照抗原，用于免疫。,Figure 3. Sucrose gradient analysis. Lysates were layered onto 10 50% sucrose gradients for ultracentrifugation. Ten fractions were taken from top to bottom. (A) SDS-PAGE analysis. The ten fractions were separated by SDS-PAGE, and subsequently stained with Coomassie Blue dye. M, protein marker. EV, purified whole virion EV71 standard from Hualan Inc.,(B) Western blotting with anti-VP0 polyclonal antibody. (C) Western blotting with anti-VP1 polyclonal antibody. (D) Western blotting with anti-VP3 polyclonal antibody.,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(68 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 373 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(1105/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 nave mice were used as the control. Results of triplicate wells are shown. (C) ELISPOT analysis of EV71-specific antibody-secreting cells in mouse spleens. Samples from nave mice were used as the control. Results of triplicate wells are shown.,十一、中和能力 血清样本用 含有2% FBS 的DMEM培养基以2倍倍比稀释。 EV71稀释到 2 TCID50/ul的工作浓度。 中和试验在96孔板中进行，50ul 稀释的抗血清与50ul 含有100 TCID50 的EV71混合加入到每孔中371 h。 然后，100ul中含有 15,000 RD细胞的细胞悬液 was 加入到含有病毒与抗血清混合物的孔中， 5% CO2中 37 培养。 3天之后观察并评价CPE。 能够完全保护细胞没有出现CPE的最高血清稀释度为中和作用的滴度。,Figure 6. VLP-immunized sera efficiently neutralized EV71 infection in vitro. Neutralization titers of the antisera were determined by TCID50 reduction assay. The antisera of the Sf9 group did not show any neutralization at 1:32 (the lowest dilution tested) and were therefore assigned a titer of 16 for GMT computation. Each symbol represents a mouse, and the line indicates the GMT of the group. Results are representative of two independent experiments,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9106TCID50的EV71基因亚型C4中，37孵育1h。 混合物加入到3.5105/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 9106 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