EV病毒C亚型重组病毒样

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 monoclonalantibodies 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 ELISAwith (A) anti-VP0, (B) anti-VP1, or (C) anti-VP3polyclonal 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 lysatewas 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Westernblotting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 1050% sucrose gradients for ultracentrifugation. Ten fractions were takenfrom top to bottom. (A) SDS-PAGE analysis. The ten fractions wereseparated by SDS-PAGE, and subsequently stained with Coomassie Bluedye. M, protein marker. EV, purified whole virion EV71 standard fromHualan Inc.,(B) Western blotting with anti-VP0 polyclonal antibody. (C)Western blotting with anti-VP1 polyclonal antibody. (D) Westernblotting 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 cellsin mouse bone marrow. Samples from nave mice were used as thecontrol. Results of triplicate wells are shown. (C) ELISPOT analysis ofEV71-specific antibody-secreting cells in mouse spleens. Samples fromnave mice were used as the control. Results of triplicate wells areshown.,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 EV71infection in vitro. Neutralization titers of the antisera were determined by TCID50 reduction assay. The antisera of the Sf9 group did not showany 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 predominantlytarget 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 neutralizationtesting. 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 attachmentto cells. Antisera at different dilutions (1:50, 1:500, and 1:5,000) weremixed with 400 ml of EV71 containing 9106 TCID50, and incubated for1 hour at 37 . The mixtures were added to (A) RD or (B) Vero cells forincubation for 1 hour at 4 to allow virus attachment to the cells. Afterincubation, the cells were washed with cold PBS three times, collected,and subjected to