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1.
研究灭活SARS病毒的免疫原性。SARS病毒F6 9株经甲醛灭活后 ,加入氢氧化铝佐剂 ,以 3种剂量接种BALB/c小鼠 ,定时采血 ,测定特异性抗体的滴度及其中和活性 ,同时用化学发光酶联免疫法测定抗血清与SARS病毒结构蛋白的反应特异性及其相对强度。结果发现 ,小鼠接种疫苗 4d后 ,血清中可检测到IgM抗体 ,直至 2 6d后逐渐下降 ;IgG抗体在初次免疫 8d后出现 ,4 7d时达最高峰 ,6 3d后进入稳定期 ;不同剂量组的抗体滴度具有明显剂效关系 ,低剂量组和中剂量组滴度峰值为 1∶192 0 0 ,高剂量组滴度峰值为 1∶384 0 0。中和实验结果表明 ,小鼠所产生的抗体具有中和病毒活性 ,在 6 3d时 ,低剂量组和中剂量组血清的中和效价为 1∶12 80 ,高剂量组血清的中和效价为 1∶5 12 0。抗体分类结果表明 ,小鼠抗血清中含有针对多种SARS病毒结构蛋白的特异性抗体 ,其中 ,针对N蛋白、S4蛋白和S2蛋白的抗体水平相对较高 ,而抗M抗体、抗 3CL抗体的水平相对较低。上述结果说明 ,SARS病毒F6 9株经甲醛灭活后 ,各主要结构蛋白仍保持较强免疫原性 ;免疫小鼠后 ,可以诱导产生高滴度的特异性混合抗体 ,在体外可以保护敏感细胞不受SARS病毒攻击  相似文献   

2.
SARS-CoV核衣壳蛋白抗原性鉴定和基因免疫研究   总被引:1,自引:1,他引:0  
目的 了解SARS冠状病毒 (SARS CoV)核衣壳蛋白 (N蛋白 )的抗原性及其基因疫苗的免疫原性。方法 用大肠杆菌表达SARS CoV的N蛋白 ,用SARS患者恢复期血清对其进行抗原性鉴定。再构建N蛋白基因疫苗 ,肌肉注射接种小鼠 ,检测小鼠血清中的抗N蛋白IgG抗体、脾细胞增殖和迟发型超敏反应。结果 大肠杆菌重组表达的SARS CoVN蛋白具有强抗原性 ,其基因疫苗能在小鼠有效诱导N蛋白特异性抗体和CD4 、CD8 T淋巴细胞免疫应答。结论 SARS CoV的N蛋白可作为重要的SARS血清学诊断抗原 ,并对于SARS的特异性预防和治疗有潜在的应用价值。  相似文献   

3.
目的:研究含不同佐剂的SARS灭活疫苗免疫小鼠后,小鼠的早期细胞免疫和体液免疫反应。方法:灭活的SAPS冠状病毒F69株分别与弗氏佐剂、氢氧化铝、CpG佐剂配伍,制备灭活疫苗。接种6周龄SPF’级BALB/C小鼠。定时采血,分析小鼠外周血中CD4^ 、CD8^ T细胞亚群动态变化,同时测定鼠血清中特异性IgG抗体及抗体的病毒中和活性。结果:由3种佐剂制备的SARS灭活疫苗免疫小鼠后,CD4^ 、CD8^ T细胞百分比升高或无明显改变,CD4/CD8比值无显著性改变。其中,弗氏佐剂疫苗免疫小鼠的CD4^ T细胞百分比升高较明显,且在一段时间内,维持在一个较高的水平;铝佐剂疫苗免疫小鼠后,未观察到明显T细胞亚群改变;而CoG佐剂疫苗免疫小鼠的CD8^ T细胞百分比改变较其它佐剂疫苗改变更明显。与此相异的是,3种疫苗均可诱导小鼠产生较高滴度特异性IgG抗体,并且所产生的抗体具有中和活性。在初免后第34天,3种佐剂组的IgG抗体滴度分别为1:12800、1:3200和1:1600,中和效价分别为1:2560、1:960和1:320。结论:SARS灭活疫苗接种小鼠后,可诱导较强的体液免疫反应,同时轻度上调CD4^ 、CD8^ T细胞活性,程度因佐剂而异。  相似文献   

4.
目的:构建针对SAILS相关冠状病毒M蛋白基因的重组核酸疫苗,观察其免疫小鼠后肌体的抗体产生情况,探讨其作为抗SAILS病毒疫苗的可能性。方法:通过分子生物学的方法构建SARS相关冠状病毒M蛋白基因真核表达质粒pcDNA3.1/M。经肌注免疫健康BALB/c小鼠,在免疫后的2.4、6周取小鼠血清,用ELISA法检测其中的抗体。结果:接种含M基因的真核表达质粒pcDNA3.1/M的低剂量组和高剂量组的小鼠血清在接种2周后就可检测出SARS-CoV特异性IgG,第4周时,这种特异性IgG水平有升高趋势,第6周时,血清中的抗体含量与4周时无大的差异。高剂量组产生抗体与低剂量组产生抗体无显著差异。pcDNA3.1空载体接种的对照组未检测出特异性抗体(其OD值低于0.18)。结论:构建的真核表达质粒pcDNA3.1/M能诱导小鼠产生了针对SARS的抗体。  相似文献   

5.
目的:初步探究血管内皮生长因子(VEGF)/碱性成纤维细胞生长因子(b FGF)复合多肽疫苗(VEGF/b FGF complex peptide vaccine,VBP3)对雌性小鼠的毒性及其生殖的影响。方法:通过镍离子亲和层析柱纯化VBP3蛋白,将纯化获得的VBP3免疫雌性BALB/c小鼠,酶联免疫吸附实验(ELISA)方法检测小鼠血清抗体效价和特异性,监测亲本小鼠体重,测定亲本小鼠脏器重量并对小鼠脏器进行HE染色观察;将免疫小鼠与未免疫雄性小鼠交配后,测定F1代小鼠存活率、体重、相关脏器重量,并对相关脏器进行HE染色观察。结果:间接ELISA检测结果显示免疫小鼠血清中抗VEGF、抗b FGF抗体滴度分别为1∶3 000、1∶20 000,且免疫组F1代小鼠能检测出低滴度的抗b FGF抗体,但抗VEGF抗体不能检出。在小鼠生产率实验中,免疫组与对照组崽鼠数目未见明显差异,但免疫组F1代小鼠存活率较对照组降低(P0.05)。在亲本小鼠中,免疫组小鼠各脏器重量与对照组比较均未有差异,而在F1代小鼠中,2个组别的小鼠肝脏重量存在差异,但其它脏器重量未有差异;HE染色显示,在亲本小鼠中,2个组别小鼠的各脏器形态未有明显差异;在F1代小鼠中,免疫组F1代小鼠的肝脏与对照组相比存在形态差异。结论:VEGF/b FGF复合多肽疫苗未对亲本小鼠主要脏器造成直接损伤,但对亲本小鼠的生殖及F1代小鼠健康具有一定的影响。  相似文献   

6.
目的:构建抗原基因为SARS冠状病毒(Severe acute respiratory syndrome coronavirus,SARS-CoY)S蛋白C端片段基因的DNA疫苗,采用肌注和滴鼻的方法接种小鼠后观察肌肉注射途径和黏膜途径免疫使机体产生免疫应答的情况。方法:将S蛋白C端片段克隆至真核表达载体pcDNA3.1(-),随之将重组质粒进行小鼠肌肉和黏膜免疫,定期检测外周血中抗SARS-CoV的病毒特异性抗体水平,流式细胞仪观察其淋巴细胞表型变化,免疫组化检测抗原表达分布,脾细胞增殖实验评价CTL效果。结果:疫苗注射后15天就能在血清中检测出病毒特异性抗体。随着时问的延续,抗体水平逐步升高,至30天后达到稳定,以CTL为主的CD8^+T淋巴细胞的百分比含量增加极显著,引起强大的体液免疫和细胞免疫;疫苗滴鼻后45天可在鼻黏膜检测到抗原表达;而空载体质粒对照组未检测出明显的特异性免疫应答。结论:以S蛋白C端片段基因为抗原基因的DNA疫苗通过肌注和滴鼻能诱导小鼠针对SARS病毒强大的免疫应答。  相似文献   

7.
SARS病毒N蛋白的表达与DNA疫苗的构建   总被引:3,自引:1,他引:2  
目的:在大肠杆菌中表达SARS冠状病毒核衣壳N蛋白,并构建其DNA疫苗。方法:构建含N基因的原核表达载体pQEN,并在大肠杆菌M15中表达N蛋白。采用NP亲和层析法纯化目的蛋白。将N基因克隆入真核表达载体pSecTagB中,构建真核重组质粒pSecN。以其免疫小鼠制备抗血清,并用ELISA法检测其与大肠杆菌中表达的重组N蛋白及天然全病毒N蛋白的反应性。结果:重组N蛋白能与DNA疫苗免疫的小鼠血清以及SARS患者血清发生特异性反应;SARS—CoV病毒颗粒也可与DNA疫苗免疫的小鼠血清发生特异性反应。结论:重组N蛋白保留了病毒的一些特异性抗原表位,可作为用ELISA法检测SARS—CoV的抗原。构建的DNA疫苗可在小鼠体内产生高效价的抗SARS病毒N蛋白的特异性抗体,从而为该疫苗的开发奠定了基础。  相似文献   

8.
目的:构建人精子特异性乳酸脱氢酶DNA疫苗,并免疫雌雄两性BALB/c小鼠,以观察特异性抗体的产生。方法:采用分子克隆的方法构建pVAX1-hLDHC DNA疫苗。双酶切及正反向测序对该DNA疫苗进行鉴定。肌肉注射方法免疫雌雄两性BALB/c小鼠,ELISA和Western blot方法检测小鼠血清中的特异性抗体,常规病理学方法检测雄性实验小鼠的睾丸组织。结果:双酶切及正反向测序结果表明:插入片段的序列以及插入的位置和方向均正确。肌肉注射免疫BALB/c小鼠后可诱导明显的体液免疫反应,且在雄性实验小鼠中未观察到睾丸炎的发生。结论:成功构建了具有一定免疫效果的hLDHC DNA疫苗,为今后进行更深入的研究奠定了基础。  相似文献   

9.
目的 构建SARS病毒spike基因片段真核表达质粒DNA疫苗;检测spike基因片段的免疫原性;筛选SARS病毒疫苗构建的理想靶抗原。方法 采用RT PCR从SARS冠状病毒北京0 1(BJ0 1)株cDNA获取了spike基因cDNA的全长D12、编码N末端氨基酸的cDNA片段D14和编码C末端氨基酸的cDNA片段D2 3;构建重组真核表达质粒pcDNA3 /D12、pcDNA3 D14、pcDNA3 D2 /3;用3种重组质粒和pcDNA3空质粒载体(对照)DNA皮下注射免疫Wistar大鼠;ELISA检测免疫后大鼠血清S蛋白特异性抗体IgG的产生;同位素51 Cr实验检测特异性CTL应答;ELISPOT检测单细胞水平IFN -γ分泌。结果 pcDNA3 /D2 3疫苗免疫组大鼠血清有S蛋白阳性抗体IgG产生、抗体滴度>10 0 0 ;脾淋巴细胞可诱导特异的CTL应答和IFN γ分泌,pcDNA3 /D2 3疫苗组与其它组之间统计分析P <0 .0 5。结论SARS冠状病毒S蛋白的C末端具有较强的免疫原性,是疫苗构建的理想候选靶抗原,本研究结果为SARS病毒的疫苗研究提供了资料。  相似文献   

10.
以甲醛灭活SARS冠状病毒F6 9株 ,分别与福氏佐剂、氢氧化铝、CpG佐剂配伍 ,制备灭活疫苗 ,免疫实验动物。SPF级BALB c小鼠 ,雌雄各半 ,随机分为高、中、低 3个剂量组 ;初次免疫时 ,疫苗接种体积分别为 0 .3ml、0 .2ml和0 .1ml,腹腔 皮下接种小鼠 ;14d后进行第 2次免疫 ,免疫剂量减半。 2次免疫10d后 ,以不含佐剂的疫苗进行加强免疫。初次免疫后第 4、8、12、19、2 6、34天 ,各组小鼠取 3只断尾取血 ,肝素抗凝 ,流式细胞仪分析CD4 + 、CD8+ T细胞百分比。结果表明 ,中剂量含福氏佐剂的SARS灭活试验疫苗免疫小鼠后 ,外周血中CD4 + …  相似文献   

11.
It is believed today that nucleocapsid protein (N) of severe acute respiratory syndrome (SARS)-CoV is one of the most promising antigen candidates for vaccine design. In this study, three fragments [N1 (residues: 1-422); N2 (residues: 1-109); N3 (residues: 110-422)] of N protein of SARS-CoV were expressed in Escherichia coli and analyzed by pooled sera of convalescence phase of SARS patients. Three gene fragments [N1 (1-1269 nt), N2 (1-327 nt) and N3 (328-1269 nt)-expressing the same proteins of N1, N2 and N3, respectively] of SARS-N were cloned into pVAX-1 and used to immunize BALB/c mice by electroporation. Humoral (by enzyme-linked immunosorbent assay, ELISA) and cellular (by cell proliferation and CD4(+):CD8(+) assay) immunity was detected by using recombinant N1 and N3 specific antigen. Results showed that N1 and N3 fragments of N protein expressed by E. coli were able to react with sera of SARS patients but N2 could not. Specific humoral and cellular immunity in mice could be induced significantly by inoculating SARS-CoV N1 and N3 DNA vaccine. In addition, the immune response levels in N3 were significantly higher for antibody responses (IgG and IgG1 but not IgG2a) and cell proliferation but not in CD4(+):CD8(+) assay compared to N1 vaccine. The identification of antigenic N protein fragments has implications to provide basic information for the design of DNA vaccine against SARS-CoV. The present results not only suggest that DNA immunization with pVax-N3 could be used as potential DNA vaccination approaches to induce antibody in BALB/c mice, but also illustrates that gene immunization with these SARS DNA vaccines can generate different immune responses.  相似文献   

12.
目的筛选SARS病毒S蛋白的B细胞表位。方法使用SARS-CoV S DNA疫苗免疫BALB/c小鼠,获得SARS病毒S蛋白的免疫血清。人工合成包含169条部分氨基酸序列重叠的SARS-CoV S蛋白的多肽库。将多肽片段包被ELISA板,利用免疫小鼠血清,通过抗体结合试验来筛选SARS病毒S蛋白的线性B细胞表位。并将筛选结果与使用B细胞表位分析软件预测的结果进行比较。结果使用重叠肽合成法筛选到SARS.CoV蛋白两条多肽片段S335—352和S442—458.能与免疫动物血清特异性结合,与使用Bcipep数据库预测B细胞表位的结果相一致。结论鉴定了两个新的SARS病毒S蛋白B细胞表位。  相似文献   

13.
目的 以严重急性呼吸综合征冠状病毒(SARS-CoV)密码子优化的S、S1和S2基因分别构建其真核表达质粒,免疫BALB/c小鼠,以初步评价其诱导特异性体液免疫的效果。方法将人工合成密码子优化的S、S1和S2基因克隆入pcDNA4/HisMax-TOPO表达载体,重组质粒转染293T细胞,Western blot和免疫组化检测其真核表达,重组质粒免疫BALB/c小鼠,酶联免疫(ELISA)检测抗S蛋白抗体,伪病毒中和试验、细胞融合抑制试验检测中和抗体。结果3种重组质粒均可在真核细胞中获得表达,免疫小鼠后可诱导针对S蛋白的特异性抗体,抗体在12周观察期内呈持续上升趋势;其中,仅S和S1蛋白重组质粒能够诱导中和抗体的产生,以S蛋白的效价为高。结论密码子优化S和S1蛋白重组质粒可以有效诱导BALB/c小鼠产生中和抗体,其抗体可能具有阻断SARS-CoV侵袭易感细胞的能力。该结果为进一步研究SARS-CoV DNA疫苗提供了参考依据。  相似文献   

14.
OBJECTIVE: To study the preventive and therapeutic effects of recombinant IFN-alpha2b for nasal spray on SARS-CoV infection in Macaca mulata (rhesus monkey). METHODS: Ten rhesus monkeys were divided into two groups, 5 in interferon group, and 5 in control group. Before and after SARS-CoV attack, the virus was detected in samples such as pharyngeal swab in all the two groups by Real-time PCR (RT-PCR) and virus isolation was performed. RESULTS: After virus attack, the level of SARS-CoV-specific IgG and neutralizing antibody were induced by SARS-CoV in the interferon group was weaker than in control group. Hematology items showed no apparent changes after virus attack in treated group. Through pathological examination, the morphology of the lung tissues of two Macaques in the treated group was normal, while the other three displayed the interstitial pneumonia with the thickened septum and infiltration with mononuclear cells. Among which, one monkey showed part of thickened septum fused with each other. These lesions in the interferon treated animals were similar to those seen in the animals in control group, but with smaller scope of pathological changes. No significant abnormity was detected in other organs. CONCLUSION: Recombinant IFN-alpha2b could effectively interdict or weaken SARS-CoV injury in monkeys.  相似文献   

15.
目的: 研究严重急性呼吸综合征冠状病毒(SARS-CoV)的S蛋白作用于小鼠发生的免疫和病理变化,试图揭示S蛋白引起急性肺损伤的可能机制。方法: 将BALB/c小鼠随机分为PBS空白对照组、S蛋白组和γ-干扰素可诱导的10 kD蛋白(IP-10)组,分别经气管滴注、尾静脉注射的途径注入,观察其临床症状、死亡率;称取双肺湿重,进行HE染色和免疫荧光方法检测肺组织中IP-10的表达。结果: 经气管滴注或尾静脉注射S蛋白均可导致小鼠出现呼吸急促症状,甚至死亡,肺脏湿重增加,显微镜下可见肺泡正常结构破坏,间质增厚,炎性细胞浸润,上皮细胞和血管内皮细胞的肿胀脱落,小鼠肺内气管上皮细胞和血管内皮细胞均强阳性表达IP-10。经气管滴注小鼠IP-10蛋白小鼠出现上述类似的肺损伤表现。结论: (1)SARS-CoV的S蛋白引起小鼠急性肺损伤。(2)SARS-CoV的 S蛋白可诱导小鼠肺组织表达IP-10。(3)经气管滴注小鼠IP-10可引起小鼠肺损伤。  相似文献   

16.
Inactivated severe acute respiratory syndrome-associated coronavirus (SARS-CoV) has been tested as a candidate vaccine against the re-emergence of SARS. In order to understand the efficacy and safety of this approach, it is important to know the antibody specificities generated with inactivated SARS-CoV. In the current study, a panel of twelve monoclonal antibodies (mAbs) was established by immunizing Balb/c mice with the inactivated BJ01 strain of SARS-CoV isolated from the lung tissue of a SARS-infected Chinese patient. These mAbs could recognize SARS-CoV-infected cells by immunofluorescence analysis (IFA). Seven of them were mapped to the specific segments of recombinant spike (S) protein: six on S1 subunit (aa 12-798) and one on S2 subunit (aa 797-1192). High neutralizing titers against SARS-CoV were detected with two mAbs (1A5 and 2C5) targeting at a subdomain of S protein (aa 310-535), consistent with the previous report that this segment of S protein contains the major neutralizing domain. Some of these S-specific mAbs were able to recognize cleaved products of S protein in SARS-CoV-infected Vero E6 cells. None of the remaining five mAbs could recognize either of the recombinant S, N, M, or E antigens by ELISA. This study demonstrated that the inactivated SARS-CoV was able to preserve the immunogenicity of S protein including its major neutralizing domain. The relative ease with which these mAbs were generated against SARS-CoV virions further supports that subunit vaccination with S constructs may also be able to protect animals and perhaps humans. It is somewhat unexpected that no N-specific mAbs were identified albeit anti-N IgG was easily identified in SARS-CoV-infected patients. The availability of this panel of mAbs also provided potentially useful agents with applications in therapy, diagnosis, and basic research of SARS-CoV.  相似文献   

17.
Gai W  Zou W  Lei L  Luo J  Tu H  Zhang Y  Wang K  Tien P  Yan H 《Viral immunology》2008,21(1):27-37
Severe acute respiratory syndrome (SARS) is a deadly and highly infectious disease caused by SARS Coronavirus (SARS-CoV). Inactivated SARS-CoV has been explored as a vaccine against SARS-CoV; however, current knowledge of inactivated SARS-CoV vaccine is quite limited. We attempted to investigate the effects of different immunization protocols and adjuvant on the antibody responses to inactivated SARS-CoV vaccine. With an intraperitoneal (IP) immunization protocol, inactivated SARS-CoV alone induced significant amounts of SARS-CoV-specific IgG antibodies in sera and a small quantity of SARS-CoV-specific IgA antibodies in the genital tract and feces, but failed to induce any detectable SARS-CoV-specific IgA antibodies in sera, saliva, lung, and intestine, and the addition of CpG ODN 2006 had only a marginal effect on antibody production. In contrast, with an intranasal (IN) immunization protocol, inactivated SARS-CoV alone failed to induce any detectable SARS-CoV-specific IgA antibodies in sera, saliva, lung, and intestine, except for a small quantity of IgA antibodies in fecal extracts and the genital tract, along with IgG antibodies in sera, but when given with adjuvant CpG ODN 2006, inactivated SARS-CoV induced significant amounts of SARS-CoV-specific IgG antibodies in sera, and a detectable amount of SARS-CoV-specific IgA antibodies in sera and all tested mucosal secretions and tissues (i.e., saliva, the genital tract, fecal extract, lung, and intestine). On a neutralization assay, neutralizing activity with the IP immunization protocol was detected in sera and mucosal secretions (from the saliva and genital tract), but sera from the IN protocol failed to show any neutralizing activity. Our study demonstrated that inactivated SARS-CoV vaccine is promising, and our data provide a sound foundation for the development of an effective inactivated SARS-CoV vaccine.  相似文献   

18.
Immunogenicity of SARS inactivated vaccine in BALB/c mice   总被引:3,自引:0,他引:3  
Severe acute respiratory syndrome (SARS) is a serious infectious threat to public health. To create a novel trial vaccine and evaluate its potency, we attempted to generate a SARS inactivated vaccine using SARS coronavirus (SARS-CoV) strain F69 treated with formaldehyde and mixed with Al(OH)3. Three doses of the vaccine were used to challenge three groups of BALB/c mice. We found that the mice exhibited specific IgM on day 4 and IgG on day 8. The peak titers of IgG were at day 47 in low-dose group (1:19,200) and high-dose group (1:38,400) whereas in middle-dose group (1:19,200), the peak was at day 40. On day 63, the IgG levels reached a plateau. Neutralization assay demonstrated that the antisera could protect Vero-E6 cells from SARS-CoV's infection. Analysis of the antibody specificity revealed that the mouse antisera contained a mixture of antibodies specifically against the structure proteins of SARS-CoV. Furthermore, the mouse antisera conferred higher amount of antibodies against protein N, polypeptide S4 and S2 than those of proteins M and 3CL. These findings suggest that the inactivated SARS-CoV could preserve its antigenicity and the inactivated vaccine can stimulate mice to produce high levels of antibodies with neutralization activity. Results also suggest that polypeptides originating from protein N or S might be a potential target for the generation of a recombinant SARS vaccine.  相似文献   

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