SARS冠状病毒S基因重组DNA诱导的体液免疫反应

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。     

SARS病毒S蛋白受体结合区DNA疫苗及免疫效果研究

目的 研究SARS冠状病毒(SARS-CoV)靶细胞受体结合区所构建之DNA疫苗的免疫效果,为进一步的SARS-CoV免疫机理研究及疫苗研制奠定基础.方法 选取SARS-CoV S基因包含靶细胞受体结合区和S1亚单位C端2个基因片段作为目的基因,构建真核表达质粒pVAX-RBD(receptor binding domain)、pVAX-S1C作为DNA疫苗免疫BALB/c小鼠,检测其特异性体液免疫及细胞免疫情况.结果 体液免疫方面,以SARS全病毒裂解产物和原核表达的RBD蛋白作为诊断抗原,用ELISA均可检测到高滴度的小鼠血清抗体IgG的产生.而且,血清中和试验显示pVAX-RBD质粒激发了小鼠保护性中和抗体的产生.通过流式细胞分析和酶联免疫斑点实验(ELISPOT)检测,pVAX-RBD和pVAX-S1C两组质粒均诱导免疫小鼠产生了特异性细胞免疫反应.结论 证明SARS-CoV S蛋白受体结合区上中和表位的存在;体液免疫在抗SARS-CoV感染方面起到重要作用.     

SARS相关冠状病毒M蛋白基因重组核酸疫苗的实验研究

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。     

SARS冠状病毒S蛋白C端片段基因的克隆及其DNA疫苗的免疫学研究

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。     

应用合成的SARS病毒S蛋白重叠肽筛选B细胞表位

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。     

靶向SARS-CoV spike中和抗体的检测

目的 建立靶向SARS-CoV spike中和抗体的检测模型.方法 构建优化的全基因SARS-CoV spike质粒免疫小鼠产生S1190抗血清,与合成的结合受体ACE2的S318-510蛋白进行作用,通过荧光显微镜观察对SARS假病毒感染抑制作用的变化以确证中和抗体的高效性.结果 SARS-CoV spike质粒诱导产生的S蛋白全长抗血清不但可以有效中和SARS假病毒进入HEK293E/ACE2-Myc细胞,S318-510蛋白的预处理可以降低抗血清对SARS假病毒的进入抑制效应.结论 受体结合部(RBD)的S318-510蛋白靶向SARS-CoV spike中和抗体的检测模型得到有效的验证.可以作为直观迅速判断中和抗体是否高效的模型推广至不具备P3级别的实验室.     

抗SARS冠状病毒中和活性单克隆抗体的筛选及其结合位点分析

目的 建立能分泌具有中和活性的抗SARS-CoV单克隆抗体（McAb）细胞株，制备有中和活性的抗SARS-CoV MeAb，用于SARS-CoV感染的早期特异诊断和进行SARS-CoV结构蛋白的功能研究。方法 用灭活纯化sARS-c0V（BJ01株）免疫BALB／c小鼠，通过细胞融合技术，建立能稳定分泌SAILS病毒MeAb的杂交瘤细胞系，然后用中和试验进一步筛选分泌具有中和活性的抗SARS-CoV MeAb细胞株。利用SARS-CoV的S和N蛋白的不同长度肽段通过EusA鉴定McAb的特异结合区域，分析S和N蛋白不同区域诱导产生抗体的能力和特性。结果 建立了12株能稳定分泌抗SARS-CoV McAb的杂交瘤细胞株，间接免疫荧光法检测抗体效价在1：320～1：20 480之间，其中6株具有较好的中和SARS-CoV的能力。具有中和活性的McAb中，3株是针对S蛋白的，2株是针对N蛋白的。其中抗S蛋白的中和抗体活性比抗N蛋白的中和抗体活性高，另外1株可能是针对SARS-CoV的其他结构蛋白的。进一步对6株抗S蛋白的McAb的特异抗原结合表位分析，发现其中3株结合位点在S蛋白第12～311氨基酸之间，2株在第310～535氨基酸之间，后者中和效价高于前者，另1株是针对S2蛋白的，SARS病毒的受体结合区正位于第310～535氨基酸这一区段。具有中和活性的McAb通过间接免疫荧光、ELISA、酶标免疫组化法和胶体金方法对SARS病毒进行检测，都显示出高度的特异性和良好亲和力。结论 成功制备了具中和活性的抗SARS-CoV单克隆抗体，并分别测定了McAb对S蛋白和N蛋白的特异抗原结合活性，初步确定了S蛋白的中和表位。为今后SARS病毒的特异诊断、结构蛋白的功能分析和重组疫苗的研制奠定了较好的基础。     

严重急性呼吸综合征冠状病毒2(SARS-CoV-2)入胞机制及基于刺突蛋白的疫苗研制策略

严重急性呼吸综合征冠状病毒2(SARS-CoV-2)利用其刺突(S)吸附与穿入细胞,冠状病毒S蛋白为同源三聚体,每一条肽链分为S1和S2两个区,S1通过其受体结合域(RBD)与细胞膜上的受体结合,S2介导病毒包膜与细胞膜或内体膜融合使病毒核衣壳进入细胞质。冠状病毒疫苗设计的主要出发点是诱导机体产生针对S蛋白的中和抗体,从而阻止病毒对细胞的吸附与穿入。动物实验和临床试验都已证实,冠状病毒S蛋白能刺激机体产生中和抗体,后者对机体具有保护作用。基于S蛋白的新冠病毒疫苗可以是病毒载体疫苗、重组蛋白疫苗、 DNA疫苗、 mRNA疫苗或病毒样颗粒疫苗,可以针对S蛋白的全长设计或针对RBD或S蛋白的其他区域设计。     

SARS冠状病毒spike基因DNA疫苗的初步研究

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。     

SARS冠状病毒刺突蛋白的研究进展

SARS冠状病毒S蛋白是病毒颗粒最大的结构蛋白和最重要的表面抗原。该蛋白在介导病毒颗粒与宿主细胞受体结合、融合的过程中起着关键作用。它是病毒中和性抗体的靶点,在疫苗研究和抗病毒治疗研究中均占有重要地位。本文就SARS流行以来有关S蛋白的研究作一综述。     

SARS病毒S蛋白片段与细胞结合作用分析

目的研究SARS冠状病毒(SARSCoV)S蛋白片段与SARSCoV敏感细胞Vero的相互作用,明确S蛋白的受体结合位点。方法在E.coli中表达S蛋白第260～600位氨基酸(S260600)和397～796位氨基酸(S397796)片段,通过Westernblot对蛋白表达进行确认,用NiSepharose螯合层析对重组蛋白进行纯化。将纯化的S260600和S397796蛋白与Vero细胞4℃共同孵育1h后,先后与SARS患者血清及FITC标记的抗人IgG作用,通过流式细胞仪检测蛋白与细胞表面受体的结合情况。结果成功构建了原核表达质粒pET30a/S260600和pET30a/S397796,并表达、纯化出S260600和S397796重组蛋白。分别用SARS患者血清和抗6×His单克隆抗体进行Westernblot检测,证实重组蛋白得到正确表达。流式细胞仪分析显示S260600和S397796重组蛋白均可与Vero细胞发生结合,但S397796的结合力要弱于S260600。同时发现S260600重组蛋白与SARSCoV非敏感细胞NIH3T3细胞不能结合,进一步证明S260600重组蛋白与Vero细胞表面受体的结合是特异性的。结论重组蛋白S397796和S260600具有受体结合能力,尤其S260600包含了重要的受体结合位点,对进一步研究介导SARSCoV感染的细胞表面受体、开展疫苗和抗病毒药物的筛选均具有重要意义。     

Identification of a critical neutralization determinant of severe acute respiratory syndrome (SARS)-associated coronavirus: importance for designing SARS vaccines

The spike (S) protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is not only responsible for receptor binding, but also a major antigenic determinant capable of inducing protective immunity. In this study, we demonstrated that the receptor-binding domain (RBD) of S protein is an important immunogenic site in patients with SARS and rabbits immunized with inactivated SARS-CoV. Serum samples from convalescent SARS patients and immunized rabbits had potent neutralizing activities against infection by pseudovirus expressing SARS-CoV S protein. Depletion of RBD-specific antibodies from patient or rabbit immune sera by immunoadsorption significantly reduced serum-mediated neutralizing activity, while affinity-purified anti-RBD antibodies had relatively higher potency neutralizing infectivity of SARS pseudovirus, indicating that the RBD of S protein is a critical neutralization determinant of SARS-CoV during viral infection and immunization. Two monoclonal antibodies (1A5 and 2C5) targeting at the RBD of S protein were isolated from mice immunized with inactivated SARS-CoV. Both 1A5 and 2C5 possessed potent neutralizing activities, although they directed against distinct conformation-dependant epitopes as shown by ELISA and binding competition assay. We further demonstrated that 2C5, but not 1A5, was able to block binding of the RBD to angiotensin-converting enzyme 2 (ACE2), the functional receptor on targeted cells. These data provide important information for understanding the antigenicity and immunogenicity of SARS-CoV and for designing SARS vaccines.     

Endocytosis of the receptor-binding domain of SARS-CoV spike protein together with virus receptor ACE2

Cell entry of severe acute respiratory syndrome coronavirus (SARS-CoV) is mediated by the viral spike (S) protein. Amino acids 319-510 on the S protein have been mapped as the receptor-binding domain (RBD), which mediates binding to the SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) on SARS-CoV susceptible cells. In this study, we expressed a fusion protein containing the human codon-optimized RBD of the SARS-CoV spike protein linked to the Fc portion of human IgG1 (named RBD-Fc) in HEK293 cells. The RBD-Fc protein was purified by affinity chromatography. The flow cytometry assay showed that the purified RBD-Fc protein could bind to ACE2. We demonstrated that the RBD spike protein alone could be internalized into SARS-CoV susceptible cells together with ACE2. We also showed that the removal of N-glycans from the RBD spike protein did not abolish this phenomenon. Our discoveries may have some implications for the development of the SARS vaccine.     

Selection of SARS-coronavirus-specific B cell epitopes by phage peptide library screening and evaluation of the immunological effect of epitope-based peptides on mice

Antibodies to SARS-Coronavirus (SARS-CoV)-specific B cell epitopes might recognize the pathogen and interrupt its adherence to and penetration of host cells. Hence, these epitopes could be useful for diagnosis and as vaccine constituents. Using the phage-displayed peptide library screening method and purified Fab fragments of immunoglobulin G (IgG Fab) from normal human sera and convalescent sera from SARS-CoV-infected patients as targets, 11 B cell epitopes of SARS-CoV spike glycoprotein (S protein) and membrane protein (M protein) were screened. After a bioinformatics tool was used to analyze these epitopes, four epitope-based S protein dodecapeptides corresponding to the predominant epitopes were chosen for synthesis. Their antigenic specificities and immunogenicities were studied in vitro and in vivo. Flow cytometry and ELISPOT analysis of lymphocytes as well as a serologic analysis of antibody showed that these peptides could trigger a rapid, highly effective, and relatively safe immune response in BALB/c mice. These findings might aid development of SARS diagnostics and vaccines. Moreover, the role of S and M proteins as important surface antigens is confirmed.     

A subcutaneously injected UV-inactivated SARS coronavirus vaccine elicits systemic humoral immunity in mice

The recent emergence of severe acute respiratory syndrome (SARS) was caused by a novel coronavirus, SARS-CoV. It spread rapidly to many countries and developing a SARS vaccine is now urgently required. In order to study the immunogenicity of UV-inactivated purified SARS-CoV virion as a vaccine candidate, we subcutaneously immunized mice with UV-inactivated SARS-CoV with or without an adjuvant. We chose aluminum hydroxide gel (alum) as an adjuvant, because of its long safety history for human use. We observed that the UV-inactivated SARS-CoV virion elicited a high level of humoral immunity, resulting in the generation of long-term antibody secreting and memory B cells. With the addition of alum to the vaccine formula, serum IgG production was augmented and reached a level similar to that found in hyper-immunized mice, though it was still insufficient to elicit serum IgA antibodies. Notably, the SARS-CoV virion itself was able to induce long-term antibody production even without an adjuvant. Anti-SARS-CoV antibodies elicited in mice recognized both the spike and nucleocapsid proteins of the virus and were able to neutralize the virus. Furthermore, the UV-inactivated virion induced regional lymph node T-cell proliferation and significant levels of cytokine production (IL-2, IL-4, IL-5, IFN-gamma and TNF-alpha) upon restimulation with inactivated SARS-CoV virion in vitro. Thus, a whole killed virion could serve as a candidate antigen for a SARS vaccine to elicit both humoral and cellular immunity.     

SARS病毒N蛋白的表达与DNA疫苗的构建

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。     

SARS-CoV S蛋白表位的表达及鉴定

目的:制备SARS冠状病毒S蛋白串联表位重组蛋白,为SARS的防治提供新型抗原蛋白。方法:应用抗原表位分析软件分析S蛋白的表位。选取其中16个表位,设计并合成表位串联重组蛋白编码基因Z,构建其原核细胞表达重组体,在大肠杆菌BL21(DE3)表达该重组表位蛋白Z,应用Ni离子亲合层析法纯化重组蛋白Z做抗原,采用皮下注射法免疫新西兰白兔。斑点杂交法测定抗Z-血清中S蛋白的抗体,ELISA法检测Z蛋白的抗原性。结果:构建了S蛋白重组表位蛋白Z的原核表达体,在BL21菌中表达了Z蛋白,Z蛋白免疫新西兰白兔后获得了抗Z血清。斑点杂交分析显示,抗Z血清识别哺乳动物细胞中表达的S蛋白。ELISA检测结果显示,抗Z血清识别SARS冠状病毒抗原。结论:建立了制备SARS冠状病毒S蛋白重组表位蛋白的方法,为防治SARS提供了新型抗原蛋白Z。     

Expression of SARS coronavirus nucleocapsid protein and construction of its DNA vaccine]

AIM: To express the nucleocapsid (N) protein of SARS coronavirus (SARS-CoV) in E. coli and construct its DNA vaccine. METHODS: The prokaryotic expression vector pQEN containing N gene was constructed and transformed into the E. coli. The recombinant N protein was then expressed and purified by Ni(2+)-NTA affinity resin. In addition, the N gene was cloned into the eukaryotic expression plasmid pSecTagB and the eukaryotic recombinant expression vector pSecN was obtained. The DNA vaccine pSecN was injected to immunize the BALB/c mice to produce the antiserum against N protein of SARS-CoV. Subsequently, the reactivity of the antiserum with recombinant N protein and SARS-CoV particles was assayed by ELISA. RESULTS: Recombinant N protein reacted strongly and specifically with the sera from immunized mice and SARS patients. Similarly, the sera of immunized mice could also react specifically with SARS-CoV particles. CONCLUSION: The recombinant N protein could be used as a good antigen to detect SARS. The DNA vaccine pSecN could also efficiently induce the production of IgG against N protein of SARS-CoV, which offered clues to the development of a potential DNA vaccine.