基于立即早期蛋白IE62建立水痘-带状疱疹病毒感染性滴度检测方法

目的:基于水痘-带状疱疹病毒(VZV)立即早期蛋白IE62与酶联免疫斑点检测技术(ELISpot)建立一种新型的VZV感染性滴度的快速检测方法。方法:应用生物信息学方法设计并合成VZV-IE62蛋白的多肽抗原,牛血清白蛋白偶联后免疫BALB/c小鼠,筛选和制备抗VZV-IE62单克隆抗体,应用Western blot和免疫荧光法等开展抗体性能评价,继而应用酶联免疫斑点技术(ELISpot)结合生物素-亲和素放大法建立新型的VZV感染性滴度检测方法,并与经典空斑计数法进行比较。结果:获得抗VZV-IE62的单克隆抗体1B7,应用于ELISpot方法可特异识别VZV感染后的细胞,以之为基础建立了新型的VZV感染性滴度检测方法。相比经典空斑计数法,该方法可显著缩短检测时间(从5至7 d缩短至32 h)检测结果具有较好的一致性。结论:本研究建立了一种新型的基于VZV立即早期蛋白IE62与酶联免疫斑点技术的VZV感染性滴度检测方法(VZV-IE62 ELISpot),具有潜在的转化应用前景,可为VZV的防治研究提供支持。国家自然科学基金项目(81601762)资

Preparation and Application of Soluble Human Squamous Cell Carcinoma Antigen Expressed by Escherichia coli

旨在建立基于大肠杆菌表达系统的高效可溶性表达人鳞状上皮细胞癌抗原(SCCAg)方法,获得具有较好活性的重组SCCAg抗原并应用于建立抗原检测方法; 。基于pGEX-6P-l载体和大肠杆菌E. coli; ER2566菌株开展重组SCCAg抗原可溶性表达纯化方法研究,评价纯化抗原活性,筛选特异性单克隆抗体,初步建立并评价SCCAg抗原检测方法。结果; 显示,pGEX-6P-l载体和E coli; ER2566菌株可用于建立较高效的可溶性表达和纯化SCCAg抗原的方法,获得了具有较高纯度和活性的重组SCCAg抗原,筛选获得特异性单克隆抗体并; 初步建立了 SCCAg管式化学发光检测方法。建立了有效的基于大肠杆菌表达系统的可溶性表达和纯化SCCAg的方法。The aims of this study are to establish a method for efficient soluble; expression of human squamous cell carcinoma antigen(SCCAg ) based on; Escherichia coli expression system and obtain the recombinant SCCAg; antigen in fine activity, then apply it in the detection method; establishment of antigen. The study on the method of soluble expression; and purification of recombinant SCCAg antigen was conducted based on; pGEX-6P-l vector and E. coli ER2566 strain. The activity of the purified; antigen was evaluated by Abbott Kit and the specific monoclonal antibody; was screened by indirect ELISA. It was proved that PGEX-6P-1 vector and; E. coli strain ER2566 could be used to establish efficient soluble; expression and purification method for recombinant SCCAg antigen.; Moreover, the recombinant SCCAg antigen was proved to be in high purity; and activity. Thus,the SCCAg detection method of chemical luminous tube; was established with the specific monoclonal antibodies. In conclusion,; an effective method for the expression and purification of SCCAg, which; is based on the E. coli expression system, is established.国家高技术研究发展计划(863计划

水痘-带状疱疹病毒主要衣壳蛋白ORF40单克隆抗体的制备及初步应用

目的:制备抗水痘-带状疱疹病毒(VZV)主要衣壳蛋白ORF40的单克隆抗体,对该蛋白在VZV感染细胞内的表达、分布以及在VZV病毒颗粒中的包含情况进行初步探究。方法:设计并合成VZV ORF40多肽片段,偶联...国家自然科学基金项目(81601762)资

Near-atomic cryo-electron microscopy structures of varicella-zoster virus capsids

VZV是一种广泛存在并且具有高度传染性的人类α-疱疹病毒。初次感染VZV可导致水痘，人群普遍易感（感染率约为61%～100%）。该病毒可在背根神经节潜伏感染，持续终生。夏宁邵教授团队长期开展VZV相关基础与新型疫苗研究，通过系统和精细探索建立了高效的VZV规模化培养和病毒颗粒纯化技术体系，成功获得高质量的VZV颗粒样品。首次揭示了疱疹病毒α家族的水痘-带状疱疹病毒（VZV）不同类型核衣壳的近原子分辨率结构，阐明了VZV核衣壳不同组成蛋白的相互作用网络与衣壳装配机制，可为进一步开展新型载体疫苗设计及抗病毒药物等研究提供重要支持。 我校博士后王玮、高级工程师郑清炳、博士生潘德全和俞海副教授为该论文共同第一作者，我校夏宁邵教授、程通副教授、李少伟教授以及美国罗格斯大学朱桦（Hua Zhu）教授、加利福尼亚大学洛杉矶分校周正洪（Z. Hong Zhou）教授为该论文的共同通讯作者。【Abstract】Varicella-zoster virus (VZV) is a medically important human herpesvirus that causes chickenpox and shingles, but its cell-associated nature has hindered structure studies. Here we report the cryo-electron microscopy structures of purified VZV A-capsid and C-capsid, as well as of the DNA-containing capsid inside the virion. Atomic models derived from these structures show that, despite enclosing a genome that is substantially smaller than those of other human herpesviruses, VZV has a similarly sized capsid, consisting of 955 major capsid protein (MCP), 900 small capsid protein (SCP), 640 triplex dimer (Tri2) and 320 triplex monomer (Tri1) subunits. The VZV capsid has high thermal stability, although with relatively fewer intra- and inter-capsid protein interactions and less stably associated tegument proteins compared with other human herpesviruses. Analysis with antibodies targeting the N and C termini of the VZV SCP indicates that the hexon-capping SCP—the largest among human herpesviruses—uses its N-terminal half to bridge hexon MCP subunits and possesses a C-terminal flexible half emanating from the inner rim of the upper hexon channel into the tegument layer. Correlation of these structural features and functional observations provide insights into VZV assembly and pathogenesis and should help efforts to engineer gene delivery and anticancer vectors based on the currently available VZV vaccine.This research was supported by grants from the National Science and Technology Major Projects for Major New Drugs Innovation and Development (no. 2018ZX09711003-005-003), the National Science and Technology Major Project of Infectious Diseases (no. 2017ZX10304402), the National Natural Science Foundation of China (no. 81871648, 81601762), the Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences (no. 2019RU022) and the US National Institutes of Health (DE025567/028583). 该研究获得了国家自然科学基金、新药创制国家科技重大专项和传染病防治国家科技重大专项等资助

Preparation of Monoclonal Antibodies against Glycoprotein N of Varicella-zoster Virus and a Preliminary Study

水痘-带状疱疹病毒(VZV)是水痘和带状疱疹的共同致病原,其糖蛋白N(gN)在子代病毒的组装、成熟和出胞过程中发挥重要作用。但是目前关于VZV; gN的研究还很少,其蛋白性质和具体分子功能仍不清楚。本研究目的是制备抗VZV; gN的单克隆抗体,对该蛋白在VZV感染细胞和人皮肤组织内的表达和分布情况进行初步探究。本研究选择抗原性和亲水性较强的gN区段基因,构建rGST-; gN融合蛋白的原核表达质粒,将鉴定正确的重组质粒转化大肠杆菌后用IPTG诱导融合蛋白表达,经亲和层析柱纯化rGST-gN融合蛋白后使用PSP酶切; 除GST标签,将获得的重组蛋白rgN免疫BALB/c小鼠,使用杂交瘤技术制备其单克隆抗体;共获得24株稳定分泌抗VZV; gN单克隆抗体的杂交瘤细胞株,选取其中一株性能最优的12E10进行单克隆抗体生产纯化和抗体鉴定;单克隆抗体12E10为IgG2a亚型,其ELIS; A效价达到1∶10 000;通过Western blot、免疫荧光及免疫组织化学染色方法鉴定,该抗体能够特异性识别细胞和组织中表达的VZV; gN;本研究应用该抗体首次确定了gN在VZV感染细胞中的反式高尔基体定位,并且确定了gN在VZV感染人皮肤组织中的表达。以上工作为今后深入研究V; ZV gN功能奠定了基础。Varicella-zoster virus(VZV)is the causative agent for both; varicella(chicken pox)and herpes zoster(shingles).Glycoprotein; N(gN)plays an important role in VZV virion assembly,maturation and; egress.However,properties and the precise molecular functions of VZV gN; are still unclear.This research is to prepare monoclonal; antibodies(mAbs)specific for VZV gN,and study its expression and; localization in VZV-infected cells and human skin; tissues.Firstly,apotentially antigenic and hydrophilic region of VZV gN; was selected to construct its prokaryotic expression plasmid.Then,the; correct recombinant plasmid was transformed into E.coli,and IPTG was; used to induce the expression of the fusion protein rGST-gN.After; purification by affinity chromatography,rGST-gN was cut with PreScission; protease(PSP)to remove GST tag,and the resulting protein rgN was used to; immunize BALB/c mice.MAbs were prepared by hybridoma technique.A total; of 24 hybridoma cell lines secreting mAbs against VZV gN were; obtained,and one of them,12E10,was selected for mAb production and; characterization.The results showed that the Ig subclass of 12E10 was; IgG2 a,and it exhibited an ELISA titer of 1∶10,000.12E10 can be used to; detect expression of VZV gN in cell lines and tissues in Western; blot,immunofluorescence and immunohistochemistry.In addition,our study; for the first time determined the trans-Golgi network(TGN)localization; of gN in VZV-infected cells,and we confirmed the expression of gN in; VZV-infected human skin tissues.Overall,the above work laid the; foundation for further research on the function of VZV gN.国家自然科学基