Study of assembly associated domain of hepatitis E virus capsid protein in vitro

蛋白质－蛋白质相互作用主要是通过作用结构域来实现的，结构域是研究蛋白质结构和功能关系的桥梁，相互作用结构域的研究是当前功能蛋白质组学的研究热点。病毒颗粒的组装是蛋白质－蛋白质相互作用的典型，类病毒颗粒（VLP）结构的解析和组装机理的研究对探讨病毒的感染、发病和致病机理有着重要的理论和实际意义。本论文研究主要利用原核系统表达HEV衣壳蛋白，获得衣壳组装的中间态复合物和体外组装的HEV类病毒颗粒，并借助多种实验技术对HEV衣壳蛋白的体外折叠、相互作用和VLP体外组装的相关结构域进行研究，得到了初步的HEVORF2衣壳蛋白的功能结构域分布图，并提出HEV类病毒颗粒可能的体外组装机理。 SDS-PA...Interface domains play a very important role in protein-protein interaction, and build a bridge between protein’s structure and function. A hot theme on functional proteomics is investigating into interface domains of proteins. Self-assembly of virion is a paradigm of protein-protein interaction. The unwrapped structures and elucidated assembly mechanisms of some virus-like particles (VLPs) always...学位：理学博士院系专业：生命科学学院生物学系_动物学学号：B20002600

Virus-like particle-based vaccine development using Escherichia coli expression system

重组病毒样颗粒是病毒衣壳蛋白外源表达的重要形式,形态结构与天然病毒高度相似,位于纳米尺度的大小易于被免疫系统识别,可激发机体产生保护性免疫反应,且不含有病毒基因,因此,是一种理想的疫苗形式,也是基于结构进行疫苗设计的重要结构载体。目前已上市的乙型肝炎疫苗、人乳头瘤病毒疫苗和戊型肝炎疫苗等基因工程疫苗均采用病毒样颗粒形式。大肠杆菌表达系统被广泛用于基因工程药物的生产,具有安全性好、生产周期短、易于放大生产等优点,在病毒样颗粒疫苗应用上具有良好前景。本文综述了利用大肠杆菌研制戊型肝炎疫苗和人乳头瘤病毒疫苗的进展,特别是这些病毒样颗粒疫苗的表达及组装、表位结构特征和临床试验结果。Virus-like particles(VLPs) are generated by the self-assembly of viral structural protein using various expression systems.VLPs ensemble native virus particles in morphology and maintain key immune epitopes as authentic virus.In light of nanometer-sized particles with diameters of 20- 60 nm, VLPs were shown to be a passport to immune recognition, thus being capable of eliciting strong protective immune responses.Recombinant VLP-based vaccines have superior safety profiles due to lack of any viral genome.There are several licensed and highly successful VLP-based vaccines produced using recombinant DNA technology, such as recombinant hepatitis B, human papillomavirus and hepatitis E vaccine.E.coli expression system was well-established in production of biotherapeutics.This production platform for recombinant VLP-based vaccines has many advantages, such as rapid replication cycle and amenability for scale-up for commercial scale production.This review outlines the success of hepatitis E and human papillomavirus vaccines derived from E.coli.We highlight the protein expression, particle assembly, key epitope structure and clinical trials of these VLP-based vaccines.国家自然科学基金项目(30925030;81172885

Monoclonal antibodies against HPV11 virus-like particles: functional characteristics and application on quality assessment

目的分析和鉴定抗HPV11病毒样颗粒(virus-like particle,VLP)鼠源单克隆抗体的性质,筛选性质和生物学活性较优的抗体,并初步应用于抗原或疫苗的质量分析。方法分别利用间接ELISA法和Western blot对HPV11的22株单克隆抗体的亚类、与HPV11 VLP的结合能力和构象敏感性进行检测;采用血凝抑制实验对单克隆抗体的血凝抑制活性进行分析;运用基于假病毒的抗体中和实验对单克隆抗体的中和活性进行鉴定,选出中和活性高的单抗进行两两配对,采用双抗夹心ELISA法捕获单抗并筛选合适的配对双抗。结果对22株单抗的性质进行了详细和完整的鉴定,并根据构象敏感性进行排序,筛选出6株型别特异、结合活性强且中和活性高的单抗(2A2、4A1-3、16G7、14A6、9C12和19C7);成功建立了基于单抗的双抗夹心(14A6∶Ag∶9C12-HRP)ELISA定量分析方法。结论获得了较全面的HPV11 VLP单抗性质信息,建立了重组HPV11抗原质量分析的双抗夹心ELISA法,为HPV11抗原的生命周期管理或尖锐湿疣疫苗的研发、工艺优化、产品放行和稳定性研究等提供了技术支持。Objective To quantitatively analyze the characteristics of a panel of murine anti-human papillomavirus( HPV) 11 L1-derived virus-like particle( VLP) monoclonal antibodies( m Abs) and establish the m Ab-based methods for antigen quality analysis. Methods A panel of 22 murine anti-HPV11 m Abs were characterized in details with their isotype,and binding affinity,conformational sensitivity were examined quantitatively in the direct binding ELISA and Western blot. The hemagglutination inhibition activity of m Abs were identified using the hemagglutination inhibition assay and the pseudovirus( Ps V) neutralization efficiency were examined quantitatively using the Ps V-based neutralization assay. The type-specific, highly conformational sensitive and neutralizing m Abs were selected to be used in the sandwich ELISA assay. Results Based on the quantitative and semi-quantitative results,six type-specific,highly conformational sensitive and neutralizing m Abs( 2A2,4A1-3,16G7,14A6,9C1 and 19C7) were identified. These m Abs,along with 10D6 were screened as the capture m Ab or as the detection m Ab in the sandwich ELISA. Conclusion The binding affinity,conformational sensitivity and neutralization efficiency of anti-HPV11 m Abs were characterized in details. A m Ab-based sandwich ELISA assay( 14A6∶Ag∶9C12-HRP) were developed,which could be used in the in vitro potency analysis of HPV11 VLP-based vaccine.重大新药创制(2015ZX09101034

Intensity Distribution Design for Laser-Induced Thermal Loading Based on Numerical Simulation

To accomplish laser-induced thermal loading simulation tests for pistons,the Gaussian beam was modulated into multi-circular beam with specific intensity distribution.A reverse method was proposed to design the intensity distribution for the laser-induced thermal loading based on finite element(FE) analysis.Firstly,the FE model is improved by alternating parameters of boundary conditions and thermal-physical properties of piston material in a reasonable range,therefore it can simulate the experimental resul..

二种多管水母光蛋白基因的分离、表达及生物活性初步研究

分别从厦门东海域的大型多管水母和细小多管水母中分离到了新的光蛋白基因aeqxm和aeqxxm,并在大肠杆菌中进行了表达.aeqxm和aeqxxmDNA序列的编码区总长均为585bp,无内含子序列,推导的氨基酸序列总长均为195个氨基酸.aeqxm,aeqxxmDNA和AEVAQ440XcDNA之间的核苷酸序列同源性分别为80 7%,85 1%,aeqxm和aeqxxmDNA的核苷酸序列同源性为87 2%.原光蛋白apoaeqxm,apoaeqxxm与AEVAQ440X编码的氨基酸序列之间的同源性分别为84 7%,84 2%,apoaeqxm和apoaeqxxm的氨基酸序列之间的同源性为94 4%.分别将aeqxm和aeqxxm基因克隆至pTO-T7表达载体,apoaeqxm,apoaeqxxm在大肠杆菌中的表达量都达到40%左右.取菌体超声上清与腔肠动物荧光素f、巯基乙醇混合再生后,加入CaCl2,用荧光全谱仪检测到470nm处的瞬时发光,表明表达的apoaeqxm,apoaeqxxm具有正常的生物学功能

疫苗技术的研究进展和分析

接种疫苗是预防、控制甚至消灭传染病最为有效的方法。疫苗在发展过程中逐渐形成了一系列疫苗品种和疫苗研究技术。随着结构生物学、反向遗传学、生物信息学等学科和技术的快速发展,疫苗研发正逐步迈入新抗原发现及免疫原精准设计的崭新阶段。经过近几十年的发展,我国疫苗研究技术逐步提升,质量水平持续提高,研发品种更为丰富,与国外差距逐渐缩小,初步形成了基础研究技术库、应用转化和产业化的全链条体系。但与发达国家相比,我国在疫苗研发技术和疫苗新品种方面还存在一定的差距。为全面提升我国疫苗研究水平,今后要深耕疫苗基础研究,重点规划疫苗研发和产业化共性技术问题攻关,合理布局疫苗重点研发任务,逐步实现疫苗强国的宏伟目标

HIV-1 Env三聚体抗原改造研究进展

HIV-1疫苗研发是当前艾滋病研究的一大热点,其病毒表面包膜糖蛋白Env三聚体介导病毒与细胞融合,是HIV-1疫苗研究的重要靶点。因此,设计并在体外表达类天然Env三聚体对HIV-1疫苗的研发具有重要的意义。近年来,Env三聚体研究取得了显著的进展。SOSIP、NFL2P、UFO等抗原改造方法实现了类天然Env三聚体的体外表达,逐步解决了改造抗原产量低、结构不稳定等问题,且表达的Env三聚体抗原能在动物免疫中诱导机体产生较高的中和抗体水平。Env三聚体改造方法促进了HIV-1疫苗的研究。文中综述了SOSIP、NFL2P、UFO三种HIV-1 Env三聚体抗原改造方法,对比各个改造方法优缺点,并结合自身工作提出相关建议,为后续HIV-1抗原的相关设计提供指导。国家自然科学基金(No.81671645)资助~

分子对接预测病毒表位的研究进展

分子对接技术是基于相互作用分子的结构特征,预测蛋白质或酶与配体相互作用细节和反应机制的方法。与一般的实验方法比较,其具有快捷、自动化、高通量、低成本和信息丰富等特点。至今,由于局限于有限的结构信息,特别是分子的相互作用,人们对诸多病毒被受体识别并感染细胞过程和产生免疫保护仍然缺乏详细的理论基础,同时许多病毒导致的疾病仍缺乏有效的疫苗或药物。另一方面利用实验方法进行病原体的研究需要消耗大量的资源和时间,如何加速病毒学相关的基因组学信息分析和新发突发病毒的药物开发等过程,分子对接技术起到了不可或缺的作用。本文主要概述了分子对接技术的理论、搜索算法、打分函数、准确性及其在病毒表位预测中的应用,为计算机模拟的病毒表位预测及疫苗或药物研发提供参考。国家自然科学基金(81571996);;\n福建省自然科学基金(2017J07005

Comparison of methods for determination of immunogenicity of recombinant human papillomavirus vaccine

目的评价重组人乳头瘤病毒(HuMAn PAPIllOMAVIruS,HPV)疫苗免疫原性2种检测方法的相关性。方法应用间接酶联免疫吸附(ElISA)法和假病毒中和(PSEudOVIrIOn-bASEd nEuTrAlIzATIOn ASSAy,PbnS)法对26份阴性血清样本、34份自然感染HPV16或HPV18的血清样本及30份接种HPV16/18型双价疫苗后7个月的血清样本进行抗体滴度检测,并采用PEArMAn进行相关性分析。结果 3组样本的抗HPV16型和HPV18型抗体滴度的2次检测结果比值显示,试验室内重复性较好;2种方法检测血清样本抗HPV16型抗体滴度的总体相关系数为0.826,抗HPV18型为0.921;自然感染HPV的血清样本抗HPV16型抗体滴度的相关系数为0.519,抗HPV18型为0.613;接种HPV16/18型双价疫苗后的血清样本抗HPV16型抗体滴度的相关系数为0.671,抗HPV18型为0.879。结论间接ElISA法和PbnS法在检测血清样本中抗HPV16/18型抗体滴度时均具有较好的重复性和相关性,其中以接种疫苗后的血清样本中抗体滴度相关性最佳,为HPV疫苗免疫原性的检测及剂量探索试验提供了参考依据。Objective To assess the correlation between two methods for determination of immunogenicity of recombinant human papillomavirus(HPV) vaccine.Methods Enzyme-linked immunosorbent assay(ELISA) and pseudovirion-based neutralization assay(PBNS) were used to measure the anti-HPV 16 or anti-HPV 18 antibody levels in 26 negative serum samples,34 serum samples naturally infected with HPV16 or HPV18 and 30 serum samples 7 months after immunization with HPV16 / 18 bivalent vaccine,between which the correlation was analyzed by Pearman method.Results The ratio of results of two tests for HPV16 and HPV18 antibody titers in three groups of samples showed high intra-reproducibility.The total correlation coefficient of test results of HPV16 antibody titers by two methods was 0.826,while that of HPV18 antibody was 0.921.The correlation coefficient for HPV 16 antibody titer in naturally infected samples was 0.519,while that for HPV18 antibody titer was 0.613.However,the correlation coefficient for HPV 16 antibody titer in serum samples after immunization with HPV16 / 18 bivalent vaccine was 0.671,while that for HPV 18 antibody titer was 0.879.Conclusion Both ELISA and PBNS showed high reproducibility and correlation for determination of HPV16 / 18 antibody titers in serum samples,especially for those in samples after vaccination.It provided a reference for determination of immunogenicity and optimization of dosage of HPV vaccine.国家高技术研究发展计划(2012AA02A402); Themajorpro-jectofInternationalScienceandTechnologyCollaborativeProgram(2010DFB30100

HIV-1 CAP2NC蛋白的表达及体外自组装

构建并表达HIV-1 CAP2NC蛋白,探索其体外自组装条件。通过PCR技术扩增HIV-1(NL4-3毒株)CAP2NC基因片段,并将其连接到原核表达载体pTO-T7,获得重组质粒pTO-T7-CAP2NC,然后转化至大肠杆菌BL21(DE3)菌株,经疏水层析纯化后获得重组蛋白CAP2NC。SDS-PAGE结果表明,重组蛋白CAP2NC可在大肠杆菌可溶高效表达,经纯化后纯度约为95%。ELISA检测表明重组蛋白CAP2NC可被HIV-1衣壳蛋白特异性单克隆抗体识别,具有较好反应活性。重组蛋白透析后在非原性SDS-PAGE中呈现为多种聚体形式。分子筛排阻层析分析CAP2NC蛋白透析后可进行组装,负染电镜进一步观察显示CAP2NC蛋白在RNA存在条件下,可形成空心管状颗粒,其形态结构与HIV-1病毒衣壳体外自组装形成的类似。上述结果表明HIV-1 CAP2NC蛋白具有体外自组装的性质,为进一步在体外研究非成熟病毒样颗粒结构奠定基础。国家自然科学基金(Nos.81671645,81371818)资助~