仿病毒衣壳自组装体及其生物医学应用

仿病毒衣壳结构自组装体具有重要的基础研究和应用价值,是化学、材料、生物医学等多学科前沿交叉领域.本文从天然病毒衣壳的基本结构和特征出发,立足于从结构仿生到功能仿生的角度,综述了以天然病毒衣壳蛋白质和人工合成材料为组装基元构建仿病毒衣壳自组装体的策略,及其形态结构的调控和功能优化等.重点论述了近年来合成肽类分子在仿病毒衣壳自组装体结构和功能方面的进展.同时,就仿病毒衣壳自组装体在药物控释、基因传递等生物医学领域的应用也做了论述.     

模拟构筑二十面体病毒衣壳结构的研究进展

许多病毒具有二十面体对称结构,二十面体病毒衣壳结构的模拟构筑已成为物质结构的研究目标之一.介绍了二十面体病毒衣壳结构的几种模型,对各种模型的特点及应用范围进行了归纳,并对衣壳结构模拟构筑的意义进行了简要介绍.     

二十面体病毒及其衣壳蛋白质

许多病毒具有二十面体结构。本文对已经发现的各种二十面体病毒进行了分类,分析了二十面体病毒衣壳的结构特征,阐述了二十面体病毒的结构蛋白。     

二十面体壳层结构：纪念C60发现10周年

圆顶建筑、病毒衣壳和Ｃ６０分子笼的生成机制和尺寸差别很大，但都属二十面体壳层，遵循同一几何规律。圆顶建筑和病毒衣壳由近似一等的三角面构成，有１２个五键加３＾５顶，１０（Ｔ－１）个六键连３＾６顶，其中三角面数Ｔ＝ｈ＾２＋ｈｋ＋ｋ＾２是二十面体的一个三角面中的小三角面数，ｈ，ｋ是六角坐标系中３＾５顶的坐标。另一方面，全碳分子笼Ｃｎ（包括其原型Ｃ６０），由１２个五角面与１０（Ｔ－１）个六角面围成，全是三     

DNA-silver Nanocluster Binary Probes for Ratiometric Fluorescent Detection of HPV-related DNA

Herein, we described a ratiometric strategy based on "chameleon" DNA-silver nanoclusters( DNA-AgNCs) fluorescent binary probes. The strategy was applied to detect high-risk human papillomavirus( HPV) DNA sequences, HPV-16. First, DNA-AgNCs were synthesized by a simple reduction method. The obtained nanoprobes showed typical yellow and red fluorescence of AgNCs. Upon the addition of HPV-16 DNA, the yellow fluorescence of AgNCs was reduced greatly, whereas tlie red fluorescence of AgNCs was increased. The concentration of HPV-16 DNA in the samples was characterized by the ratio of fluorescence intensity at 570 and 630 nm. Tlie ratiometric nanoprobes showed good selectivity for HPV-16 DNA, and the detection limit was 2 ninol/L. In addition, the practical applicability of this strategy was demonstrated by analysing the HPV-16 DNA in hiunan serum, illustrating its potential promise for clinical diagnosis.     

重组HEV衣壳蛋白截短体的表达及鉴定

戊型肝炎(HE)是由戊型肝炎病毒(HEV)感染引起的肠道病毒性传染病. HEV是一种无囊膜的单股正链RNA病毒, 其编码区由3个开放阅读框(ORF)组成, 属戊型肝炎病毒科. HEV衣壳蛋白由ORF2编码. 本研究根据编码HEV ORF2 aa382~aa674的核苷酸序列克隆了p293基因, 并将其克隆入原核表达载体pET28a, 利用大肠杆菌BL21(DE3)对HEV衣壳蛋白截短体(p293)进行了表达. SDS-PAGE和Western blot检测结果表明, 在优化的表达条件下(1 mmol/L IPTG, 250 r/min, 37℃, 5 h), 重组蛋白p293能够在大肠杆菌内有效表达, 目的蛋白约占总蛋白的66.15%. TEM检测结果显示, 原核表达的p293能够在体外形成约30~40 nm的病毒样颗粒. 免疫印迹和免疫荧光检测结果表明, p293与HEV标准阳性血清具有良好的反应原性和反应特异性. 实验结果表明, p293可应用于HEV宿主吸附和病毒装配研究, 为HEV的预防与诊断研究奠定了基础.     

二十面体壳层结构——纪念C60发现10周年

圆顶建筑、病毒衣壳和C₆₀分子·笼的生成机制和尺寸差别很大，但都属二十面体壳层，遵循同一几何规律。圆顶建筑和病毒衣壳由近似全等的三角面构成，有12个五键连3⁵顶，10（T-1)个六键连3⁶顶，其中三角面数T=h²+hk十k²是二十面体的一个三角中的小三角面数，h,k是六角坐标系中3⁵顶的坐标。另一方面，全碳分子笼C_n(包括其原型C₆₀)由12个五角面与10(T-1)个六角面围成，全是三键连顶(h,k是一个五角面中心的坐标)。显然，这两类二十面体壳层是对偶的，也就是它们的面、顶有互换关系。本文讨论这些二十面体壳层的几何特征及富勒烯的多样性，强调不同学科间的联系和科学研究的继承与发展的关系。     

我国人乳头瘤病毒6型新变种基因组克隆及L1晚期结构基因在大肠杆菌中表达

本文从一例中国妇女外阴尖锐湿疣组织标本中提取DNA,BamHI酶切,以pAT153为载体,成功地克隆了我国人乳头瘤病毒6型(HPV-6BV)基因组.经South-ern杂交,酶谱及部分DNA序列分析,发现该克隆的HPV6基因组有明显变异,可视为我国发现的第一个HPV6的新变种.本文进一步分离了HPV6BV的L1基因,以pUR288为载体,在大肠杆菌中表达了β-半乳糖苷酶/HPV6BVL1N融合蛋白.经免疫转印试验证明,它既能与β-半乳糖苷酶抗血清反应,又能与HPV抗体反应,具有融合前各自的抗体亲和活性.该蛋白可用作诊断试剂及流行病学研究.     

基于比色荧光纳米球的新冠病毒N蛋白侧向层析检测北大核心CSCD

新型冠状病毒传播速率快,且能导致严重呼吸综合征,危及生命安全。及时检测该病毒尤为重要。目前的检测手段各有优势,但仍需进一步提高。本文制备了一种比色荧光纳米球用于新冠病毒核衣壳蛋白(N protein)的侧向层析检测。该纳米球内有多个量子点可用于荧光定量检测,外有多个金纳米粒子用于比色定性检测。荧光检测限可达2.1 nmol/L。该方法重现性和特异性好,具有较强抗干扰能力。本研究为新冠病毒的快速检测提供了一种新方法。     

HIV-1病毒DNA与整合酶结合后的构象变化

用分子动力学(MD)模拟方法优化了HIV-1病毒DNA与整合酶(IN)二聚体(IN2)复合物模型结构, 并分析了HIV-1病毒DNA结合IN2后的构象变化. 结果表明, 按照HIV-1病毒DNA与IN2结合能力的强度, 病毒DNA可分为五个区域: 非结合区、强结合区1、弱结合区、强结合区2和反应区, 并用结合自由能计算验证了该分区的合理性. 与未结合IN2的病毒DNA相比, 复合物模型中病毒DNA除了非结合区碱基外, 其它四个区域的碱基构象变化较大. 复合物模型中病毒DNA主链较大程度地偏离标准B型DNA以及结合部位的小沟变宽都是识别IN的结构基础. 模拟结果与实验数据吻合较好, 为基于HIV-1 IN的药物分子设计提供了一定的结构信息.     

The differences in heparin binding for the C-terminal basic-sequence-rich peptides of HPV-16 and HPV-18 capsid protein L1

The high-risk types of human papillomaviruses (HPV) HPV-16 and -18 are the predominant types associated with cervical cancer. HPV-16 and -18 account for about 50% and 20%, respectively, of cervical cancers worldwide. While the reason and molecular mechanism of the distinct prevalence and distributions between them remain poorly understood, the binding affinity of cell surface receptor with capsid proteins, especially L1, may be involved. We examined heparin binding with two synthetic peptides corresponding to the 14 amino acid C-terminal peptides of HPV-16 and -18 L1 with the goal of comparing the equivalent residues in different HPV types. Using isothermal titration calorimetry (ITC) and static right-angle light scattering (SLS), we determined the binding constant K, reaction enthalpy ΔH, and other thermodynamic parameters in the interaction. Especially, we assessed the role of specific residues in binding with heparin by comparing the NMR spectra of free and heparin-bound peptides.     

Study of intact virus‐like particles of human papillomavirus by capillary electrophoresis

Virus‐like particles of human papillomavirus (HPV‐VLP), resulting from the self‐assembly of the capsid proteins (L1 or L1 and L2), have been widely used to study HPV as they are similar to the native virion. Moreover, two prophylactic vaccines, Gardasil^® and Cervarix^®, are based on HPV‐VLP L1. Analytical techniques currently used to characterize HPV‐VLP, such as SDS‐PAGE, Western blot, ELISA, are time‐consuming and semiquantitative. In this study, CE was evaluated for the analysis of intact HPV16‐VLP. The usefulness of capillary inner wall coating as well as various BGEs, pH, and detergent additives were investigated. Reproducible HPV‐VLP analysis in CE was achieved using poly(ethylene oxide)‐coated capillary and a BGE containing high salt concentration and low SDS concentration. The developed method enables HPV‐VLP detection in less than 10 min (migration times RSD: 1.6%). The identity of HPV‐VLP peak was confirmed by comparison with a sample obtained from a wild‐type baculovirus and with VLP‐based vaccine, Gardasil^®, after adjuvant dissolution. Finally, we applied the developed methodology to VLP‐based vaccines, demonstrating that CE could be successfully used for vaccine quality control.     

体积排阻色谱法定量检测9种型别人乳头瘤病毒原液中病毒样颗粒

据统计,5%以上的人类癌症由人乳头瘤病毒(HPV)导致。HPV疫苗的使用,尤其是多价HPV疫苗的使用,可有效预防HPV感染和肿瘤的发生。例如,9价HPV疫苗可有效预防90%以上HPV相关癌前病变。人乳头瘤病毒样颗粒(VLP)是HPV疫苗的唯一抗原。VLP由360份衣壳蛋白L1组成。VLP的含量测定对HPV原液和HPV疫苗的质量评价至关重要。该文发展了一种以体积排阻色谱(SEC)为基础的9种型别人乳头病毒样颗粒的定量方法。实验优化了包括色谱柱类型、色谱柱孔径、流动相离子强度和流动相pH值在内的色谱条件。经过考察,以SHIMSEN Ankylo SEC-300色谱柱(300 mm×7.8 mm, 3 μm)为固定相,以含有300 mmol/L NaCl和50 mmol/L磷酸盐(pH 7.0)的缓冲溶液为流动相时,VLP的色谱峰更窄,从而可获得更高的响应和更好的灵敏度,因此选择该色谱条件用于VLP与基质的分离。优化所得的方法具有较宽的线性范围,良好的重复性(峰面积的相对标准偏差不大于5.0%)和灵敏度(定量限为4.58~15.24 μg/mL)。将方法用于HPV原液中VLP的含量测定,监测VLP的稳定性。结果显示,HPV原液中VLP颗粒不稳定,于4 ℃放置一周后,VLP含量与生产后立即测得的含量相比存在一定程度的降解。此外,方法还可用于疫苗上清液中游离蛋白质的分析,监测铝佐剂对VLP的吸附情况。被测厂家的铝佐剂可较好的吸附VLP,无明显残余蛋白质检出。与传统的蛋白质定量方法相比,如Folin-酚法(Lowry法),该法具有操作简单、自动化程度高、分析通量高等优点,可实现VLP含量的批量化分析。     

Improved immunodetection of human papillomavirus E7

Human papillomavirus E7 (HPV E7) is a viral oncoprotein that plays an important role in cervical carcinogenesis through binding with retinoblastoma protein (Rb). Inactivation of Rb by E7 is necessary but not sufficient for cellular transformation, suggesting other protein-protein interactions are required for E7-mediated cellular transformation aside from the interaction with Rb. However, studies on the oncogenic function of HPV E7 have been limited by its poor immunoreactivity. In this report, we show that the fixation of purified recombinant HPV E7 on blotted nitrocellulose membrane with glutaldehyde markedly enhanced the immunoreactivity of HPV E7 protein. Using HeLa and Caski cell lines which are infected with HPV 18 and HPV 16, respectively, we demonstrated that native HPV E7 proteins also could be detected by this method. These results therefore can provide the experimental conditions for detection of HPV E7 proteins with greater sensitivity and may help to analyze E7 functions.     

Ultrasensitive Electrochemical Biosensor for HPV16 Oncogene Based on Y-shaped DNA Catalytic Hairpin Assembly and Template-free DNA Extension Reaction

An ultrasensitive electrochemical biosensor for HPV16 oncogene was explored. Hairpin DNA-1, which can specifically bind with HPV16 oncogene, was fixed on the surface of gold electrode. Two hairpin DNAs underwent catalytic hairpin assembling with hairpin DNA-1 to construct Y-shaped DNA nanostructure, liberating HPV16 oncogene for target recycling. The 3’ terminus of Y-shaped DNA nanostructure was prolonged under the catalysis of terminal deoxynucleotidyl transferase. Methylene blue was adsorbed onto DNA nanostructure to generate characteristic differential pulse voltammetry signal. This signal was increased with the concentration of HPV16 oncogene, and the detection limit of HPV16 oncogene was as low as 0.19 fM.     

The inhibitory properties of acidic functionalised calix[4]arenes on human papillomavirus pentamer formation

ABSTRACT

Human Papillomavirus (HPV) is the leading cause of cervical cancer, with only some HPV types prevented with vaccines and no treatments for the viral infection itself. One way to target viral infection is by inhibiting the assembly of the L1 monomer into a pentamer, which forms the viral capsid. Four calix[4]arene compounds functionalised with D- and L-aspartic and glutamic acid and an iminodiacetic functionalised calix[4]arene were synthesised and tested for L1 pentamer formation inhibition. The amino acid functionalised calix[4]arene derivatives showed millimolar inhibition (IC₅₀ = 0.72 to 2.67 mM) of pentamer formation, with little difference between the stereoisomers. The iminodiacetic acid calix[4]arene derivative showed no inhibitory properties, despite sharing structural similarities with the four other calix[4]arenes. Confirmation of binding the negatively charged compounds to the positive residues of the L1 protein was achieved by trypsin digestion. This study is helpful in the development of cost-effective inhibitors to prevent HPV assembly.     

Motions on the millisecond time scale and multiple conformations of HIV-1 capsid protein: implications for structural polymorphism of CA assemblies

The capsid protein (CA) of human immunodeficiency virus 1 (HIV-1) assembles into a cone-like structure that encloses the viral RNA genome. Interestingly, significant heterogeneity in shape and organization of capsids can be observed in mature HIV-1 virions. In vitro, CA also exhibits structural polymorphism and can assemble into various morphologies, such as cones, tubes, and spheres. Many intermolecular contacts that are critical for CA assembly are formed by its C-terminal domain (CTD), a dimerization domain, which was found to adopt different orientations in several X-ray and NMR structures of the CTD dimer and full-length CA proteins. Tyr145 (Y145), residue two in our CTD construct used for NMR structure determination, but not present in the crystallographic constructs, was found to be crucial for infectivity and engaged in numerous interactions at the CTD dimer interface. Here we investigate the origin of CA structural plasticity using solid-state NMR and solution NMR spectroscopy. In the solid state, the hinge region connecting the NTD and CTD is flexible on the millisecond time scale, as evidenced by the backbone motions of Y145 in CA conical assemblies and in two CTD constructs (137-231 and 142-231), allowing the protein to access multiple conformations essential for pleimorphic capsid assemblies. In solution, the CTD dimer exists as two major conformers, whose relative populations differ for the different CTD constructs. In the longer CTD (144-231) construct that contains the hinge region between the NTD and CTD, the populations of the two conformers are likely determined by the protonation state of the E175 side chain that is located at the dimer interface and within hydrogen-bonding distance of the W184 side chain on the other monomer. At pH 6.5, the major conformer exhibits the same dimer interface as full-length CA. In the short CTD (150-231) construct, no pH-dependent conformational shift is observed. These findings suggest that the presence of structural plasticity at the CTD dimer interface permits pleiotropic HIV-1 capsid assembly, resulting in varied capsid morphologies.     

High‐Mass MALDI‐MS Analysis for the Investigation of Protein Encapsulation within an Engineered Capsid Forming Protein

Chemical cross‐linking combined with MALDI ‐MS was applied to structural analysis of a protein nanocontainer. Specifically, an engineered variant of lumazine synthase from Aquifex aeolicus (AaLS ‐13) was investigated that self‐assembles into a capsid‐like structure and is known to encapsulate other proteins by Coulombic attraction. Two complementary soft ionization techniques, MALDI ‐MS and native ESI ‐MS , were utilized to map the subunit stoichiometry of the high molecular weight capsid. In accordance with the previously reported cryo‐electron microscopy structure of this protein container, only pentameric subunits were detected. This study highlights the possibility to map subunit stoichiometry via chemical cross‐linking with glutaraldehyde followed by MALDI ‐MS . The same approach was used to study protein‐protein interactions during encapsulation of GFP (+36) by the AaLS ‐13 capsid. Heterocomplexes between GFP (+36) and AaLS ‐13 multimers were not observed when mixed at maximal loading capacity (AalS‐13 monomer:GFP (+36) 4:1). This is in agreement with the known fast encapsulation of GFP (+36) by the protein capsid, which essentially removes any free GFP (+36) from the solution. Exceeding the maximal loading capacity by addition of excess GFP (+36) results in aggregation.     

In Silico Comparison of Low- and High-Risk Human Papillomavirus Proteins

Human papillomavirus (HPV) is an important pathogen which is classified into two, high- and low-risk groups. The proteins of high-risk and low-risk HPV types have different functions. Therefore, there is a need to develop a computational method for predicting these two groups. In the present study, the physiochemical properties of all early (E1, E2, E4, E5, E6, and E7) and late (L1 and L2) proteins in high- and low-risk HPV types have been studied. The concept of receiver operating characteristic analysis and support vector machines methods has been used for comparison of high- and low-risk HPV types. The results demonstrate that amino acid composition, physiochemical, and secondary structure of E2 protein are significantly different between these two groups. The results demonstrate that in silico properties can create useful information to predict high-risk and low-risk HPV types.