Purification,Crystaliziation of broad spectum Anti-H5 AIV Therapeutic Mab 13D4 and Prediction of its Epitope

高致病性禽流感（Avianinfluenza,AI）是由A型流感病毒H5N1引起的烈性传染病。近年来，该疾病随着候鸟迁徙不断向各国传播，在造成巨大的经济损失的同时，也引起了多例人感染禽流感的病例，因此，对禽流感防治手段的研究引起了广泛重视。中和单抗是防治病毒性疾病的一种很有效手段。但是由于禽流感病毒基因突变频率高，尤其以血凝素（HA）基因为最,因此针对禽流感病毒的中和单抗中和谱较窄，往往只能中和少数遗传变异亚系的病毒，这就极大限制了这些中和单抗的应用。 本实验室所制备的单抗13D4具有广谱中和活性，能有效中和41株不同遗传变异亚系的H5N1病毒代表株，在禽流感的防治中具有广阔的应用前景。本研...Highly pathogenic avian influenza (AI), one of syndrome disease caused by influenza A virus H5N1, has recently spread to many countries by the migratory birds. It severely led heavy loss of national economy in these countries, and even caused fatal disease on human. Study on prevention and treatment of avian influenza has aroused extensive attention. Neutralization antibody becomes one of effectiv...学位：理学硕士院系专业：生命科学学院生物学系_生物化学与分子生物学学号：2012005130210

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

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目的 研究高危型人乳头瘤病毒（HR-HPV）感染相关的宫颈病变程度与抗人乳头瘤状病毒（HPV）抗体水平的相关性,探讨HPV致病的免疫学问题。方法 选取2012年1月-2013年6月在该院妇产科门诊就诊的女性中选取60例病理确诊为女性宫颈病变（CINⅠ及以上）,且宫颈脱落细胞的DNA检测为HR-HPV阳性者为研究组,根据组织学检测结果将研究组进一步分为低度病变组30例（CINⅠ）和高度病变组30例（CINⅡ或Ⅲ）。对照组为经核酸检测无HPV感染且病理诊断未见癌前病变的门诊患者60例,按年龄进一步分为低年龄组和高年龄组。低年龄组和高年龄组与低度病变组和高度病变组的年龄差异无统计学意义（P〉0.05）。分别抽取研究组及对照组外周血,检测其抗HR-HPV L1 Ig G抗体及中和抗体滴度,比较研究组与对照组抗体阳性率及抗体滴度水平的差别。结果 研究组血清中的抗HR-HPV L1 Ig G抗体阳性率及抗体滴度均高于对照组（P〈0.05）;低度病变组患者血清中的抗HR-HPV的中和抗体滴度高于相应对照组（P〈0.05）,而高度病变组的抗HR-HPV的中和抗体阳性率及抗体水平与相应对照组比较差异无统计学意义（P〉0.05）。结论 宫颈病变患者血清中抗HR-HPV L1 Ig G抗体升高,说明HPV感染可引起机体的体液免疫反应。低度病变组的中和抗体水平相对较高,可能是机体产生的中和抗体阻止了病毒的进一步感染。高度病变组的Ig G抗体和中和抗体均低下,说明进入CINⅡ/Ⅲ后HPV已经逃避了免疫系统的监视。基金项目：厦门市科技局项目（3502220124050）;2011年福建省科技计划项目资助计划,青年创新项目（20111）00310517

抗H5亚型禽流感病毒单链抗体在毕赤酵母中的分泌型表达和生物活性分析

在本实验室研制出的多株针对H5N1亚型禽流感血凝素单抗中,13D4单抗对所有H5亚型病毒均有血凝抑制和中和活性,具有特异性高、反应性强和识别广的特点,且在小鼠实验中显示了对各种代表株禽流感的感染和发病均具有良好的治疗效果。在此研究基础上,本实验通过基因工程构建含有13D4单链抗体(scFv)基因的毕赤酵母表达载体,实现目的蛋白的分泌性表达和纯化。经过竞争法和血凝抑制检测其活性,表明获得的单链抗体具有与原始鼠源抗体相近的反应活性和相同的识别表位。H5N1广谱中和单抗13D4的单链抗体的成功构建,为进一步研制针对H5N1禽流感病毒的治疗性抗体奠定了基础

人乳头瘤病毒16型病毒样颗粒的制备及其免疫原性研究

利用PCR技术从HPV16阳性阴道分泌物标本中获得HPV16 L1基因片段,并将其插入表达载体pTO-T7中,构建重组表达质粒pTO-T7-HPV16-L1;以该重组质粒转化大肠杆菌ER2566并表达HPV16 L1蛋白;所表达的HPV16 L1蛋白经过硫酸铵沉淀、离子交换层析和疏水相互作用层析等纯化步骤后,HPV16 L1纯度达到98%以上,并可在体外装配为直径50nm的病毒样颗粒;动物免疫原性研究结果显示,该病毒样颗粒可诱导高滴度的针对HPV16的中和抗体。上述研究结果表明通过大肠杆菌表达系统制备的HPV16病毒样颗粒具有纯度高,与天然病毒颗粒形态高度相似的特点,并具有高度免疫原性,可以应用于HPV16病毒样颗粒的结构功能研究及HPV16疫苗研发等领域

大肠杆菌来源的人乳头瘤病毒11型病毒样颗粒的制备及其免疫原性

【目的】利用大肠杆菌表达系统制备人乳头瘤病毒11型病毒样颗粒(HPV11VLPs),并对其免疫原性和所诱导中和抗体的型交叉反应性进行研究。【方法】在大肠杆菌ER2566中非融合表达HPV11-L1蛋白,并通过离子交换层析,疏水相互作用层析其进行纯化。纯化后的HPV11-L1经体外组装形成病毒样颗粒,通过动态光散射,透射电镜检测其形态,并通过多种HPV型别假病毒中和实验评价HPV11VLPs的免疫原性及型交叉反应性。【结果】HPV11-L1蛋白在大肠杆菌中可以以可溶形式表达。经过硫酸铵沉淀、离子交换和疏水相互作用色谱纯化,目的蛋白纯度能够达到95%以上。纯化的HPV11-L1蛋白去除还原剂DTT后,能够在体外自发组装成为直径约50nm、与天然病毒颗粒形态高度相似的VLPs。动物实验结果显示,该病毒样颗粒在小鼠体内的中和抗体半数有效剂量(ED50)为0.031μg,在小鼠体内可诱导高达106的中和抗体滴度,这些中和抗体与HPV6有较好的交叉反应,与HPV18有一定的交叉反应,而与HPV16没有明显的交叉反应,这一结果与分子进化树的分析相一致。【结论】本研究利用原核表达系统获得了具有较高免疫原性的HPV11VLPs,为HPV11预防性疫苗的研制奠定了良好的基础

Prediction of Energy Resolution in the JUNO Experiment

International audienceThis paper presents the energy resolution study in the JUNO experiment, incorporating the latest knowledge acquired during the detector construction phase. The determination of neutrino mass ordering in JUNO requires an exceptional energy resolution better than 3% at 1 MeV. To achieve this ambitious goal, significant efforts have been undertaken in the design and production of the key components of the JUNO detector. Various factors affecting the detection of inverse beta decay signals have an impact on the energy resolution, extending beyond the statistical fluctuations of the detected number of photons, such as the properties of liquid scintillator, performance of photomultiplier tubes, and the energy reconstruction algorithm. To account for these effects, a full JUNO simulation and reconstruction approach is employed. This enables the modeling of all relevant effects and the evaluation of associated inputs to accurately estimate the energy resolution. The study reveals an energy resolution of 2.95% at 1 MeV. Furthermore, the study assesses the contribution of major effects to the overall energy resolution budget. This analysis serves as a reference for interpreting future measurements of energy resolution during JUNO data taking. Moreover, it provides a guideline in comprehending the energy resolution characteristics of liquid scintillator-based detectors

JUNO Sensitivity on Proton Decay p→νˉK+p\to \bar\nu K^+ Searches

The Jiangmen Underground Neutrino Observatory (JUNO) is a large liquid scintillator detector designed to explore many topics in fundamental physics. In this paper, the potential on searching for proton decay in $p\to \bar\nu K^+$ mode with JUNO is investigated.The kaon and its decay particles feature a clear three-fold coincidence signature that results in a high efficiency for identification. Moreover, the excellent energy resolution of JUNO permits to suppress the sizable background caused by other delayed signals. Based on these advantages, the detection efficiency for the proton decay via $p\to \bar\nu K^+$ is 36.9% with a background level of 0.2 events after 10 years of data taking. The estimated sensitivity based on 200 kton-years exposure is $9.6 \times 10^{33}$ years, competitive with the current best limits on the proton lifetime in this channel

JUNO sensitivity on proton decay p → ν K + searches*

The Jiangmen Underground Neutrino Observatory (JUNO) is a large liquid scintillator detector designed to explore many topics in fundamental physics. In this study, the potential of searching for proton decay in the $p\to \bar{\nu} K^+$ mode with JUNO is investigated. The kaon and its decay particles feature a clear three-fold coincidence signature that results in a high efficiency for identification. Moreover, the excellent energy resolution of JUNO permits suppression of the sizable background caused by other delayed signals. Based on these advantages, the detection efficiency for the proton decay via $p\to \bar{\nu} K^+$ is 36.9% ± 4.9% with a background level of $0.2\pm 0.05({\rm syst})\pm$$0.2({\rm stat})$ events after 10 years of data collection. The estimated sensitivity based on 200 kton-years of exposure is $9.6 \times 10^{33}$ years, which is competitive with the current best limits on the proton lifetime in this channel and complements the use of different detection technologies