噬菌体展示疫苗的研究进展

噬菌体是感染细菌、真菌、放线菌和螺旋体等微生物的病毒,因其固有的免疫原性、遗传可塑性、稳定性及安全性等优势,使其在疫苗研发中具有独特的潜力。目前,有较多利用其构建疫苗递送平台的研究,主要包括噬菌体展示疫苗、噬菌体DNA疫苗及杂交噬菌体DNA疫苗3种形式,其中研究最为广泛的是噬菌体展示疫苗。噬菌体展示技术是一种新型疫苗制备技术,是以噬菌体为载体,通过将外源多肽或蛋白基因整合至噬菌体基因中,以融合蛋白的形式展示在噬菌体表面的分子生物学技术。本文主要就噬菌体展示疫苗的免疫学基础、展示系统及其在疾病预防应用的研究进展作一综述,以期为噬菌体展示疫苗的研发与应用提供参考。     

噬菌体展示技术在纳米材料合成中的应用

建立在化学、生物学和材料学等交叉学科基础上的噬菌体展示技术,为合成、组装新颖纳米材料提供了一条新的途径.噬菌体作为一种信息载体,能模拟自然进化过程产生特异性多肽,从而在分子水平上识别靶材料并进行自组装.噬菌体的单分散性和长杆状外形,造就了特定的识别部位,使各种纳米材料有序地组装成规则的层次结构.通过噬菌体展示技术筛选出来的特异性识别肽,可以指导多肽介导的矿化过程,从而合成具有应用价值的一系列无机和有机纳米材料,进一步制成的纳米装置将应用于电子、光学、生物技术和医学领域.     

噬菌体展示抗体库技术的研究进展

噬菌体展示抗体库技术是一种将抗体组合文库与噬菌体表面展示技术相结合所形成的新技术,可以将抗体分子展示在噬菌体表面,且保持了抗体的天然构象和生物学活性,为人源抗体的制备提供了良好的技术平台。本文对噬菌体展示抗体库技术的原理、特点、类型及其研究进展作一综述。     

噬菌体展示颗粒疫苗的研究进展

噬菌体是以细菌为宿主的病毒,具有严格的宿主特异性,只"寄生"在易感宿主菌体内,对动植物细胞无毒性。噬菌体结构简单,基因数少,是分子生物学与基因工程的良好操作系统。建立在噬菌体展示技术基础上的噬菌体颗粒,因其安全可靠,免疫效果好,在预防性和治疗性疫苗的研究中取得良好效果,表明噬菌体作为疫苗载体用于疫苗学的研究是一种新的发展方向,对新型疫苗的开发具有重要意义。本文就噬菌体颗粒用于疫苗研究的优势、研究进展及其应用前景作一综述。     

噬菌体展示技术与肽疫苗的设计

近年来,噬菌体肽库的构建及筛选技术得到迅速发展和推广,现已广泛应用于基因定位、分子识别、疫苗设计等相关领域,尤其在非常规疫苗设计方面具有巨大的潜力。该疫苗设计策略是在分子免疫学抗原呈递的机制研究基础上提出的,具有安全、分子小、周期短、特异性高、易于大规模制备等特点,可以代替传统疫苗。本文就噬菌体展示技术用于肽疫苗设计的原理及前景作一综述。     

全人源单链抗体库噬菌体展示技术的建立

目的建立全人源单链抗体(single-chain antibody fragment,scFv)库的噬菌体展示技术。方法采集100名健康老年志愿者的外周血,5 mL/人,混合所有全血并分离外周血单个核细胞(peripheral blood mononuclear cells,PBMCs),抽提RNA,反转录合成cDNA,以其为模板,IgG类抗体基因为引物,PCR扩增获得重链可变区片段(V_H)和轻链可变区片段(V_L),然后以V_H和V_L为模板,经重叠PCR获得scFv,经sfiⅠ酶切,插入pComb3xss载体,电转后获得一定容量的抗体库;加入辅助噬菌体VCSM13过夜培养,上清液经PEG-NaCl沉淀,无菌PBS重悬噬菌体沉淀,即完成噬菌体抗体库的构建;采用Phage ELISA法进行噬菌体单克隆抗体的特异性鉴定。结果成功构建了库容为2. 6×10~9的噬菌体抗体库;随机挑选的100个噬菌体单克隆中共筛选到2个疑似H1N1株HA抗原的噬菌体抗体。结论成功构建了噬菌体抗体库的技术平台,可用于各类功能抗体的筛选。     

噬菌体展示技术开发兔源抗体的工艺优化

特异性抗体可以与蛋白相结合,监控靶点蛋白在生物体内的变化,比如质量和定位等变化,因此可以揭示蛋白在生命活动中所起的功能,在科学研究中起着关键作用。单克隆抗体因其特异性和稳定性越来越受到重视,筛选单克隆抗体有多种方法,本文以噬菌体展示技术为核心,阐述了抗CDT1蛋白（蛋白号：Q9H211）单克隆抗体的开发工艺,通过对该工艺流程的两点优化方案,构建了噬菌体scFv文库,成功地淘选到抗CDT1蛋白的噬菌体scFv,通过酶联免疫吸附测定（Enzyme Linked Immunosorbent Assay,ELISA）和免疫印迹检测确定其特异性和亲和性后,将此scFv转换成完整抗体IgG,可以应用于免疫印迹和免疫荧光试验,表明此工艺优化的可行性。     

Cult3D技术及其在陶瓷产品展示中的应用

与传统的产品二维图像展示相比,基于Web3D技术的产品三维展示以其独有的优势正成为目前国内外企业推广产品的主要方式。为此,文章概述了Web3D技术的基本概念和特点,研究了Cult3D的构成、关键技术和开发流程,并以实例的方式阐述了Cult3D技术在陶瓷产品展示中的应用。     

冷冻干燥法制备1型糖尿病噬菌体展示疫苗

目的采用冷冻干燥法制备1型糖尿病噬菌体展示疫苗。方法将正交试验筛选的不同冻干保护剂与1型糖尿病噬菌体展示疫苗混合,优化冻干曲线进行冻干。检测冻干前后噬菌体疫苗的滴度,并通过各项指标对冻干后的疫苗进行评价。结果经筛选,最佳保护剂配方为:10.85%海藻糖+17.37%甘氨酸(w/v);最佳冻干曲线为:S1:-40℃4h,1.5℃/min;S2:-15℃5h,1.5℃/min;S3:25℃4h,1.5℃/min;真空度:0.02mbar。冻干后疫苗滴度下降不超过0.5pfu/ml,外观及电镜观察疫苗样品形态均较好,玻璃态转化温度能达到220℃以上,含水量小于3%。结论以海藻糖、甘氨酸为保护剂,采用冷冻干燥法制备的1型糖尿病噬菌体展示疫苗具有保护剂组成成分少、热稳定性强、含水量低、冻干曲线简化、保存时间长等特点。     

噬菌体在污水处理系统中的应用

近年来,伴随复杂多样的水媒型传染病的不断爆发以及多重耐药性超级细菌的全球蔓延,噬菌体以其在消灭病原菌上具有高度特异性、指数增殖性等特点,受到科研工作者们的广泛重视。该文综述了噬菌体的基本特征、研究应用中存在的技术优势与局限以及噬菌体在污水处理系统中的应用研究进展,论述了利用噬菌体技术防治污水中病原菌的现状及前景。     

从半合成噬菌体抗体库筛选抗狂犬病毒人单链抗体

目的　应用纯化的狂犬病毒抗原从半合成噬菌体抗体库中筛选针对狂犬病毒的人单链抗体(ScFv)。方法　用固相化的狂犬病毒抗原对半合成抗体库进行 3轮“吸附 洗脱 扩增”的筛选 ,从第 3轮洗脱下来的克隆中获得一株有可溶性表达且特异性结合狂犬病毒抗原的ScFv ,并进行基因序列测定。结果　所获氨基酸序列经blast数据库搜索 ,与一种抗狂犬病毒免疫球蛋白的氨基酸序列同源性最高 ( 82 % )。经检索kabat数据库 ,发现其轻、重链可变区分别属于VkⅠ型、VHⅢ型。结论　从噬菌体抗体库可以方便快捷地分离到针对狂犬病毒的单链抗体 ,对狂犬病毒的预防具有重要意义     

DNA疫苗及其在传染病控制中的应用

疫苗在人类抵御传染性疾病的过程中扮演着重要角色,DNA疫苗在应对突发性新型传染病方面具有独特的优势。近年来,随着对DNA疫苗研究的不断深入,DNA疫苗的安全性和有效性均得到了极大的提高。虽然目前尚无人用DNA疫苗应用于临床,但在畜牧养殖业已有了初步的探索。本文对DNA疫苗的发展及其作用机制、优势、安全性、改进及其临床应用情况作一综述。     

白细胞介素-10在感染性疾病中的作用

白细胞介素-10是一种具有多种生物学功能的细胞因子,在疾病的治疗及诊断中具有重要的作用。本文对白细胞介素-10的分子结构特点、在感染性疾病中的作用及其临床应用作一综述。     

Phage Display Screening of Bovine Antibodies to Foot-and-Mouth Disease Virus and Their Application in a Competitive ELISA for Serodiagnosis

For serodiagnosis of foot-and-mouth disease virus (FMDV), monoclonal antibody (MAb)-based competitive ELISA (cELISA) is commonly used since it allows simple and reproducible detection of antibody response to FMDV. However, the use of mouse-origin MAb as a detection reagent is questionable, as antibody responses to FMDV in mice may differ in epitope structure and preference from those in natural hosts such as cattle and pigs. To take advantage of natural host-derived antibodies, a phage-displayed scFv library was constructed from FMDV-immune cattle and subjected to two separate pannings against inactivated FMDV type O and A. Subsequent ELISA screening revealed high-affinity scFv antibodies specific to a serotype (O or A) as well as those with pan-serotype specificity. When BvO17, an scFv antibody specific to FMDV type O, was tested as a detection reagent in cELISA, it successfully detected FMDV type O antibodies for both serum samples from vaccinated cattle and virus-challenged pigs with even higher sensitivity than a mouse MAb-based commercial FMDV type O antibody detection kit. These results demonstrate the feasibility of using natural host-derived antibodies such as bovine scFv instead of mouse MAb in cELISA for serological detection of antibody response to FMDV in the susceptible animals.     

Design and Characterization of a New pVII Combinatorial Phage Display Peptide Library for Protease Substrate Mining Using Factor VII Activating Protease (FSAP) as Model

We describe a novel, easy and efficient combinatorial phage display peptide substrate-mining method to map the substrate specificity of proteases. The peptide library is displayed on the pVII capsid of the M13 bacteriophage, which renders pIII necessary for infectivity and efficient retrieval, in an unmodified state. As capture module, the 3XFLAG was chosen due to its very high binding efficiency to anti-FLAG mAbs and its independency of any post-translational modification. This library was tested with Factor-VII activating protease (WT-FSAP) and its single-nucleotide polymorphism variant Marburg-I (MI)-FSAP. The WT-FSAP results confirmed the previously reported Arg/Lys centered FSAP cleavage site consensus as dominant, as well as reinforcing MI-FSAP as a loss-of-function mutant. Surprisingly, rare substrate clones devoid of basic amino acids were also identified. Indeed one of these peptides was cleaved as free peptide, thus suggesting a broader range of WT-FSAP substrates than previously anticipated.     

从噬菌体表面展示肽库中筛选边缘无浆体膜表面蛋白5单克隆抗体识别的抗原表位

目的从噬菌体表面展示肽库中筛选边缘无浆体膜表面蛋白5(Membrane surface protein5,MSP5)单克隆抗体识别的抗原表位。方法用MSP5单克隆抗体1D8作为靶标,对噬菌体展示随机12肽库进行筛选,通过ELISA和竞争抑制ELISA鉴定筛选克隆的结合特性,并提取阳性克隆的单链DNA,进行测序分析。结果从表面展示随机肽序列的噬菌体文库中筛选到与MSP5单抗1D8特异结合的噬菌体克隆,其一致序列为LING。竞争抑制试验表明,含特异序列的克隆能与MSP5重组蛋白抗原竞争。结论初步确定MSP5单克隆抗体1D8的抗原表位为线性表位,为进一步研究其在边缘无浆体诊断及新型疫苗研制中的作用奠定了基础。     

乙肝乙脑麻疹多表位噬菌体疫苗的构建及其免疫原性

目的构建乙肝乙脑麻疹多表位噬菌体疫苗,并检测其免疫原性。方法将PCR方法合成的乙肝乙脑麻疹多表位基因COM插入T7噬菌体基因组中,使其与噬菌体10B蛋白融合,展示在T7噬菌体表面,构建成多表位噬菌体疫苗COM/T7。分别以1010pfu/只和1012pfu/只两种剂量,通过腹腔、皮下和鼻腔3种途径免疫昆明小鼠,ELISA检测小鼠特异性抗体水平。结果重组噬菌体COM/T7经PCR鉴定证明构建正确。乙肝乙脑麻疹多表位噬菌体疫苗能诱导小鼠产生针对各表位的特异性IgG抗体。在3种免疫途径中,腹腔免疫效果最好。免疫剂量与诱导的抗体水平在一定范围内呈正相关。多表位噬菌体疫苗诱导的抗-HBs和抗-JEV水平高于常规疫苗,而抗-MV水平低于常规疫苗。结论构建的乙肝乙脑麻疹多表位噬菌体疫苗具有良好的免疫原性。     

The Screening of Therapeutic Peptides for Anti-Inflammation through Phage Display Technology

For the treatment of inflammatory illnesses such as rheumatoid arthritis and carditis, as well as cancer, several anti-inflammatory medications have been created over the years to lower the concentrations of inflammatory mediators in the body. Peptides are a class of medication with the advantages of weak immunogenicity and strong activity, and the phage display technique is an effective method for screening various therapeutic peptides, with a high affinity and selectivity, including anti-inflammation peptides. It enables the selection of high-affinity target-binding peptides from a complex pool of billions of peptides displayed on phages in a combinatorial library. In this review, we will discuss the regular process of using phage display technology to screen therapeutic peptides, and the peptides screened for anti-inflammation properties in recent years according to the target. We will describe how these peptides were screened and how they worked in vitro and in vivo. We will also discuss the current challenges and future outlook of using phage display to obtain anti-inflammatory therapeutic peptides.     

Endocrine-Disrupting Chemicals and Infectious Diseases: From Endocrine Disruption to Immunosuppression

Endocrine-disrupting chemicals (EDCs) are hormonally active compounds in the environment that interfere with the body’s endocrine system and consequently produce adverse health effects. Despite persistent public health concerns, EDCs remain important components of common consumer products, thus representing ubiquitous contaminants to humans. While scientific evidence confirmed their contribution to the severity of Influenza A virus (H1N1) in the animal model, their roles in susceptibility and clinical outcome of the coronavirus disease (COVID-19) cannot be underestimated. Since its emergence in late 2019, clinical reports on COVID-19 have confirmed that severe disease and death occur in persons aged ≥65 years and those with underlying comorbidities. Major comorbidities of COVID-19 include diabetes, obesity, cardiovascular disease, hypertension, cancer, and kidney and liver diseases. Meanwhile, long-term exposure to EDCs contributes significantly to the onset and progression of these comorbid diseases. Besides, EDCs play vital roles in the disruption of the body’s immune system. Here, we review the recent literature on the roles of EDCs in comorbidities contributing to COVID-19 mortality, impacts of EDCs on the immune system, and recent articles linking EDCs to COVID-19 risks. We also recommend methodologies that could be adopted to comprehensively study the role of EDCs in COVID-19 risk.