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。     

构建大容量噬菌体抗体库及获得人源抗体的策略

构建大容量噬菌体抗体库,可实现不经免疫直接制备针对任意抗原的人源性抗体。本文综述了噬菌体抗体库技术的原理、抗体库的构建方法、不经免疫制备人源抗体的策略以及提高抗体体外亲和力的方法。     

丙型肝炎病毒人源单克隆抗体的初步筛选

目的构建天然人源噬菌体抗体库,初步筛选人源抗丙型肝炎病毒(hepatitis C virus,HCV)多肽抗原的单克隆重组噬菌体抗体。方法以健康成人外周血淋巴细胞cDNA为模板,采用PCR技术扩增轻(VL)、重链(VH)可变区基因;重叠PCR法拼接生成单链抗体可变区基因片段(single chain fragment variable,scFv),并克隆至噬菌体载体p CANTAB5e,得到初级抗体库;使用HCV多肽混合抗原,经3轮淘筛富集噬菌体抗体;Phage ELISA法初步筛选HCV重组噬菌体抗体。结果成功构建了天然人源噬菌体抗体库。scFv(VH-κ)库的库容为6.0×10~7 PFU/mL,scFv(VH-λ)库的库容为4.28×10~9 PFU/m L;经初步淘筛,成功获得HCV三级抗体库;Phage ELISA得到了3株候选重组噬菌体抗体。结论初步得到HCV多肽抗原重组噬菌体抗体,为今后进一步筛选高亲和力噬菌体抗体和可溶抗体奠定了基础。     

抗药性基因克隆技术研究进展

综述了近几年来抗药性基因克隆技术的研究进展。对RAPD-PCR、单链构象多态性(SSCP)、差异显示PCR(DDRT-PCR)、代表性差异分析技术(representational difference analysis，RDA)、抑制消减杂交法(suppression subtractive hybridization，SSH)、获得全长基因的消减杂交法(full length gene obtainable subtractive hybridization)、基因芯片技术、基因表达系统分析(serial analysis of gene expression，SAGE)的技术和从蛋白质水平克隆抗药性基因的双向电泳技术(two-dimensional gel electrophoresis)、噬菌体表面全套抗体库技术(phage display antibody repertoire library technique)、核糖体展示抗体库技术等方法的特点、优缺点及适用性进行了评述。     

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。     

噬菌体展示抗乙型肝炎病毒表面抗原Fab抗体分子的筛选和序列分析

目的　从自建的免疫噬菌体展示Fab抗体库中获得抗乙型肝炎病毒表面抗原Fab抗体分子 ,并进行基因序列分析。方法　采用纯化的乙型肝炎病毒表面抗原作为包被抗原 ,对自建的 4× 10 5Fab噬菌体抗体库进行富集筛选 ,从阳性强的次级库中挑选单个克隆菌 ,分别进行噬菌体ELISA、PCR和限制性内切酶鉴定后 ,再选取三者均为阳性的克隆菌进行基因序列分析。结果　共挑选了 6 0个单克隆菌株 ,其中噬菌体ELISA阳性有 2 7个 ,阳性率为 4 5 % ;PCR鉴定均为相应大小片段 ;限制性内切酶鉴定表明 2 7个阳性克隆菌中有 13个具有约 15 0 0bp的片段 ,其余均为约 75 0bp大小 ;BstOI酶切鉴定表明各克隆菌的酶切方式不同 ;选取 5个含约 15 0 0bp片段和 2个含约75 0bp片段的克隆菌株 ,经测序均为人免疫球蛋白基因 ,且重链分别属于VH3、VH4、VH5家族 ,轻链分别属于L5、L6、L8和 0 12 /0 2家族。结论　从自建的免疫噬菌体展示Fab抗体库中成功地获得了抗乙型肝炎病毒表面抗原Fab抗体分子。表明所构建的抗乙型肝炎病毒表面抗原的Fab抗体库的抗体基因具有多样性。     

人源性抗甲型肝炎病毒Fab噬菌体抗体分子的初步筛选

目的从自建的大容量非免疫Fab噬菌体抗体库中筛选具有抗甲型肝炎病毒(HAV)抗原活性的噬菌体抗体,并进行鉴定。方法采用纯化的甲型肝炎病毒作为包被抗原,对自建的容量为1.26×1011pfu/ml的Fab噬菌体抗体库进行筛选和富集,分别对原始库和筛选后的抗体库进行Phage-ELISA和活性检测,经酶切鉴定和DNA测序分析后进行诱导表达。结果经2轮筛选,抗-HAV抗原的噬菌体抗体得到了明显富集,ELISA检测证实其具有良好的抗甲肝病毒抗原的特异性,抑制率为38.61%。酶切鉴定表明抗体库重组较稳定。SDS-PAGE检测显示Fab抗体蛋白在E.coliDH5α中得到了表达。结论从自建的非免疫Fab噬菌体抗体库中成功获得了抗甲肝病毒噬菌体抗体,所构建的非免疫抗体库可用于人源性抗体的筛选。     

2018诺贝尔化学奖:进化控制摘得桂冠

正近日,瑞典皇家科学院宣布,将授予美国科学家弗朗西斯·阿诺德、美国科学家乔治·史密斯及英国科学家格雷戈里·温特2018年诺贝尔化学奖,以表彰他们在酶进化控制研究等领域的贡献。弗朗西斯·阿诺德的成果首次实现了酶的定向进化。乔治·史密斯的主要贡献是研制一种叫做噬菌体展示的实验室技术。格雷戈里·温特是治疗性单克隆抗体的先驱者,研制的噬菌体展示技术应用于抗体     

仙台病毒Tianjin株噬菌体Fab抗体库的构建及初步筛选

目的构建仙台病毒Tianjin株噬菌体Fab抗体库,并进行初步筛选。方法用仙台病毒Tianjin株免疫小鼠,制备脾细胞悬液,提取小鼠脾细胞总RNA,RT-PCR扩增鼠抗体轻链(κ链)和重链Fd基因,将轻链基因和噬菌粒p3MH经SacⅠ和XbaⅠ双酶切后纯化回收,经T4 DNA连接酶连接,电转化E.coli XL1-Blue,构建轻链库;将重链Fd段基因和轻链库p3MH-κ经SpeⅠ和XhoⅠ双酶切后,纯化回收并连接,电转化E.coli XL1-Blue,获得噬菌体抗体库,计算重组率和抗体库滴度。以仙台病毒Tianjin株重组蛋白HN2为抗原进行初步筛选,制备噬菌体抗体,并进行测序。结果构建的免疫噬菌体抗体库库容为1.18×107,重组率为90%,制备的噬菌体抗体滴度为1011pfu/ml;经初步筛选,噬菌体富集了约63.6倍,获得2株与重组蛋白HN2有结合活性的阳性克隆,经NCBI BLAST进行同源性分析,显示为鼠源性抗体,经IMGT分析,显示轻链基因与Vk9亚群基因的同源性为94.87%,重链基因与VH7亚群基因的同源性为97.85%。结论成功构建了仙台病毒Tianjin株噬菌体Fab抗体库,为该病毒株的诊断、疾病治疗及致病机制等研究奠定了基础。     

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。     

Antigen selection from an HIV-1 immune antibody library displayed on yeast yields many novel antibodies compared to selection from the same library displayed on phage

Phage display of antibody libraries has been widely used for over a decade to generate monoclonal antibodies. Yeast display has been developed more recently. Here the two approaches were directly compared using the same HIV-1 immune scFv cDNA library expressed in phage and yeast display vectors and using the same selecting antigen (HIV-1 gp120). Yeast display was shown to sample the immune antibody repertoire considerably more fully than phage display, selecting all the scFv identified by phage display and twice as many novel antibodies. Positive phage display selection appeared to largely reflect those antibodies that as phage-scFv gave the highest signal in phage ELISAs assessing antigen binding. This signal is thought to reflect the efficiency of expression of folded scFv at the phage surface. Increased access to immune repertoires may increase the rescue of novel antibodies of therapeutic or analytical value that often form a minor part of a typical antibody response.     

Antibody phage display libraries: contributions to oncology

Since the advent of phage display technology, dating back to 1985, antibody libraries displayed on filamentous phage surfaces have been used to identify specific binders for many different purposes, including the recognition of tumors. Phage display represents a high-throughput technique for screening billions of random fusion antibodies against virtually any target on the surface or inside cancer cells, or even soluble markers found in patient serum. Many phage display derived binders targeting important tumor markers have been identified. Selection directed to tumoral cells' surfaces lead to the identification of unknown tumoral markers. Also the improvement of methods that require smaller amounts of cells has opened the possibility to use this approach on patient samples. Robust techniques combining an antibody library displayed on the phage surface and protein microarray allowed the identification of auto antibodies recognized by patient sera. Many Ab molecules directly or indirectly targeting angiogenesis have been identified, and one of them, ramucirumab, has been tested in 27 phase I-III clinical trials in a broad array of cancers. Examples of such antibodies will be discussed here with emphasis on those used as probes for molecular imaging and other clinical trials.     

Parallel selection of antibody libraries on phage and yeast surfaces via a cross-species display

We created a cross-species display system that allows the display of the same antibody libraries on both prokaryotic phage and eukaryotic yeast without the need for molecular cloning. Using this cross-display system, a large, diverse library can be constructed once and subsequently used for display and selection in both phage and yeast systems. In this article, we performed the parallel phage and yeast selection of an antibody maturation library using this cross-display platform. This parallel selection allowed us to isolate more unique hits than single-species selection, with 162 unique clones from phage and 107 unique clones from yeast. In addition, we were able to shuttle yeast hits back to Escherichia coli cells for affinity characterization at a higher throughput.     

Generation of a Highly Reactive Chicken-Derived Single-Chain Variable Fragment against Fusarium verticillioides by Phage Display

Fusarium verticillioides is the primary causal agent of Fusarium ear and kernel rot in maize, producing fumonisin mycotoxins that are toxic to humans and domestic animals. Rapid detection and monitoring of fumonisin-producing fungi are pivotally important for the prevention of mycotoxins from entering into food/feed products. Chicken-derived single-chain variable fragments (scFvs) against cell wall-bound proteins from F. verticillioides were isolated from an immunocompetent phage display library. Comparative phage enzyme-linked immunosorbant assays (ELISAs) and sequencing analyses identified four different scFv antibodies with high sensitivity. Soluble antibody ELISAs identified two highly sensitive scFv antibodies, FvCA3 and FvCA4, with the latter being slightly more sensitive. Three-dimensional modeling revealed that the FvCA4 may hold a better overall structure with CDRH3, CDRL1 and CDRL3 centered in the core region of antibody surface compared with that of other scFvs. Immunofluorescence labeling revealed that the binding of FvCA4 antibody was localized to the cell walls of conidiospores and hyphae of F. verticillioides, confirming the specificity of this antibody for a surface target. This scFv antibody was able to detect the fungal mycelium as low as 10(-2) μg/mL and contaminating mycelium at a quantity of 10(-2) mg/g maize. This is the first report that scFv antibodies derived from phage display have a wide application for rapid and accurate detection and monitoring of fumonisin-producing pathogens in agricultural samples.     

Efficient production of antibodies against a mammalian integral membrane protein by phage display

The application of phage display technology to mammalian proteins with multiple transmembrane regions has had limited success due to the difficulty in generating these proteins in sufficient amounts and purity. We report here a method that can be easily and generally applied to sorting of phage display libraries with multispan protein targets solubilized in detergent. A key feature of this approach is the production of biotinylated multispan proteins in virions of a baculovirus vector that allows library panning without prior purification of the target protein. We obtained Fab fragments from a na?ve synthetic antibody phage library that, when engineered into full-length immunoglobulin (Ig)G, specifically bind cells expressing claudin-1, a protein with four transmembrane regions that is used as an entry co-receptor by the hepatitis C virus (HCV). Affinity-matured variants of one of these antibodies efficiently inhibited HCV infection. The use of baculovirus particles as a source of mammalian multispan protein facilitates the application of phage display to this difficult class of proteins.     

人源抗狂犬病病毒scFv噬菌体抗体库的构建及筛选

目的构建人源抗狂犬病病毒单链抗体(Single-chain fragment variable,scFv)噬菌体抗体库,并进行筛选。方法以接种过狂犬病疫苗的21份高效价的健康人外周血为原料,提取淋巴细胞总RNA,PCR扩增VH、Vκ、Vλ基因,重叠延伸PCR扩增获得scFv基因,克隆入噬菌粒pS100,电转化E.coli TG1,建立scFv初级抗体库,并进行基因序列分析及鉴定。以灭活的狂犬病病毒为抗原,进行3轮scFv噬菌体抗体库的富集筛选,随机挑取单克隆,制备噬菌体抗体颗粒,并进行ELISA分析,阳性克隆进行序列分析,采用快速荧光灶抑制试验(Rapid fluorescent facus inhibition test,RFFIT)检测噬菌体抗体颗粒的中和活性。结果获得了库容为1.7×108的人源抗狂犬病病毒scFv噬菌体抗体库,该抗体库具有较好的序列正确率和多样性,经3轮筛选后,ELISA分析显示,共获得24个序列各不相同的针对狂犬病病毒的阳性克隆,对其中7个克隆的噬菌体抗体颗粒进行RFFIT检测,均具有中和活性。结论已成功构建了人源抗狂犬病病毒scFv噬菌体抗体库,并建立了筛选方法。     

A Novel Synthetic Antibody Library with Complementarity-Determining Region Diversities Designed for an Improved Amplification Profile

Antibody discovery by phage display consists of two phases, i.e., the binding phase and the amplification phase. Ideally, the selection process is dominated by the former, and all the retrieved clones are amplified equally during the latter. In reality, the amplification efficiency of antibody fragments varies widely among different sequences and, after a few rounds of phage display panning, the output repertoire often includes rapidly amplified sequences with low or no binding activity, significantly diminishing the efficiency of antibody isolation. In this work, a novel synthetic single-chain variable fragment (scFv) library with complementarity-determining region (CDR) diversities aimed at improved amplification efficiency was designed and constructed. A previously reported synthetic scFv library with low, non-combinatorial CDR diversities was panned against protein A superantigen, and the library repertoires before and after the panning were analyzed by next generation sequencing. The enrichment or depletion patterns of CDR sequences after panning served as the basis for the design of the new library. Especially for CDR-H3 with a higher and more random diversity, a machine learning method was applied to predict potential fast-amplified sequences among a simulated sequence repertoire. In a direct comparison with the previous generation library, the new library performed better against a panel of antigens in terms of the number of binders isolated, the number of unique sequences, and/or the speed of binder enrichment. Our results suggest that the amplification-centric design of sequence diversity is a valid strategy for the construction of highly functional phage display antibody libraries.     

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