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1.
本研究旨在利用噬菌体展示技术构建人源性天然抗体库,以可溶性Aβ1-42寡聚体对抗体库进行筛选获得针对低分子量Aβ1-42寡聚体的特异性单链抗体.利用RT-PCR法从10个健康人外周血淋巴细胞中得到全套人抗体VH和VL基因,经过重叠延伸PCR将VH和VL连接得到scFv片段,将scFv片段酶切后克隆至pCANTAB5E噬菌体载体,电转化TG1感受态菌,获得库容为2.5×109单链抗体库.经辅助噬菌体M13K07拯救,以可溶性Aβ1-42寡聚体为抗原,对抗体库进行4轮筛选,ELISA法筛选特异性识别Aβ1-42寡聚体的阳性克隆,将筛选到的阳性克隆B19转化至E coliHB2151菌,诱导表达可溶性scFv抗体.SDS-PAGE及Western blotting分析结果显示可溶性scFv抗体获得了正确表达,且能够与Aβ1-42三聚体及纤维特异性结合,亲和力(Kd)为9×10-6 mol/L.Aβ1-42寡聚体特异性单链抗体的获得为老年性痴呆(AD)的治疗研究奠定了基础.  相似文献   

2.
本研究旨在利用噬菌体展示技术构建人源性天然抗体库,以可溶性Aβ1-42寡聚体对抗体库进行筛选获得针对低分子量Aβ1-42寡聚体的特异性单链抗体。利用RT-PCR法从10个健康人外周血淋巴细胞中得到全套人抗体VH和VL基因,经过重叠延伸PCR将VH和VL连接得到scFv片段,将scFv片段酶切后克隆至pCANTAB5E噬菌体载体,电转化TG1感受态菌,获得库容为2.5×109单链抗体库。经辅助噬菌体M13K07拯救,以可溶性Aβ1-42寡聚体为抗原,对抗体库进行4轮筛选,ELISA法筛选特异性识别Aβ1-42寡聚体的阳性克隆,将筛选到的阳性克隆B19转化至E.coli HB2151菌,诱导表达可溶性scFv抗体。SDS-PAGE及Western blotting分析结果显示可溶性scFv抗体获得了正确表达,且能够与Aβ1-42三聚体及纤维特异性结合,亲和力(Kd)为9×10-6 mol/L。Aβ1-42寡聚体特异性单链抗体的获得为老年性痴呆(AD)的治疗研究奠定了基础。  相似文献   

3.
噬菌体抗体库技术是获得治疗性抗体的一条重要途径。以20份健康人外周血为样本,通过提取淋巴细胞、逆转录-PCR(RT PCR)、抗体可变区基因的扩增、重叠PCR获得单链抗体(ScFv)基因,将ScFv克隆入噬粒载体,通过近300次的电转化获得了库容量为1.3×109的全人源天然ScFv噬菌体抗体库。通过随机挑克隆测序和用5种不同抗原筛选对抗体库进行了初步验证。随机测序表明抗体库具有较好的多样性,用5种不同抗原对其进行筛选,均获得了特异性噬菌体抗体的不同富集,表明成功构建了一个多样性良好的人源天然ScFv噬菌体抗体库。  相似文献   

4.
噬菌体抗体库技术是获得人源抗体的重要方法,此文探讨大容量天然噬菌体抗体库在筛选人源抗体中的实际应用价值。从正常人外周血分离淋巴细胞,通过基因工程方法获得抗体基因并且构建到含有重组位点的载体pDF中获得初级噬菌体抗体库,初级库在重组系统中重组之后获得了容量为9×1010的天然Fab噬菌体抗体库,通过序列测定和重组蛋白筛选来对抗体库的质量进行初步鉴定。对随机挑取的96个克隆测序序列分析表明各种型别比例基本合适;用四种抗原筛选有明显的富集,并且获得了针对其中一种抗原的阳性抗体克隆。  相似文献   

5.
目的:从大容量噬菌体抗体库中筛选人源性抗呼吸道合胞病毒F蛋白的单链抗体。方法:以RSV F蛋白为靶抗原,通过“吸附-洗涤-洗脱-扩增”过程从天然人源性噬菌体抗体库中筛选特异性抗F蛋白单链抗体。5轮筛选后,单克隆经ELISA检测,阳性克隆进行核酸序列分析,并将阳性克隆噬菌体感染E.coli HB2151,经IPTG诱导,制备抗RSV F蛋白的可溶性单链抗体,并进行Western及Dot blot分析。结果:经过筛选,获得了18株能与F蛋白特异性结合的阳性克隆,取OD值最高的克隆E4经测序并检索Kabat数据库分析,显示其基因与人免疫球蛋白可变区基因具有高度同源性,Western及Dot blot分析表明为单链抗体。结论:利用天然人源性噬菌体抗体库技术制备出高特异性的人源性抗RSV F蛋白单链抗体。  相似文献   

6.
目的:从天然的大容量噬菌体抗体库中筛选特异的抗结核分枝杆菌晶体蛋白( alpha-crystallin Acr)的人源抗体.方法:以结核分枝杆菌Acr蛋白包被免疫管,通过对噬菌体抗体库进行4轮“吸附-洗脱-扩增”的过程从大容量抗体库中筛选特异性抗结核分枝杆菌Acr蛋白的抗体,并对可变区序列进行了测序分析.将特异性的噬菌体抗体感染HB2151菌,经IPTG诱导表达,制备了抗结核分枝杆菌Acr蛋白的可溶性单链抗体;对其序列和抗原结合活性进行分析鉴定.结果:经过4轮筛选,获得了43个与结核分枝杆菌Acr蛋白结合的阳性克隆,其中29个特异结合的克隆;测序分析有26不同的可变区片段;通过可溶性单链抗体(scFv)表达筛选到14株特异性结合Acr蛋白的可溶性单链抗体克隆;经过基因测序,分析了可变区基因的亚群.成功制备了可溶性单链抗体.Westren blotting分析证实筛选的人源单链抗体能与天然蛋白结合.结论:利用单链大容量抗体库获得抗结核分枝杆菌Acr蛋白的噬菌体抗体并且成功制备抗结核分枝杆菌Acr天然蛋白的可溶性单链抗体,为今后的研究和应用奠定基础.  相似文献   

7.
目的:构建鼠源E型肉毒毒素(BoNT/E)免疫噬菌体单链抗体库,筛选BoNT/E特异性抗体。方法:从E型肉毒类毒素免疫小鼠的脾细胞中提取总RNA,反转录成cDNA,分别扩增出小鼠重链可变区基因和轻链可变区基因;通过重叠延伸PCR将重链可变区基因和轻链可变区基因组装成scFv基因,重组于噬粒pS100中,电转化大肠杆菌TG_1,合并所有克隆成初级库;随机挑取克隆进行核苷酸序列测定,对初级库序列多样性进行分析;在辅助噬菌体M_(13)K_(07)的拯救下,构建成scFv噬菌体抗体库;用纯化的BoNT/E对鼠源BoNT/E免疫噬菌体单链抗体库进行3轮富集筛选,制备单克隆的噬菌体抗体颗粒进行酶联免疫吸附试验,阳性克隆进行核苷酸序列测定。结果:鼠源BoNT/E免疫噬菌体单链抗体库的库容为7.09×10~7,随机挑取的20个克隆序列各不相同,序列正确率为85%,基本覆盖了IgHV、IgKV、IgLV的优势家族;纯化的BoNT/E作为抗原通过3轮筛选,噬菌体抗体富集了66倍,第3轮筛选后随机挑取90个克隆制备噬菌体抗体颗粒,酶联免疫吸附试验分析有88个呈现阳性反应,序列比对得到了24个不同序列的BoNT/E特异性抗体。结论:构建了库容量达7.09×10~7的鼠源BoNT/E免疫噬菌体单链抗体库,筛选得到了24个不同序列的BoNT/E特异性抗体。  相似文献   

8.
利用分子生物学技术,构建表达丙型肝炎病毒(HCV)包膜蛋白E2的人源单链可变区抗体(ScFv)的原核表达载体,并在大肠杆菌JM109中表达可溶性的HCV-E2-ScFv.以重组的HCVE2蛋白为包被抗原,利用噬菌体抗体库的表面展示技术,筛选到含有HCV-E2-ScFv基因的噬菌体克隆,从噬菌体抗体阳性克隆中提取质粒,经Ncol/NotI酶切鉴定后,该ScFv基因由750bp组成,将其亚克隆到pCANTAB5E载体中,转化大肠杆菌JM109,提取质粒进行DNA序列测定,符合ScFv的基因结构特点.IPTG诱导转化的大肠杆菌JM109,在其培养上清中获得了可溶性HCVE2单链可变区抗体的表达.酶联免疫吸附法(ELISA)证实表达的HCV-E2-ScFv具有与重组HCVE2蛋白的反应活性和特异性,对转化的JM109大肠杆菌上清中表述的HCV-E2-ScFv进行聚丙烯酰胺凝胶电泳(PAGE),证实表达的HCV-E2-ScFv的分子量为28kD.为应用HCV-E2-ScFv进行肝组织免疫组织化学和细胞内免疫基因治疗研究奠定了基础.  相似文献   

9.
利用分子生物学技术,构建表达丙型肝炎病毒(HCV)包膜蛋白E2的人源单链可变区抗体(ScFv)的原核表达载体,并在大肠杆菌JM109中表达可溶性的HCV-E2-ScFv。以重组的HCV E2蛋白为包被抗原,利用噬菌体抗体库的表面展示技术,筛选含有HCV-E2-ScFv基因的噬菌体克隆,从噬菌体抗体阳性克隆中提取质粒,经Ncol/NotⅠ酶切鉴定后,该ScFv基因由750bp组成,将菜亚克隆到pCANTAB5E载体中,转化大肠杆菌JM109,在其培养上清中获得了可溶性HCV E2单链可变区抗体的表达。酶联免疫吸附法(ELISA)证实表达的HCV-E2-ScFv进行聚丙烯酰胺凝胶电泳(PAGE),证实表达的HCV-E2-ScFv的分子量为28KD,为应用HCV-E2-ScFv进行肝组织免疫组织化学和细胞内免疫基因治疗研究奠定了基础。  相似文献   

10.
目的:构建天然兔源噬菌体单链抗体库。方法:采用RT-PCR法从未免疫的兔子脾脏中克隆得到抗体重链可变区(VH)与轻链可变区(VL)基因,重叠PCR将VH和VL拼接成scFv片段,将scFv连接到噬菌粒pComb3XSS上,电转入XL1-Blue菌中,得到单链抗体库,并用此抗体库筛选抗肌酸激酶抗体。结果:构建了容量为4×108,基因重组率95%的单链抗体库,DNA指纹图谱显示抗体库多样性良好。以肌酸激酶为抗原,从该库中筛到3株抗肌酸激酶的抗体。结论:分析表明构建的天然兔源单链抗体库质量良好,可用于快速筛选、制备多种单链抗体。  相似文献   

11.
治疗性单克隆抗体研究进展   总被引:4,自引:0,他引:4  
杂交瘤技术使鼠源单克隆抗体(鼠单抗)被广泛用于人类疾病的诊断和研究,建立了治疗性抗体的第一个里程碑。但随后出现的人抗鼠抗体等副作用极大地限制了鼠单抗的临床应用。随着生物学技术的发展和抗体基因结构的阐明,应用DNA重组技术和抗体库技术对鼠单抗进行人源化改造,先后出现了嵌合抗体、改型抗体和全人抗体,同时也涌现了各种单抗衍生物,它们从不同角度克服了鼠单抗临床应用的不足,未人类疾病治疗带来新的曙光。我们就上述治疗性抗体人源化的研究进展做简要综述。  相似文献   

12.
基因工程抗体研究进展   总被引:3,自引:0,他引:3  
临床治疗中人抗鼠抗体反应的出现使鼠源性单克隆抗体的应用受到了极大的限制。为降低其免疫原性,人们利用基因工程技术对鼠源抗体进行改造,以减少其鼠源成分。简要概述了目前研究比较多的几种基因工程抗体及其临床应用。  相似文献   

13.
The recognition that few human diseases are thoroughly addressed by mono-specific, monoclonal antibodies (mAbs) continues to drive the development of antibody therapeutics with additional specificities and enhanced activity. Historically, efforts to engineer additional antigen recognition into molecules have relied predominantly on the reformatting of immunoglobulin domains. In this report we describe a series of fully functional mAbs to which additional specificities have been imparted through the recombinant fusion of relatively short polypeptides sequences. The sequences are selected for binding to a particular target from combinatorial libraries that express linear, disulfide-constrained, or domain-based structures. The potential for fusion of peptides to the N- and C- termini of both the heavy and light chains affords the bivalent expression of up to four different peptides. The resulting molecules, called zybodies, can gain up to four additional specificities, while retaining the original functionality and specificity of the scaffold antibody. We explore the use of two clinically significant oncology antibodies, trastuzumab and cetuximab, as zybody scaffolds and demonstrate functional enhancements in each case. The affect of fusion position on both peptide and scaffold function is explored, and penta-specific zybodies are demonstrated to simultaneously engage five targets (ErbB2, EGFR, IGF-1R, Ang2 and integrin αvβ3). Bispecific, trastuzumab-based zybodies targeting ErbB2 and Ang2 are shown to exhibit superior efficacy to trastuzumab in an angiogenesis-dependent xenograft tumor model. A cetuximab-based bispecific zybody that targeting EGFR and ErbB3 simultaneously disrupted multiple intracellular signaling pathways; inhibited tumor cell proliferation; and showed efficacy superior to that of cetuximab in a xenograft tumor model.  相似文献   

14.
林周  黎燕 《生物技术通讯》2001,12(4):300-301
抗体在疾病诊断,治疗和预防中发挥着重要作用,近几年来将基因工程应用到抗体生产的基因工程抗体技术发展迅速,本就基因工程抗体中的重要组成部分-嵌合抗体的研究进展加以综述。  相似文献   

15.
It is well established that all camelids have unique antibodies circulating in their blood. Unlike antibodies from all other species, these special antibodies are devoid of light chains, and are composed of a heavy chain homodimer. These so-called heavy-chain antibodies (HCAbs) are expressed after a V-D-J rearrangement and require dedicated constant gamma genes. An immune response is raised in these HCAbs following a classical immunization protocol. These HCAbs are easily purified from serum, and their antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies. The antigen binding site of the dromedary HCAb comprises one single domain, referred to as VHH or nanobody (Nb), therefore, a strategy was designed to clone the Nb repertoire of an immunized dromedary and to select the Nb with specificity for our target antigens. The monoclonal Nb is produced well in bacteria, is very stable and highly soluble, and it binds the antigen with high affinity and specificity. Currently, the recombinant Nb has been developed successfully for research purposes, as a probe in biosensors, to diagnose infections, or to treat diseases such as cancer or trypanosomiasis.  相似文献   

16.
17.
Antibodies are essential in modern life sciences biotechnology. Their architecture and diversity allow for high specificity and affinity to a wide array of biochemicals. Combining monoclonal antibody (mAb) technology with recombinant DNA and protein expression links antibody genotype with phenotype. Yet, the ability to select and screen for high affinity binders from recombinantly-displayed, combinatorial libraries unleashes the true power of mAbs and a flood of clinical applications. The identification of novel antibodies can be accomplished by a myriad of in vitro display technologies from the proven (e.g. phage) to the emerging (e.g. mammalian cell and cell-free) based on affinity binding as well as function. Lead candidates can be further engineered for increased affinity and half-life, reduced immunogenicity and/or enhanced manufacturing, and storage capabilities. This review begins with antibody biology and how the structure and genetic machinery relate to function, diversity, and in vivo affinity maturation and follows with the general requirements of (therapeutic) antibody discovery and engineering with an emphasis on in vitro display technologies. Throughout, we highlight where antibody biology inspires technology development and where high-throughput, “big data” and in silico strategies are playing an increasing role. Antibodies dominate the growing class of targeted therapeutics, alone or as bioconjugates. However, their versatility extends to research, diagnostics, and beyond.  相似文献   

18.
梁琍 《生物技术通讯》2006,17(5):799-802
作为一种具有靶向性的生物大分子,单克隆抗体始终是人们关注的热点之一,被广泛用于治疗肿瘤、病毒感染和抗移植排斥等。但鼠源单克隆抗体的临床应用受限于诱导产生人抗鼠抗体、肿瘤渗入量低、亲和力低和半衰期短等。随着分子生物学技术的发展及其向各学科的渗透,通过基因操作技术对抗体进行改造,可使其适用于多种疾病的治疗。抗体人源化已经成为治疗性抗体的发展趋势,同时各种抗体衍生物也不断涌现,它们从不同角度克服了抗体本身的应用局限,也为治疗人类疾病提供了利器。本文简要介绍上述技术的基本原理、特点和治疗性抗体的研究进展。  相似文献   

19.
噬菌体抗体库技术及其应用研究进展   总被引:1,自引:0,他引:1  
潘博  童贻刚 《生物技术通讯》2010,21(4):581-585,589
噬菌体呈现抗体库是近年发展的一项分子生物学新技术,它的建立是抗体技术领域中的一次革命性进展。它以其独特的构建和筛选系统,彻底改变了抗体制备的传统途径,使抗体工程技术进入了一个新的发展阶段,并对生物学领域中许多技术的发展起到了巨大的推动作用。该技术是迄今发展最成熟、应用最广泛的制备抗体技术。我们简要综述此项技术的研究应用进展。  相似文献   

20.
Bispecific immunoglobulin‐like antibodies capable of engaging multiple antigens represent a promising new class of therapeutic agents. Engineering of these molecules requires optimization of the molecular properties of one of the domain components. Here, we present a detailed crystallographic and computational characterization of the stabilization patterns in the lymphotoxin‐beta receptor (LTβR) binding Fv domain of an anti‐LTβR/anti‐TNF‐related apoptosis inducing ligand receptor‐2 (TRAIL‐R2) bispecific immunoglobulin‐like antibody. We further describe a new hierarchical structure‐guided approach toward engineering of antibody‐like molecules to enhance their thermal and chemical stability. Proteins 2009. © 2009 Wiley‐Liss, Inc.  相似文献   

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