Construction of human Fab library and screening of a single-domain antibody of amyloid-beta 42 oligomers

Screening humanized antibodies from a human Fab phage display library is an effective and quick method to obtain beta-amyloid oligomers. Thus, the present study prepared amyloid-beta 42 oli- gomers and constructed a have human Fab phage display library based on blood samples from six healthy people. After three rounds of biopanning in vitro, a human single-domain antibody that spe- cifically recognized amyloid-beta 42 oligomers was identified. Western blot and enzyme-linked im- munosorbent assay demonstrated this antibody bound specifically to human amyloid-beta 42 tetramer and nonamer, but not the monomer or high molecular weight oligomers. This study suc- cessfully constructed a human phage display library and screened a single-domain antibody that specifically recognized amyloid-beta 42 oligomers.

Construction of normal human IgE phage antibody library and the screening of the anti-trichosanthin IgE

Allergen specific IgE response is the major cause of immediate hypersensitivity. However the number of IgEproducing B cells and the amount of IgE, especially the specific IgE, are so low, it greatly impedes the study of the allergic-specifc antibody responses. Here we report the construction of a normal human IgE combinatorial library The repertoire of IgE VH genes and of K genes were separately amplified from normal human peripheral blood lymphocytes through RT-PCR, and were then constructed to form the phage surface display human Fab(IgEVH) library. A plant protein allergen, trichosanthin(TCS), was used to affinity-enrich and to screen the anti-TCS phage HuFab clones from the library. Human IgE(Fab) to TCS were detected.

Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer

AIM: To construct the natural immune Fab antibody phage display libraries of colorectal cancer and to select antibodies related with colorectal cancer. METHODS: Extract total RNA from tissue of local cancer metastasis lymph nodes of patients with colorectal cancer. RT-PCR was used to amplify the heavy chain Fd and light chain kappa and the amplification products were inserted successively into the vector pComb3 to construct the human libraries of Fab antibodies. They were then panned by phage display technology. By means of Dot immunoblotting and ELISA, the libraries were identified and the Fab phage antibodies binding with antigens of colorectal cancer were selected. RESULTS: The amplified fragments of Fd and kappa gained by RT-PCR were about 650 bp. Fd and kappa PCR products were subsequently inserted into the vector pComb3, resulting in a recombination rate of 40% and the volume of Fab phage display library reached 1.48 x 10(6).The libraries were enriched about 120-fold by 3 cycles of adsorption-elution-multiplication (panning). Dot immunoblotting showed Fab expressions on the phage libraries and ELISA showed 5 clones of Fab phage antibodies which had binding activities with antigens of colorectal cancer. CONCLUSION: The natural immune Fab antibody phage display libraries of colorectal cancer were constructed. They could be used to select the relative antibodies of colorectal cancer.

Rapid Selection of Phage Se-scFv with GPX Activity via Combination of Phage Display Antibody Library with Chemical Modification

Glutathione peroxidase（GPX） plays an important role in scavenging reactive oxygen species. A series of catalytic antibodies with GPX activity have been generated by the authors of＇ this study. To obtain humanized catalytic antibodies, the phage-displayed human antibody library was used to select novel antibodies by repetitive screening, Phage antibodies, scFv-B8 and scFv-H6 with the GSH-binding site, were obtained from the library by enzyme-linked immu- nosorbent assay（ELISA） analysis with 4 rounds of scelection against their respective haptens, S-2,4-dinitriphenyl t-butyl ester（GStI-s-DNP-Bu） and S-2,4-dinit,-iphenyl t-hexyl ester（GSH-s-I）NP-He）. Nevertheless, several studies need to be condueted to determine whether scFv-B8 and seFv-tI6 possess GPX activity. 1＇o enhance the speed of the selection, selenocysteine（Sec, the catalytic group of GPX） was incorporated directly into the phages, scFv-B8 and seFv-H6, by chemical mutation to form the phages Se-scFv-B8 and Se-scFv-H6. The GPX activities were found to be 3012 units/μmol and 2102 units/μmol, respectively. To improve the GPX activity of the phage Se-scFv-B8, DNA shuffling was used to construct a secondary library and another positive phage antibody scFv-B9 was screened out by another panning against GSH-s-DNP-Bu. When Sec was incorporated via chemical mutation into the phage antibody scFv-B9, its GPX activity reached 3560 units/μmol, which is 1.17-fold higher than the phage antibody Se-scFv-B8 and almost approached the order of magnitude of native GPX. The rapid selection is the prerequisite for generating humanized Se-seFv with GPX activity.

Selection of humanized antibody against HBsAg and analysis of gene encoding its heavy chain

Objective: To select a human phage antibody against HBsAg and sequence the heavy chain and light chain gene. Methods: Human immunoglobulin combinatorial library was generated by using phage surface display expression system, from which phage antibodies (Fab fragments ) against HBsAg were screened, and their antigenbinding activity and specificity were assayed by ELISA and competition inhibition ELISA. The heavy chain gene was seqllenced by 373A automatic DNA sequence analysis machine. Results: A human phage antibody against HBsAg was obtained. It’s heavy chain gene was whole, but it’s light chain gene was lost. Conclusion: The VH gene of the selected antibody belonged to VH I subgroup. The antibody fragment with whole heavy chain and withoutlight chain also showed strong binding activity to HBsAg.

Selection,Expression and Purification of Human Abzyme Containing Selenium with Type Ⅰ Thyroxine Deiodinase Activity

Single-chain fragment variable（ScFv） antibodies with the substrate binding sites were obtained by repetitive selections from a semi-synthetic phage display antibody library against two haptens——thyroxine（T4） and O-methyl-T4（O-CH3-T4）.The positive phage clones were determined by ELISA and then cloned into vector pET30a（＋）.The recombined plasmids were identified by DNA sequence analysis and transformed into E.coli BL21（DE3）.The expressed proteins were isolated,purified,identified by Western blot analysis,and finally the catalytic group——selenocysteine was incorporated into the antibodies binding sites by chemical mutation.The type·thyroxine deiodinase activities of the abzymes were 0.006 and 0.008 pg·mL-1·min-1,respectively,which were about one tenth that of the mouse intact antibody that was obtained from hybridoma.

噬菌体展示技术在全人源性抗体发现中的应用

噬菌体展示技术是目前应用最广泛的体外抗体筛选技术,它以噬菌体为载体,将外源抗体库基因插入到噬菌体衣壳蛋白基因中,在噬菌体表面表达衣壳蛋白的同时也展示了抗体分子。抗体药物因靶向优势在肿瘤免疫和病原生物感染中都发挥着重要作用,是其成为医药研发领域热点的重要推动力。因此,本文对噬菌体展示技术的研发背景、基本原理、抗体库构建类型及抗体展示片段类型进行综述,并对基于噬菌体展示的全人源性抗体的最新进展和应用前景进行了展望。

Selection and characterization of the novel anti-human PD-1 FV78 antibody from a targeted epitope mammalian cell-displayed antibody library

Currently,display-based methods are well established and widely used in antibody engineering for affinity maturation and structural stability improvement.We obtained a novel anti-human programmed death 1(PD-1)antibody using computer-aided design and a mammalian cell display technology platform.We used computer-aided modeling and distance geometry methods to predict and assign the key residues that contributed to the binding of human PD-L1 to PD-1.Then,we analyzed the sequence of nivolumab(an anti-human PD-1 antibody,referred to as MIL75 in the article)to determine the template for antibody design and library construction.We identified a series of potential substitutions on the obtained template and constructed a virtual epitope-targeted antibody library based on the physicochemical properties and each possible location of the assigned key residues.The virtual antibody libraries were displayed on the surface of mammalian cells as the antigen-binding fragments of full-length immunoglobulin G.Then,we used flow cytometry and sequencing approaches to sort and screen the candidates.Finally,we obtained a novel anti-human PD-1 antibody named FV78.FV78 competitively recognized the PD-1 epitopes that interacted with MIL75 and possessed an affinity comparable to MIL75.Our results implied that FV78 possessed equivalent bioactivity in vitro and in vivo compared with MIL75,which highlighted the probability and prospect of FV78 becoming a new potential antibody therapy.

Human antibodies from a single-chain Fv fusion phage library

THAT the bacteriophages can display human antibody fragments on their surfaces equivalent tothe mammalian immune repertoire provides a new and powerful means of making human anti-bodies for immunotherapy. This technique combines production, selection and affinity matura-tion in numerous imaginative ways. The phage antibody display systems have an advantageover the conventional hybridoma technique in the generation of human antibody and the development of an affinity maturation process that mimics the immune system. Since the first

抗SARS-CoV抗原的人源Fab段噬菌体抗体库的构建

目的 :利用抗SARS冠状病毒IgG抗体阳性的SARS康复患者外周血淋巴细胞 ,构建人源Fab段抗体文库。方法 :制备外周血淋巴细胞总RNA ,逆转录成cDNA。以其为模板 ,利用针对家族特异性Ig基因的引物扩增重链Fd段和轻链基因 ,并重组到噬菌粒载体pComb3中 ,将重组噬菌粒载体电转化大肠杆菌XL 1Blue,酶切鉴定抗体库的重组率 ,并测定噬菌体抗体库的库容量。结果 :构建了源于SARS康复患者血清中抗Fab段的抗体文库 ,轻链、重链Fd段基因的重组率分别为91%和 75 % ,库容量为 7.2 3× 10 7。结论 :成功地构建了抗SARS

从半合成噬菌体抗体库中筛选抗角蛋白人抗体

目的：从半合成噬菌体抗体库中筛选人源性抗角蛋白抗体并进行鉴定。方法：以表皮角蛋白为抗原，通过吸附－洗脱－扩增过程从半合成噬菌体抗体库中筛选特异性抗角蛋白抗体，对其抗原结合活性和序列进行分析鉴定。结果：经过４轮筛选，获得２０个能与角蛋白结合的阳性克隆，其中可产生特异性抗角蛋白抗体的克隆１８个。经ＤＮＡ指纹分析，判断所获克隆分别包含４个不同的Ｆａｂ段基因。经切除基因Ⅲ后１个克隆表达出可与角蛋白特异性结合的可溶性Ｆａｂ，序列分析表明其Ｖ和ＶＨ分别属于人Ｖ１亚群和ＶＨ１亚群，ＶＨＣＤＲ３序列符合半合成抗体库构建时的引物设计。结论：利用噬菌体抗体库技术可以不经免疫制备出高特异性的人源性抗角蛋白抗体。

人源性天然抗体库的构建及初步鉴定

目的 构建较大容量的人源性天然抗体库。方法 以未经铭疫的健康人外周血单个核细胞为基因来源，扩增多样性的Ｋ轻链基因和 ＩｇＧ／ＩｇＭ重链Ｆｄ段基因，分别构建铡库和重链库，然后组合为Ｆａｂ段面展示的噬菌体抗体库。通过多次重组积累达到理想的库容，对抗体库进行筛选和鉴定。结果 经过５次轻、重链基因的重组和转化，获得了库容为１×1０～９的人源性天然抗体库。该抗体库性能良好，用３种抗原进行“亲和吸附－洗脱－扩增”淘筛，获得针对其中两种抗原的７株特异性噬菌体抗体。结论天然抗体库为用于不经免疫制备人源性单克隆抗体创造了良好的条件。

从半合成抗体库克隆抗TNF-α人单链抗体

通过噬菌体抗体库技术，不经免疫克隆抗ＴＮＦ－α人单链抗体，用固相化的人重组ＴＮＦ－α抗原对半合成噬菌体抗体库进行了４轮“吸附－洗脱－扩增”的筛选，可见到明显富集，从第３轮和第４轮洗脱下来的克隆中获得两株可特异结合ＴＮＦ－α的克隆，其可变区分别属于ＶＨ１、ＶＨ３和Ｖλ１亚群。

人源噬菌体抗体库的构建及抗人NH-LBP抗体的筛选与鉴定

目的: 构建人源噬菌体抗体库并制备抗人脂多糖结合蛋白氨基端片段 (NH- LBP)的单克隆抗体 (mAb)。方法:以噬菌体展示系统(pComb3H/VCSM13)建立人源噬菌体抗体库(Fab), 并以昆虫细胞sf21在无血清培养基 (SF 900Ⅱ )中,通过BAC- TO- BAC杆状病毒表达系统来表达NH -LBP。再以NH -LBP为抗原, 从人源噬菌体抗体库中筛选可产生抗NH- LBPmAb的菌株并进行鉴定。结果: 昆虫细胞sf21可表达人源NH- LBP, 经亲和纯化柱芯 (TALON)有效纯化后, 获得约8mg的NH- LBP。成功地建立人源噬菌体抗体库, 库容达5. 0×108 CFU。经 8轮筛选后, 抗体库被富集 1. 85×104 倍。经ELISA法鉴定, 获得 3株可产生抗NH -LBPmAb的菌株(核酸序列及氨基酸序列见GenBank中的: AY337713,AY337714 )。结论: 以昆虫细胞sf21表达NH LBP及以其为抗原制备噬菌体mAb是可行的。本研究为进一步建立抗NH- LBP的二硫键稳定的Fv抗体 ( disulfidestabilizedFvfrag ments, dsFv), 研究人体内LBP的变化规律和过度炎症反应的防治奠定了基础。

从人源性噬菌体抗体库分离出1株含有异常序列的抗HBsAg Fab克隆

曾从人源性噬菌体抗体库中筛选出１株抗人乙肝表面抗原（ＨＢｓＡｇ）的Ｆａｂ克隆，为了筛选出新的抗ＨＢｓＡｇＦａｂ段，采用抗原屏蔽法，用已得到的Ｆａｂ段封闭相应的抗原决定基，对该抗体库进行了再次筛选，得到了１株新的人抗ＨＢｓＡｇＦａｂ段克隆，经序列分析发现其轻链可变区基因来源于Ｖκ１亚群和Ｊκ４基因，重链可变区基因来源于ＶＨ１亚群和ＪＨ４基因，但在ＶＨ第７７位和第７８位氨基酸残基之间出现了７个多余的氨基酸残基，经对基因数据库检索未能查到该序列的来源，但显然这段序列未影响抗体的抗原结合活性。

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 。

采用哺乳动物细胞表面展示技术构建全长人源抗肾癌抗体基因库

目的采用哺乳动物细胞表面展示技术构建全长人源抗肾癌抗体基因库。方法分离肾癌患者的外周血淋巴细胞(PBMC),提取PBMC的总RNA,采用RT-PCR的方法扩增抗体全长Kappa型轻链(LCκ)和重链可变区(VH)基因,分别插入载体pDGB-HC-TM,电击转化感受态大肠杆菌TOPO10,构建轻、重链抗体基因库,然后将轻、重链抗体基因库联合转染293T细胞,流式细胞仪分析全长人源抗体在293T细胞表面的表达。结果成功构建IgG1-Kappa型抗肾癌抗体基因库,随机挑选的克隆经DNA序列分析显示轻、重链库的序列正确性达90%(9/10)和80%(8/10),流式细胞仪分析显示来自轻、重链库的上述克隆各有7个可检测到相应基因在293T细胞表面的表达,可表达抗体库库容量高达7.5×1010。结论 IgG1-Kappa型抗肾癌抗体基因库转染293T细胞后能够在细胞表面表达全长人源抗体,抗体的多样性为下一步筛选特异性抗体奠定良好的基础。

快速构建哺乳动物细胞表面展示的全长人抗体基因库

目的快速构建哺乳动物细胞表面展示的全长人抗体基因库。方法分离外周血淋巴细胞(PBMC),提取PBMC的总RNA,采用RT-PCR的方法扩增抗体重链可变区(VH)和全长Kappa型轻链(LCκ)基因库,分别插入载体pDGB-HC-TM,化学转化感受态大肠杆菌DH5α,构建抗体的轻、重链基因库,然后将其联合转染CHO细胞,流式细胞仪分析全长人源抗体在CHO细胞表面的表达。结果构建的非特异性IgG1-Kappa抗体基因库,重链库容量达2.6×105,轻链库容量达2.0×105,理论库容量高达5.2×1010。随机从重链库和轻链库各挑选10个克隆送测序,重链10个克隆的DNA序列分析显示8个含有正确的阅读框架,轻链10个克隆的DNA序列分析显示全部含有正确的阅读框架。流式细胞仪分析显示来自轻链库的10个克隆、重链库的8个克隆均可检测到抗体在CHO细胞表面的表达。用所构建的轻重链抗体库共转染CHO细胞,流式细胞仪分析可检测到抗体在细胞表面的表达,阳性细胞比例达到31.5%,说明所构建的抗体库能够在CHO细胞表面成功展示全长抗体。结论采用本研究建立的哺乳动物细胞展示技术,可在2周内成功构建库容量大且多样性好的全人源抗体基因库,用于高亲和力特异性抗体的筛选。

人源脂多糖结合蛋白单克隆抗体的筛选及初步鉴定

目的制备人源抗脂多糖结合蛋白(LBP)单克隆抗体,并对该抗体的效价进行初步鉴定。方法以人脂多糖结合蛋白(human lipopolysaccharide binding protein,hLBP)为抗原,以人源噬菌体抗体库进行筛选,经5轮“吸附洗脱扩增”的富集反应后,筛选出3株抗人LBP阳性克隆株,抽提其质粒、切除geneⅢ、自身环化并电转入XL1blue后,以IPTG诱导分泌表达可溶性抗体,通过ELISA法测定抗体效价。结果抗体库被浓缩8.16×104倍,获得3个与LBP结合能力较强的单克隆菌株,其中结合力最强者产生抗体活性为106。结论成功获得针对人LBP的Fab抗体,经初步鉴定具有较高效价,为下一步研究该抗体功能奠定基础。

人源抗狂犬病毒免疫型抗体库的构建及特异性抗体筛选与鉴定

目的:利用噬菌体展示技术,构建人源抗狂犬病毒单链抗体库,筛选特异性的抗狂犬病毒糖蛋白人源单链抗体(scFv)并对其进行初步鉴定。方法:从接种狂犬疫苗的志愿者外周血中提取总RNA,RT-PCR扩增VH和VL基因,并利用重叠扩增PCR将VH和VL拼接为scFv。将纯化后的scFv克隆至噬菌体载体pComb3XSS构建人源抗狂犬病毒单链抗体库,以狂犬病毒糖蛋白(RABVG)为抗原,从抗体库中筛选抗RABVG的scFv。通过phage-ELISA验证噬菌体单链抗体的结合特异性,将阳性克隆转化E.coliTOP10F′进行表达。结果:构建了人源抗狂犬病毒抗体库,抗体库的库容为3.29×109。经过5轮筛选,获得43株与RABVG特异结合的噬菌体单链抗体,其中15个A450值较高的克隆序列分析结果显示,有3个正确的单链抗体基因。单链抗体基因转化TOP10F′构建工程菌,表达纯化后的单链抗体,经ELISA检测能够与RABVG特异性结合。结论:从构建的人源免疫型抗狂犬病毒单链抗体库筛选出的能与RABVG特异性结合的scFv,为进一步制备抗RABVG的治疗性人源化抗体奠定了基础。