Generation of a Recombinant Full-Length Human Antibody Binding to Botulinum Neurotoxin A

In order to develop a recombinant full-length human anti-botulinum neurotoxin A (BoNT/A) antibody, human peripheral blood mononuclear cells (PBMC) were collected from three healthy volunteers and induced for BoNT/A-specific immune response by in vitro immunization. The genes encoding human Fd fragment, consisting of antibody heavy chain variable region and constant region 1 with the genes encoding antibody light chain, were cloned from the immunized PBMC. Afterwards, one combinatory human antigen-binding fragment (Fab) library was constructed using a lambda phage vector system. The size of the constructed library was approximately 10⁵ Escherichia coli transformants. After screening the library by BoNT/A antigen using a plaque lifting with immunostaining approach, 55 clones were identified as positive. The Fab gene of the most reactive clone exhibiting particularly strong BoNT/A binding signal was further subcloned into a full-length human IgG1 antibody gene template in an adenoviral expression vector, in which the heavy and light chains were linked by a foot-and-mouth-disease virus-derived 2A self-cleavage peptide under a single promoter. After the full-length human IgG1 was expressed in mammalian cells and purified with protein L column, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the heavy and light chains of the antibody were cleaved completely. The affinity expressed as the dissociation constant (K _d) for the recombinant human antibody to bind to BoNT/A was determined by indirect enzyme-linked immunosorbent assay and results confirmed that the recombinant full-length human antibody retained BoNT/A-binding specificity with K _d value of 10⁻⁷ M.     

Screening of scFvs against cTnI from Phage Display Antibody Library and Their Expression in E.coli Rosetta

Introduction CardiactroponinI(cTnI),aspecificproteinof cardiacmusclecells,showsa40%dissimilarity withskeletaltroponinI(sTnI)inaminoacidse- quence.Moreover,humancardiacTnIhas31addi- tionalresiduesonitsN-terminalend,whichare notpresentinskeletalforms,thusprovidingahigh potentialforobtainingcardiac-specificantibod- ies[1,2].Themolecularweightofthisproteinis29 kDaandtherefore,itwillbereleasedreasonably rapidlyafteracutemyocardialinfarction(AMI). CTnIoftenappearsinbloodwithinafewhoursaf- ter…     

具有谷胱甘肽过氧化物酶活性的含硒人源单链抗体的制备

以谷胱甘肽(GSH)为靶抗原, 从噬菌体展示人源单链抗体库中筛选人源单链抗体(scFv). 经3轮筛选后, 用ELISA方法检测出5个(2, 11, 16, 24, 32 )可以和GSH结合的克隆. PCR产物的电泳和测序结果表明, 只有3个克隆(11, 16, 24)具有完整的scFv编码基因. 选取和GSH结合力高的克隆11的scFv 编码基因组装到表达载体pPELB上, 在大肠杆菌Rosetta中进行可溶性表达, 用Ni2+螯合亲和层析纯化scFv-11, 免疫点印迹结果证实该抗体能与GSH特异结合. 通过化学突变将scFv-11的丝氨酸转变成硒代半胱氨酸(Sec)后, 获得了具有谷胱甘肽过氧化物酶(GPX)活力的含硒(Se)人源单链抗体(Se-scFv-11), 其活力为351 U/μmol.     

High-throughput screening of surface displayed gene products

With the human genome project approaching completion, there is a growing interest in functional analysis of gene products. The characterization of large numbers of proteins, their expression patterns and in vivo localisations, demands the use of automated technology that maintains a logistic link to the encoding genes. As a complementary approach, phage display is used for recombinant protein expression and the selection of interacting (binding) molecules. Cloning of libraries in filamentous bacteriophage or phage mid vectors provides a physical link between the expressed protein and its encoding DNA sequence. High-throughput technology for automated library handling and phage display selection has been developed using picking-spotting robots and a module for pin-based magnetic particle handling. This system enables simultaneous interaction screening of libraries and the selection of binders to different target molecules at high throughput. Target molecules are either displayed on high-density filter membranes (protein filters) or tag-bound to magnetic particles and can be handled as native ligands. Binding activity is confirmed by magnetic particle ELISA in the microtitre format. The whole procedure from immobilisation of target molecules to confirmed clones of binders is automatable. Using this technology, we have selected human scFv antibody fragments against expression products of human cDNA libraries.     

Selecting folded proteins from a library of secondary structural elements

A protein evolution strategy is described by which double-stranded DNA fragments encoding defined Escherichia coli protein secondary structural elements (alpha-helices, beta-strands, and loops) are assembled semirandomly into sequences comprised of as many as 800 amino acid residues. A library of novel polypeptides generated from this system was inserted into an enhanced green fluorescent protein (EGFP) fusion vector. Library members were screened by fluorescence activated cell sorting (FACS) to identify those polypeptides that fold into soluble, stable structures in vivo that comprised a subset of shorter sequences ( approximately 60 to 100 residues) from the semirandom sequence library. Approximately 108 clones were screened by FACS, a set of 1149 high fluorescence colonies were characterized by dPCR, and four soluble clones with varying amounts of secondary structure were identified. One of these is highly homologous to a domain of aspartate racemase from a marine bacterium (Polaromonas sp.) but is not homologous to any E. coli protein sequence. Several other selected polypeptides have no global sequence homology to any known protein but show significant alpha-helical content, limited dispersion in 1D nuclear magnetic resonance spectra, pH sensitive ANS binding and reversible folding into soluble structures. These results demonstrate that this strategy can generate novel polypeptide sequences containing secondary structure.     

Isolation of rice allergenic cDNA clones from a rice cDNA library by immunoscreening with a polyclonal antibody specific to 16 kD rice allergenic protein

Clinical cases of type-1 hypersensitive reaction to rice (Oryza sativa) have been reported in western countries as well as in Japan. Among rice proteins, 14-16 kD globulin proteins encoded by multiple gene family have been identified as major rice allergens. In this study, a rice cDNA library was constructed using lambda UniZap vector and screened with a rat anti-16 kD globulin protein polyclonal antibody in order to isolate Korean rice allergenic cDNA clones. Five independent cDNA clones, termed RAK1-5, were obtained after second rounds of plaque assay and immunoblot analysis. These clones encoded 13-19 kD recombinant proteins upon IPTG induction, which were identified by the polyclonal antibody in immunoblot analysis. DNA sequencing analysis showed that RAK1-4 have 99% sequence homology with RA5b, and RAK5 is closely related with RA14c. This result indicated that RA5b gene is widely distributed in our cDNA library among other possible rice allergenic genes, and more study is needed to isolate heterogeneous or novel rice allergen genes.     

Engineered Fully Human Single-Chain Monoclonal Antibodies to PIM2 Kinase

Proviral integration site of Moloney virus-2 (PIM2) is overexpressed in multiple human cancer cells and high level is related to poor prognosis; thus, PIM2 kinase is a rational target of anti-cancer therapeutics. Several chemical inhibitors targeting PIMs/PIM2 or their downstream signaling molecules have been developed for treatment of different cancers. However, their off-target toxicity is common in clinical trials, so they could not be advanced to official approval for clinical application. Here, we produced human single-chain antibody fragments (HuscFvs) to PIM2 by using phage display library, which was constructed in a way that a portion of phages in the library carried HuscFvs against human own proteins on their surface with the respective antibody genes in the phage genome. Bacterial derived-recombinant PIM2 (rPIM2) was used as an antigenic bait to fish out the rPIM2-bound phages from the library. Three E. coli clones transfected with the HuscFv genes derived from the rPIM2-bound phages expressed HuscFvs that bound also to native PIM2 from cancer cells. The HuscFvs presumptively interact with the PIM2 at the ATP binding pocket and kinase active loop. They were as effective as small chemical drug inhibitor (AZD1208, which is an ATP competitive inhibitor of all PIM isoforms for ex vivo use) in inhibiting PIM kinase activity. The HuscFvs should be engineered into a cell-penetrating format and tested further towards clinical application as a novel and safe pan-anti-cancer therapeutics.     

量子点与人源抗谷胱甘肽单链抗体的连接与表征

用已构建的表达载体pPELB-B3, 在大肠杆菌Rosetta中可溶性表达人源抗谷胱甘肽(GSH)单链抗体B3(scFv-B3), 经Ni2+螯合亲和层析纯化后, 用点印迹法验证了其与GSH结合的特异性. 将水相合成的半导体纳米粒子(半导体量子点, QDs)在N-羟基琥珀酰亚胺(NHS)和1-乙基-3-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC)的作用下, 与scFvs连接. 光谱分析和膜印迹结果表明, scFvs成功地共价连接到QDs表面, 所得的QD-scFvs复合物能够较好地识别GSH. 荧光显微镜观察QD-scFvs与人乳腺癌细胞MCF-7的作用结果, 初步判断QD-scFvs能够跨膜进入细胞.     

Antibody synthesis induced by endogenous internal images

In this study, immunization with a vaccine consisting of multiple F(abt’)₂ fragments of affinity-purified antitetanus toxoid antibodies covalently bound to a carrier protein successfully induced antitetanus toxoid antibodies. Further studies showed that this vaccine preparation contained no biologically detectable tetanus antigen. The induced antitetanus antibody (Ab1t’) titer was higher than the titer of antibodies binding control antigens. The immunizing F(abt’)₂ preparation did not elicit a secondary antitetanus response from mice primed with tetanus toxoid and, hence, appeared free of tetanus epitopes. The specificity of Ab1t’ was established by absorption and inhibition with antigen. Immunization with antitetanus F(abt’)₂ (Ab1t’) fragments appears to have elicited naturally occurring autologous antitetanus toxoid antibody (Ab1t’) through an idiotypic pathway. As predicted by network theory, anti-idiotype (Ab2) and antitetanus (Ab1t’) cycled reciprocally. Clonotypic characterization of Ab1t’ using isoelectric focusing and affinity immunoblotting showed increases in Ab1t’ titer to be the result of increased synthesis by limited subsets of antitetanus toxoid B-cell clones and not increased synthesis by multiple clones, as is characteristic of antigen-driven Ab1 responses. Many Ab1 and Ab1t’ clonotypes had identical pIs, suggesting that they either share V region genes or are the product of the same B-cell clones. These findings indicate that immunization with polyclonal multivalent Ab1 preparations can trigger active synthesis of antibodies with the same specificity. The results provide further evidence for naturally occurring idiotypic cascades that could be exploited for studies of catalytic antibodies.     

Combined use of ESI–QqTOF-MS and ESI–QqTOF-MS/MS with mass-spectral library search for qualitative analysis of drugs

The potential of the combined use of ESI–QqTOF-MS and ESI–QqTOF-MS/MS with mass-spectral library search for the identification of therapeutic and illicit drugs has been evaluated. Reserpine was used for standardizing experimental conditions and for characterization of the performance of the applied mass spectrometric system. Experiments revealed that because of the mass accuracy, the stability of calibration, and the reproducibility of fragmentation, the QqTOF mass spectrometer is an appropriate platform for establishment of a tandem-mass-spectral library. Three-hundred and nineteen substances were used as reference samples to build the spectral library. For each reference compound, product-ion spectra were acquired at ten different collision-energy values between 5 eV and 50 eV. For identification of unknown compounds, a library search algorithm was developed. The closeness of matching between a measured product-ion spectrum and a spectrum stored in the library was characterized by a value called “match probability”, which took into account the number of matched fragment ions, the number of fragment ions observed in the two spectra, and the sum of the intensity differences calculated for matching fragments. A large value for the match probability indicated a close match between the measured and the reference spectrum. A unique feature of the library search algorithm—an implemented spectral purification option—enables characterization of multi-contributor fragment-ion spectra. With the aid of this software feature, substances comprising only 1.0% of the total amount of binary mixtures were unequivocally assigned, in addition to the isobaric main contributors. The spectral library was successfully applied to the characterization of 39 forensic casework samples. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible to authorized users.     

Library selection approaches to engineering enhanced retroviral and lentiviral vectors

Retroviral and lentiviral based gene delivery vectors have been used in numerous pre-clinical studies and clinical trials due to their advantages, including stable and prolonged expression of therapeutic transgenes and minimal immune responses against the vector. Despite such advantages, however, retroviral vectors also have several limitations for gene therapy applications. For example, they can suffer from a lack of efficient or targeted gene delivery to key cell types. In addition, retroviral vector stability can be compromised by their envelope proteins. This review briefly describes how such limitations have been overcome by recently developed library selection approaches that borrow a lesson from nature: the ability of evolution to generate biomolecules with novel function. These library selection approaches are based on the construction of retroviral libraries where the sequences encoding natural viral components are partially randomized using a variety of methods in order to generate diverse libraries that can be selected to create improved or novel functions. These high throughput, library-based approaches provide a strong complement to rational engineering of viral components for the rapid development of efficient and safe retroviral and lentiviral vector systems for gene therapy.     

Phage display of antibody fragments

In recent years, phage display of peptides and proteins has become a very popular method in oncology, immunology, protein engineering and ligand-receptor studies among others. Antibody fragments, as Fabs or single chain Fv, have been among the first proteins to be displayed on the surface of a filamentous bacteriophage with a procedure initially described in 1990 by McCafferty et al. (Nature, 348, 552-554). From that time, molecular biology techniques have allowed the creation of large repertoires of antibody fragments from antibody V genes, bypassing hybrydoma technology and even immunisation. A large number of phage antibody libraries, from which molecules of the desired functional properties can be rapidly selected, have been built and distributed in many laboratories world-wide. Antibody fragments recovered from phage libraries generally show moderate binding strength; with different systems of biopanning binders can be obtained with dissociation constant ranged between 10-(5) to 10-(8) M. Nevertheless, antibody fragments can be furtherly modified to improve affinity or avidity, respectively by mutating crucial residues of complementarity determining regions or by increasing the number of binding sites making dimeric, trimeric or multimeric molecules. Here, we summarise the latest progress in this field, with particular reference to applications of scFv in the diagnosis and therapy of solid tumours and in the molecular mimicry of viral antigens and membrane receptors. In fact, the production of artificial protein epitopes by phage antibodies is becoming a valid system to overcome problems caused by difficult cloning and low expression of particular recombinant proteins.     

Suppressive Subtractive Hybridization Approach Revealed Differential Expression of Hypersensitive Response and Reactive Oxygen Species Production Genes in Tea (Camellia sinensis (L.) O. Kuntze) Leaves during Pestalotiopsis thea Infection

Tea (Camellia sinensis (L.) O. Kuntze) is an economically important plant cultivated for its leaves. Infection of Pestalotiopsis theae in leaves causes gray blight disease and enormous loss to the tea industry. We used suppressive subtractive hybridization (SSH) technique to unravel the differential gene expression pattern during gray blight disease development in tea. Complementary DNA from P. theae-infected and uninfected leaves of disease tolerant cultivar UPASI-10 was used as tester and driver populations respectively. Subtraction efficiency was confirmed by comparing abundance of ??-actin gene. A total of 377 and 720 clones with insert size >250?bp from forward and reverse library respectively were sequenced and analyzed. Basic Local Alignment Search Tool analysis revealed 17 sequences in forward SSH library have high degree of similarity with disease and hypersensitive response related genes and 20 sequences with hypothetical proteins while in reverse SSH library, 23 sequences have high degree of similarity with disease and stress response-related genes and 15 sequences with hypothetical proteins. Functional analysis indicated unknown (61 and 59?%) or hypothetical functions (23 and 18?%) for most of the differentially regulated genes in forward and reverse SSH library, respectively, while others have important role in different cellular activities. Majority of the upregulated genes are related to hypersensitive response and reactive oxygen species production. Based on these expressed sequence tag data, putative role of differentially expressed genes were discussed in relation to disease. We also demonstrated the efficiency of SSH as a tool in enriching gray blight disease related up- and downregulated genes in tea. The present study revealed that many genes related to disease resistance were suppressed during P. theae infection and enhancing these genes by the application of inducers may impart better disease tolerance to the plants.     

Single-Chain Fv Antibody Fragments Retain Binding Properties of the Monoclonal Antibody Raised Against Peptide P1 of the Human Prion Protein

Prion diseases are incurable neurodegenerative diseases that affect both humans and animals. The infectious agent is a pathogenic form of the prion protein that accumulates in brain as amyloids. Currently, there is neither cure nor reliable preclinical diagnostics on the market available. The growing number of reports shows that passive immunisation is one of the most promising strategies for prion disease therapy, where antibodies against prions may prevent and even cure the infection. Since antibodies are large molecules and, thus, might not be suitable for the therapy, different antibody fragments are a good alternative. Therefore, we have designed and prepared single-chain antibody fragments (scFvs) derived from the PrP^Sc-specific murine monoclonal antibody V5B2. Using a new expression vector pMD204, we produced scFvs in two opposing chain orientations in the periplasm of Escherichia coli. Both recombinant antibody fragments retained the specificity of the parent antibody and one of these exhibited binding properties comparable to the corresponding murine Fab fragments with the affinity in nM range. Our monovalent antibody fragments are of special interest in view of possible therapeutic reagents for prion diseases as well as for development of a new generation of diagnostics.     

Genome-wide high-throughput mining of natural-product biosynthetic gene clusters by phage display

We have developed a phage-display method for high-throughput mining of bacterial gene clusters encoding the natural-product biosynthetic enzymes, polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). This method uses the phosphopantetheinyl transferase activity of Sfp to specifically biotinylate NRPS and PKS carrier-protein domains expressed from a library of random genome fragments fused to a gene encoding a phage coat protein. Subsequently, the biotinylated phages are enriched through selection on streptavidin-coated plates. Using this method, we isolated phage clones from the multiple NRPS and PKS gene clusters encoded in the genomes of Bacillus subtilis and Myxococcus xanthus. Due to the rapid and unambiguous identification of carrier domains, this method will provide an efficient tool for high-throughput cloning of NRPS and PKS gene clusters from many individual bacterial genomes and multigenome environmental DNA.     

Construction of Human Nonimmune Library and Selection of scFvs Against IL-33

Interleukin (IL) 33 plays very important roles in inflammatory and allergic diseases. To select human single-chain Fv fragments (scFvs) against IL-33, a nonimmune phage library system was constructed. The full-length cDNA library was synthesized for amplification of the variable heavy chain (V_H) and variable light chain (V_L). By overlapping extension PCR for splicing V_H and V_L, the full-length scFv library DNA were amplified and then transformed into Escherichia coli TG1. The scFv library was constructed successfully which contained 2.5?×?10⁸ independent clones with full-length scFv inserts. The results of fingerprint maps of the scFvs by BstN I and DNA sequencing from the library at random proved that the library was diverse. The human IL-33 was amplified, expressed, and purified. The purified IL-33 with bioactivity was biotinylated and used as antigen for selection of scFv library by phage display. After three rounds of affinity selection, about 30?% of clones have specific binding activity with IL-33. Five of those with good binding activity were transformed into E. coli strain HB2151 for soluble expression. The selected scFvs were further identified by western blot and sequencing. Those selected scFvs could be used for further research of their effect on inflammatory and allergic diseases such as asthma by blockade of IL-33.     

高密度噬菌体抗体芯片对细胞表面蛋白的识别

采用正常人和白血病患者的白细胞对人源噬菌体抗体库进行淘选, 以获得对两种细胞表面蛋白特异的抗体. 通过pVIII展示系统, 使抗体以多价展示于重组噬菌体颗粒表面, 从上述两组中各挑选出48个克隆分别固定于环氧基片上, 并以空白噬菌体和牛血清白蛋白作为对照, 制成高密度噬菌体抗体芯片. 取来自3名正常人和3名白血病患者的白细胞裂解物样品, 用荧光染料Cy3标记, 与噬菌体抗体芯片反应, 对微阵共聚焦扫描得到的荧光图谱进行分析. 在白血病白细胞表面蛋白的识别图谱中有8组斑点显著不同于正常图谱. 由此表明, 噬菌体抗体芯片可用于识别细胞表面蛋白.     

Generation of anti-colorectal cancer fab phage display libraries with a high percentage of diverse antigen-reactive clones

A combinatorial Fab phage display library was generated from the antibody variable region genes of each of 2 BALB/c mice immunized with the human colorectal cancer cell lines SW480, SW948, and SW837. These libraries were shown to be diverse by nucleotide sequencing and diagnostic restriction enzyme digestion (fingerprinting) of individual members. The two libraries were combined and selected for binding to a suspension of formaldehyde-fixed human colorectal cancer cells in two successive rounds of selection and phage amplification by infection of bacteria. Analysis of the selected libraries as well as individual library clones by ELISA, showed binding to the cancer cell lines in both formaldehyde-fixed and native forms. Fifty five percent and 94% of library clones were positive for colorectal cancer cell binding after the first and second rounds of selection, respectively. Fingerprinting of individual clones showed the first round selected library to be very diverse and the second round selected library to be of more limited diversity. After absorption with normal human cell types, these anti-cancer selected libraries could be used to develop therapeutic and/or diagnostic agents.