Antioxidant Effect of Human Selenium-containing Single-chain Fv in Rat Cardiac Myocytes

Reactive oxygen species（ROS） plays a key role in human heart diseases. Glutathione peroxidase（GPX） functions as an antioxidant as it catalyzes the reduction of hydroperoxide. In order to investigate the antioxidant effect of human selenium-containing single-chain Fv（Se-scFv-B3）, a new mimic of GPX, a model system of hydrogen peroxide（H202）-induced rat cardiac myocyte damage was established. The cardiac myocyte damage was characte- rized in terms of cell viability, lipid peroxidation, cell membrane integrity, and intracellular H202 level. The Se-scFv-B3 significantly reduced H2O2-induced cell damage as shown by the increase of cell viability, the decline of malondialdehyde（MDA） production, lactate dehydrogenase（LDH） release, and intracellular H2O2 level. So Se-scFv-B3 may have a great potential in the treatment of human heart diseases induced by ROS.

Screening and evaluation of human single-chain fragment variable antibody against hepatitis B virus surface antigen

BACKGROUND: Phage display technology has become a vital tool in studies aimed at identifying molecules binding to a specific target. It enables the rapid generation and selection of high affinity, fully human antibody product candidates to essentially any disease target appropriate for antibody therapy. In this study, we prepared the recombinant single-chain fragment variable ( ScFv) antibody to hepatitis B virus surface antigen (HBsAg) by the phage display technology for obtaining a virus-targeting mediator. METHODS: mRNA was isolated from B-lymphocytes from a healthy volunteer and converted into cDNA. The fragment variables of heavy and light chain were amplified separately and assembled into ScFv DNA with a specially constructed DNA linker by polymerase chain reaction. The ScFv DNA was ligated into the phagmid vector pCANT-AB5E and the ligated sample was transformed into competent E. coli TG1. The transformed cells were infected with M13K07 helper phage to form a human recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by bacterial colony count and restriction analysis. After two rounds of panning with HBsAg. the phage clones displaying ScFv of the antibody were selected by enzyme-linked immunosorbant assay ( ELISA) from the enriched phage clones. The antigen binding affinity of the positive clone was detected by competition ELISA. HB2151 E. coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the anti-HBsAg ScFv. ELISA assay was used to detect the antigen binding affinity of the soluble anti-HBsAg ScFv. Finally, the relative molecular mass of soluble anti-HBsAg ScFv was measured by SDS-PAGE. RESULTS: The variable heavy ( VH ) and variable light (VL) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 × 106 and 8 of 10 random clones were recombinants. Two phage clones could strongly compete with the original HBsAb for binding to HBsAg. Within 2 strong positive phage clones, the soluble anti-HBsAg ScFv from one clone was found to have the binding activity with HBsAg. SDS-PAGE showed that the relative molecular weight of soluble anti-HBsAg ScFv was 32 kDa. CONCLUSION: The anti-HBsAg ScFv successfully produced by phage antibody technology may be useful for broadening the scope of application of the antibody.

Construction and Analysis of Three-dimensional Graphic Model of Single-chain Fv Derived from an Anti-human Placental Acidic Isoferritin Monoclonal Antibody by Computer

Summary: A three-dimensional (3D) graphic model of a single-chain Fv (scFv) which was derived from an anti-human placental acidic isoferritin (PAF) monoclonal antibody (MAb) was construct- ed by a homologous protein-predicting computer algorithm on Silicon graphic computer station. The structure, surface static electricity and hydrophobicity of scFv were investigated. Computer graphic modelling indicated that all regions of scFv including the linker, variable regions of the heavy (VH) and light (VL) chains were suitable. The VH region and the VL region were involved in composing the 'hydrophobic pocket'. The linker was drifted away VH and VL regions. The complementarity determining regions (CDRs) of VH and VL regions surrounded the 'hydrophobic pocket'. This study provides a theory basis for improving antibody affinity, investigating antibody structure and analyzing the functions of VH and VL regions in antibody activity.

Anti-amyloid beta single-chain Fv ameliorates behavioral impairment in Alzheimer's disease mice via adeno-associated virus delivery

Intracranial delivery of human Fc-deleted antibody specific to amyloid-β peptide （Aβ, anti-Aβ single-chain Fv, scFv） via adeno-associated virus （AAV） inhibits amyloid deposition in transgenic mice. However, the effects of AAV-mediated Fc-deleted antibody on animal behavior remain unclear. In this study, the anti-Aβ scFv antibody gone, isolated from phage display, was fused to the 5＇ end of the scFv antibody gone for antibody secretion by 2 rounds of polymerase chain reaction amplification. The fused antibody cDNA was cloned into a pSNAV2 plasmid under the control of the cytomegalovirus promoter. The sequence verified expression vector pSNAV2/scFv was transferred to BHK-21 ceils, and stable transfected BHK-21/scFv cells were established by G418 selection and infected with the recombinant herpes simplex virus rHSV/repcap for AAV production. Recombinant AAV was injected into the left quadriceps femoris of PDAPP transgenic mice. After 3 months, Morris water-maze results confirmed significantly improved cognitive function in a mouse model of Alzheimer＇s disease. Key Words： Alzheimer＇s disease; adeno-associated virus; amyloid-β peptide; single-chain antibody; neurodegenerative diseases; neural regeneration

Bioinformatics-led design of single-chain antibody molecules targeting DNA sequences for retinoblastoma

The development of single-chain Fv antibody (scFv) by recombinant gene expression is an important milestone for cancer therapy. Single-chain antibodies are reconstructed for cancer-targeted therapy to provide good penetration into tumor tissue and to improve their pharmacokinetics in vivo, offering a clinically valuable application. The relationship needs to be analyzed that there may be some variations between the structure and function of the fusion proteins, and the relationship between the structure and function of protein molecules was obtained through analyzing relevant literature at home and abroad as well as modeling analysis. Through our analysis of the interaction region between antibody and antigen, and of the binding sites for molecular conformation, it is clear that existing antibodies need to be modified at the DNA sequence level, enhancing the biological activity of the antibodies. Based on the view that bio-molecular computer models are closely integrated with biological experiments, a bio-molecular structure-activity relationship model can be established in terms of molecular conformation, physical and chemical properties and the biological activity of single-chain antibodies. Two enlightenments are obtained from our analysis. On one hand, the structure-activity relationship is clear for new immune molecules at the gene expression level. On the other hand, a single-chain antibody molecule can be designed and optimized for the cancer-oriented treatment. In this article, we provided the theoretical and experimental basis for the development of single-chain antibodies appropriate for retinoblastoma therapy.

Novel Selenium-containing Human Single-chain Variable Fragment with Glutathione Peroxidase Activity from Computer-aided Molecular Design

In order to enhance the glutathione peroxidase（GPX） catalytic activity of the selenium-containing single-chain variable fragments（Se-scFv）, a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis, three-dimensional（3D） model building, binding-site analysis and docking. The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b（＋）, then the scFv protein was expressed in soluble form in Escherichia coli BL21（DE3） and purified by Ni2＋-immobilized metal affinity chromatography（IMAC）. The serine residue of scFv in the active site was converted into selenocysteine（Sec） with the chemical modification method, thus, the human Se-scFv with GPX activity was obtained. The GPX activity of the Se-scFv protein was characterized. Compared with other Se-scFv, the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione. It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mechanism.

MORPHOLOGICAL OBSERVATION OF SINGLE-CHAIN POLY (METHYL METHACRYLATE) PARTICLES

Tapping mode atomic force microscope has been applied to observe single-chain PMMA particles which were stored for six months at room temperature after sprayed hom very dilute solutions in good solvents, good/poor mixed solvents, and a theta solvent. Monodisperse PMMA standards of molecular weights ranging from 7.90 x 10(4) to 1.3 x 10(6) were used to investigate the effect of molecular weight on the size of the single-chain PMMA particles. These single-chain particles showed close to spherical shapes. The morphology of single-chain PMMA particles of a given molecular weight was found to be identical in spite of different solvents used for solution spraying. Molecular weight dependence of the particle dimension was also found. The diameters of single-chain PMMA particles after correction of tip-geometry effect were compared to the values estimated from molecular weight and density.

Fusion protein of single-chain variable domain fragments for treatment of myasthenia gravis

Single-chain variable domain fragment （scFv） 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion pro-tein was expressed in Pichia pastoris. The afifnity of scFv-human serum albumin fusion protein to bind to acetylcholine receptor at the neuromuscular junction of human intercostal muscles was detected by immunolfuorescence staining. The ability of the fusion protein to block myas-thenia gravis patient sera binding to acetylcholine receptors and its stability in healthy serum were measured by competitive ELISA. The results showed that the inhibition rate was 2.0-77.4%, and the stability of fusion protein in static healthy sera was about 3 days. This approach suggests the scFv-human serum albumin is a potential candidate for speciifc immunosuppressive therapy of myasthenia gravis.

Expression of secreted human single-chain fragment variable antibody against human amyloid beta peptide in Pichia pastoris

BACKGROUND：Studies have shown that monoclonal or polyclonal antibody injections of amyloid β peptide are effective in removing amyloid β peptide overload in the brain. OBJECTIVE： Based on successful screening of a human single-chain fragment variable antibody specific to amyloidβpeptide, this paper aimed to express recombinant human single-chain variable antibody against amyloid β peptide. DESIGN, TIME AND SETTING： A single sample experiment was performed at the Department of Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Hospital （Beijing, China） from January to July 2006. MATERIALS： Human single-chain fragment variable antibody gene against amyloid β peptide was screened from a human phage-display antibody library. METHODS： Human single-chain fragment variable antibody gene was mutated to eliminate a BamHI restriction site and cloned into a T easy plasmid for pT-scFvAβ construction, which was identified by PCR amplification and endonuclease digestion. Plasmid pT-scFvAβ was cut by EcoRI and NotI endonucleases, and the antibody gene was cloned into pPIC9K plasmid to construct pPIC9K-scFvAβ expression vector, which was confirmed by gene sequencing. Linearized pPIC9K-scFvAβ was used to transform a Pichia pastoris GS115 cell line, and the recombinant was induced by 0.5% methanol to express human single-chain fragment variable antibody specific to amyloid β peptide. MAIN OUTCOME MEASURES： Protein electrophoresis was used to identify PCR products, gene sequencing was used to verify the pPIC9K-scFvA sequence, and SDS-PAGE was used to detect recombinant expression of human single-chain fragment variable antibody specific to amyloid β peptide in Pichia pastoris. RESULTS： Gene sequencing confirmed pPIC9K-scFvAβ orientation. Recombinants were obtained by linearized pPIC9K-scFvAβ transformation. After induction with 0.5% methanol, the recombinant yeast cells secreted proteins of 33-ku size. CONCLUSION： The expression vector pPIC9K-scFvAβ was successfully constructed. Human single-chain fragment variable antibody specific to amyloid β peptide was recombinantly expressed in Pichia pastoris.

Ligand Modified and Light Switched On/Off Single-Chain Magnets of {Fe_(2)Co} Coordination Polymers via Metal-to-Metal Charge Transfer

It is still a formidable challenge to simultaneously switch single-chain magnet(SCM)behavior via ligand modification and light irradiation in the field of molecular spintronics.Herein,we present a ligandbridged layer{[pzTpFe(CN)3]4Co2(Bib)4}·3H2O(1;pzTp,tetra-kis(1-pyrazolyl)borate;Bib,1,4-bis-(1Himidazol-1-yl)benzene)and a well-isolated double chain{[pzTpFe(CN)3]2Co(Bpi)2}·CH3CN·4H2O(2;Bpi,1-Biphenyl-4-yl-1H-imidazole)that display reversible metal-to-metal charge transfer(MMCT)between FeIII LS(μ-CN)CoII HS(μ-NC)FeIII LS(LS,low spin;HS,high spin)and FeIII LS(μ-CN)CoIII LS(μ-NC)FeII LS linkages under alternating irradiation with 808 and 532 nm lasers.The bidirectional light irradiations induces significant changes in anisotropy and intrachain magnetic interactions,resulting in the on/off switching of SCM behavior with observable hysteresis loops by 808 and 532 nm light irradiations for both compounds.Because of the ligand modification,the SCM property of 2 with the monodentate ligand is greatly improved with a correlation length increased to 83,which is the largest correlation length among all reported light actuated SCMs.Furthermore,the influence of ligand modification on their thermally induced MMCT is also discussed.This study provides a rational approach for the swift and reversible control of SCM behavior via ligand modified and light induced MMCT,which is crucial to the future technological demand for high-density data storage and processing.

Construction of expression vectors and study on single-chain antibody and reshaping single-domain antibody against CD3

Two vectors, pWA180 and pROH80, for expression of single-chain Fv fragments (ScFv) were con-siruciea. (?)ne anti-CD3 VH and VL genes were amplified from UCHTl cells by RT-PCR and sequenced. Both genes were cloned in pWA180 to express native ScFv and pROH80 for GST-ScFv fusion protein expression. The expression products were analysed by ELISA and Western blot. The combining site of OKT3 was modeled. Human [g LS1 and Nd were selected as acceptors of CDRs of OKT3 VL and VH to construct a reshaping antibody against CD3. By com-paring OKT3, LS1 and Nd with their own family sequences, some residues were changed and the reshaping VL and VH genes were designed. The full VH gene was assembled in three steps with eight chemically synthesized oligonu-cleotide fragments using overlapping PCR and sequenced. The VH gene was expressed as active protein in pCOMB3 and as inclusion bodies in pGEX-4T-l by ELISA and Western blot analysis.

Cloning,expression and characterization of a single-chain antibody PS-9 targeted to pancreatic cancer

Genes encoding single-chain antibodies have been first constructed,which consist of the heavyand light chain variable domains of antibody PS-9 joined together by a flexible peptide linker.The geneswere cloned into coat protein g3p genes of pCANTAB5 phagemids,and expressed as fusion proteins on thephage tips.Immunological assay demonstrated that the engineered antibodies specifically bound to cancer cellsLS-174-T as well as to pure bovine submaxillary gland mucin.Their specificity and affinity appeared the sameas their parent antibodies.Our results supposed that the single-chain antibodies will be a target for thediagnosis and treatment of cancer.

Platinum Atoms Dispersed in Single-chain Polymer Nanoparticles

The intramolecular cross-linking of single polymer chains can form single-chain nanoparticles(SCNPs),which have many applications.In this study,styrenic copolymers with pendent triphenylphosphine as the coordination site for platinum ions(Pt(Ⅱ))and benzocyclobutene as the latent reactive groups are synthesized.Triphenylphosphine groups in the chains can coordinate Pt(Ⅱ)and aid slight single-chain folding in dilute solution.The intramolecular cross-linking caused by the ring-open reaction of benzocyclobutene completes the single-chain collapse and forms stable SCNPs in dilute solution.Pt(Ⅱ)embedded in SCNPs can be further reduced to platinum atoms(Pt(0)).Pt(0)steadily and atomically dispersed in SCNPs exhibits better catalytic properties than normal polymer carried platinum particles do for the reduction of p-nitrophenol to p-aminophenol.

Selection and design of high affinity DNA ligands for mutant single-chain derivatives of the bacteriophage 434 repressor

Single-chain repressor RRTRES is a derivative of bacteriophage 434 repressor, which contains covalently dimerized DNA-binding domains (amino acids 1-69) of the phage 434 repressor. In this single-chain molecule, the wild type domain R is connected to the mutant domain RTRES by a recombinant linker in a head-to-tail arrangement. The DNA-contacting amino acids of RTRES at the -1, 1, 2, and 5 positions of the a3 helix are T, R, E, S respectively. By using a randomized DNA pool containing the central sequence -CATACAAGAAAGNNNNNNTTT-, a cyclic, in vitro DNA-binding site selection was performed. The selected population was cloned and the individual members were characterized by determining their binding affinities to RRTRES. The results showed that the optimal operators contained the TTAC or TTCC sequences in the underlined positions as above, and that the Kd values were in the 1×10-12 mol/L-1×10-11mol/L concentration range. Since the affinity of the natural 434 repressor to its natural operator sites is in the 1×10-9 mol/L range, the observed binding affinity increase is remarkable. It was also found that binding affinity was strongly affected by the flanking bases of the optimal tetramer binding sites, especially by the base at the 5′ position. We constructed a new homodimeric single-chain repressor RTRESRTRES and its DNA-binding specificity was tested by using a series of new operators designed according to the recog-nition properties previously determined for the RTRES domain. These operators containing the con-sensus sequence GTAAGAAARNTTACN or GGAAGAAARNTTCCN (R is A or G) were recognized by RTRESRTRES specifically, and with high binding affinity. Thus, by using a combination of random selection and rational design principles, we have discovered novel, high affinity protein-DNA inter-actions with new specificity. This method can potentially be used to obtain new binding specificity for other DNA-binding proteins.

Supramolecular assemblies from single-chain hydrocarbon fluorocarbon hybrid bolaamphiphiles

Three types of single-chain hydrocarbon fluorocarbon hybrid bolaamphiphiles were synthesized. They readily formed different kinds of organized supramolecular assemblies, including vesicles, tapes, and micellar fibers, in aqueous solution. The aggregates morphology was characterized by transmission electron microscopy (TEM) after negative staining. The superstructures of these aggregates seemed to be determined by the geometry and the head group's properties of the corresponding amphiphiles..

Multi-Functional Nano-Sciences of Advanced Metal Complexes:Photo-Induced Switching between Single-Chain Quantum Magnet and Paramagnet,and Conducting Single-Molecule Quantum Magnets

1 Results In the fields of the molecule-based magnets,the quantum molecular magnets have been attracting much attention. While the bulk magnets or classic magnets are based on the 3D ferro- or ferrimagnetic interaction,the quantum molecular magnets are based on the double-well potential barrier defined with DS2,where D and S are uni-axial anisotropy and spin quantum number,respectively.Therefore,while the memory capacity of the bulk magnets such as floppy disc is 109 bits,the quantum magnets may have th...

Controlling the Chain Folding for the Synthesis of Single-Chain Polymer Nanoparticles Using Thermoresponsive Polymers

Synthetic polymer single-chain nanoparticles(SCNPs)are an emerging new class of nanomaterials that possess similar folded structures as natural proteins.However,most SCNPs reported so far are packed loosely in their interior,resembling those of intrinsically disordered proteins(IDPs).Here,we report a facile strategy to synthesize SCNPs with controllable folding in aqueous solution.

Inhibitory effect of humanized anti-VEGFR-2 ScFv-As_2O_3-stealth nanoparticles conjugate on growth of human hepatocellular carcinoma:in vitro and in vivo studies

Objective:To investigate the inhibitory effect of humanized anti-VEGFR-2 ScFv-As_2O_3-stealth nanoparticles conjugate on growth of human hepatocellular carcinoma both in vitro and in vivo,which may be a potential agents with sensitivity and targeting ability for human hepatocellular cancer.Methods:Humanized anli-VECFR-2 ScFv-As_2O_3-stealth nanoparticles conjugate was previously constructed using ribosome display technology and antibody conjugate technology.In this combined in vitro and in vivo study,the inhibitory effects of anti-VEGFR-2 ScFv-As_2O_3-stealth nanoparticles conjugate on tumor growth,invasion,and metastasis was observed with human liver carcinoma cell line Bel7402 and normal cell L02 by MTT assay,Tanswell assay,Hochest33258 staining,and DNA ladder analysis.The anticancer activity and distribution of anti-VEGFR-2 ScFv-As_2O_3-stealth nanoparticles was then verified in a mouse model of Bel7402xenografts.Results:Anti-VEGFR-2 ScFv-As_2O_3-stealth nanoparticles significantly inhibited the proliferation of Bel7402 in the 3-(4,5-dimethylthiazol-2-yh-2,5-diphenyltetrazolium bromide assay while had almost no effects on L02 cells.And the apoptosis inducing effects were proved by Hochest33258 staining and DNA ladder analysis.Transwell assay found that the drug also inhibited the metastasis ability of tumor cells.Furthermore,anti-VEGFR-2 ScFv-As^-stealth nanoparticles significantly delayed the growth of Bel7402 xenografts after administration(92.9%),followed by As_2O_3-stealth nanoparticles,anti-VEGFR-2 ScFv,and As203(61.4%,58.8%,20.5%,P<0.05).The concentration of As_2O_3 in anti-VEGFR-2 ScFv-As_2O_3-steallh nanoparticles group was more selectively.Conclusions:Anti-VEGFR-2 ScFv-As_2O_3-stealth nanoparticles is a potent and selective anti-hepatocellular carcinoma agent which could inhibit the growth of liver cancer as a targeting agent both in vitro and in vivo and also significantly inhibit angiogenesis.

Generation of high affinity human single-hain antibody against PreSl of hepatitis B virus from immune phage-display antibody library

BACKGROUND: A single-chain antibody ( ScFv) phage display library was created by cloning antigen-binding re- gions of VH (variable domain) and VL gene repertoires as fusion proteins with a minor coat protein of filamentous phage, from which high affinity completely humanized ScFv against PreS1 of hepatitis B virus could be screened and characterized. METHODS: A combinatorial library of phage-display hu- man ScFv genes, which were derived from peripheral blood lymphocytes immunized by peptide PreS1 in vitro, was constructed. The library contained 7 × 108 clones. RESULTS: After 3 rounds panning, a high affinity (K = 10-7-10-8 mol/L) ScFv specific to PreS1 was obtained. Sequence analysis showed that the VH belonged to the VH4 family and Vλ to Vλ4. CONCLUSIONS: The described ScFv may provide a more satisfactory therapy. This application further illustrates that the method of in vitro antigen stimulation is expeditious for the source of human immune antibody library.

Selection for Anti-transferrin Receptor Bispecific T-cell Engager in Different Molecular Formats

Selecting an ideal molecular format from diverse structures is a major challenge in developing a bispecific antibody(BsAb).To choose an ideal format of anti-CD3 x anti-transferrin receptor(TfR)bispecific antibodies for clinical application,we constructed TfR bispecific T-cell engager(BiTE)in two extensively applied formats,including single-chain tandem singlechain variable fragments(scFvs)and double-chain diabodies,and evaluated their functional characterizations in vitro.Results demonstrated that TfR-BiTE in both formats directed potent killing of TfR+HepG2 cells.However,compared to two・chain diabodies,scFvs were more efficient in antigen binding and TfR target killing.Furthermore,different domain orders in scFvs would also be evaluated because single-TfR-CD3-His was preferable to single-CD3-TfR-His in immunotherapeutic strategies.Thus,the single-chain tandem TfR-CD3 format was favored for further investigation in cancer therapy.