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.     

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.     

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…     

Construction,Expression, and Characterization of a Single-Chain Variable Fragment Antibody Against 2,4-Dichlorophenoxyacetic Acid in the Hemolymph of Silkworm Larvae

A single-chain variable fragment antibody against herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D-scFv) has been successfully expressed in the hemolymph of silkworm larvae using a rapid Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Variable heavy- and light-chain domains were cloned directly from the cDNA of the hybridoma cell line 2C4 and assembled together with flexible peptide linker (Gly₄Ser)₃ between two domains. The yield of functional 2,4-D-scFv after purification was 640 μg per 30 ml of hemolymph, which is equivalent to 21.3 mg per liter of hemolymph. The characterization of 2,4-D-scFv using an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that it has wide cross-reactivities against 2,4,5-trichlorophenoxyacetic acid (65.5%), 2,4-dichlorophenol (47.9%), and 2,4-dichlorobenzoic acid (26.0%), making it possible to apply 2,4-D-scFv to icELISA for detecting/determining 2,4-D and its metabolites. Judging from its cost and time requirements and its ease of handling, this BmNPV bacmid DNA expression system is more useful for expressing functional scFv than bacterial systems, which frequently require costly and time-consuming refolding.     

高效液相色谱法分离纯化人胎盘泌乳素酶切片段

高效液相色谱法分离纯化人胎盘泌乳素酶切片段杨煜,杨波,麦荫乔（白求恩医科大学长春１３００２１）１前言人胎盘泌乳素（Ｈｕｍａｎｐｌａｃｅｎｔａｌｌａｃｔｏｇｅｎ,ＨＰＬ）是由胎盘绒毛滋养层的合体细胞分泌的一种单链多肽类激素。它主要有催乳作用￣［１］及促...     

Isolation and Characterization of Two Novel Gene Encoded Alkaline Esterases from an Alkaline Soil Metagenomic Library

<正>The development of industrial biotechnology has created an increasing demand for alkaline lipolytic enzymes with functional diversity.In this study,an alkaline soil metagenomic library was constructed to search for new lipolytic enzymes.Two novel gene encoded alkaline esterases(designated as estA and estB) were isolated by functional screening from the library.The estA gene consisted of 834 bp and coded for 277 amino acids with a molecular mass of 29998.Amino acid sequence homology analysis indicates that EstA belongs toα/βhydrolase fold family 4.4(abH4.4),with EstA being the smallest member of that family yet reported.The estB gene consisted of 1185 bp and encoded 394 amino acids with a theoretical molecular mass of 40090.Its conserved domain analysis shows that EstB belongs to the GDSL hydrolase superfamily.Both EstA and EstB exhibit only moderate identity(38%) in amino acid sequence to the known lipolytic enzyme genes in the database.The two genes were respectively expressed in Escherichia coli and the protein products were purified for functional characterization.While the expressed EstA did not exhibit the functional properties that were superior to those of other esterases previously reported,the EstB was stable at temperature up to 45℃and its maximum activity was measured to be 53.6 U/mg at pH=10.Both the enzymes have further enriched the diversity of the lipolytic enzymes database and also appear to be promising biocatalysts for potential biotechnological application.     

Isolation and Characterization of Heparan Sulfate from Human Lung Tissues

Glycosaminoglycans are a class of linear, highly negatively charged, O-linked polysaccharides that are involved in many (patho)physiological processes. In vitro experimental investigations of such processes typically involve porcine-derived heparan sulfate (HS). Structural information about human, particularly organ-specific heparan sulfate, and how it compares with HS from other organisms, is very limited. In this study, heparan sulfate was isolated from human lung tissues derived from five donors and was characterized for their overall size distribution and disaccharide composition. The expression profiles of proteoglycans and HS-modifying enzymes was quantified in order to identify the major core proteins for HS. In addition, the binding affinities of human HS to two chemokines—CXCL8 and CCL2—were investigated, which represent important inflammatory mediators in lung pathologies. Our data revealed that syndecans are the predominant proteoglycan class in human lungs and that the disaccharide composition varies among individuals according to sex, age, and health stage (one of the donor lungs was accidentally discovered to contain a solid tumor). The compositional difference of the five human lung HS preparations affected chemokine binding affinities to various degrees, indicating selective immune cell responses depending on the relative chemokine–glycan affinities. This represents important new insights that could be translated into novel therapeutic concepts for individually treating lung immunological disorders via HS targets.     

Isolation and Characterization of an Antinicrobial Polypeptide from Loach

A novel antimicrobial polypeptidc was isolated and charaeterized from loach,Misgurmis anguillieaudatus, The polypcptide, named MAPP, is a single-chain polypcptide with Mw about 9,800 Dalton and p1 about 4.78; the N-tag ofMAPP was CFGWN. MAPP showed good inhibition against various bacteria including Bacillus subtilis, Eseherichia coli and Staphylococcus aurens. MAPP could be used as a lead compound in antibiotics drug discovery.     

Isolation and Characterization of an Antimicrobial Polypeptide from Loach

Hydrobionts formed their special defense systems during evolution. One such system is that of non-specific immunity which comprises a wide variety of peptides with potent antimicrobial activities1. The mechanism of action of most antimicrobial peptides was reported as that a few peptide molecules formed a channel on cell membrane, and the cell was then died of the outflowing of cellular contents. The above mechanism was different from that of antibiotics2, 3. It is a promising area to disc…     

Isolation and Characterization of Adipose-derived Mesenchymal Stem Cells (ADSCs) from Cattle

Adipose-derived mesenchymal stem cells (ADSCs) were isolated from the adult adipose tissue of 2-year-old cattle, and then characterized by immunofluorescence and RT-PCR. We found that primary bADSCs could be expanded for 25 passages. Expression of β-integrin, CD44, and CD73 was observed by immunofluorescence and RT-PCR. Passage 3 bADSCs were successfully induced to differentiate into osteoblasts and adipocytes. The results indicate the potential for multi-lineage differentiation of bADSCs that may represent an ideal candidate for cellular transplantation therapy.     

Screening and Identification of a Targeting Peptide to nGLP-1R from Phage Display Peptide Library

<正>In order to provide the structure information for designing new exendin-4 analogues,a phage display peptide library was screened by targeting the N-terminal extracellular domain of GLP-1R(nGLP-1R).After four rounds of selection,nine sequences were obtained,four of them have higher affinity for nGLP-1R than the others.We chose two of them named X and Y peptides.Isletβ-cell proliferation assay suggested that X and Y peptides didn't have any activity to increase isletβ-cell proliferation.In other words,X and Y peptides were not agonists to GLP-1R.However, the conservative motifs of X and Y peptides provided us useful information to design new exendin-4 analogues.     

Structural and Dynamic Characterization of the Molecular Hub Early Region 1A (E1A) from Human Adenovirus

The small‐DNA human adenovirus encodes one of the most versatile molecular hubs, the E1A protein. This protein is essential for productive viral infection in human cells and a vast amount of biologically relevant data are available on its interactions with host proteins. Up to now, however, no high‐resolution structural and dynamic information on E1A is available despite its important biological role. Among the different spliced variants of E1A, two are expressed at high level in the early stage of infection. These are 243 and 289 residues isoforms. Herein, we present their NMR characterization, showing that they are both highly disordered, but also demonstrate a certain heterogeneous behavior in terms of structural and dynamic properties. Furthermore, we present the characterization of the isolated domain of the longer variant, known as CR3. This study opens the way to understanding at the molecular level how E1A functions.     

Isolation and Characterization of a Bismuth(II) Radical

More than 80 years after Paneth’s report of dimethyl bismuth, the first monomeric Bi^II radical that is stable in the solid state has been isolated and characterized. Reduction of the diamidobismuth(III) chloride Bi(NON^Ar)Cl (NON^Ar=[O(SiMe₂NAr)₂]²⁻; Ar=2,6‐iPr₂C₆H₃) with magnesium affords the Bi^II radical ^.Bi(NON^Ar). X‐ray crystallographic measurements are consistent with a two‐coordinate bismuth in the +2 oxidation state with no short intermolecular contacts, and solid‐state SQUID magnetic measurements indicate a paramagnetic compound with a single unpaired electron. EPR and density functional calculations show a metal‐centered radical with >90 % spin density in a p‐type orbital on bismuth.     

Identification of a Neutralizing scFv Binding to Human Vascular Endothelial Growth Factor 165 (VEGF165) Using a Phage Display Antibody Library

Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that plays a major role in angiogenesis. Alternative splicing causes the production of several different isoforms (VEGF121, 145, 165, 183, 189, 206). VEGF is essential for tumor angiogenesis, and several studies have correlated elevated VEGF levels with tumor stage, metastases, and progression. We now report the isolation by phage display of human single-chain antibody fragment (scFv) anti-VEGF165. After four rounds of panning against VEGF165, 40 out of 90 phage clones displayed VEGF165-binding activity. One of the positive clones, designated B8, bound to VEGF165 with relatively high affinity and neutralized VEGF165 bioactivity in vitro. The B8 clone was expressed in the soluble form in Escherichia coli HB2151 and purified by immobilized metal affinity chromatography. The purified scFv recognized VEGF165 with the K _D of 1.80 × 10⁻⁸ M without cross-reaction to VEGF121. In addition to binding, the purified scFv could does-dependently inhibit VEGF165-induced human umbilical vein-derived endothelial cells proliferation. Together with its fully human mature, B8 scFv may have therapeutic implications in therapy of angiogenesis-dependent diseases.     

Isolation and Characterization of Sulfur-Containing Polycyclic Aromatic Compounds (Thia-Arenes) from Complex Environmental Mixtures

Abstract

A multi-dimensional chromatographic method was developed and applied to the separation of thia-arenes and polycyclic aromatic hydrocarbons (PAH) derived from coal tar. A thia-arene-rich fraction, prepared using a combination of alumina chromatography and palladium chloride/silica gel chromatography, was further separated using normal phase HPLC to isolate fractions containing thia-arenes with molecular masses ranging from 184 amu to 284 amu. These fractions were analysed using gas chromatography-mass spectrometry; approximately one-half of the thia-arenes in a coal tar extract were retained and separated by the palladium chloride-silica gel step. This methodology has been successfully applied to coal tar, air particulate material, sediments and biological samples.     

SmxY1-x(Sal)3(phen)转光剂的合成与性能表征

在钐中掺入钇,以水杨酸(Hsal)、邻菲咯啉(Phen)为有机配体合成了一系列新的三元有机配合物,研究了该系列配合物的荧光性能、紫外/可见光谱、红外光谱及热效应等性能,并且从中探讨了配合物性能随Y3+掺入量变化的规律.结果表明,荧光惰性离子Y3+的适量掺入,不仅会改变中心离子Sm3+与有机配体之间的配位能力,还能增强Sm3+的红光发光强度,在Y3+掺入量为90%(摩尔分数)时达到最佳.同时,该系列配合物的主要荧光发射峰位645 nm与植物光合作用吸收谱较为吻合.     

Isolation of an oxychloroniobium(IV) complex from hydrochloric acid solution

Summary An oxychloroniobium(IV) complex was isolated for the first time by zinc reduction of NbCl₅ in concentrated HCl solution. The product, an extremely moisture- and air-sensitive, intense blue solid was assigned a dimeric structure (H₃O)₂[Nb₂O₂Cl₆] based on elemental analysis, i.r. and u.v.-vis spectroscopy as well as magnetic and conductometric measurements.