基于细胞的抗单增李斯特菌单域重链抗体的筛选及鉴定

【目的】获得针对单增李斯特菌的特异性单域重链抗体,并对筛选过程中特异性克隆的富集规律进行分析,为筛选具有种属特异性的噬菌体展示抗体提供参考。【方法】采用固相筛选技术,以热灭活的单增李斯特菌菌体为抗原,通过四轮常规筛选和一轮消减筛选,从驼源天然噬菌体展示文库中筛选针对单增李斯特菌的单域重链抗体。采用Phage-ELISA法,对后四轮筛选洗脱物中随机挑选的噬菌体进行鉴定,阳性克隆进行基因测序及结合特异性分析。通过多序列比对分析将获得的基因序列进行分组和统计。【结果】成功筛选到2株单增李斯特菌特异性的单域重链抗体。【结论】在优化的筛选条件下,基于全细胞的筛选方法能够获得特异性识别单增李斯特菌的单域重链抗体,消减筛选对于去除非特异性克隆是有效的和必要的。     

Evaluation of the Applied Biosystems automated Taqman polymerase chain reaction system for the detection of meningococcal DNA

In a period where the proportion of culture confirmed cases in the UK has been steadily declining, diagnosis by PCR has been used to increase the number of confirmed cases and provide additional epidemiological data. This report presents a comparative evaluation of the fluorogenic probe-based 5' exonuclease assay (Taqman) using the Perkin-Elmer Applied Biosystems automated sequence detection system 7700 with previously reported polymerase chain reaction enzyme-linked immunosorbent (PCR ELISA) assays for the detection of meningococcal DNA in CSF, plasma and serum samples. Taqman assays developed were based on the detection of a meningococcal capsular transfer gene (ctrA), the insertion sequence IS1106 and the sialytransferase gene (siaD) for serogroup B and C determination and compared with similar assays in a PCR ELISA format. The Taqman ctrA assay was specific for Neisseria meningitidis, however the IS1106 assay gave false positive reactions with a number of non-meningococcal isolates. Sensitivity of the Taqman ctrA, IS1106 and siaD assays testing samples from culture-confirmed cases were 64, 69 and 50%, respectively, compared with 26, 67 and 43% for the corresponding PCR ELISA assays. Improvements to the DNA extraction procedure has increased the sensitivity to 93 and 91% for the TaqMan ctrA and siaD assays, respectively, compared to culture confirmed cases. Since the introduction of Taqman PCR a 56% increase in laboratory confirmed cases of meningococcal disease has been observed compared to culture only confirmed cases. The developed Taqman assays for the diagnosis of meningococcal disease enables a high throughput, rapid turnaround of samples with considerable reduced risk of contamination.     

Identification and characterization of single chain anti-cocaine catalytic antibodies

Cocaine is a powerful and addictive stimulant whose abuse remains a prevalent health and societal crisis. Unfortunately, no pharmacological therapies exist and therefore alternative protein-based therapies have been examined. One such approach is immunopharmacotherapy, wherein antibodies are utilized to either bind or hydrolyze cocaine thereby blocking it from exerting its euphoric effect. Towards this end, antibodies capable of binding and hydrolyzing cocaine were identified by phage display from a biased single chain antibody library generated from the spleens of mice previously immunized with a cocaine phosphonate transition state analog hapten. Two classes of antibodies emerged based on sequence homology and mode of action. Alanine scanning mutagenesis and kinetic analysis revealed that residues H97, H99, and L96 are crucial for antibodies 3F5 and 3H9 to accelerate the hydrolysis of cocaine. Antibodies 3F1 through 3F4, which are similar to our previously identified 3A6 class of antibodies, catalyze hydrolysis through transition state stabilization by tyrosine or histidine residues H50 and L94. Mutation of either one or both tyrosine residues to histidine conferred hydrolytic activity on previously inactive antibody 3F4. Mutational analysis of residue H50 of antibody 3F3 resulted in a glutamine mutant with a rate enhancement three times greater than wild-type. A double mutant, containing glutamineH50 and lysineH52, showed a tenfold rate enhancement over wild-type. These results indicate the power of initial selection of catalytic antibodies from a biased antibody library in both rapid generation and screening of mutants for improved catalysis.     

Construction of a large phage-displayed human antibody domain library with a scaffold based on a newly identified highly soluble, stable heavy chain variable domain

Currently, almost all U.S. Food and Drug Administration-approved therapeutic antibodies and the vast majority of those in clinical trials are full-size antibodies mostly in an immunoglobulin G1 format of about 150 kDa in size. Two fundamental problems for such large molecules are their poor penetration into tissues (e.g., solid tumors) and poor or absent binding to regions on the surface of some molecules [e.g., on the human immunodeficiency virus envelope glycoprotein (Env)] that are accessible by molecules of smaller size. We have identified a phage-displayed heavy chain-only antibody by panning of a large (size, ∼ 1.5 × 10¹⁰) human naive Fab (antigen-binding fragment) library against an Env and found that the heavy chain variable domain (V_H) of this antibody, designated as m0, was independently folded, stable, highly soluble, monomeric, and expressed at high levels in bacteria. m0 was used as a scaffold to construct a large (size, ∼ 2.5 × 10¹⁰), highly diversified phage-displayed human V_H library by grafting naturally occurring complementarity-determining regions (CDRs) 2 and 3 of heavy chains from five human antibody Fab libraries and by randomly mutating four putative solvent-accessible residues in CDR1 to A, D, S, or Y. The sequence diversity of all CDRs was determined from 143 randomly selected clones. Most of these V_Hs were with different CDR2 origins (six of seven groups of V_H germlines) or CDR3 lengths (ranging from 7 to 24 residues) and could be purified directly from the soluble fraction of the Escherichia coli periplasm. The quality of the library was also validated by successful selection of high-affinity V_Hs against viral and cancer-related antigens; all selected V_Hs were monomeric, easily expressed, and purified with high solubility and yield. This library could be a valuable source of antibodies targeting size-restricted epitopes and antigens in obstructed locations where efficient penetration could be critical for successful treatment.     

Modulation of in vivo IgG crystallization in the secretory pathway by heavy chain isotype class switching and N-linked glycosylation

Crystalline bodies (CBs) can develop in the endoplasmic reticulum (ER) of antibody-producing cells. Although this phenotype is often reported in association with plasma cell dyscrasias and other hematological disorders, the details of CB biogenesis and CB's roles in pathophysiology remain poorly understood. Using an imaging-based screening method, we identified a secretion-competent human IgG2/λ clone that develops spindle-shaped intracellular crystals in transiently-transfected HEK293 cells upon Brefeldin A treatment. When stably overexpressed from CHO cells, the IgG2/λ clone spontaneously produced spindle-shaped CBs in the ER. Some CBs were released to the extracellular space while remaining enclosed by the membranes of secretory pathway origin. Structural modeling on the variable-region did not uncover prominent surface characteristics such as charge clusters. In contrast, alterations to the constant domain-encoded properties revealed their modulatory roles in CB-inducing propensities and CB morphology. For example, deletion of the entire Fc domain changed the morphology of CBs into thin filaments. Elimination of an N-linked glycan by a N297A mutation promoted Russell body biogenesis accompanied by marked reduction in IgG secretion. Isotype class switching from the original IgG2 to IgG1 and IgG4 changed the crystal morphology from spindle-shaped to long needle and acicular shaped, respectively. The IgG3 version, in contrast, suppressed the CB formation. Either the HC or LC alone or the Fc-domain alone did not trigger CB biogenesis. An IgG's in vivo crystal morphology and crystallization propensity can thus be modulated by the properties genetically and biochemically encoded in the HC constant region.     

Asia I型口蹄疫病毒中和表位与羊IgG重链恒定区的融合表达及免疫原性测定

VP1蛋白是口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)诱导机体产生抗病毒感染免疫的主要蛋白,含有病毒的若干中和表位。本研究设计和合成了由AsiaI型FMDVVP1蛋白136~160aa和198~211aa两个表位组成的重复串联表位的编码基因,并克隆了羊IgG重链恒定区编码基因。利用BamHI、EcoRI和XhoI位点将2个基因片段依次克隆到pPROExHTb载体,构建成重组质粒pPRO-FshIgG,将其转化大肠杆菌BL21(DE3)感受态细胞,以IPTG诱导表达得到融合蛋白FshIgG。100μgFshIgG蛋白免疫豚鼠后刺激豚鼠产生了高效价的FMDV中和抗体,而且使这些免疫豚鼠在用200ID50剂量FMDV攻击时得到了完全保护。由此证明,羊IgG重链恒定区蛋白能够作为FMDV表位肽的载体,而融合蛋白FshIgG可成为一种口蹄疫表位疫苗候选物用于口蹄疫的预防。     

Commercial enzyme-linked immunosorbent assay versus polymerase chain reaction for the diagnosis of chronic Chagas disease: a systematic review and meta-analysis

Chronic Chagas disease diagnosis relies on laboratory tests due to its clinicalcharacteristics. The aim of this research was to review commercial enzyme-linkedimmunosorbent assay (ELISA) and polymerase chain reaction (PCR) diagnostic testperformance. Performance of commercial ELISA or PCR for the diagnosis of chronicChagas disease were systematically searched in PubMed, Scopus, Embase, ISI Web, andLILACS through the bibliography from 1980-2014 and by contact with the manufacturers.The risk of bias was assessed with QUADAS-2. Heterogeneity was estimated with theI² statistic. Accuracies provided by the manufacturers usuallyoverestimate the accuracy provided by academia. The risk of bias is high in mosttests and in most QUADAS dimensions. Heterogeneity is high in either sensitivity,specificity, or both. The evidence regarding commercial ELISA and ELISA-recsensitivity and specificity indicates that there is overestimation. The currentrecommendation to use two simultaneous serological tests can be supported by the riskof bias analysis and the amount of heterogeneity but not by the observed accuracies.The usefulness of PCR tests are debatable and health care providers should not orderthem on a routine basis. PCR may be used in selected cases due to its potential todetect seronegative subjects.     

灭癌素链霉菌中磷硫酰化修饰DNA结合结构域的功能分析

[目的]DNA磷硫酰化(phosphorothioation,PT)是由硫原子取代DNA骨架磷原子上的非桥联氧原子形成的一种新型DNA修饰.PT修饰除参与组成限制修饰系统外,其更为广泛的生物学功能仍有待揭示.PT修饰现有的检测方法操作复杂、成本高、耗时长,而具有操作简便、成本低、耗时短等特点的酶联免疫检测(enzyme...     

The amyloid fibrils of the constant domain of immunoglobulin light chain

Light chain-associated (AL) amyloidosis is characterized by dominant fibril deposition of the variable domain (VL) of an immunoglobulin light chain, and thus its constant domain (CL) has been considered not to be amyloidogenic. We examined the in vitro fibril formation of the isolated CL in comparison with β₂-microglobulin (β2-m), an immunoglobulin domain-like amyloidogenic protein responsible for dialysis-related amyloidosis. Two methods useful for β2-m at neutral pH also induced amyloid fibrils of CL, which were monitored by thioflavin-T binding and electron microscopy (EM). These results suggest that CL plays an important role, more than previously assumed, in the development of AL-amyloidosis.     

Structural differences in the motor domain of temperature-associated myosin heavy chain isoforms from grass carp fast skeletal muscle

We determined coding sequences for three types of grass carp myosin subfragment-1 (S1) heavy chain by extending 5′-regions of the three known genes encoding light meromyosin isoforms (10 °C, intermediate and 30 °C types). The primary structures of these three S1 heavy chain isoforms showed 81.4%, 81.2%, and 97.8% identities between the 10 °C and intermediate types, between the 10 °C and 30 °C types, and between the intermediate and 30 °C types, respectively. Isoform-specific differences were clearly observed between the 10 °C type and the other two types in 97 amino acid residues. Furthermore, among these amino acid mutations, 51 mutations occurred at the conserved residue sites of S1 heavy chain from fish and homoiotherm. Additionally, the 10 °C type showed striking differences compared with the other two types in the two surface loops, loop 1 located near the ATP-binding pocket and loop 2, which is one of the actin-binding sites, suggesting that such structural differences possibly affect their motor functions. Interestingly, this 10 °C-type myosin heavy chain isolated from adult grass carp skeletal muscle was surprisingly similar to the embryonic fast-type myosin heavy chain from juvenile silver carp in the structure of S1 heavy chain, indicating that it may also function as embryonic fast-type myosin heavy chain in juvenile stage.     

Interaction between the antigen and antibody is controlled by the constant domains: normal mode dynamics of the HEL-HyHEL-10 complex

The antigen binding fragment (Fab) of a monoclonal antibody (HyHEL-10) consists of variable domains (Fv) and constant domains (CL-CH1). Normal modes have been calculated from the three-dimensional structures of hen egg lysozyme (HEL) with Fab, those of HEL with Fv, and so on. Only a small structural change was found between HEL-Fab and HEL-Fv complexes. However, HEL-Fv had a one order of magnitude lower dissociation constant than HEL-Fab. The Calpha fluctuations of HEL-Fab differed from those of HEL-Fv with normal mode calculation, and the dynamics can be thought to be related to the protein-protein interactions. CL-CH1 may have influence not only around local interfaces between CL-CH1 and Fv, but also around the interacting regions between HEL and Fv, which are longitudinally distant. Eighteen water molecules were found in HEL-Fv around the interface between HEL and Fv compared with one water molecule in HEL-Fab. These solvent molecules may occupy the holes and channels, which may occur due to imperfect complementarity of the complex. Therefore, the suppression of atomic vibration around the interface between Fv and HEL can be thought to be related to favorable and compact interface formation by complete desolvation. It is suggested that the ability to control the antigen-antibody affinity is obtained from modifying the CL-CH1. The second upper loop in the constant domain of the light chain (UL2-CL), which is a conserved gene in several light chains, showed the most remarkable fluctuation changes. UL2-CL could play an important role and could be attractive for modification in protein engineering.     

Dissection of the protein G B1 domain binding site for human IgG Fc fragment.

The contribution to the free energy of binding of each of the residues forming the binding site for a human IgG Fc fragment on the surface of the B1 domain of protein G was determined by alanine-scanning mutagenesis. The interface between these two proteins is atypical in that it is smaller than usual, polar in character, and involves two well-defined "knobs-into-holes" interactions. The bulk of the free energy of binding is contributed by three central residues, which make hydrogen bonds across the interface. Of these, the most critical interaction is formed by Glu27, which acts as a charged knob on the surface of the B1 domain, inserting into a polar hole on the Fc fragment. A single alanine mutation of this residue virtually abolishes stable complex formation. Formation of a stable interface between these two proteins is therefore dominated by a small, polar "hot spot."     

Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies

The somatic mutations accumulated in variable and framework regions of antibodies produce structural changes that increase the affinity towards the antigen. This implies conformational and non covalent bonding changes at the paratope, as well as possible quaternary structure changes and rearrangements at the V_H-V_L interface. The consequences of the affinity maturation on the stability of the Fv domain were studied in a system composed of two closely related antibodies, F10.6.6 and D44.1, which recognize the same hen egg-white lysozyme (HEL) epitope. The mAb F10.6.6 has an affinity constant 700 times higher than D44.1, due to a higher surface complementarity to HEL. The structure of the free form of the Fab F10.6.6 presented here allows a comparative study of the conformational changes produced upon binding to antigen. By means of structural comparison, kinetics and thermodynamics of binding and stability studies on Fab and Fv fragments of both antibodies, we have determined that the affinity maturation process of anti-protein antibodies affects the shape of the combining site and the secondary structure content of the variable domain, stabilizes the V_H-V_L interaction, and consequently produces an increase of the Fv domain stability, improving the binding to antigen.     

Influence of the internal disulfide bridge on the folding pathway of the CL antibody domain

Disulfide bridges are one of the most important factors stabilizing the native structure of a protein. Whereas the basis for their stabilizing effect is well understood, their role in a protein folding reaction still seems to require further attention. We used the constant domain of the antibody light chain (C(L)), a representative of the ubiquitous immunoglobulin (Ig)-superfamily, to delineate the kinetic role of its single buried disulfide bridge. Independent of its redox state, the monomeric C(L) domain adopts a typical Ig-fold under native conditions and does not retain significant structural elements when unfolded. Interestingly, its folding pathway is strongly influenced by the disulfide bridge. The more stable oxidized protein folds via a highly structured on-pathway intermediate, whereas the destabilized reduced protein populates a misfolded off-pathway species on its way to the native state. In both cases, the formation of the intermediate species is shown to be independent of the isomerization state of the Tyr(141)-Pro(142) bond. Our results demonstrate that the internal disulfide bridge in an antibody domain restricts the folding pathway by bringing residues of the folding nucleus into proximity thus facilitating the way to the native state.     

Structural Basis of Enhanced Crystallizability Induced by a Molecular Chaperone for Antibody Antigen-Binding Fragments

Monoclonal antibodies constitute one of the largest groups of drugs to treat cancers and immune disorders, and are guiding the design of vaccines against infectious diseases. Fragments antigen-binding (Fabs) have been preferred over monoclonal antibodies for the structural characterization of antibody–antigen complexes due to their relatively low flexibility. Nonetheless, Fabs often remain challenging to crystallize because of the surface characteristics of complementary determining regions and the residual flexibility in the hinge region between the variable and constant domains. Here, we used a variable heavy-chain (V_HH) domain specific for the human kappa light chain to assist in the structure determination of three therapeutic Fabs that were recalcitrant to crystallization on their own. We show that this ligand alters the surface properties of the antibody–ligand complex and lowers its aggregation temperature to favor crystallization. The V_HH crystallization chaperone also restricts the flexible hinge of Fabs to a narrow range of angles, and so independently of the variable region. Our findings contribute a valuable approach to antibody structure determination and provide biophysical insight into the principles that govern the crystallization of macromolecules.     

Redesigning of anti‐c‐Met single chain Fv antibody for the cytoplasmic folding and its structural analysis

Typically, single chain Fv antibodies are unable to fold properly under a reducing cytoplasm because of the reduction of disulfide bonds. The inability to fold limits both the production of the functional scFvs and their targeting against antigens, which are generally executed in a reducing cytoplasm. In this study, the target scFv CDR was grafted with stable human consensus framework sequences, which enabled the generation of a foldable scFv in a reducing cytoplasm of Escherichia coli. Additionally, the structural features affecting the folding efficiency of the engineered scFv were identified by analyzing the predicted structure. An anti‐c‐Met scFv, which was a cytoplasmic non‐foldable protein, was redesigned as the model system. This study confirmed that the engineered anti‐c‐Met scFv was folded into its native form in the cytoplasm of E. coli BL21(DE3) without a significant loss in the specific binding activity against c‐Met antigen. The structures of the wild‐type anti‐c‐Met scFv and the engineered scFv were predicted using homology modeling. A comparative analysis based on the sequence and structure showed that the hydrophobicity of 12 solvent exposed residues decreased, and two newly formed salt bridges might have improved the folding efficiency of the engineered scFv under the reducing condition. Biotechnol. Bioeng. 2010; 106: 367–375. © 2010 Wiley Periodicals, Inc.     

Structure-based stability engineering of the mouse IgG1 Fab fragment by modifying constant domains

A semi-rational approach based on structural data was exploited in a search for CH1 and CL domains with improved intrinsic thermodynamic stabilities. Structural and amino acid level comparisons were carried out against known biophysically well-behaving and thermodynamically beneficial scFv and Fab fragments. A number of mutant Fab fragments were constructed by site-directed mutagenesis of regions in the CH1 and CL domains expected to be most sensitive under physical stress conditions. These mutations were located on three sites in the Fab constant domains; a mobile loop in the CH1 domain, residues surrounding the two largest solvated hydrophobic cavities located in the interface of the CH1 and CL domains and the hydrophobic core regions of both CH1 and CL. Expression levels of functional Fab fragments, denaturant-induced unfolding equilibria and circular dichroism spectroscopy were used to evaluate the relative stabilities of the wild-type and the mutant Fab fragments. The highest thermodynamic stability was reached through the mutation strategy, where the hydrophobicity and the packing density of the solvated hydrophobic cavity in the CH1/CL interface was increased by the replacement of the hydrophilic Thr178 in the CL domain by a more hydrophobic residue, valine or isoleucine. The midpoint of the transition curve from native to unfolded states of the protein, measured by fluorescence emission, occurred at concentrations of guanidine hydrochloride of 2.4 M and 2.6 M for the wild-type Fab and the most stable mutants, respectively. Our results illustrate that point mutations targeted to the CH1/CL interface were advantageous for the overall thermodynamic stability of the Fab fragment.     

Structure of Fab fragment of malaria transmission blocking antibody 2A8 against P. vivax P25 protein

Understanding the structural basis of recognition between antigen and antibody requires the structural comparison of free and complexed components. Previously, we have reported the crystal structure of the complex between Fab fragment of murine monoclonal antibody 2A8 (Fab2A8) and Plasmodium vivax P25 protein (Pvs25) at 3.2 Å resolution. We report here the crystallization and X-ray structure of native Fab2A8 at 4.0 Å resolution. The 2A8 antibody generated against Pvs25 prevents the formation of P. vivax oocysts in the mosquito, when assayed in membrane feeding experiment.Comparison of native Fab2A8 structure with antigen bound Fab2A8 structure indicates the significant conformational changes in CDR-H1 and CDR-H3 regions of V_H domain and CDR-L3 region of V_L domain of Fab2A8. Upon complex formation, the relative orientation between V_L and V_H domains of Fab2A8 is conserved, while significant differences are observed in elbow angles of heavy and light chains. The combing site residues of complexed Fab2A8 exhibited the reduced temperature factor compared to native Fab2A8, suggesting a loss of conformational entropy upon antigen binding.     

Mouse immunoglobulin A: nucleotide sequence of the structural gene for the alpha heavy chain derived from cloned cDNAs

The cDNAs complementary to mouse immunoglobulin alpha heavy chain mRNAs have been cloned into the PstI site of the plasmid vector pBR322. Recombinant plasmids have been identified by hybrid-arrested translation and purification of alpha heavy chain mRNA on DNA-DBM filters. The nucleotide sequence of the inserts encodes the constant and 3' untranslated regions of the alpha heavy chain mRNA. The CH3 domains of human and mouse alpha chains are highly homologous, including a 36 amino acid fragment not reported in the protein sequence (Robinson and Appella, 1980). As in the case of the mu secreted heavy chain, the alpha heavy chain contains a carboxy terminal piece of 20 amino acids.