Structural analysis of the mouse T-cell receptor Tcra V2 subfamily

Cosmid clones containing T-cell receptor Tcra V2 subfamily gene segments have been isolated from a BALB/c cosmid library and subjected to DNA sequence analysis. The V gene segments in the Tcra V2 subfamily differ from each other by 3%–7% at the nucleotide level and 5%–16% at the amino acid level. T-cell receptor Tcra V2 gene segment polymorphisms have been identified in the B10.PL and PL/J mouse strains with a Tcra V2 subfamily-specific probe. These V gene segment polymorphisms may cause the differential Tcra V gene usage in induced experimental allergic encephalomyelitis between B10.PL and PL/J mice.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide database and have been assigned the accession numbers U04312 and U04622-U04626     

An anti-sulfatide antibody O4 immunoprecipitates sulfatide rafts including Fyn, Lyn and the G protein α subunit in rat primary immature oligodendrocytes

The association of sulfatide with specific proteins in oligodendrocytes was examined by co-immunoprecipitation with an anti-sulfatide antibody. Protein kinase activity was detected in precipitates with a monoclonal antibody to sulfatide (O4) from the rat primary immature oligodendrocytes. We conducted in vitro kinase assay of tyrosine phosphorylated proteins of 80, 59, 56, 53 and 40 kDa by gel electrophoresis. Of these proteins, the proteins of 59 kDa and 53/56 kDa were identified as the Src family tyrosine kinases Fyn and Lyn on the basis of their sequential immunoprecipitation with anti-Fyn and anti-Lyn antibodies, respectively. The 40 kDa protein was identified as the α subunit of the heterotrimeric G protein. These observations suggest that O4 immunoprecipitates sulfatide rafts including Fyn, Lyn and the α subunit of the heterotrimeric G protein.     

A simple and rapid immunochromatographic test strip for detection of pathogenic isolates of Vibrio harveyi

Mouse monoclonal antibodies (MAbs) and rabbit polyclonal antibody (PAb) against Vibrio harveyi were generated from immunization of mice and rabbits with highly virulent isolate of V. harveyi. Two MAbs specific to virulent isolates of V. harveyi were obtained and one of them (VH4) was selected to conjugate with colloidal gold as the detector antibody was laid on a sample pad. Rabbit polyclonal antibody was used as the capture antibody at the test line (T) and goat anti-mouse IgG antibody (GAM) was used as the capture antibody at the control line (C) of nitrocellulose strip. The ready-to-use strip was held in a plastic case and then stored in a desiccated plastic bag. A sample volume of 100 μl of bacterial suspension from various sources mixed with application buffer was applied to the sample chamber at one end of the strip and allowed to flow by chromatography through the nitrocellulose membrane to the other end. In test samples containing virulent isolates of V. harveyi, the bacteria would bind to the monoclonal antibody conjugated with colloidal gold and the resulting complex would be captured by the antibodies at the test line to give a reddish-purple band. Any unbound monoclonal antibody conjugated with colloidal gold moved across the test line would be captured by the GAM and form a band at the control line (C). In sample without V. harveyi or with V. harveyi below the limit (< 10⁶ CFU/ml) of detection for the kit, only the control line band was observed. If the test sample was pre-enriched in tryptic soy broth (TSB) for 6 h before application to the strip, the sensitivity would increase to 1–10 CFU/ml which is comparable to that of PCR. This method could be used to detect pathogenic isolates of V. harveyi in pond water or infected shrimp in order to monitor and to reduce the risk of V. harveyi outbreak in the shrimp culture. The beneficial features of this kit are that simple, convenient and quick results (within 15 min) can be obtained without the requirement of sophisticated tools or special equipments and skills.     

Monoclonal antibodies with specificities for Streptococcus pneumoniae group 9 capsular polysaccharides

Streptococcus pneumoniae group 9 includes four capsular polysaccharide types: 9A, 9L, 9N and 9V. We have generated four mouse monoclonal antibodies against group 9 polysaccharide using heat-treated S. pneumoniae strains of different capsular polysaccharides types as immunogens. The specificities of the monoclonal antibodies were determined by ELISA using capsular polysaccharide directly coated to the wells as antigens and by dot blotting with heat-treated bacteria. Two groups of monoclonal antibodies were found. The first group included two monoclonal antibodies which were found to be capsular type specific. The second group was monoclonal antibodies that bound to epitopes shared by two or three pneumococcal group 9 types. The monoclonal antibody 204,A-4 (IgM) was found to be specific for S. pneumoniae type 9N. The binding of the type 9V specific monoclonal antibody 206,F-5 (IgG1) was found to be dependent upon O-acetyl groups. Monoclonal antibody 205,F-3 (IgM) reacted also with type 9V, but was found to cross-react with types 9A and 9L. The binding of this monoclonal antibody to polysaccharide 9V was not dependent upon O-acetyl moieties. The fourth monoclonal antibody (214,G-5, isotype IgM) did not show any correlation between reactivity with isolated polysaccharides and dot blotting with relevant bacteria. The monoclonal antibody reacted with polysaccharides 9A and 9L in ELISA, but not with the homologous bacteria.     

Both VH and VL regions contribute to the antigenicity of anti-idiotypic antibody that mimics melanoma associated ganglioside GM3

Previously we developed a murine monoclonal anti-idiotype (antiid) antibody (4C10) that mimics the melanoma-associated ganglioside antigen GM₃, that is, it carries the internal image of GM₃. 4C10 was made against the human monoclonal antibody (HuMAb) L612, which reacts with several types of human cancer cells, including melanoma and breast cancer. To reduce mouse components of 4C10, the constant region was replaced by a human constant domain to form the murine/human chimeric anti-id antibody TVE-1. In the present study, we sought to determine which chain (V_H or V_L) of the anti-id is responsible for the antigenicity of GM₃. The TVE-1 V_H and V_L expression vectors were simultaneously transfected with either the V_H or V_L expression vector of a murine-human chimeric IgG antidansyl haptenic antibody, resulting in the construction of three different combinations of V_H and V_L chimeric antibodies. These IgG molecules were produced from the transfectomas, and their reactivity to HuMAb L612 was tested. Neither of the IgG proteins that had cross-combined the V_H-V_L pair showed positive results, suggesting that both heavy and light chains are required to express the antigenicity. The in vivo antigenicity of this chimeric anti-id was confirmed by skin tests in melanoma patients receiving active specific immuntherapy.     

Tissue distribution of P2X<Subscript>4</Subscript> receptors studied with an ectodomain antibody

A monoclonal antibody was developed to the extracellular domain of the rat P2X₄ receptor. The antibody was highly selective among all rat P2X receptor subunits, and recognised only the oligomeric, non-denatured form of the P2X₄ receptor. Immunohistochemistry showed an extensive pattern of distribution throughout the central and peripheral nervous systems, the epithelia of ducted glands and airways, smooth muscle of bladder, gastrointestinal tract, uterus, and arteries, uterine endometrium and fat cells. The protein was identified by Western blotting in membrane extracts of these tissues, and the ectodomain antibody immunoprecipitated a protein that was recognised with a P2X₄ receptor C terminus antibody. The findings indicate that the P2X₄ receptor subunit has a very extensive distribution among mammalian tissues, and this suggests possible new functional roles.The work was supported by the Wellcome Trust (R.S., R.A.N.) and the British Heart Foundation (G.B.)     

Localization and characterization of a specific linear epitope of the Brucella DnaK protein

We have previously produced and characterized four monoclonal antibodies to the Brucella DnaK protein which were derived from mice infected with B. melitensis or immunized with the B. melitensis cell wall fraction. By use of a recombinant DNA technique, we have localized a linear epitope, recognized by two of these monoclonal antibodies (V78/07B01/G11 and V78/09D04/D08), in the last 21 amino acids of the C-terminal region of the Brucella DnaK protein. The C-terminal region has been reported to be the most variable region among DnaK proteins. The two other monoclonal antibodies (A53/09G03/D02 and A53/01C10/A10) failed to react with the recombinant clones and might recognize discontinuous epitopes of the Brucella DnaK protein. The four monoclonal antibodies reacted with all recognized Brucella species and biovars in immunoblotting after SDS-PAGE. Monoclonal antibodies V78/07B01/G11 and V78/09D04/D08 did not react with reported cross-reacting bacteria nor with bacteria of the α-2 subdivision of the class Proteobacteria for which a close genetic relationship with Brucella spp. has been reported. However, monoclonal antibodies A53/09G03/D02 and A53/01C10/A10 reacted with Phyllobacterium rubiacearum and/or Ochrobactrum anthropi, both bacteria of the α-2 subdivision of the class Proteobacteria. The Brucella genus DnaK specific epitopes could be of importance for diagnostic purposes.     

Maintenance of Sex-Related Genes and the Co-Occurrence of Both Mating Types in Verticillium dahliae

Verticillium dahliae is a cosmopolitan, soilborne fungus that causes a significant wilt disease on a wide variety of plant hosts including economically important crops, ornamentals, and timber species. Clonal expansion through asexual reproduction plays a vital role in recurring plant epidemics caused by this pathogen. The recent discovery of recombination between clonal lineages and preliminary investigations of the meiotic gene inventory of V. dahliae suggest that cryptic sex appears to be rare in this species. Here we expanded on previous findings on the sexual nature of V. dahliae. Only 1% of isolates in a global collection of 1120 phytopathogenic V. dahliae isolates contained the MAT1-1 idiomorph, whereas 99% contained MAT1-2. Nine unique multilocus microsatellite types comprised isolates of both mating types, eight of which were collected from the same substrate at the same time. Orthologs of 88 previously characterized sex-related genes from fungal model systems in the Ascoymycota were identified in the genome of V. dahliae, out of 93 genes investigated. Results of RT-PCR experiments using both mating types revealed that 10 arbitrarily chosen sex-related genes, including MAT1-1-1 and MAT1-2-1, were constitutively expressed in V. dahliae cultures grown under laboratory conditions. Ratios of non-synonymous (amino-acid altering) to synonymous (silent) substitutions in V. dahliae MAT1-1-1 and MAT1-2-1 sequences were indistinguishable from the ratios observed in the MAT genes of sexual fungi in the Pezizomycotina. Patterns consistent with strong purifying selection were also observed in 18 other arbitrarily chosen V. dahliae sex-related genes, relative to the patterns in orthologs from fungi with known sexual stages. This study builds upon recent findings from other laboratories and mounts further evidence for an ancestral or cryptic sexual stage in V. dahliae.     

An Inhibitory Antibody Blocks Interactions between Components of the Malarial Invasion Machinery

Host cell invasion by apicomplexan pathogens such as the malaria parasite Plasmodium spp. and Toxoplasma gondii involves discharge of proteins from secretory organelles called micronemes and rhoptries. In Toxoplasma a protein complex comprising the microneme apical membrane antigen 1 (AMA1), two rhoptry neck proteins, and a protein called Ts4705, localises to the moving junction, a region of close apposition between parasite and host cell during invasion. Antibodies against AMA1 prevent invasion and are protective in vivo, and so AMA1 is of widespread interest as a malaria vaccine candidate. Here we report that the AMA1 complex identified in Toxoplasma is conserved in Plasmodium falciparum. We demonstrate that the invasion-inhibitory monoclonal antibody (mAb) 4G2, which recognises P. falciparum AMA1 (PfAMA1), cannot bind when PfAMA1 is in a complex with its partner proteins. We further show that a single completely conserved PfAMA1 residue, Tyr251, lying within a conserved hydrophobic groove adjacent to the mAb 4G2 epitope, is required for complex formation. We propose that mAb 4G2 inhibits invasion by preventing PfAMA1 from interacting with other components of the invasion complex. Our findings should aid the rational design of subunit malaria vaccines based on PfAMA1.     

Reduced Na+ Affinity Increases Turnover of Salmonella enterica Serovar Typhimurium MelB

The melibiose permease of Salmonella enterica serovar Typhimurium (MelB_St) catalyzes symport of melibiose with Na⁺, Li⁺, or H⁺. Bioinformatics and mutational analyses indicate that a conserved Gly117 (helix IV) is a component of the Na⁺-binding site. In this study, Gly117 was mutated to Ser, Asn, or Cys. All three mutations increase the maximum rate (V_max) for melibiose transport in Escherichia coli DW2 and greatly decrease Na⁺ affinity, indicating that intracellular release of Na⁺ is facilitated. Rapid melibiose transport, particularly by the G117N mutant, triggers osmotic lysis in the lag phase of growth. The findings support the previous conclusion that Gly117 plays an important role in cation binding and translocation. Furthermore, a spontaneous second-site mutation (P148L between loop_4-5 and helix V) in the G117C mutant prevents cell lysis. This mutation significantly decreases V_max with little effect on cosubstrate binding in G117C, G117S, and G117N mutants. Thus, the P148L mutation specifically inhibits transport velocity and thereby blocks the lethal effect of elevated melibiose transport in the Gly117 mutants.     

Detection of G-quadruplex DNA in mammalian cells

It has been proposed that guanine-rich DNA forms four-stranded structures in vivo called G-quadruplexes or G4 DNA. G4 DNA has been implicated in several biological processes, but tools to study G4 DNA structures in cells are limited. Here we report the development of novel murine monoclonal antibodies specific for different G4 DNA structures. We show that one of these antibodies designated 1H6 exhibits strong nuclear staining in most human and murine cells. Staining intensity increased on treatment of cells with agents that stabilize G4 DNA and, strikingly, cells deficient in FANCJ, a G4 DNA-specific helicase, showed stronger nuclear staining than controls. Our data strongly support the existence of G4 DNA structures in mammalian cells and indicate that the abundance of such structures is increased in the absence of FANCJ. We conclude that monoclonal antibody 1H6 is a valuable tool for further studies on the role of G4 DNA in cell and molecular biology.     

Stepwise Engineering of Heterodimeric Single Domain Camelid VHH Antibodies That Passively Protect Mice from Ricin Toxin

In an effort to engineer countermeasures for the category B toxin ricin, we produced and characterized a collection of epitopic tagged, heavy chain-only antibody V_H domains (V_HHs) specific for the ricin enzymatic (RTA) and binding (RTB) subunits. Among the 20 unique ricin-specific V_HHs we identified, six had toxin-neutralizing activity: five specific for RTA and one specific for RTB. Three neutralizing RTA-specific V_HHs were each linked via a short peptide spacer to the sole neutralizing anti-RTB V_HH to create V_HH “heterodimers.” As compared with equimolar concentrations of their respective monovalent monomers, all three V_HH heterodimers had higher affinities for ricin and, in the case of heterodimer D10/B7, a 6-fold increase in in vitro toxin-neutralizing activity. When passively administered to mice at a 4:1 heterodimer:toxin ratio, D10/B7 conferred 100% survival in response to a 10 × LD₅₀ ricin challenge, whereas a 2:1 heterodimer:toxin ratio conferred 20% survival. However, complete survival was achievable when the low dose of D10/B7 was combined with an IgG1 anti-epitopic tag monoclonal antibody, possibly because decorating the toxin with up to four IgGs promoted serum clearance. The two additional ricin-specific heterodimers, when tested in vivo, provided equal or greater passive protection than D10/B7, thereby warranting further investigation of all three heterodimers as possible therapeutics.     

Characterization of the Molecular Mechanism Underlying Gibberellin Perception Complex Formation in Rice

The DELLA protein SLENDER RICE1 (SLR1) is a repressor of gibberellin (GA) signaling in rice (Oryza sativa), and most of the GA-associated responses are induced upon SLR1 degradation. It is assumed that interaction between GIBBERELLIN INSENSITIVE DWARF1 (GID1) and the N-terminal DELLA/TVHYNP motif of SLR1 triggers F-box protein GID2-mediated SLR1 degradation. We identified a semidominant dwarf mutant, Slr1-d4, which contains a mutation in the region encoding the C-terminal GRAS domain of SLR1 (SLR1^G576V). The GA-dependent degradation of SLR1^G576V was reduced in Slr1-d4, and compared with SLR1, SLR1^G576V showed reduced interaction with GID1 and almost none with GID2 when tested in yeast cells. Surface plasmon resonance of GID1-SLR1 and GID1-SLR1^G576V interactions revealed that the GRAS domain of SLR1 functions to stabilize the GID1-SLR1 interaction by reducing its dissociation rate and that the G576V substitution in SLR1 diminishes this stability. These results suggest that the stable interaction of GID1-SLR1 through the GRAS domain is essential for the recognition of SLR1 by GID2. We propose that when the DELLA/TVHYNP motif of SLR1 binds with GID1, it enables the GRAS domain of SLR1 to interact with GID1 and that the stable GID1-SLR1 complex is efficiently recognized by GID2.     

Specific identification of tissue kallikrein in exocrine tissues and in cell-free translation products with monoclonal antibodies.

A panel of six mouse monoclonal antibodies (IgG1) has been prepared against purified rat urinary kallikrein (EC 3.4.21.35) and characterized. In radioimmunoassay, the antibody titres of ascitic fluid giving 50% binding to 125I-kallikrein range from 1:2 X 10(3) to 1:1 X 10(6). Antibodies from four of the clones show no cross-reactivity with human urinary kallikrein, rat urinary esterase A or tonin. However, antibodies from a fifth clone cross-react with tonin and, from a sixth, with both urinary esterase A and tonin. Three of the kallikrein affinity-purified monoclonal antibodies inhibited, whereas one of the antibodies stimulated, kallikrein activity. Tissue kallikrein from rat submandibular-gland and pancreatic extracts and urine were labelled with [14C]di-isopropyl phosphofluoridate, immunoprecipitated with each of the six monoclonal antibodies and identified to be 38 kDa proteins, similar in size to purified rat urinary kallikrein. Western-blot analysis shows that 125I-labelled kallikrein monoclonal antibodies (V4D11) bind directly to a 38 kDa protein in submandibular-gland and pancreatic extracts and urine. Cell-free translation products of submandibular-gland polyadenylylated[poly(A)+]mRNA were immunoprecipitated with affinity-purified sheep anti-kallikrein antibodies and three monoclonal antibodies (V4D11, V4G6 and V1C3). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these immunoprecipitates revealed that two kallikrein precursors with Mr values of 37 000 and 35 000 are encoded by submandibular-gland mRNA. The third monoclonal antibody, V1C3, which binds to active kallikrein, did not recognize either precursor form. Collectively, the data show that these monoclonal antibodies comprise a set of powerful and specific reagents for studies of tissue kallikreins.     

Human Vκ immunoglobulin gene number: Implications for the origin of antibody diversity

To assess the relative contributions of germline versus somatically mutated genes in the human immune system, we have examined the size of the kappa light-chain variable region (V_κ) gene pool. Two cloned kappa subgroup 1 (V_κ1) gene probes detected the same family of 15 to 20 crosshybridizing restriction fragments in human DNA, whereas flanking region probes detected fewer hybridizing fragments. Most of the hybridizing bands represent single-copy genes, as judged by a “gene titration” experiment. Furthermore, the number of hybridization bands is a good estimate of the haploid gene number, since we observed little polymorphism of restriction sites in the V_κ locus of eight unrelated people. A cloned V_κ3 probe hybridized to essentially the same 15–20 genes in human DNA as the V_κ1 probes. These results strongly suggest that a discrete family of 15–20 genes constitutes a large proportion of the V genes from three of the four V_κ subgroups. The small number of V_κ genes in the human genome supports the idea that somatic mutation plays a major role in the origin of antibody diversity in man.     

Atrial fibrillation-associated Connexin40 mutants make hemichannels and synergistically form gap junction channels with novel properties

Mutations of Cx40 (GJA5) have been identified in people with lone chronic atrial fibrillation including G38D and M163V which were found in the same patient. We used dual whole cell patch clamp procedures to examine the transjunctional voltage (V_j) gating and channel conductance properties of these two rare mutants. Each mutant exhibited slight alterations of V_j gating properties and increased the gap junction channel conductance (γ_j) by 20–30 pS. While co-expression of the two mutations had similar effects on V_j gating, it synergistically increased γ_j by 50%. Unlike WTCx40 or M163V, G38D induced activity of a dominant 271 pS hemichannel.     

Molecular cloning of the 31 kDa cytosolic phospholipase A2, as an antigen recognized by the lung cancer-specific human monoclonal antibody, AE6F4

The human monoclonal antibody AE6F4 specifically reacts with human lung cancer tissues but does not with normal tissues. This monoclonal antibody recognizes a cytosolic 31 kDa antigen in the cancer cells. In a previous study, we elucidated that the 31 kDa antigen belonged to a family of proteins collectively designated as 14-3-3 proteins, which were known as protein kinase-dependent activators of tyrosine/trytophan hydroxylases, or protein kinase C inhibitor proteins. Here we report molecular cloning of the 31 kDa antigen from the human lung adenocarcinoma cell line, A549. Sequencing analysis indicates that the cloned cDNA is identical to that of previously reported human placental cytosolic phospholipase A₂ (cPLA₂), which is also a member of the 14-3-3 protein family. Western analysis demonstrated that a 31 kDa recombinant cPLA₂ expressed in monkey COS cells was recognized by the AE6F4 monoclonal antibody. Binding of the monoclonal antibody to the recombinant cPLA₂ was abolished when treated with sodium periodate, suggesting that not only are carbohydrate chains associated with the cPLA₂, but they also play a crucial role in antigen recognition by the monoclonal antibody.     

Coevolution of Antibody Stability and Vκ CDR-L3 Canonical Structure

Antibodies recognize antigens through six hypervariable loops, five of which have a limited set of conformations known as canonical structures. For κ light chains, the majority of CDR-L3 [the third hypervariable loop of the light chain variable domain (V_L)] adopts the type 1 canonical structure (CS1), with a cis-proline at position 95. Here, we present the design and structural studies of the monoclonal antibody mAb15 and related mutants that contained a series of progressively germline mutations only in the heavy chain variable domain (V_H) that ultimately led to an increase of more than 11 °C in the melting temperature (T_m) of the antigen-binding fragment (Fab). The all-trans CDR-L3 structure in the wild type is significantly different from any known CDR-L3 canonical structures. In the thermally stable mutants, the L94^L-S95^L peptide bond adopts an energetically unfavorable non-X-proline cis conformation, but the overall CDR-L3 loop converted to CS1. The stabilized V_H appears to function as a specific molecular chaperone that facilitated the trans-cis isomerization of S95^L. Thus, it is plausible that proline is the evolutionary choice to maintain overall structure and stability for V_L. These results provide new insights into the evolution of CS1 and suggest a potential molecular switch mechanism at position 95 that links CDR-L3 structural diversity and antibody stability and will have implications for antibody engineering.     

Vitex negundo Modulates Selenite-Induced Opacification and Cataractogensis in Rat Pups

Recently, much interest has been generated in the search for phytochemical therapeutics, as they are largely free from adverse side effects and economical. The goal of this study was to determine the efficacy of Vitex negundo in modulating the selenite-induced oxidative stress in vivo model. Sprague–Dawley rat pups of 8 days old were used for the study and divided into control (G I), selenite induced (G II), and selenite + V. negundo treated (G III). Cataract was induced by the single subcutaneous injection of sodium selenite (4 mg/kg body weight) on the tenth day and V. negundo (2.5 mg/Kg body weight) administered intraperitoneally from eighth to 15th day. Morphological examination of the rat lenses revealed no opacification in G I and mild opacification in G III whereas dense opacification in G II (stages 4–6). Levels of selenium in G II and G III showed no significant changes. The activities of superoxide dismutase, catalase, and Ca²⁺ATPase were significantly increased in G III compared to G II (p?<?0.05), while lower level of reactive oxygen species, Ca²⁺, and thiobarbituric acid reactive substances were observed in G III compared G II (p?<?0.05). These results indicate the therapeutic potential of methanolic extract of V. negundo on modulating biochemical parameters against selenite-induced cataract, which have been reported in this paper for the first time.     

A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses

Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (V_H) and light (V_L) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (V_L-V_H) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli.