Screening for a Single-Chain Variable-Fragment Antibody That CanEffectively Neutralize the Cytotoxicity of the Vibrio parahaemolyticusThermolabile Hemolysin
Rongzhi Wang,a Sui Fang,a Dinglong Wu,a Junwei Lian,a Jue Fan,a Yanfeng Zhang,b Shihua Wang,a and Wenxiong Lina
The Ministry of Education Key Laboratory of Biopesticide and Chemical Biology and the College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou,China,a and Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USAb
Vibrio parahaemolyticus is a halophilic bacterium that is widely distributed in water resources. The bacterium causes lethalfood-borne diseases and poses a serious threat to human and animal health all over the world. The major pathogenic factor of V.parahaemolyticus is thermolabile hemolysin (TLH), encoded by the tlh gene, but its toxicity mechanisms are unknown. A high-affinity antibody that can neutralize TLH activity effectively is not available. In this study, we successfully expressed and purifiedthe TLH antigen and discovered a high-affinity antibody to TLH, named scFv-LA3, by phage display screening. Cytotoxicity anal-ysis showed that scFv-LA3 has strong neutralization effects on TLH-induced cell toxicity.
Vibrio parahaemolyticus, a Gram-negative motile bacteriumthat inhabits marine and estuarine environments throughout
the world (7, 23, 36), is a major food-borne pathogen that causeslife-threatening diseases in humans through the consumption ofraw or undercooked seafood (5, 21, 34). V. parahaemolyticus in-fection can also occur through open wounds. The U.S. Centers forDisease Control and Prevention (CDC) reported cases of deathresulting from wound infections by V. parahaemolyticus in 2005(15). Given the harmful effects and prevalence of V. parahaemo-lyticus, it is very important to investigate its pathogenesis.
It is well known that V. parahaemolyticus contains many dif-ferent kinds of toxins, such as thermostable direct hemolysin(TDH), TDH-related hemolysin (TRH), and some noncharacter-ized proteins. TDH is considered one of the major virulence fac-tors of V. parahaemolyticus, and its function has been well charac-terized and discussed (25). TRH is another hemolysin of V.parahaemolyticus that can also lyse red blood cells, and it has highsequence homology with TDH (24, 42). However, identifying thepathogenic serovars of V. parahaemolyticus by use of only thesetwo toxins is not sufficiently accurate, as other hemolysins maytake part in the pathogenicity of V. parahaemolyticus. Thermo-labile hemolysin (TLH), a toxin encoded by the tlh gene of V.parahaemolyticus and present in almost every clinical and envi-ronmental V. parahaemolyticus strain (14, 37), has been suggestedas a promising target for pathogen detection (30, 35, 44). Al-though TLH has hemolytic activity and can lyse red blood cells, itscytotoxic and biochemical mechanisms of action are still notclearly understood (3, 26, 31). Since TLH may be as important asTDH and TRH (6), it is necessary to investigate its function duringthe process of infection.
Single-chain variable-fragment (scFv) antibody generation is aversatile technology for generating antibodies that are specific fora given antigen (40). It has also been used for selective moleculartargeting in cancer research for conditions such as lymphatic in-vasion vessels, colon cancer, and hepatocarcinoma (27, 29, 33,43). Furthermore, scFv antibody generation has been used exten-sively to generate ligands for detecting pathogenic germs in vitroand in vivo (8, 22, 38, 39). Compared to polyclonal antibodies orhybridoma technology, scFv antibodies can easily be manipulated
genetically to improve their specificity and affinity, reducing pro-duction costs. In addition, they can be fused with molecular mark-ers for immunological detection of pathogenic bacteria (10, 28).scFv antibody generation has also been used extensively in vitroand in animal models to generate ligands and to detect pathogenicgerms (8, 22, 38, 39). It has the power to mimic the features ofimmune diversity and selection, and it is possible to synthesizescFv antibodies in virtually unlimited quantities. Combining scFvantibody generation with selection panning strategies provides auseful tool that allows the selection of antibodies against specificantigens. Using this tool, we have been able to characterize thebinding properties of single-chain antibodies and to investigatetheir potential use as diagnostic tools or therapeutic agents(12, 13).
In the present study, we successfully used phage display screen-ing technology to discover an scFv antibody, named scFv-LA3,that can inhibit the cytotoxicity of TLH. The results demonstratethat phage display technology is a feasible method for generating aspecific and high-affinity antibody that could protect againstpathogen infection.
MATERIALS AND METHODSMaterials. V. parahaemolyticus strain XM01 (tlh� tdh�) was generouslyprovided by Xuanxian Peng (Xiamen University, China). V. parahaemo-lyticus strains CGMCC 1.1615 and CGMCC 1.1616 were purchased fromthe Institute of Microbiology, Chinese Academy of Sciences (Beijing,China). Other strains were stored in our lab. HeLa, Changliver, andRAW264.7 cells were purchased from the cell bank of the Chinese Acad-emy of Sciences (Shanghai, China). BALB/c mice were purchased fromthe Shanghai Laboratory Animal Center, and all animal work was per-formed according to relevant national and international guidelines. All
animal experiments were approved by the Animal Ethics Committee ofthe Fujian Agriculture and Forestry University. DNA restriction enzymes,mRNA isolation kits, and reverse transcription kits were purchased fromPromega. Taq DNA polymerase and T4 DNA ligase were purchased fromTaKaRa (Dalian, China). Horseradish peroxidase (HRP)-labeled goatanti-mouse IgG was purchased from Boster Biological Technology Co.(Wuhan, China). Ampicillins, kanamycin sulfate, bovine serum albumin(BSA), and isopropyl-�-D-thiogalactopyranoside (IPTG) were purchasedfrom Sigma Chemical Co. All oligonucleotides listed in Table 1 and allother reagents used were of analytical reagent grade.
Expression and purification of recombinant TLH antigen. The V.parahaemolyticus genome was used as a template to amplify the tlh gene.Primers with BamHI and HindIII restriction enzyme sites were designedfor cloning the tlh gene into the pET32a(�) and pET28a(�) vectors, andthe identity of the cloned tlh amplicon was verified by sequencing. Forprotein expression, the recombinant plasmid was transformed into Esch-erichia coli BL21 by electroporation, and a single colony from the selectionplate was inoculated into 5 ml LB liquid medium containing 100 �g/mlampicillin or 50 �g/ml kanamycin. The culture was incubated overnightwith shaking at 37°C and then transferred to a larger scale in LB medium(500 �l of culture was transferred to 50 ml of fresh LB). Expression of thetarget protein was induced by adding 1 mM IPTG when the culturereached an optical density at 600 nm (OD600) of 0.8. Cells were grown foran additional 6 h at 28°C and then harvested by centrifugation. Proteinpurification was performed using Ni2� affinity chromatography. Ex-pressed and purified proteins were visualized by SDS-PAGE using 12%(vol/vol) polyacrylamide gels, and protein concentration was determinedby the bicinchoninic acid (BCA) protein assay.
Cell culture and cytotoxicity analysis. HeLa, Changliver, andRAW264.7 cells were used to evaluate the cytotoxicity of TLH with typicalMTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bro-mide] and fluorescence-activated cell sorter (FACS) methods (1, 41).Briefly, cells were cultured in RPMI 1640 medium supplemented with10% fetal bovine serum (FBS) and penicillin-streptomycin in 96-wellplates (triplicate wells) and were adjusted to a final concentration of 106
cells/ml. The purified recombinant TLH protein was sterilized by filtra-tion with a 0.22-�m filter, and TLH proteins of different concentrations(100 �l/well) were added to the wells. BSA was used as a negative control.Plates were incubated at 37°C and 5% CO2 for 24 h. MTT solution wasadded to the mixture at a 1:10 ratio (by volume) and incubated at 37°Cand 5% CO2 for a further 4 h. Viable cells were evaluated by measuring theconversion of soluble MTT to insoluble blue formazan crystals (41). ForFACS analysis, cells were incubated as described above, and test cells werestained with fluorescein isothiocyanate (FITC)-conjugated annexin andpropidium iodide (PI) (1).
Immunoassay. Purified TLH was used to immunize four BALB/cmice (100 �g/mouse) four times, at intervals of 20 days, and serum titerswere measured by indirect enzyme-linked immunosorbent assay (ELISA).Blood from both control and immunized mice was sampled 7 days afterthe third immunization. Once high serum titers were obtained, animalswere sacrificed 5 days after the last immunization, and spleens were col-lected for immunological assays. Total mRNA was extracted from isolatedspleens by use of TRIzol (Promega Biotech).
Construction of a phage-displayed anti-TLH scFv antibody library.First-strand cDNA was synthesized by reverse transcription-PCR (RT-PCR) with reverse transcriptase and random hexadeoxyribonucleotideprimers, using the isolated mRNA as a template. The coding sequences forthe variable regions of the heavy chain (VH) and light chain (VL) were thenamplified from first-strand cDNA through primary PCR amplification.To construct an scFv antibody fragment, a special 93-bp DNA encoding a(Gly4Ser)3 protein sequence was designed as a linker connecting the VH
and VL fragments and was also amplified by PCR. The assembly reactionwas performed by gene splicing by overlap extension-PCR (SOE-PCR) ata molecular ratio of VH to VL to linker DNA of approximately 3:3:1.Another round of PCR was performed to add SfiI and NotI restrictionsites to either end of the scFv antibody fragment for subsequent cloninginto the phage plasmid vector pCANTAB-5. The product was then ligatedwith a precut vector and transformed into E. coli TG1 competent cells byelectroporation. To count colonies, 100 �l of diluted transformed cells oruntransformed negative-control cells was plated onto SOB-AG plates(SOB medium containing 100 �g/ml ampicillin and 2% glucose) andincubated at 30°C overnight. The helper phage M13KO7 was used torescue the recombinant phagemid. All clones were transferred to a sepa-rate tube for phage rescue, and recombinant phage scFv antibody cloneswere obtained by collecting the supernatants by centrifugation (10,000 �g, 20 min, 4°C).
Panning of the phage display library. A 96-well microtiter plate wascoated with diluted antigen (2.5 �g/ml) in phosphate-buffered saline(PBS) (100 �l/well) and incubated at 4°C overnight. The plate was thenwashed three times with PBS and blocked with PBSM (PBS containing 4%nonfat milk). An uncoated (containing only PBS), PBSM-blocked wellwas set as a control at the same time. To enhance panning efficiency,recombinant phage was precipitated from solution by use of polyethyleneglycol (PEG)-NaCl to remove any soluble antibodies that might react withthe TLH antigen. The recombinant phage was then diluted and added to apretreated plate (100 �l/well). After incubation at 37°C for 2 h, the platewas washed 20 times with PBS and then 20 times with PBST (PBS containing0.05% Tween 20) to remove unbound phage. Phage which reacted with TLHwas eluted with 10 ml triethylamine followed by 10 ml of Tris-HCl (pH 7.4) toneutralize the reaction. Eluted phage was used to infect log-phase E. coli TG1
TABLE 1 Primer sequences for amplification of the VH, VL, scFv, and tlh genes
cells, and 10 �l of infected E. coli cells was plated at a 10� serial dilution ontoSOB-AG plates for screening of individual colonies.
Screening of clones with binding specificity from enriched clones.Enriched clones were individually transformed and cultured in a tubewith M13KO7 for rescue. To test the specific binding activity by phageELISA, a 96-well plate was coated with purified TLH antigen at 5 �g/ml inPBS (100 �l/well), followed by blocking with PBSM and subsequentwashing. Phage dissolved in PBSM was added to the plate and incubatedfor 2 h at 37°C. After washing with PBST and PBS, the bound phage wasdetected with an anti-M13 antibody conjugated to HRP at a 1:4,000 dilu-
tion. 3,3=,5,5=-Tetramethylbenzidine (TMB) (100 �g/ml) was then usedfor color development, and the reaction was stopped after 30 min byaddition of 2 M H2SO4. Absorbance at 450 nm was measured with amicroplate reader. Binding activity was evaluated by the ratio of values forthe sample and the negative control (S/N ratio). An S/N ratio of 2 was usedas the cutoff value to indicate positive clones. Specificity analysis of se-lected anti-TLH clones, such as scFv-LA3, was also performed as de-scribed above.
Analysis of anti-TLH scFv-LA3 specificity. Phage ELISA was per-formed to determine the specificity of the anti-TLH scFv-LA3 clone. BSA
FIG 1 Expression of recombinant TLH in E. coli. SDS-PAGE analysis was performed with recombinant TLH expressed by plasmids pET32a(�) (A) andpET28a(�) (B). Lanes: M, protein molecular mass markers; 1, negative control (empty vector); 2, total cell lysate; 3, TLH purified using a Ni2�-nitrilotriaceticacid (Ni2�-NTA) column.
FIG 2 TLH induces cytotoxicity in cultured cells. (A) HeLa cells (a and b), Changliver cells (c and d), and RAW264.7 cells (e and f) were treated with or without20 �g/ml TLH. Cell morphology was observed under a microscope and photographed after 24 h. Magnification, �100. (B) Dose-response analysis of TLH-mediated cytotoxicity. HeLa, Changliver, and RAW264.7 cells were exposed to different concentrations of TLH for 24 h, and cell viability was determined usingthe MTT assay. Data are presented as means � standard deviations (SD) for three separate experiments. (C) Time course of TLH-mediated cytotoxicity. HeLa,Changliver, and RAW264.7 cells were incubated with 20 �g/ml of TLH for the indicated times. Cell growth was determined using the MTT assay. (D) Serum titerassay. ELISA was used to determine the serum titer after four immunizations. The serum titer was evaluated by the S/N ratio (signal �1.0); the cutoff value fora positive clone was an S/N ratio of �2.
and associated V. parahaemolyticus antigens (TDH, TLH, and YscF) wereused to coat 96-well plates in triplicate (5 �g/ml; 100 �l/well). After block-ing with PBSM, secreted recombinant phage particles were added to thereaction wells and incubated at 37°C for 2 h. The specificity of the scFv-LA3 clone was detected with the anti-M13 HRP-conjugated antibody. Theenzyme reaction was then performed with TMB as a substrate, and colordevelopment was terminated with 2 M H2SO4 for 30 min. Absorbance at450 nm was measured using a microplate reader.
Bioinformatics analysis of the scFv gene. Homology of the sequenceof the scFv gene with known murine genes from the GenBank/EMBL andV-Quest databases was determined using BLAST. The scFv-LA3 clonesequence was analyzed using V-Quest IMGT (International ImMuno-GeneTics Information System [http://www.imgt.org]) to identify thegerm line origin of the VH and VL regions (32).
Soluble expression and affinity determination. To express the anti-TLH antibody, log-phase HB2151 cells were infected with a positive scFv-LA3 clone and grown in 2� YT-AG medium (YT medium containing 100�g/ml ampicillin and 2% glucose) at 37°C overnight. The culture was thendiluted at a ratio of 1:100 in 100 ml fresh 2� YT medium containing 100�g/ml ampicillin and grown at 37°C until the OD600 reached 0.8. IPTG (1mM) was added to induce the expression of the scFv clone, and the culturewas incubated at 30°C with shaking at 200 rpm for 6 h before centrifuga-tion at 2,000 � g for 20 min. To extract the expressed soluble antibody, cellculture pellets were resuspended in 10 ml ice-cold PPB buffer (0.2 MTris-HCl, pH 8.0; 0.5 mM EDTA; 0.5 M sucrose) and incubated on ice for30 min, followed by centrifugation at 12,000 rpm for 20 min (4°C). Thesupernatant was carefully discarded, and the pellet was resuspended in 2ml MgSO4 (5 mM) and incubated at 25°C for 10 min. Cell debris was
pelleted as described above, and the supernatant was analyzed by ELISAand SDS-PAGE for the presence of soluble antibody. The affinity constant(Kaff) of the scFv clone against TLH was determined using the equationKaff � (n � 1)/(n[Ab2] � [Ab1]) as described previously (11), where[Ab1] and [Ab2] represent the respective scFv antibody concentrationsrequired to achieve 50% of the maximum absorbance between two differ-ent concentrations of coated antigen ([Ag1] � n[Ag2]) and n is the dilu-tion factor between the concentrations of antigen used.
Western blotting. To further confirm the interactions between scFv-LA3 and the TLH antigen, Western blotting was performed as describedby Singh et al. (32), with minor modifications. Briefly, the purified TLHantigen or the total protein of V. parahaemolyticus or another associatedbacterium was transferred from an SDS-PAGE gel onto a polyvinylidenedifluoride (PVDF) membrane, and the membrane was treated with solu-ble scFv antibody by use of a standard protocol. After washing and block-ing, the membrane was subsequently incubated with HRP-conjugatedanti-E-tag antibody. Signals were visualized by enhanced chemilumines-cence (ECL).
Neutralization of TLH cytotoxicity by scFv antibody. To evaluatethe neutralization ability of the selected scFv-LA3 clone for TLH,HeLa, Changliver, and RAW264.7 cells were treated with TLH forMTT or FACS analysis using standard methods, with minor modifica-tions. Briefly, cells were cultured as described above. TLH (10 �g/ml or5 �g/ml) and a dilution series of the scFv-LA3 antibody (1010 CFU/ml;100 ml/well) were added to cells, and the mixture was incubated at37°C for 24 h in a humidified atmosphere with 5% CO2. Cells werestained with FITC-conjugated annexin and PI for FACS analysis. Cell
FIG 3 Construction of an anti-TLH scFv antibody library. (A) PCR analysis of VH and VL. Lane 1, VH gene (�340 bp); lane 2, VL gene (�325 bp); lane M,DL-2000 DNA marker. (B) Amplified fragment of the scFv gene. Lane M, DL-2000 DNA marker; lanes 1 and 2, amplified scFv gene (�750 bp). (C) Selection ofphage scFv antibody library by panning. In each round of panning, the number of input phage was kept constant, at 3.0 � 1010 CFU/ml, and phage that did notbind TLH was removed by washing. The number of phage was counted after each panning round. After three panning rounds, the number of eluted phage waskept constant at 3.0 � 106 CFU/ml. (D) ELISA analysis of the binding activities of four different anti-TLH scFv antibody clones. The graph shows the relationshipbetween OD450 values and TLH concentrations.
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viability was determined using the MTT method according to themanufacturer’s instructions.
RESULTSExpression and purification of recombinant TLH antigen. Weused two different expression vectors, pET32a(�) and pET28a(�),to obtain a high yield of TLH. The pET32a(�) vector expressed the Nterminus of TLH fused to thioredoxin (TRX) in order to enhance thesolubility of expressed TLH. pET28a(�) contained a 6�His tag forprotein purification purposes. Protein expression and purificationresults showed that TLH was highly expressed (Fig. 1). The fusionprotein encoded by the pET32a(�) vector was used as an immuno-gen to immunize BALB/c mice for titer detection, while TLH derivedfrom the pET28a(�) vector was used for cytotoxicity and ELISAanalyses.
Induction of cytotoxicity and immunization of mice withTLH. HeLa cells, Changliver cells, and RAW 264.7 macrophageswere treated with different concentrations of TLH (0.1 to 20 �g/
ml) purified after expression with the pET28a(�) vector. Cellswere observed under a microscope after 24 h to evaluate morpho-logical changes, edge atrophy, and cell viability. As shown inFig. 2A, all three types of cells exhibited signs of severe cytotoxic-ity, such as round cells, edge atrophy, and cell death, when treatedwith 2 �g/ml of TLH, and effects were dose and time dependent(Fig. 2B and C). The 50% inhibitory concentrations (IC50s) forHeLa cells, Changliver cells, and RAW 264.7 macrophages were4.25 �g/ml, 4.75 �g/ml, and 3.99 �g/ml, respectively.
To generate an antibody specific to TLH, four female BALB/cmice were immunized with the TLH protein purified frompET32a(�) expression. Mouse serum titers were determined byELISA (Fig. 2D). Two of the immunized mice had the same anti-TLH titer (1:16,000), indicating that a high-titer anti-TLH anti-body was obtained by immunization.
Phage library construction and screening of specific scFvs.The VH and VL gene fragments encoding the anti-TLH antibody
FIG 4 Molecular characterization of scFv-LA3 clone. (A) Nucleotide and amino acid sequences of the scFv-LA3 fragment. Complementarity-determiningregions are underlined. (B and C) IMGT collier de perle graphical 2-dimensional representations of the VH (B) and VL (C) regions of scFv-LA3. Asterisks indicatedifferences between scFv-LA3 and mouse germ line genes. Hydrophobic amino acids (those with positive hydropathy index values, i.e., I, V, L, F, C, M, and A)and tryptophan (W) are shown with gray circles. All proline (P) residues are also shown in gray circles. The CDR IMGT sequences are delimited by amino acidsshown in squares (anchor positions), which belong to the neighboring framework region (FR-IMGT). Dotted circles correspond to missing positions accordingto the IMGT unique numbering. Residues at positions 23, 41, 89, 104, and 118 are conserved.
FIG 5 Characterization of scFv-LA3. (A) SDS-PAGE analysis of expressed scFv-LA3. Lane M, protein marker; lane 1, negative control (empty vector); lanes 2 and3, expression product of scFv-LA3. (B) Western blot analysis of the binding activity of scFv-LA3 toward the TLH antigen. (Left) SDS-PAGE results forNi2�-NTA-purified TLH. Lane M, protein molecular mass markers; lanes 1 and 2, purified TLH. (Right) Western blotting results. Lanes 3 and 4, TLH band at47 kDa bound by scFv-LA3. The bound anti-TLH scFv-LA3 clone was detected using an anti-E-tag HRP-conjugated antibody. (C) scFv-LA3 specifically bindsthe TLH antigen. *, P 0.05. BSA and associated antigen proteins (TDH, TLH, and YscF) of Vibrio parahaemolyticus were used to coat 96-well plates in triplicate(5 �g/ml; 100 �l/well). Secreted recombinant phage particles were added to the reaction wells and incubated for 2 h at 37°C. Specificity of the scFv-LA3 clone wasdetermined using an anti-M13 HRP-conjugated antibody. (D) Western blotting. The total proteins isolated from tlh-positive and tlh-negative bacterial strainswere transferred to a PVDF membrane, and bound anti-TLH scFv-LA3 was detected using an anti-E-tag HRP-conjugated antibody. (E) Binding of scFv-LA3 todifferent concentrations of TLH antigen as determined by ELISA. The four TLH concentrations were 5 �g/ml (A), 2.5 �g/ml (B), 1.25 �g/ml (C), and 0.625�g/ml (D).
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generated from immunized mice were amplified by RT-PCR afterisolation of total mRNA from TLH-immunized mouse spleencells. The 340-bp and 325-bp products were joined by adding a(Gly4Ser)3 linker peptide (�750 bp in total) (Fig. 3B). An initialphage-displayed scFv antibody library with a size of 3 � 1010
CFU/ml was constructed. The input and output of the library areshown in Fig. 3C. About 3 � 1010 CFU/ml phage clones were usedin each panning round, and after the third round, the eluted phageclones were kept at a level of approximately 1 � 106 CFU/ml. Toestimate the size of the final library containing potential positiveclones more accurately, 54 clones from the eluted phage were se-lected randomly and identified by PCR and restriction enzymedigestion. Since more than 90% of the recombination clones werepositive, we estimated the size of the final recombinant phage-displayed antibody library to be approximately 3 � 106 CFU/ml(Fig. 3C). After six rounds of panning, four scFv antibody clonesshowing strong binding to the TLH antigen were isolated from thelibrary. The clone with the highest binding activity, scFv-LA3, wasselected for further analysis (Fig. 3D).
Bioinformatics and biochemical characterization of scFv-
LA3. The scFv-LA3 clone is 735 nucleotides long and encodes 245amino acids, including a flexible (Gly4Ser)3 linker between VH andVL (Fig. 4A). The three complementarity-determining regions(CDRs) of VH and VL were determined according to the method ofKabat et al. (19). Sequence alignment with other mouse germ linegenes by use of all available bioinformatics sources (GenBank,RefSeq Nucleotides, EMBL, DDBJ, and PDB databases) con-firmed that the VH and VL sequences of scFv-LA3 are mouse an-tibody variable region genes. Figure 4B and C show IMGT “collierde perle” (pearl necklace) graphical two-dimensional (2-D) rep-resentations of scFv-LA3. We expressed the scFv-LA3 antibody insoluble form in infected E. coli HB2151 cells to further character-ize its biochemical properties. scFv-LA3 was expressed as an �30-kDa protein (Fig. 5A), and its affinity constant for TLH (deter-mined by ELISA) was 5.39 � 108 M�1 (Fig. 5E). scFv-LA3 wasfound to be specific to TLH; there was no cross binding to anyother antigen-associated proteins such as TDH or YscF (Fig. 5Band C). In addition, the Western blotting results derived fromFig. 5D show that the scFv-LA3 antibody is active against Vibriocells possessing TLH. These results indicate that scFv-LA3 rec-
FIG 6 Neutralizing activity of scFv-LA3. (A) The neutralizing activity of scFv-LA3 for TLH-induced cell toxicity is dose dependent. HeLa, Changliver, andRAW264.7 cells were cultured in triplicate in 96-well flat-bottom plates and treated with 10 �g/ml of TLH, with or without various dilutions of phage scFv-LA3.Cell growth was evaluated using the MTT assay. (B) Time course of scFv-LA3 neutralization. HeLa, Changliver, and RAW264.7 cells were cultured in triplicatein 96-well flat-bottom plates and then treated with 10 �g/ml of TLH and the same concentration of phage scFv-LA3 (1010 CFU/ml) for different lengths of time(at intervals of 3 h) for up to 27 h. Cell growth was evaluated using the MTT assay. Data are presented as means � SD for three independent experiments. (C)FACS analysis. Changliver and RAW264.7 cells were cultured in triplicate in 6-well flat-bottom plates and treated with 5 �g/ml of TLH and the sameconcentration of phage scFv-LA3 (1010 CFU/ml). Cells were incubated for 24 h and stained with FITC-conjugated annexin and propidium iodide.
ognizes TLH specifically and can be used as an antibody reagentto detect TLH.
scFv-LA3 inhibits TLH-induced cytotoxicity. As shownabove, TLH was lethal to several types of cells, and �80% to 90%of these cells died when exposed to 10 �g/ml TLH. To test itspotential neutralizing effects, scFv-LA3 was added to cells togetherwith TLH. As shown in Fig. 6A and B, scFv-LA3 protected cellsfrom TLH-induced cytotoxicity, in a dose- and time-dependentmanner. At the highest dose (�106 CFU/ml), MTT assays dem-onstrated that the cytotoxic effects of TLH on cells could be neu-tralized by scFv-LA3, while the BSA control did not show anyprotective effects. FACS results also showed that the scFv-LA3antibody inhibited the cytotoxicity of the TLH antigen and en-hanced the viability of cells (Fig. 6C). Taken together, our resultsdemonstrate that scFv-LA3 strongly counteracts TLH-inducedcytotoxicity and is a promising candidate for antibody therapyagainst V. parahaemolyticus infection.
DISCUSSION
Recombinant phage antibodies generated by phage display tech-nology have been used widely to produce powerful reagents fortherapeutic and diagnostic purposes (16, 20). Compared to tradi-tional methods that produce antibodies from hybridoma cells,phage display technology exhibits many advantages. First, phagedisplay solves several problems associated with using hybridomacells, such as a lack of specificity, low antibody titers (32), the needfor large-scale culture, and instability of cell lines. Second, it isefficient and has the power to mimic the features of immune di-versity and selection (2, 17). Third, phage-displayed antibodiescan be manipulated genetically, and the specificity and affinity ofeach antibody for specific antigens can be enhanced greatly andimproved by mutating and rearranging antibody coding genes (4,9). Finally, the phage ELISA detection method has made phagedisplays one of the most remarkable technologies for detection ofpathogenic bacteria (18, 32, 40).
The assembly of the scFv antibody gene was an important stepfor scFv antibody library construction. To effectively improve thesituation, the molecular ratio of VH, VL, and linker DNAs wasadjusted to 3:3:1 to reduce the formation of nonspecific DNAbands and to increase the productivity of scFv antibodies. After along period of exploration, we finally amplified a better scFv bandby PCR. In addition, the linker DNA, containing 93 bp encodingthe amino acid linker (Gly4Ser)3, provided sufficient space andallowed suitable flexibility for the VH and VL subunits to interact.It is possible that flexible linkers such as (Gly4Ser)3 improve thefolding between the VH and VL subunits and enhance the affinityof scFv antibodies.
In the process of biopanning, we selected four single clones thatshowed strong binding effects on the TLH antigen, but the scFv-LA3 clone had the highest binding activity. This outcome wasattributed to the difference in scFv CDR sequence and the aminoacid variation of the antibody scaffold. In addition, in the samecase, the fusion expression with PIII tends to interfere with theproper folding of the scFv antibody, and this may also be an im-portant factor affecting the binding activity of scFv antibodies.However, the detailed mechanism of this variation in binding ac-tivity needs to be investigated further.
In conclusion, we used phage display technology as a tool toscreen for a specific antibody, and the ELISA results show thatscFv-LA3 has high binding activity toward TLH. In addition, scFv-
LA3 inhibits TLH-induced cytotoxicity and has a protective effectin multiple types of TLH-infected cells. The above results demon-strate that phage display technology is a useful tool for selecting anantibody against a specific antigen. In addition, this tool can beeffective at generating therapeutic agents in defense of disease pro-voked by a pathogen.
ACKNOWLEDGMENTS
We thank Xuanxian Peng (Xiamen University, China) for the gift of V.parahaemolyticus strains and Shuhong Luo (University of Illinois at Ur-bana-Champaign) for helpful discussions and suggestions. We thank Li-jun Bi and Fleming Joy (Institute of Biophysics, Chinese Academy ofSciences) for actively revising and editing the manuscript. We thank BingWang (Dalian Fisheries University, China) for technical discussions andthe gift of Vibrio spp.
This work was supported by the National Natural Science Founda-tion of China (grants 30700535 and 31172297), the Program for NewCentury Excellent Talents in University (grant NCET-10-0010), theFujian Fund for Distinguished Young Scientists (grant 2009J06008),the National Agricultural Achievements Transformation Fund (grant2011GB2C400012), and the Fok Ying Tong Education Foundation(grant 111032).
Rongzhi Wang, Shihua Wang, and Wenxiong Lin conceived and de-signed the experiments; Rongzhi Wang, Sui Fang, Dinglong Wu, andJunwei Lian performed the experiments; Rongzhi Wang, Jue Fan, andShihua Wang analyzed the data; and Rongzhi Wang, Yanfeng Zhang,and Shihua Wang wrote the paper.
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