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Screening a scFv Antibody Against Vibrio Parahaemolyticus TLH and Study on Its’ Function

Author: WangRongZhi
Tutor: LinWenXiong; WangShiHua
School: Fujian Agriculture and Forestry University
Course: Biochemistry and Molecular Biology
Keywords: Vibrio parahaemolyticus Thermolabile Hermolysin scFv Co-expression Function
CLC: Q939.91
Type: PhD thesis
Year: 2012
Downloads: 80
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Abstract


Vibrio parahaemolyticus is a halophilic bacterium that is widely distributed inwater resources. The bacterium causes lethal food-borne diseases and poses a seriousthreat to human and animal health all over the world. The major pathogenic factor ofV. parahaemolyticus is thermolabile hemolysin (TLH) encoded by the tlh gene and itstoxicity mechanisms is unknown. A high-affinity antibody that can neutralize TLHactivity effectively is not available. In this study, we successfully expressed andpurified a TLH antigen, and screened a high-affinity antibody to TLH namedscFv-LA3by phage display. Cytotoxicity analysis showed that scFv-LA3has strongneutralization effects on TLH-induced cell toxicity. The main contents and resultswere as follows:The V. parahaemolyticus genome was used as a template to amplify the tlh gene,and four different recombinant expression vectors were constructed. Proteinpurification was performed using Ni2+affinity chromatography. Expressed andpurified proteins were visualized by SDS-PAGE using12%(v/v) polyacrylamide gels,and protein concentration was determined by the BCA protein assay. Hela, Changliver,and RAW264.7cells were used to evaluate the cytotoxicity of TLH with the typicalMTT and FACS. These results showed that all three types of cells exhibited signs ofsevere cytotoxicity, and effects were dose and time-dependent. The IC50for Helacells, Changliver cells, and RAW264.7macrophages was4.25,4.75and3.99μg/mL,respectively. To generate an antibody specific to TLH, four Balb/c female mice wereimmunized with TLH protein purified from pET32a (+)-tlh expression. Mouse serumtiters were determined by ELISA. Two of the immunized mice had a similar anti-TLHtiter of1:16000, indicating that a high anti-TLH titer serum was obtained byimmunization.First-strand cDNA was synthesized by RT-PCR with random hexadeoxyribonucleotides primers using the isolated mRNA as a template. The variable regions ofthe heavy chain (VH) and light chain (VL) were then amplified from first-strand cDNA.To construct a scFv fragment, a special93bp DNA encoding a (Gly4Ser)3protein sequence was designed as a linker connecting the VHto VLfragments and was alsoamplified by PCR. The size of the final recombinant phage-displayed antibody librarywas approximately3×1010CFU/mL. After six rounds of panning, four scFv clonesshowing strong binding ability to the TLH antigen were isolated from the library. Theclone scFv-LA3with the highest binding activity was selected for further analysis.These results derived from ELISA and Western blotting indicated that scFv-LA3wasspecific to TLH; there was no cross binding to any other antigen-associated proteinssuch as TDH and YscF.To evaluate the neutralization ability of the selected scFv-LA3clone for TLH,Hela, Changliver and RAW264.7cells were treated with TLH for MTT or FACSanalysis using standard methods with minor modifications. MTT assays demonstratedthat the cytotoxic effects of TLH on cells could be neutralized by scFv-LA3, whilecontrol BSA did not show any protective effects. FACS results also showed that thescFv-LA3antibody inhibited the cytotoxicity of TLH antigen, and enhanced theviability of cells.Four different expression vectors were constructed, and their ability to expressthe soluble scFv protein were also test. The solubility and binding activity of thepurified protein were analyzed by SDS-PAGE and ELISA, respectively. Among offour purified protein, the Skp co-expression protein showed the highest solubility, andthe binding activity to antigen TLH was3-4folded higher than other three purifiedproteins. Besides, the scFv of Skp co-expression is specific for TLH and does notcross-react with other TLH-associated proteins. Notably, scFv of Skp co-expressioncan be used to directly detect TLH, suggesting that the pACYC-Duet-Skp vectorsystem is an effective expression system for soluble expression of scFv.

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