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Generation and Characterization of Recombinant Single Chain Fv Antibody That Recognizes Platelet Glycoprotein Iba

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Generation and Characterization of Recombinant Single Chain Fv Antibody That Recognizes Platelet Glycoprotein Iba http://www.paper.edu.cn Thrombosis Research 109 (2003) 137–144 Regular Article Generation and characterization of recombinant single chain Fv antibody that recognizes platelet glycoprotein Iba Kesheng Dai, Huaiping Zhu, Changgeng Ruan* Thrombosis and Hemostasis Research Unit, Jiangsu Institute of Hematology, The First Affiliated Hospital of Suzhou University, Suzhou 215006, China Received 2 June 2002; received in revised form 7 January 2003; accepted 3 February 2003 Abstract A recombinant single chain Fv (scFv) fragment with specific activity against platelet glycoprotein (GP) Iba was developed and characterized. The scFv was generated from the SZ-2 hybridoma, which produced an anti-platelet antibody reactive to GPIba. VH and VL gene segments were generated from the SZ-2 hybridoma by reverse transcribed-polymerase chain reaction (RT-PCR). After cloning into pUCm-T vector, the DNA sequences of both VH and VL genes were analyzed from two different clones, respectively, the same results were obtained. Comparison of SZ-2 variable region to the Kabat database showed that VH belonged to the mouse Ig heavy family XV while VL belonged to the mouse Ig kappa family XXVI. For assembly of the SZ-2 scFv, VH and VL fragments were cloned into pSW1-scFv successively. The scFv was arranged in VH–VL orientation, being joined together with a 15-amino-acid (Gly4Ser)3 linker. The scFv encoding sequence was amplified and cloned into pET22b vector in-frame with a pel B leader sequence to direct secretion of the protein. Escherichia coli strain BL-21(DE3)PlysS was transformed with the recombinant plasmid, and expression of the scFv was induced using isopropyl-h-D-thiogalactopyranoside (IPTG). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the recombinant antibody revealed a protein with apparent molecular weight of approximately 31,000. By comparing band intensity on a Coomassie brilliant blue-stained SDS-PAGE, the production yield of SZ-2 scFv was about 25% of the total cellular proteins. The recombinant SZ-2 scFv antibody was successfully purified using Ni-NTA affinity chromatography with a yield of 120 mg/l. The SZ-2 scFv antibody could bind to platelets demonstrated by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Analyzed by Western blot, it could bind to platelet GPIb. It retained the binding capacity of its parental SZ-2 monoclonal antibody (MoAb). In functional studies, SZ-2 scFv inhibited platelet agglutination and aggregation induced by ristocetin and thrombin, respectively, but had no effect on ADP- induced platelet aggregation. Therefore, SZ-2 scFv has the potential to be used as an antithrombotic agent. D 2003 Elsevier Science Ltd. All rights reserved. Keywords: Platelets; Glycoprotein Ib; Single chain Fv; von Willebrand factor; Thrombin; Thrombosis; Monoclonal antibody Thrombosis is an acute form of cardiovascular disease site for the vWF A1 domain [1,2]. GPIb, a major component wherein sudden aggregation of blood-borne platelets of the platelet membrane, consists of disulfide-linked poly- occludes the arterial blood supply, leading to tissue infarc- peptide chains (a and h), associated with GPIX and GPV to tion. Platelet binding to von Willebrand factor (vWF) at site form a non-covalent, oligomeric complex. GPIb has dual of vascular injury provides a mechanism for the arrest of functions: first, GPIb is a receptor of vWF that is essential for bleeding and also contributes to the occlusion of diseased mediating the initial attachment of platelets to the vessel wall vessels in pathologic states. The process is mediated by the at the sites of injury; second, GPIb plays a role in thrombin- glycoprotein (GP) Ib–IX–V complex in which the amino- induced platelet activation, although this role is poorly terminal domain of the GPIba-chain contains the binding understood [3]. The interaction of GPIb with vWF allows the initial tethering and rolling of platelets before their firm Abbreviations: scFv, single chain Fv; ELISA, enzyme-linked immuno- adhesion and activation [4]. If no other bonds are formed, sorbent assay; GPIb-IX, glycoprotein Ib-IX; MoAb, monoclonal antibody; tethered platelets translocate in the direction of flow, albeit at vWF, von Willebrand factor; RT-PCR, reverse transcribed-polymerase a markedly lower velocity than freely flowing blood cells chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel [1]. On reactive substrates, however, binding of vWF to electrophoresis; IPTG, isopropyl-h-D-thiogalactopyranoside. * Corresponding author. Tel.: +86-512-5101708; fax: +86-512- GPIb–IX–V transduces signals across the plasma mem- 5101708. brane, then activates the platelets. During this process, 2+ E-mail address: [email protected] (C. Ruan). agonists such as ADP are secreted, Cytosolic Ca is 0049-3848/03/$ - see front matter D 2003 Elsevier Science Ltd. All rights reserved. 转载 doi:10.1016/S0049-3848(03)00152-X 中国科技论文在线 http://www.paper.edu.cn 138 K. Dai et al. / Thrombosis Research 109 (2003) 137–144 elevated and the Ca2+-dependent integrin GPIIb–IIIa is (RT-PCR). Briefly, total RNA was extracted using TRIzol activated. These reactions mediate platelet aggregation reagent from 1 Â 107 SZ-2 hybridoma cells secreting MoAb through the binding of GPIIb–IIIa to fibrinogen and result against GPIb. Single-strain cDNA was synthesized from 5.0 in subsequently thrombus formation [4,5]. Therefore, an Ag of total RNAwith 100 pmol random primer using 10 units inhibitor of thrombin-platelet and vWF-platelet interaction of M-MLV reverse transcriptase in a final volume of 30 Al. may be useful in the prevention of thrombotic diseases. The reaction was incubated at 42 jC for 1 h. VH and VL A murine monoclonal antibody (MoAb) SZ-2 against were amplified by polymerase chain reaction (PCR) using GPIba was previously produced and characterized in our the following primers (restriction sites underlined) [11]:VH laboratory [6]. The antibody not only inhibited the ristocetin- Back, 5V-AGGTC(G)C(A)A(A)GCTGCAGGAGTCTGG-3V dependent binding of vWF to platelets and ristocetin-induced incorporates a PstI site; VH For: 5V-TGAGGAGACGGT- platelet agglutination but also inhibited platelet aggregation GACCGTGGTCCCTTGGCCCCAG-3V incorporates a induced by thrombin [6,7]. The epitope for SZ-2 lies between BstEII site; VL Back: 5V-GACATTGAGCTCACCCAGTC- residues Tyr-276 and Glu-282 of GPIba [7]. To investigate TCCA-3V incorporates a SacIsite;VLFor:5V-GTTA- the potential of engineering SZ-2 to avoid the immunores- GATCTCGAGCTTGGTCCC-3Vincorporates a XhoIsite. ponse of the human body to mouse IgG in clinical trials, a PCR amplification was performed using a reaction mix recombinant single chain Fv (scFv) antibody was generated containing 1 Al of cDNA reaction mix, 10 pmol each primer, and characterized. In this paper, we report the construction of 200mMdNTPsand2unitsPfuDNApolymerasein SZ-2 scFv by the joining of variable domains of light and 10 Â PCR reaction buffer in a final volume of 50 Al. The heavy chain gene segments derived from SZ-2 hybridoma PCR consisted of an initial reaction for 5 min at 95 jC, 30 with a 15-amino-acid (Gly4Ser)3 linker. This scFv was cycles for 30 s at 55 jC, 1 min at 72 jC and 30 s at 95 jC, produced successfully in Escherichia coli. The molecular and final extension for 7 min at 72 jC. The products were nature and binding properties of this scFv were characterized. separated by 1.5% agarose gel and recovered using the Qiaex II gel extraction kit. The SZ-2 VH and VL fragments were cloned into pUCm-T vector. Sequencing was done using the 1. Materials and methods dideoxy-chain-termination method. Comparison of DNA sequence to Kabat database was performed through the 1.1. Cells, vectors, bacterial strains, oligonucleotide internet at http://immuno.bme.nwu.edu. primers and reagents 1.3. Construction of SZ-2 scFv plasmid The hybridoma secreting the GPIb-specific monoclonal antibody SZ-2 was generated, as described previously [6], The amplified V genes were digested with the restriction and cells were grown on DMEM, 10% fetal calf serum with enzymes PstI and BstEII for the VH gene, SacI and XhoI for 5% CO2 at 37 jC. Plasmid pSW1-scFv were kindly provided the VL gene. Both digested fragments were electrophoresed by Dr. Greg Winter (MRC Lab., University of Cambridge, in a 1.5% agarose gel and purified. The VH gene fragment Cambridge, UK). Plasmid pET22b and E. coli strain BL- was ligated into a PstI and BstEII digested pSW-scFv vector 21(DE3)PlysS were obtained from Novagen (Novagen, (containing the (Gly4Ser)3 linker sequence). Then the VL Darmstadt, Germany). Qiaex II gel extraction kit, Ni-NTA gene fragment was ligated into the recombinant plasmid Resin and Penta-His antibody were purchased from QIA- containing the VH gene at the 3V-end of the (Gly4Ser)3 linker. GEN (Qiagen, Hilden, Germany). Trizol and moloney mur- The resulting pSW1-2 scFv was identified by restriction ine leukemia virus (M-MLV) reverse transcriptase were from fragment and DNA sequence analysis. The recombinant Gibco BRL (Rockville, MD, USA). Restriction or modifying scFv gene (SZ-2 scFv) was reamplified from the construct enzymes, plasmid pUCm-T, PCR kit and Pfu DNA polymer- pSW1-2 scFv using the following primers (restriction sites ase were purchased from Sangon (Unison Biotek, Canada). underlined): P1,5V-CCAGGTCGACCTGCAGGAGTCA- Oligonucleotide primers for PCR amplification were synthe- GG-3V incorporates a SalIsite;P2,5V-TATGCGGCCGC- sized on DNA synthesizer (Applied Bio-Systems). Horse- CTCGAGCTTGGTCC-3V incorporates a NotIsite.PCR radish peroxidase (HRP)-conjugated goat anti-mouse MoAb conditions were as follows: one cycle for 4 min at 95 jC, was obtained from Immunotech (Marseilles, France). Human 30 cycles for 1 min at 58 jC, 1 min at 72 jC and 1 min at a-thrombin was purchased from American Diagnostica 94 jC and final extension for 7 min at 72 jC.
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