Preparation and characterization of recombinant protein ScFv(CD11c)-TRP2 for tumor therapy from inclusion bodies in Escherichia coli

Kou, Geng; Wang, Hao; Tan, Min; Xue, Jingya; Zhang, Dapeng; Hou, Sheng; Qian, Weizhu; Wang, Shuhui; Dai, Jing; Li, Bohua; Guo, Yajun

doi:10.1016/j.pep.2006.08.007

Cited by 18 publications

(13 citation statements)

References 21 publications

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“…Recently, Castro et al showed that in vivo targeting antigens to DCs via CD11c resulted in efficient antigen processing and presentation on MHC classes I and II products, as well as robust CD4 + and CD8 + T-cell immunity (32). In addition, our previous study showed a recombinant fusion protein consisting of mouse tyrosine-related protein 2 (TRP2) peptide and CD11c-specific single-chain fragment variable (scFv CD11c ) could strongly stimulate TRP2 peptidespecific T-cell activation and proliferation in vitro (33). Hence, CD11c might be an ideal candidate for targeting antigens to DCs to induce comprehensive antitumoral immunity.…”

Section: Dendritic Cells (Dc) Are Key Initiators and Modulators Ofmentioning

confidence: 99%

Targeted Delivery of Tumor Antigens to Activated Dendritic Cells via CD11c Molecules Induces Potent Antitumor Immunity in Mice

Wei

Wang

Zhang

et al. 2009

Clinical Cancer Research

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Purpose: CD11c is an antigen receptor predominantly expressed on dendritic cells (DC), to which antigen targeting has been shown to induce robust antigen-specific immune responses. To facilitate targeted delivery of tumor antigens to DCs, we generated fusion proteins consisting of the extracellular domain of human HER or its rat homologue neu, fused to the single-chain fragment variable specific for CD11c (scFv Dendritic cells (DC) are key initiators and modulators ofadaptive T-cell responses due to their outstanding ability to capture and process antigen, to present antigen-derived peptides in the context of MHC molecules to naive T cells, and to deliver appropriate costimulatory signals that dictate either immunogenic or tolerogenic T-cell stimulation (1, 2).These unique features of DCs to regulate adaptive immunity suggest that significant clinical benefits could be gained from directly targeting antigens to DCs in vivo (3, 4). Coupling of antigens to ligands or antibodies that specifically bind to DC receptors has been widely investigated as a means of such targeting (3 -5). The outcome of the immune response induced by targeting antigens to DCs depends on multiple parameters, including the specific receptor targeting, distribution of the targeted receptor among DC subsets, and the presence or absence of coadministered adjuvant (6 -16). Using such an approach, a lowered requirement for antigen dose in stimulating immune responses in mice has been observed after targeting a variety of molecules, including MHC class II, CD11c, DEC205, DCIR2, Dectin-1/2, CD80/86, F4/80-like receptor, CIRE, mannose receptor, and CD36 (7 -16). In addition, in vitro and in vivo studies have shown that antibodies specific for the mannose receptor or DC-SIGN can effectively deliver antigen to human DCs, indicating that this strategy will also be applicable to human vaccination (17,18).Overexpression of the HER-2 receptor tyrosine kinase has been found in various human malignancies, including breast, ovarian and gastric carcinomas, non -small cell lung cancer, and salivary gland cancers,

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Section: Dendritic Cells (Dc) Are Key Initiators and Modulators Ofmentioning

confidence: 99%

Targeted Delivery of Tumor Antigens to Activated Dendritic Cells via CD11c Molecules Induces Potent Antitumor Immunity in Mice

Wei

Wang

Zhang

et al. 2009

Clinical Cancer Research

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“…After separation via SDS-PAGE, the proteins were transferred to an Immuno-Blot PVDF membrane (Bio-Rad, Hercules, CA, USA, 0.2 μm) to identify the purified anti-triazophos scFv-8C10 antibody via immunoblotting, as described in a published method [ 42 ]. Furthermore, the band of interest protein was sliced from the SDS gel and verified commercially with ProtTech via LC–ESI–MS/MS.…”

Section: Methodsmentioning

confidence: 99%

Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain

Liang

Xiang

et al. 2016

IJMS

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Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv) antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb) against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL) to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10−10 M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples.

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“…Inclusion bodies were purified as described elsewhere (9). Briefly, pellets containing inclusion bodies were washed three times with the washing buffer (50 mM Tris-HCl, pH 8.0, 10 mM Triton X-100, 2 M urea and 10 mM EDTA) and resuspended in inclusion body solubilization buffer (50 mM Tris-HCl, pH 8.0, 8 M urea, 0.5 M NaCl and 5 mM imidazole) by stirring for 30 min at room temperature.…”

Section: Expression and Purification Of The Recombinant Hhmgb4mentioning

confidence: 99%

Generation and characterization of a polyclonal antibody against human high mobility group box 4

Yang

Hong

et al. 2013

Molecular Medicine Reports

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A human high mobility group box 4 (hHMGB4) expression construct (pET‑28a/hHMGB4) was generated by cloning the hHMGB4 full‑length cDNA in the expression vector pET‑28a(+). The hHMGB4 fusion protein with His6‑Tag was prepared using E.coli BL21 (DE3) transformed with pET‑28a/hHMGB4 and purified via preparative SDS‑PAGE plus electroelution. Immunization of rabbits with the purified hHMGB4 generated polyclonal antibodies. The titer of the antiserum was determined to be 1:102,400 by ELISA analysis. Western blotting analysis showed that the antibody specifically recognized the recombinant hHMGB4 protein and also the endogenous hHMGB4 protein in prostate cancer cells. In addition, immunohistochemical staining analysis using the prepared antibody revealed marked hHMGB4 staining in the nuclei of the human prostate tissue. These data demonstrate that the anti‑hHMGB4 polyclonal antibody may be a useful reagent for the functional study of hHMGB4.

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Preparation and characterization of recombinant protein ScFv(CD11c)-TRP2 for tumor therapy from inclusion bodies in Escherichia coli

Cited by 18 publications

References 21 publications

Targeted Delivery of Tumor Antigens to Activated Dendritic Cells via CD11c Molecules Induces Potent Antitumor Immunity in Mice

Targeted Delivery of Tumor Antigens to Activated Dendritic Cells via CD11c Molecules Induces Potent Antitumor Immunity in Mice

Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain

Generation and characterization of a polyclonal antibody against human high mobility group box 4

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