OBJECTIVETo investigate which of the two immunoglobulin (Ig)-like domains, the immunoglobulin variable region homologous domain IgV (hB7.2 IgV) and the immunoglobulin constant region homologous domain IgC (hB7.2 IgC) on the human B7.2 molecule contains receptor binding sites, and to evaluate whether the B7.2 protein expressed in bacteria has biological activity in vitro.

METHODSThree fragments of hB7.2 IgV,hB7.2 IgC and the complete extracellular region of human B7.2 containing both the IgV and IgC domains,hB7.2 Ig (V+C), were amplified by PCR and subcloned into pGEM-Teasy. Three recombinants,pGEX-4T-3-hB7.2 IgV,pGEX-4T-3-hB7.2 IgC and pGEX-4T-3-hB7.2 Ig (V+C), were generated by cloning the fragments into a prokaryote expression plasmid (pGEX-4T-3) and transformed into the host strain E. coli DH5alpha. The relevant target fusion proteins consisting of GST and hB7.2 IgV,hB7.2 IgC and hB7.2 Ig (V+C), were identified by SDS-PAGE and Western blotting. With the presence of the first signal imitated by anti-CD3 antibody, T cell activation was observed by exposing purified T lymphocytes to each soluble form of the three bacterially-produced human B7.2 fusion proteins by [(3)H]-TdR incorporation.

RESULTSThree recombinant fusion proteins of human B7.2, GST-hB7.2 IgV, GST-hB7.2 IgC and GST-hB7.2 Ig (V+C) were produced and detected in inclusion body form from engineered bacteria. With the first signal present,T lymphocytes proliferated when co-stimulated by bacterially-produced either GST-hB7.2 Ig (V+C) or GST-hB7.2 IgV fusion proteins, but not by GST-hB7.2 IgC.

CONCLUSIONSFunctional human B7.2 fusion protein can be produced in bacteria. The IgV-like domain of human B7.2 is sufficient for B7.2 to interact with its counter-receptors and co-stimulate T lymphocytes.

Antigens, CD ; pharmacology ; B7-2 Antigen ; Escherichia coli ; genetics ; Humans ; Immunoglobulin Constant Regions ; pharmacology ; Immunoglobulin Variable Region ; pharmacology ; Lymphocyte Activation ; Membrane Glycoproteins ; pharmacology ; Plasmids ; Recombinant Fusion Proteins ; pharmacology ; T-Lymphocytes ; immunology

Macrolide and corticosteroid resistance has been reported in patients with Mycoplasma pneumoniae (MP) pneumonia (MPP). MP clearance is difficult to achieve through antibiotic treatment in sensitive patients with severe MPP (SMPP). SMPP in children might progress to airway remodeling and even bronchiolitis/bronchitis obliterans. Therefore, identifying serum biomarkers that indicate MPP progression and exploring new targeted drugs for SMPP treatment require urgency. In this study, serum samples were collected from patients with general MPP (GMPP) and SMPP to conduct proteomics profiling. The Fc fragment of the IgG-binding protein (FCGBP) was identified as the most promising indicator of SMPP. Biological enrichment analysis indicated uncontrolled inflammation in SMPP. ELISA results proved that the FCGBP level in patients with SMPP was substantially higher than that in patients with GMPP. Furthermore, the FCGBP levels showed a decreasing trend in patients with GMPP but the opposite trend in patients with SMPP during disease progression. Connectivity map analyses identified 25 possible targeted drugs for SMPP treatment. Among them, a mechanistic target of rapamycin kinase (mTOR) inhibitor, which is a macrolide compound and a cell proliferation inhibitor, was the most promising candidate for targeting SMPP. To our knowledge, this study was the first proteomics-based characterization of patients with SMPP and GMPP.
Biomarkers ; Carrier Proteins ; Child ; Humans ; Immunoglobulin Fc Fragments ; Immunoglobulin G ; Macrolides ; Mycoplasma pneumoniae ; Pneumonia, Mycoplasma/drug therapy* ; Proteomics

CD28 family consists of CD28, ICOS, CTLA-4 and PD-1 molecules. The former two are activation receptors and the later two are inhibition receptors. They produce co-stimulatory signals combining with the relevant molecules in B7 family, which plays important role in T cell activation and homeostasis among T subsets. Although the mechanism of signaling by CD28 and CTLA-4 has been well studied, many questions still remain to be answered. Further investigations are required for substantiating the dual-signaling model.
Animals ; Antigens, CD ; Antigens, Differentiation ; immunology ; CD28 Antigens ; immunology ; CTLA-4 Antigen ; Humans ; Immunoglobulin Fc Fragments ; immunology ; Signal Transduction

The DNA coding for the fusion protein of thromobopoietin mimetic peptide (TMP) and human IgG1 Fc fragment was amplified from recombinant plasmid pET28a/TMPFc, inserted into pPICZalphaA and transformed into Pichia pastoris using electroporation. The recombinants of correct phenotype were identified after screening on MDH and MMH culture medium. The fusion gene was verified with PCR and western blot. MTT method was used to test the activity of TMPFc in promoting the growth of Ba/ F3-mpl cell. The TMPFc with a 64 000 molecular weight was a secretary protein in the system and its expression amounted to 65% of the total protein in the medium supernatant. The TMPFc showed a promotive effect on the growth of Ba/F3-mpl in vitro. A significant portion of the secretary protein existed as dimer, which provided material for studying the dimer in future.
Amino Acid Sequence ; Blotting, Western ; Humans ; Immunoglobulin Fc Fragments ; genetics ; Immunoglobulin G ; genetics ; Molecular Sequence Data ; Pichia ; genetics ; Plasmids ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; biosynthesis ; Thrombopoietin ; genetics

OBJECTIVETo isolate single chain antibody fragments (scFv) against cell surface molecules by pathfinder selection from an anti-KG1a cell scFv phage library.

METHODSThe anti-KG1a scFv library was enriched by KGla cell panning for three rounds, or unenriched, then processed for pathfinder selection respectively using anti-CD34 monoclonal antibody as pathfinder molecule. ScFv phage clones were randomly picked and identified by binding KG1a cells using immunofluorescein and flow cytometry. The KG1a+ clones were further identified by KG1a, HL60, U937, and CEM cell lines and ELISA. Their antigenic molecules on cell surface were digested by chymopapain and analyzed by flow cytometry. DNAs from ten positive clones were sequenced. The scFv clones with different primary structure were used to analyze the molecular weight of their antigens by Western blot.

RESULTSOne hundred and two KG1a+ scFv phage clones were isolated from 144 enriched and 96 unenriched scFv phage library respectively, among which 47 bound KG1a, HL60, U937, and CEM cells, 55 bound KG1a cells exclusively. None of 28 KG1a+, HL60-, U937-, and CEM- scFv clones bound to the CD34 antigen, as confirmed by ELISA, although most of their antigens were sensitive to chymopapain digestion. DNA sequences from ten positive clones showed that they were from four different clones. They bound antigens with different molecular weight.

CONCLUSIONSOne hundred and two scFv phage clones specific for hematopoietic stem and progenitor cells have been isolated from an anti-KG1a cell scFv phage library. The pathfinder selection has showed advantages to improve the screening efficacy of scFv phage clones against antigens, which present at very low densities on the cell surface.

Antibodies, Anti-Idiotypic ; immunology ; Antibodies, Monoclonal ; genetics ; Antibody Specificity ; Bacteriophages ; genetics ; Cloning, Molecular ; Hematopoietic Stem Cells ; immunology ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; immunology ; Immunoglobulin Fragments ; genetics ; immunology ; Immunoglobulin Variable Region ; genetics ; immunology ; Peptide Library ; Single-Chain Antibodies

BACKGROUND: Anti-IgE mAb which binds circulating but not receptor-bound IgE has been shown to be effective in treatment for asthma and other allergic diseases. However, the mechanisms by which anti-IgE mAb influences the pathophysiological responses are remained to be illustrated. This study was undertaken to examine the therapeutic efficacy of non-anaphylactogenic anti-mouse IgE mAb using murine models of IgE-induced systemic fatal anaphylaxis. METHODS: Active systemic anaphylaxis was induced by either penicillin V (Pen V) or OVA and passive systemic anaphylaxis was induced by either anaphylactogenic anti-mouse IgE or a mixture of anti-chicken gamma globulin (CGG) IgG1 mAb and CGG. The binding of the Fc portion of anti-IgE to CHO-stable cell line expressing mouse FcgammaRIIb was examined using flow cytometry. Fc fragments of anti-IgE mAb were prepared using papain digestion. The expression of phosphatases in lungs were assessed by Western blotting and immunohistochemistry. RESULTS: Anti-IgE mAb prevented IgE- and IgG-induced active and passive systemic fatal reactions. In both types of anaphylaxis, anti-IgE mAb suppressed antigen-specific IgE responses, but not those of IgG. Anti-IgE mAb neither prevented anaphylaxis nor suppressed the IgE response in FcgammaRIIb-deficient mice. The Fc portion of anti-IgE mAb was bound to murine FcgammaRIIb gene-transfected CHO cells and inhibited systemic anaphylaxis. Anti-IgE mAb blocked the anaphylaxis-induced downregulation of FcgammaRIIb-associated phosphatases such as src homology 2 domain-containing inositol 5-phosphatase (SHIP) and phosphatase and tensin homologue deleted on chromosome ten (PTEN). CONCLUSION: Anti-IgE mAb prevented anaphylaxis by delivering nonspecific inhibitory signals through the inhibitory IgG receptor, FcgammaRIIb, rather than targeting IgE.
Anaphylaxis* ; Animals ; Asthma ; Blotting, Western ; Cell Line ; CHO Cells ; Cricetinae ; Digestion ; Down-Regulation ; Flow Cytometry ; gamma-Globulins ; Immunoglobulin E ; Immunoglobulin Fc Fragments ; Immunoglobulin G* ; Immunohistochemistry ; Inositol ; Lung ; Mice* ; Ovum ; Papain ; Penicillin V ; Phosphoric Monoester Hydrolases

Both interleukin-1 and IgE are important in the pathogenic mechanism of the allergy asthma. cDNA of interleukin-1receptor antagonist (IL-1ra) and IgE were cloned and a prokaryotic expression vector IL-1ra-Fcepsilon/pBV220 was constructed. The vector was transformed into Escherichia coli BL21(DE3). The fusion protein was expressed successfully in the form of inclusion body. The recombination protein of IL-1ra-Fcepsilon was highly purified by chromatography of gel filtration and ion exchange, which was identifited by Western blotting. The cell assay showed that the activity of IL-1ra-Fcepsilon was as high as IL-1ra in vitro after refolding. The pharmacokenetic profile of IL-1ra-Fcepsilon and L-1ra was analyzed, and the half time of IL-1ra-Fcepsilon is 4.78 times than that of IL-1ra.
Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Fusion ; Immunoglobulin E ; genetics ; metabolism ; Immunoglobulin Fc Fragments ; genetics ; metabolism ; Interleukin 1 Receptor Antagonist Protein ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism

ATR-Fc is a fusion protein consisting of extracellular domain of human anthrax toxin receptor (ATR) and a fragment (hinge, CH2, and CH3 domains) of the Fc of human IgG1. The aim of ATR-Fc expression is to get an antibody-like molecule binding to protective antigen (PA), a component of anthrax toxins, this fusion protein may compete with cell surface receptor for PA binding, and block the transport of lethal factor (LF) and edema factor (EF) into cells, thereby act as an antitoxin to prevent and treat anthrax infection. A DNA fragment encoding N-terminal amino acids 1-227 of ATR and human IgG1 Fc was inserted into the Hind III and Not I sites of pcDNA3.1 to generate the eukaryotic vector pcDNA3.1/ATR-Fc for expression of ATR-Fc fusion protein. Using lipofectine-mediated gene transfer technique, pcDNA3.1/ATR-Fc was transfected into CHO-K1 cells. After selected with G418, a recombinant CHO cell line, ATR-Fc-1D5, whose expression level was about 10 - 15 microg/(10(6) cells x d), was established. The recombinant protein expressed by the ATR-Fc-1D5 cells was purified with protein A chromatography. The experimental results demonstrated a direct and specific interaction between ATR-Fc and PA assessed by ELISA.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Gene Transfer Techniques ; Genetic Vectors ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; Immunoglobulin G ; biosynthesis ; genetics ; Neoplasm Proteins ; biosynthesis ; genetics ; Receptors, Cell Surface ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

To evaluate the efficiency of three in vitro refolding methods for a humanized single-chain Fv antibody against human CTLA4(CD152) expressed in E. coli, the denatured and purified inclusion bodies (IBS) were refolded by dilution, dialysis and in situ refolding via Immobilized Metal-Ion-Affinity Chromatography (IMAC), respectively. The concentration of refolded scFvs was examined by Bradford method. And the antigen binding activity of the refolded scFvs was analyzed by indirect cell-ELISA. The highest and lowest refolding yields could be obtained by dialysis and in situ refolding via IMAC, respectively. The binding activity of the refolded scFv by dialysis was 1.95-fold higher than that by dilution, 4.13-fold higher than that by in situ refolding via IMAC (GSH/GSSH excluded) and 3.63-fold higher than that by in situ refolding via IMAC (GSH/GSSH included), respectively. In conclusion, a high refolding yield and binding activity of scFv with natural conformation could be obtained by dialysis in the condition of 0. 15 mol/L sodium chloride, 50 mmol/L Tirs-HCl, pH 8. 0 buffer containing 3 mmol/L reduced glutathione and 1 mmol/L oxidized glutathione for 48 hours at 4 degrees C.
Antigens, CD ; biosynthesis ; genetics ; immunology ; Antigens, Differentiation ; biosynthesis ; genetics ; immunology ; CTLA-4 Antigen ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

OBJECTIVETo construct a recombinant adenovirus vector carrying soluble extracellular region of tumor necrosis factor alpha receptor I-IgGFc (sTNFRI-IgGFc) and express the fusion protein in human bronchial epithelial HBE135-E6E7 cells.

METHODSsTNFRI-IgGFc fusion gene was subcloned into the adenovirus shuttle plasmid pDC316, which was co-transfected with helper plasmid pBHGloxPE1,3Cre into HEK293 cells. The recombinant adenovirus (Ad-sTNFRI-IgGFc) was generated by homologous recombination of the 2 plasmids in HEK293 cells. After identification with PCR, Ad-sTNFRI-IgGFc was amplified and purified, and its titer measured using TCID50 assay. The transcription and expression of sTNFRI-IgGFc gene in the transfected HBE135-E6E7 were detected by RT-PCR and immunohistochemistry.

RESULTSAd-sTNFRI-IgGFc was successfully constructed with a viral titer of 3 x 10(10) TCID50/ml. The expression of sTNFRI-IgGFc mRNA and protein was confirmed in the transfected HBE135-E6E7 cells.

CONCLUSIONThe constructed Ad-sTNFRI-IgGFc can effectively infect HBE135-E6E7 cells for efficient expression of sTNFRI-IgGFc protein, which antagonizes the cytolytic effect of TNFalpha in L929 cells, suggesting the potential of adenovirus expressing sTNFRI-IgGFc for local treatment of asthma.

Adenoviridae ; genetics ; Bronchi ; cytology ; Epithelial Cells ; cytology ; metabolism ; Genetic Vectors ; genetics ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; Immunoglobulin G ; biosynthesis ; genetics ; Receptors, Tumor Necrosis Factor, Type I ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection