1.Proteomics study of Mycoplasma pneumoniae pneumonia reveals the Fc fragment of the IgG-binding protein as a serum biomarker and implicates potential therapeutic targets.
Jinrong LIU ; Rongfang SHEN ; Lin FENG ; Shujun CHENG ; Jun CHEN ; Ting XIAO ; Shunying ZHAO
Frontiers of Medicine 2022;16(3):378-388
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
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Carrier Proteins
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Child
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Humans
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Immunoglobulin Fc Fragments
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Immunoglobulin G
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Macrolides
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Mycoplasma pneumoniae
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Pneumonia, Mycoplasma/drug therapy*
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Proteomics
2.Progress in the study on the molecules in CD28 family.
Yun-lu FENG ; Li-ping ZHU ; Wei HE
Acta Academiae Medicinae Sinicae 2002;24(5):536-539
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
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Antigens, CD
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Antigens, Differentiation
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immunology
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CD28 Antigens
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immunology
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CTLA-4 Antigen
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Humans
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Immunoglobulin Fc Fragments
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immunology
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Signal Transduction
3.Expression of TMP Fc in Pichia pastoris and identification of its biological activity.
Xiang-Zhong YE ; Qiang GUO ; Chao LI ; Feng-Yun LIU ; Du-Sheng CHENG
Chinese Journal of Biotechnology 2004;20(1):25-29
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
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Blotting, Western
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Humans
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Immunoglobulin Fc Fragments
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genetics
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Immunoglobulin G
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genetics
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Molecular Sequence Data
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Pichia
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genetics
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Plasmids
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Polymerase Chain Reaction
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Recombinant Fusion Proteins
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biosynthesis
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Thrombopoietin
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genetics
4.Isolation of single chain antibodies against cell surface molecules by pathfinder selection.
Jun-xia LIU ; Lei MENG ; Jing XU ; Hai-rong JIA ; Zeng-xuan SONG
Acta Academiae Medicinae Sinicae 2004;26(4):405-409
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
5.Anti-IgE mAb Suppresses Systemic Anaphylaxis through the Inhibitory IgG Receptor FcgammaRIIb in Mice: Interaction between Anti-IgE and FcgammaRIIb.
Nam In KANG ; Zhe Wu JIN ; Hern Ku LEE
Immune Network 2007;7(3):141-148
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*
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Animals
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Asthma
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Blotting, Western
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Cell Line
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CHO Cells
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Cricetinae
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Digestion
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Down-Regulation
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Flow Cytometry
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gamma-Globulins
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Immunoglobulin E
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Immunoglobulin Fc Fragments
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Immunoglobulin G*
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Immunohistochemistry
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Inositol
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Lung
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Mice*
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Ovum
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Papain
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Penicillin V
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Phosphoric Monoester Hydrolases
6.Evaluation of three in-vitro refolding methods for human-derived anti-CTLA4 scFv expressed in E. coli.
Qiang HUANG ; Lihong CHEN ; Lingyu ZENG ; Lin WAN ; Shengfu LI ; Xiaofeng LU ; Jingqiu CHENG
Journal of Biomedical Engineering 2006;23(2):388-391
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
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biosynthesis
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genetics
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immunology
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Antigens, Differentiation
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biosynthesis
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genetics
;
immunology
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CTLA-4 Antigen
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Cloning, Molecular
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Escherichia coli
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genetics
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metabolism
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Humans
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Immunoglobulin Fc Fragments
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biosynthesis
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genetics
;
immunology
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Immunoglobulin Variable Region
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biosynthesis
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genetics
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immunology
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Recombinant Fusion Proteins
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biosynthesis
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genetics
;
immunology
7.Cloning, expression and identification of IL-1ra-Fcepsilon fusion gene.
Zhongcheng LIU ; Minji ZOU ; Yuanyuan WANG ; Jiaxi WANG ; Donggang XU
Chinese Journal of Biotechnology 2008;24(10):1754-1760
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
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Escherichia coli
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genetics
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metabolism
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Gene Fusion
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Immunoglobulin E
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genetics
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metabolism
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Immunoglobulin Fc Fragments
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genetics
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metabolism
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Interleukin 1 Receptor Antagonist Protein
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genetics
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metabolism
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Recombinant Fusion Proteins
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genetics
;
metabolism
8.Construction of adenoviral vector encoding soluble human TNFRI-IgGFc cDNA and its expression in human bronchial epithelial cells.
Jin SU ; Chang-xuan YOU ; Shao-xi CAI ; Li MA ; Qian WEN ; Wei LUO ; Yong-ta HUANG
Journal of Southern Medical University 2008;28(4):517-521
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
9.Expression of ATR-Fc fusion protein in CHO cells.
Li-Hua GAO ; Xian-Wen HU ; Wei CHEN ; Jun-Jie XU ; Jian ZHAO ; Hui-Peng CHEN
Chinese Journal of Biotechnology 2005;21(5):826-831
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
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CHO Cells
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Cricetinae
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Cricetulus
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Gene Transfer Techniques
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Genetic Vectors
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Humans
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Immunoglobulin Fc Fragments
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biosynthesis
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genetics
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Immunoglobulin G
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biosynthesis
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genetics
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Neoplasm Proteins
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biosynthesis
;
genetics
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Receptors, Cell Surface
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biosynthesis
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genetics
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Recombinant Fusion Proteins
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biosynthesis
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genetics
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immunology
10.Construction of anti-VEGFR-2 IgG1 like human antibody and its expression in CHO-k cells.
Zhi-Ke LI ; Yuan HE ; Juan ZHANG ; Wei XIE ; Wan-Lu CAO ; Ze-Gen WANG ; Min WANG
Acta Pharmaceutica Sinica 2013;48(10):1544-1549
Anti-angiogenesis mechanism plays a vital role in tumor targeting immunotherapy. Based on the amino acid sequence of an anti-VEGFR-2 scFv-Fc fusion antibody (AK404R-Fc), this article is aimed to generate an anti-VEGFR-2 human IgG1-like full length antibody (Mab-04). Firstly, the light chain (L-chain) and heavy chain (H-chain) were obtained by overlap PCR and then linked to eukaryotic expression vector pcDNA3.1, separately. The recombinant plasmids (pcDNA3.1-L-chain and pcDNA3.1-H-chain) were then co-transfected into CHO-k cells using liposome transient transfection. Subsequently, Mab-04 antibody was expressed and purified by Protein A affinity chromatography. Western blotting was applied to identify the expression of Mab-04 and its affinity was detected by ELISA assay. DNA sequencing revealed the successful construction of recombinant plasmids and Western blotting assay proved the successful expression of full-length antibody (1 microg x mL(-1)). Finally, ELISA assay illustrated that the binding of the antibody to its antigen was in a concentration-dependent manner (IC50: 50 nmol x L(-1)). These outcomes above indicated that Mab-04 was successfully expressed and assembled, which laid the foundation for further preparation and antineoplastic activity study.
Animals
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Antibodies, Monoclonal, Humanized
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analysis
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CHO Cells
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Cricetulus
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Genetic Vectors
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Immunoglobulin Fc Fragments
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genetics
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Immunoglobulin G
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genetics
;
immunology
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Plasmids
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Recombinant Fusion Proteins
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analysis
;
genetics
;
Single-Chain Antibodies
;
genetics
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Transfection
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Vascular Endothelial Growth Factor Receptor-2
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genetics
;
immunology