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

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 ; Antibodies, Monoclonal, Humanized ; analysis ; CHO Cells ; Cricetulus ; Genetic Vectors ; Immunoglobulin Fc Fragments ; genetics ; Immunoglobulin G ; genetics ; immunology ; Plasmids ; Recombinant Fusion Proteins ; analysis ; genetics ; Single-Chain Antibodies ; genetics ; Transfection ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; immunology

Base Sequence ; Binding Sites, Antibody ; Biochemistry ; methods ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Genetic Vectors ; Immunoglobulin Fc Fragments ; genetics ; metabolism ; Immunoglobulin G ; genetics ; metabolism ; Ligands ; Molecular Sequence Data ; Plasmids ; Protein Binding ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; Staphylococcal Protein A ; genetics ; metabolism

To investigate a baculovirus insect cell system for expressing an interferon alpha 2b (IFNa2b)/immunoglobulin G-4 (IgG4) Fc fusion protein, which has long-acting antiviral effects. Human IFNa2b and IgG4 Fc cDNAs were generated by molecular cloning and inserted into a baculovirus shuttle vector, which was then transposed into the DH10 Bac strain to form recombinant Bacmid-IFN/Fc. The Bacmid-IFN/Fc was transfected into High five insect cells, and expression of the IFN/Fc fusion protein was detected by Western blotting and its biological activity was assessed by the cytopathic effect inhibition method. The IFNa2b and IgG4 Fc cDNA fragments were successfully amplified by RT-PCR using human peripheral lymphocytes. After cloning into the baculovirus shuttle vector, pFastBac1, and transforming into DH10 Bac competent cells, screening identified positive clones carrying the recombinant Bacmid-IFN/Fc. A Bacmid-IFN/Fc clone was successfully transfected into the High five insect cells and packaged into the baculovirus for expression of the IFN/Fc fusion protein. Western blotting revealed that the fusion protein expression was specific, and yielded a protein of 45 kD in size. The in vitro antiviral activity of the IFN/Fc fusion protein was 580 IU/mL. A novel IFN/Fc fusion protein was successfully generated using a baculovirus insect cell system, which may prove useful for providing future experimental data for development of a new long-acting interferon to treat chronic viral hepatitis.
Animals ; Antiviral Agents ; metabolism ; Baculoviridae ; genetics ; Cell Line ; Cloning, Molecular ; Gene Expression ; Gene Fusion ; Genetic Vectors ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; Immunoglobulin G ; biosynthesis ; genetics ; Insecta ; Interferon-alpha ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

Follicle-stimulating hormone (FSH) is a glycoprotein which regulates the development, growth, pubertal maturation and reproductive processes of the body. Exogenous FSH has been used to promote ovarian follicular growth and maturation in female and spermatogenesis in male. The relative short elimination half life and rapid metabolic clearance of current versions of FSH require a daily or twice-daily scheduled subcutaneous injection to maintain stable FSH level being not below the threshold during ovarian stimulation. The development of recombinant long-acting FSH with enhanced biological activities may be helpful for less injection therefore to improve patient compliance, while reducing patient stress and error rates. A number of technological strategies have been explored to develop recombinant longer-acting FSH. For examples, attachment of the C-terminal peptide (CTP) of the human chorionic gonadotropin beta subunit or a sequence containing potential glycosylation sites to either subunit of FSH, creation of a single chain containing the alpha and beta subunits of FSH combined with CTP or N-linked glycosylation signal sequence as a linker, or fusion of the Fc domain of IgGi to FSH. Based on the modifiable molecular structure and pharmacokinetic and pharmacodynamic properties of recombinant FSH, it is hopeful that more FSH drugs with prolonged half-life and increased bioactivity will be developed to meet the modern clinical demands.
Animals ; Follicle Stimulating Hormone, Human ; chemistry ; genetics ; metabolism ; pharmacology ; Glycosylation ; Half-Life ; Humans ; Immunoglobulin Fc Fragments ; chemistry ; metabolism ; Ovulation Induction ; methods ; Receptors, FSH ; chemistry ; metabolism ; Recombinant Fusion Proteins ; chemistry ; genetics ; metabolism ; pharmacology ; Reproduction ; drug effects

To investigate the effects of human anti-BAFF scFv-Fc against the hsBAFF, ICR mice were randomly divided into six groups: control, hsBAFF (1 mg x kg(-1)), hsBAFF (1 mg x kg(-1)) + Ab (1 mg x kg(-1)), hsBAFF (1 mg x kg(-1)) + Ab (2 mg x kg(-1)), hsBAFF (1 mg x kg(-1)) + human IgG (1 mg x kg(-1)) and hsBAFF (1 mg x kg(-1)) + human IgG (2 mg x kg(-1)) groups. The effects of scFv-Fc administration on the proliferation of B lymphocytes were evaluated using an MTT assay. The titres of antibody in the serum and B lymphocytes differentiation were assessed by ELISA and flow cytometry, respectively. The results showed that administration of scFv-Fc to mice injected with hsBAFF significantly prevented human BAFF-induced increases in splenic B cell numbers and serum immunoglobulin levels. Furthermore, this fully human antibody would avoid inducing the human anti-mouse antibody (HAMA) response when used in humans. These findings suggest that the compact antibody may be useful in therapeutic or diagnostic application of the BAFF-associated autoimmune diseases in human.
Animals ; B-Cell Activating Factor ; immunology ; metabolism ; B-Lymphocytes ; cytology ; Body Weight ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Female ; Humans ; Immunoglobulin Fc Fragments ; immunology ; metabolism ; Immunoglobulin G ; blood ; immunology ; Immunoglobulin M ; blood ; Mice ; Mice, Inbred ICR ; Random Allocation ; Recombinant Fusion Proteins ; immunology ; metabolism ; Single-Chain Antibodies ; immunology ; metabolism ; Spleen ; cytology

We constructed the eukaryotic expression vector of human IL-35-IgG4 (Fc)-pOptiVEC-TOPO by gene recombination technique and expressed the fusion protein human IL-35-IgG4 (Fc) in CHO/DG44 cells. The two components of the newly discovered cytokine human IL-35, EBI3 and IL-12p35, were amplified by PCR from the cDNA library derived from the KG-I cells after LPS induction. The two PCR-amplified cDNA fragments of human IL-35 were linked by over-lapping PCR and then cloned into the IgG4 (Fc)-pOptiVEC-TOPO vector. The constructed plasmid with the recombinant cDNA IL-35-IgG4 (Fc) was verified by restriction enzyme digestion analysis, PCR and DNA sequencing. The verified plasmid with the recombinant cDNA was transfected into CHO/DG44 cells using Lipofectamine 2000. The success of the transfection was examined and confirmed by RT-PCR. After selection in alpha-MEM (-) medium, the IL-35-Ig G4 (Fc) positive CHO/DG44 clones were chosen and the media from these positive clones were collected to be used to purify the fusion protein. The positive CHO/DG44 clones were further cultured in increasing concentrations of MTX and the expression levels of the fusion protein IL-35-Ig G4 (Fc) were repetitively induced by MTX-induced gene amplification. The IL-35-IgG4 (Fc) fusion protein was purified from the media collected from the positive CHO/DG44 clones by protein G affinity chromatography and then identified by SDS-PAGE and Western blotting. The results showed that one protein band was found to match well with the predicted relative molecular mass of human IL-35-IgG4 (Fc) and this protein could specifically bind to anti-human IgG4 (Fc) monoclonal antibody. In conclusion, our study successfully established an IL-35-IgG4 (Fc) positive DG44 cell line which could stably express IL-35-IgG4 (Fc) fusion protein.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Gene Fusion ; genetics ; Genetic Vectors ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; Immunoglobulin G ; biosynthesis ; genetics ; Interleukins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection

We identified the critical amino-acid residues in antigen M derterminant (MAD) epitope of human cytomegalovirus protein M. On the basis of the peptide sequence of MAD, some conservative residues were mutated into the glycine residue. Then the gene fragment of mutants linked to amino terminal of Fc were cloned into the plasmid pET32-Fc and expressed by fusion with Fc. After purified by protein A affinity chromatography, the activity of mutants binding the goat polyclonal antibodies against human cytomegalovirus (HCMV) were detected by ELISA and Western blotting. Our results showed that when glutamine residue was mutated into glycine residue, the activity of MAD(Q --> G) binding the goat polyclonal antibodies against HCMV was reduced apparently. Other mutants did not have the same characteristics. The activity of MAD was closely related to the conformation of glutamine residue.
Amino Acids ; genetics ; immunology ; Animals ; Antibodies, Monoclonal ; immunology ; Antibodies, Viral ; immunology ; Antigens, Viral ; genetics ; immunology ; Base Sequence ; Cytomegalovirus ; genetics ; immunology ; Epitopes ; genetics ; immunology ; Goats ; Humans ; Immunoglobulin Fc Fragments ; genetics ; immunology ; Molecular Sequence Data ; Mutant Proteins ; genetics ; Mutation ; Viral Matrix Proteins ; genetics ; immunology

Tumour necrosis factor (TNF-a) is a pro-inflammatory cytokine that has been implicated in many aspects of the airway pathology in asthma, and which has recently been highlighted as potentially important in refractory asthma. To study the feasibility of local treatment of asthma with recombinant adenovirus vector carrying soluble extra-cellular region of TNF receptor I-IgGFc (sTNFRI-IgGFc) fusion protein, The sTNFRI-IgGFc gene was subcloned into the adenovirus shuttle plasmid pDC316, the products were co-transfected into HEK293 cell line with helper plasmid pBHGloxDeltaE1,3Cre. The recombinant adenovirus (Ad-sTNFRI-IgGFc) was produced by homologous recombination of above 2 plasmids in HEK293 cells. After identification with PCR, Ad-sTNFRI-IgGFc was amplified and purified, its titer was measured by TCID50 assay. The transcription and expression of sTNFRI-IgGFc gene in transfected human airway smooth muscle cells (HASMCs) was detected by RT-PCR, ELISA and immunological histochemistry. The anti-TNF activity assay of transfected HASMCs culture supernatant was measured by MTT. Ad-sTNFRI-IgGFc was successfully constructed with the titer of 3x10(10) TCID50/mL. Ad-sTNFRI-IgGFc can transfect HASMC with high efficacy. The transcription of sTNFRI-IgGFc mRNA and the expression of protein were confirmed in the transfected HASMCs. Moreover, the product in 100 microL expression supernatant could completely antagonize the cytolytic effect of 2ng TNFa on L929 cells, even at 1/64 dilution. This study forms the basement of the experiment study on local treatment of asthma with adenovirus expressing sTNFRI-IgGFc.
Adenoviridae ; genetics ; metabolism ; Asthma ; therapy ; Bronchi ; cytology ; Cells, Cultured ; Genetic Vectors ; genetics ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; Immunoglobulin G ; biosynthesis ; genetics ; Myocytes, Smooth Muscle ; cytology ; metabolism ; Receptors, Tumor Necrosis Factor ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Transfection ; Tumor Necrosis Factor-alpha ; antagonists & inhibitors

In order to construct a pichia pastoris expression vector containing the extracellular domain of human Jagged1 and the Fc fragment of human IgG1 fusion gene, or containing only the Fc fragment of human IgG1 and to express them in pichia pastoris. The extracellular domain of human Jagged1 gene was cloned from normal human bone marrow cells. After DNA sequencing, the extracellular domain of Jagged1 gene was inserted into pIC-Fc vector constructed previously, which is Pichia pastoris expression vector containing only the Fc fragment of human IgG1. The constructed plasmid was transformed into yeast GS115 by means of electroporation. The recombinant transformants with a high copy number of the plasmid were selected on MD plate with G418. The expression of protein was induced by addition of methanol. Then, protein expression was analyzed by SDS-PAGE. The results indicated that the extracellular domain of human Jagged1 gene was effectively amplified. The DNA sequencing result showed that the constructed plasmid containing hJagged1(ext)-Fc fusion gene was the same as designed. The fusion protein was successfully expressed in Pichia pastoris. It is concluded that the hJagged1(ext) gene has been successfully cloned and expressed, which provides a new fusion protein for further experiments, the hJagged1(ext)-Fc fusion protein can be used as a new stimulator for proliferation of hematopoietic stem/progenitor cells in vitro.
Calcium-Binding Proteins ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; Immunoglobulin G ; biosynthesis ; genetics ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; genetics ; Jagged-1 Protein ; Membrane Proteins ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Serrate-Jagged Proteins ; Transfection