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1.
Electron. j. biotechnol ; 41: 56-59, sept. 2019. tab, graf
Article in English | LILACS | ID: biblio-1087166

ABSTRACT

Background: Chinese hamster ovary (CHO) cells are the most dependable mammalian cells for the production of recombinant proteins. Replication-incompetent retroviral vector (retrovector) is an efficient tool to generate stable cell lines. Multiple copies of integrated genes by retrovector transduction results in improved recombinant protein yield. HEK-293 and their genetic derivatives are principal cells for retrovector production. Retrovectors packaged in HEK-293 cells pose a risk of infectious agent transmission, such as viruses and mycoplasmas, from serum and packaging cells. Results: In this report, retrovectors were packaged in CHO cells cultured in chemically defined (CD) media. The retrovectors were then used to transduce CHO cells. This method can block potential transmission of infectious agents from serum and packaging cells. With this method, we generated glucagon-like protein-1 Fc fusion protein (GLP-1-Fc) stable expression CHO cell lines. Productivity of GLP-1-Fc can reach 3.15 g/L. The GLP-1-Fc protein produced by this method has comparable bioactivity to that of dulaglutide (Trulicity). These stable cell lines retain 95­100% of productivity after 40 days of continuous culture (~48­56 generations). Conclusions: Suspension CHO cells are clean, safe, and reliable cells for retrovector packaging. Retrovectors packaged from this system could be used to generate CHO stable cell lines for recombinant protein expression.


Subject(s)
Retroviridae , Recombinant Proteins/metabolism , CHO Cells/metabolism , Immunoglobulin Fc Fragments , Cell Line , Chromatography, Gel/methods , Disease Vectors , Glucagon-Like Peptide 1 , Tandem Mass Spectrometry , Batch Cell Culture Techniques
2.
Acta Pharmaceutica Sinica ; (12): 1544-1549, 2013.
Article in Chinese | WPRIM | ID: wpr-298046

ABSTRACT

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.


Subject(s)
Animals , Antibodies, Monoclonal, Humanized , CHO Cells , Cricetulus , Genetic Vectors , Immunoglobulin Fc Fragments , Genetics , Immunoglobulin G , Genetics , Allergy and Immunology , Plasmids , Recombinant Fusion Proteins , Genetics , Single-Chain Antibodies , Genetics , Transfection , Vascular Endothelial Growth Factor Receptor-2 , Genetics , Allergy and Immunology
3.
Acta Pharmaceutica Sinica ; (12): 421-426, 2012.
Article in Chinese | WPRIM | ID: wpr-323025

ABSTRACT

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.


Subject(s)
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
4.
Chinese Journal of Hepatology ; (12): 617-620, 2012.
Article in Chinese | WPRIM | ID: wpr-296838

ABSTRACT

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.


Subject(s)
Animals , Antiviral Agents , Metabolism , Baculoviridae , Genetics , Cell Line , Cloning, Molecular , Gene Expression , Gene Fusion , Genetic Vectors , Humans , Immunoglobulin Fc Fragments , Genetics , Immunoglobulin G , Genetics , Insecta , Interferon-alpha , Genetics , Recombinant Fusion Proteins , Genetics , Recombinant Proteins , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Transfection
5.
Acta Pharmaceutica Sinica ; (12): 1336-1340, 2012.
Article in Chinese | WPRIM | ID: wpr-274657

ABSTRACT

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.


Subject(s)
Animals , B-Cell Activating Factor , Allergy and Immunology , Metabolism , B-Lymphocytes , Cell Biology , Body Weight , Cell Differentiation , Cell Proliferation , Cells, Cultured , Female , Humans , Immunoglobulin Fc Fragments , Allergy and Immunology , Metabolism , Immunoglobulin G , Blood , Allergy and Immunology , Immunoglobulin M , Blood , Mice , Mice, Inbred ICR , Random Allocation , Recombinant Fusion Proteins , Allergy and Immunology , Metabolism , Single-Chain Antibodies , Allergy and Immunology , Metabolism , Spleen , Cell Biology
6.
Chinese Journal of Biotechnology ; (12): 109-115, 2009.
Article in Chinese | WPRIM | ID: wpr-302847

ABSTRACT

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.


Subject(s)
Animals , CHO Cells , Cricetinae , Cricetulus , Gene Fusion , Genetics , Genetic Vectors , Humans , Immunoglobulin Fc Fragments , Genetics , Immunoglobulin G , Genetics , Interleukins , Genetics , Recombinant Fusion Proteins , Genetics , Transfection
7.
Chinese Journal of Biotechnology ; (12): 774-779, 2008.
Article in Chinese | WPRIM | ID: wpr-342837

ABSTRACT

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.


Subject(s)
Adenoviridae , Genetics , Metabolism , Asthma , Therapeutics , Bronchi , Cell Biology , Cells, Cultured , Genetic Vectors , Genetics , Humans , Immunoglobulin Fc Fragments , Genetics , Immunoglobulin G , Genetics , Myocytes, Smooth Muscle , Cell Biology , Metabolism , Receptors, Tumor Necrosis Factor , Genetics , Recombinant Fusion Proteins , Genetics , Pharmacology , Transfection , Tumor Necrosis Factor-alpha
8.
Chinese Journal of Biotechnology ; (12): 1128-1132, 2008.
Article in Chinese | WPRIM | ID: wpr-342780

ABSTRACT

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.


Subject(s)
Amino Acids , Genetics , Allergy and Immunology , Animals , Antibodies, Monoclonal , Allergy and Immunology , Antibodies, Viral , Allergy and Immunology , Antigens, Viral , Genetics , Allergy and Immunology , Base Sequence , Cytomegalovirus , Genetics , Allergy and Immunology , Epitopes , Genetics , Allergy and Immunology , Goats , Humans , Immunoglobulin Fc Fragments , Genetics , Allergy and Immunology , Molecular Sequence Data , Mutant Proteins , Genetics , Mutation , Viral Matrix Proteins , Genetics , Allergy and Immunology
9.
Article in Chinese | WPRIM | ID: wpr-321747

ABSTRACT

<p><b>OBJECTIVE</b>To construct a recombinant adenoviral vector harboring human transforming growth factor-beta type II receptor-IgG1Fc (TbetaRII-IgG1Fc) fusion gene.</p><p><b>METHODS</b>The cDNA fragments of human TbetaRII and IgG1Fc genes were amplified by RT-PCR and fused with overlap PCR to obtain the fusion gene TbetaRKK-IgG1Fc. The TbetaRII-IgG1Fc gene was cloned into the shuttle plasmid pAdTrack-CMV, which was linearized and transfected into E.coli BJ 5183 strain containing the adenoviral backbone vector. The recombinant adenovirus vector was constructed by homologous recombination. The recombinant adenoviral plasmid was linearized and transfected into 293 cells, followed by amplification and purification of the virus and detection of TbetaRII-IgG1Fc mRNA expression by RT-PCR. The functional activity of the recombinant adenoviral plasmid was assessed using enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>The results of restriction endonuclease digestion and DNA sequencing indicated correct sequence of the target TbetaRII-IgG1Fc fusion gene. The recombinant adenoviral plasmid expressed hTbetaRII-IgG1Fc and neutralized TGF-beta1 in vitro after infection of the human lung fibroblasts (HLF), as confirmed by RT-PCR and ELISA.</p><p><b>CONCLUSIONS</b>The recombinant adenoviral plasmid capable of neutralizing TGF-beta1 in vitro is constructed successfully.</p>


Subject(s)
Adenoviridae , Genetics , Metabolism , Escherichia coli , Genetics , Metabolism , Fibroblasts , Cell Biology , Genetic Vectors , Genetics , Humans , Immunoglobulin Fc Fragments , Genetics , Metabolism , Immunoglobulin G , Genetics , Metabolism , Lung , Cell Biology , Protein-Serine-Threonine Kinases , Genetics , Metabolism , RNA, Messenger , Genetics , Metabolism , Receptors, Transforming Growth Factor beta , Genetics , Metabolism , Recombinant Fusion Proteins , Genetics , Metabolism , Transfection
10.
Chinese Journal of Biotechnology ; (12): 1754-1760, 2008.
Article in Chinese | WPRIM | ID: wpr-275344

ABSTRACT

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.


Subject(s)
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
11.
Chinese Journal of Biotechnology ; (12): 1874-1879, 2008.
Article in Chinese | WPRIM | ID: wpr-302898

ABSTRACT

To prolong serum half-life of human Erythropoietin for better efficacy, a new form of recombinant human erythropoietin (rhEpo-L-Fc) was generated by fusion of a full length human erythropoietin gene and the Fc fragment of human IgG1 with flexible linker sequence. The fusion gene rhEPO-L-Fc was constructed by PCR, then inserted into expression vector pOptiVEC-TOPO, and expressed in Chinese Hamster Ovary cells deficient in the DHFR enzyme(CHO-dhfr-). The chimeric protein was purified by Protein A affinity chromatography, showed expected molecular weight and demonstrated a similar bioactivity compared to that of the native recombinant human erythropoietin (rhEPO) in an EPO-dependent cell-based assay. In vivo pharmacokinetic studies showed that the rhEPO-L-Fc had an elimination half-life of 27 h. In vivo efficacy studies showed that a single dose administration of rhEPO-L-Fc in rats increased the reticulocyte number in the peripheral blood significantly. These results demonstrated that the new engineered rhEPO-L-Fc may become alternative therapeutic approach to extend the half-time of rhEPO to treat anemia.


Subject(s)
Animals , CHO Cells , Cricetinae , Cricetulus , Erythropoietin , Chemistry , Genetics , Metabolism , Pharmacokinetics , Female , Humans , Immunoglobulin Fc Fragments , Genetics , Male , Rats , Rats, Sprague-Dawley , Recombinant Fusion Proteins , Chemistry , Genetics , Pharmacokinetics , Recombinant Proteins
12.
Chinese Journal of Biotechnology ; (12): 1918-1923, 2008.
Article in Chinese | WPRIM | ID: wpr-302891

ABSTRACT

We studied the aggregation of a recombinant engineering antibody (chA21). Anti-ErbB2 antibody chA21 was produced by fusing single-chain Fv (scFv) with human IgG1 Fc fragment, and it was proved to be a drug candidate for cancer therapy. We characterized the aggregation of chA21 by high performance sized-exclusive chromatography (HPSEC), dynamic light scattering (DLS), SDS-PAGE, indirect ELISA assay, and compared the influence of temperature and additive on the level of aggregation and binding activity. Conformation changes of different levels of aggregation were also analyzed via circular dichroism (CD). Finally, we analyzed which part of chA21 was involved in aggregation by cleaving it into scFv and Fc fragments. The results showed that chA21 could form aggregates in the storage solution. The aggregates interacted through non-covalent bonds and remained binding activity. Temperature and additive could slightly affect the level of aggregation and binding activity, while the conformations of chA21 were stable. Aggregation propensity of scFv fragment was almost same as chA21, indicating that scFv may be the major part to form the aggregates. The research on aggregation may be helpful to develop a suitable formulation for chA21 clinical application as well as provide direction for future antibody design and reconstruction.


Subject(s)
Antibodies , Chemistry , Metabolism , Humans , Immunoglobulin Fc Fragments , Chemistry , Metabolism , Immunoglobulin Variable Region , Chemistry , Metabolism , Protein Conformation , Protein Engineering , Methods , Receptor Aggregation , Allergy and Immunology , Receptor, ErbB-2 , Chemistry , Allergy and Immunology , Recombinant Proteins , Chemistry , Genetics , Allergy and Immunology
13.
Article in Chinese | WPRIM | ID: wpr-267861

ABSTRACT

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.


Subject(s)
Calcium-Binding Proteins , Genetics , Genetic Vectors , Genetics , Humans , Immunoglobulin Fc Fragments , Genetics , Immunoglobulin G , Genetics , Intercellular Signaling Peptides and Proteins , Genetics , Jagged-1 Protein , Membrane Proteins , Genetics , Pichia , Genetics , Metabolism , Recombinant Fusion Proteins , Genetics , Serrate-Jagged Proteins , Transfection
14.
Article in Chinese | WPRIM | ID: wpr-280159

ABSTRACT

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>sTNFRI-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.</p><p><b>RESULTS</b>Ad-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.</p><p><b>CONCLUSION</b>The 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.</p>


Subject(s)
Adenoviridae , Genetics , Bronchi , Cell Biology , Epithelial Cells , Cell Biology , Metabolism , Genetic Vectors , Genetics , Immunoglobulin Fc Fragments , Genetics , Immunoglobulin G , Genetics , Receptors, Tumor Necrosis Factor, Type I , Genetics , Recombinant Fusion Proteins , Genetics , Transfection
15.
Article in Chinese | WPRIM | ID: wpr-813940

ABSTRACT

OBJECTIVE@#To evaluate the expression of hCTLA4-Ig and their biological function in newborn porcine islets (NPIs) transfected with AAV-hCTLA4-Ig.@*METHODS@#Cultured NPIs were transfected with AAV-hCTLA4-Ig. The expression of CTLA4-Ig in these NPIs was assayed by RT-PCR and immunocytochemistry. The levels of IL-2, IFN-gamma, and TNF-alpha in the culture medium were assayed by ELISA after these cells the co-cultured with human. The response of glucose-stimulated insulin secretion was observed in the transgene group and the control group.@*RESULTS@#The expressions of CTLA4-Ig mRNA and protein were detected in the transgene group. The levels of cytokines were obviously lower in the transgene group than those in the control group (P0.05).@*CONCLUSION@#AAV mediated hCTLA4-Ig expression in NPIs could inhibit T lymphocyte to produce cytokines, while the endocrine functions of the NPIs were not significantly affected.


Subject(s)
Animals , Animals, Newborn , Antigens, CD , Genetics , Antigens, Differentiation , Genetics , CTLA-4 Antigen , Cells, Cultured , Dependovirus , Genetics , Enzyme-Linked Immunosorbent Assay , Gene Expression , Humans , Immunoglobulin Fc Fragments , Genetics , Immunohistochemistry , Interferon-gamma , Interleukin-2 , Islets of Langerhans , Cell Biology , Allergy and Immunology , Metabolism , Recombinant Fusion Proteins , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Swine , Transfection , Tumor Necrosis Factor-alpha
16.
Article in English | WPRIM | ID: wpr-201420

ABSTRACT

Asthma was induced by the sensitization and challenge with ovalbumin (OVA) in mice. B-cell activating factor (BAFF) plays a role in mature B cell generation and maintenance. Here, we investigated whether, BAFF expression was changed in OVA-induced mice and whether the control of BAFF expression level alleviates the symptom of bronchial asthma. BAFF expression was detected in alveolar-associated cells surrounding bronchi of OVA-induced mouse lung tissues. BAFF protein was also increased in OVA-induced mouse serum. The increased BAFF transcripts was detected in OVA-induced mouse splenocytes. OVA-induced asthma was associated with the increased number of eosinophils in bronchoalveolar lavage fluid (BALF). When TACI:Fc scavenging soluble BAFF was injected to OVA-induced mice, a significant inhibition was detected in the thickness of airway smooth muscle and glycol-containing cellular elements in airway that were visualized by hematoxylin/eosin Y and periodic acid-Schiff staining, respectively. In addition, when mice were treated with TACI:Fc protein, BAFF protein level was decreased in alveolar-associated cells surrounding bronchi of OVA-induced mouse lung tissues compared to control mice. When compared to OVA-induced control, TACI:Fc treatment reduced the percentage of non-lymphoid cells and no changes were detected in lymphoid cell population. Hypodiploid cell formation in BALF was decreased by OVA-challenge but it was recovered by TACI:Fc treatment. Collectively, data suggest that asthmatic symptom could be alleviated by scavenging BAFF and then BAFF could be a novel target for the develpoment of anti-asthmatic agents.


Subject(s)
Animals , Apoptosis , Asthma/chemically induced , B-Cell Activating Factor/biosynthesis , Bronchi/metabolism , Bronchoalveolar Lavage Fluid/cytology , Eosinophils/pathology , Female , Humans , Immunoglobulin Fc Fragments/genetics , Immunoglobulin G/genetics , Lymphocytes/pathology , Mice , Mice, Inbred BALB C , Ovalbumin , Pulmonary Alveoli/metabolism , Recombinant Fusion Proteins/genetics , Spleen/metabolism , Transmembrane Activator and CAML Interactor Protein/genetics
17.
Chinese Journal of Cardiology ; (12): 28-32, 2007.
Article in Chinese | WPRIM | ID: wpr-304974

ABSTRACT

<p><b>OBJECTIVE</b>To explore the effects of adenovirus vector-mediated gene transfer of ICOSIg fusion protein on experimental autoimmune myocarditis (EAM) in Lewis rats.</p><p><b>METHODS</b>Expression vector containing ICOSIg (p-Adeno-ICOSIg) was constructed by fusion of human ICOS and IgGFc segment. Adenovirus vector was digested by PacI enzyme and transfected into HEK 293 cells. Adenovirus expressing ICOSIg was produced. EGFP was constructed into adenovirus vector and used as control. EAM was induced in Lewis rats by injection of porcine cardiac myosin. All immunized Lewis rats were divided into 4 groups. Group A (n = 15) and B (n = 15) received adenovirus containing ICOSIg on day 0 and day 14 respectively to study the effects of costimulatory molecules gene therapy on T cell activation and inflammation; group C (n = 10) and group D (n = 10) received adenovirus containing EGFP on day 0 and day 14 respectively as controls. Group E (n = 10) was normal controls that did not receive immunization. On day 28, all rats were killed after echocardiography examination. Histopathological examination was performed to observe myocardial inflammation. Protein levels of ICOS, ICOSL, B7-1 and B7-2 were detected by Western blot. INF-gamma, IL-2 and IL-4 mRNA were determined by realtime RT-PCR.</p><p><b>RESULTS</b>On day 28, cardiac function was significantly improved and myocardial inflammation significantly attenuated in group B compared to group A, C and D (all P < 0.05). B7-1 expression at protein level was significantly lower in group B than that of group C (P < 0.05). ICOS and ICOSL expressions at protein level were significantly decreased in both group A and B compared with group C and D (P < 0.05). IFN-gamma mRNA level significantly decreased and IL-4 mRNA significantly increased in group A and B compared to group C and D (P < 0.05).</p><p><b>CONCLUSIONS</b>Blockade of costimulatory pathway with gene therapy of ICOSIg alleviated autoimmune inflammatory damage and improved cardiac function in Lewis rats with EAM. Down-regulated costimulatory molecules in the myocardium and reduced inflammatory cytokine secretion might be responsible for the beneficial effects of ICOSIg in this model.</p>


Subject(s)
Adenoviridae , Genetics , Animals , Antigens, Differentiation, T-Lymphocyte , Genetics , Autoimmune Diseases , Allergy and Immunology , Pathology , Therapeutics , Disease Models, Animal , Gene Transfer Techniques , Genetic Therapy , Genetic Vectors , Immunoglobulin Fc Fragments , Genetics , Inducible T-Cell Co-Stimulator Protein , Male , Myocarditis , Allergy and Immunology , Pathology , Therapeutics , Rats , Rats, Inbred Lew , Recombinant Fusion Proteins , Genetics
18.
Immune Network ; : 141-148, 2007.
Article in English | WPRIM | ID: wpr-86070

ABSTRACT

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.


Subject(s)
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
19.
Article in Chinese | WPRIM | ID: wpr-249593

ABSTRACT

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.


Subject(s)
Antigens, CD , Genetics , Allergy and Immunology , Antigens, Differentiation , Genetics , Allergy and Immunology , CTLA-4 Antigen , Cloning, Molecular , Escherichia coli , Genetics , Metabolism , Humans , Immunoglobulin Fc Fragments , Genetics , Allergy and Immunology , Immunoglobulin Variable Region , Genetics , Allergy and Immunology , Recombinant Fusion Proteins , Genetics , Allergy and Immunology
20.
Chinese Journal of Biotechnology ; (12): 826-831, 2005.
Article in Chinese | WPRIM | ID: wpr-237066

ABSTRACT

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.


Subject(s)
Animals , CHO Cells , Cricetinae , Cricetulus , Gene Transfer Techniques , Genetic Vectors , Humans , Immunoglobulin Fc Fragments , Genetics , Immunoglobulin G , Genetics , Neoplasm Proteins , Genetics , Receptors, Cell Surface , Genetics , Recombinant Fusion Proteins , Genetics , Allergy and Immunology
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