[Expression of mOX40-Ig and its biological activity study].
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AIM To construct a recombinant eukaryotic expression vector containing pcDNA3.1-mOX40-Ig fusion gene and obtain mOX40-Ig fusion protein with bioactivity by transfecting CHO cells. METHODS The gene fragment encoding the human IgG1Fc was amplified by RT-PCR and the eukaryotic expression vector pcDNA3.1-hIgG1Fc was constructed. After sequencing, mOX40 extracellular gene was cloned from pIRES2-EGFP-OX40 by PCR and then inserted into the recombinant vector pcDNA3.1-hIgG1Fc. The right recombinant was transfected into CHO cells with lipofectin Ragent and its expression was detected by RT-PCR and sandwich-ELISA. After purified by protein A affinity column chromatography, the mOX40-Ig fusion protein was identified by SDS-PAGE and its effect on the proliferation of B cells in vitro was studied by (3)H-TdR method. RESULTS The hIgG1Fc, mOX40 extracellular gene and mOX40-Ig gene were consistent with DNA sequencing.The expression of mOX40-Ig fusion protein in CHO cells was confirmed by RT-PCR, sandwich-ELISA and SDS-PAGE. (3)H-TdR analysis showed the mOX40-Ig fusion protein stimulated the proliferation of B cells in vitro. CONCLUSION A eukaryotic expression vector containing pcDNA3.1-mOX40-Ig has been constructed successfully and the stable expression of mOX40-Ig fusion protein with bioactivity has been acquired, which lays a solid basis for further study of the application related to OX40.Keywords:
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AIM: To construct the eukaryotic expression vector CD80-IgG by fusing the cDNA encoding extracellular portion of murine CD80 to the 5'-terminus of cDNA encoding Fc fragment of murine immunoglobulin G1 and to express the fusion protein in Chinese hamster ovary (CHO) cells. METHODS: The two cDNAs was amplified by PCR respectively from plasmid pcDNA/B7 containing the full-length cDNA of murine CD80 from murine spleen cells, and cloned to the eukaryotic expression vector pcDNA3.0 by directional cloning. The resultant recombinant plasmid pcDNA/CD80-IgG was transfected into CHO cells with liposome transfection reagent. The stably expressing cells were obtained by G418 screening. Western blot, Dot ELISA, and flow cytometry were used to detect the expression of the fusion protein and its immunological activity. RESULTS: DNA sequencing verified the correction of the construction of recombinant plasmid pcDNA/CD80-IgG. The expressed fusion protein was detected in the supernatant of transfected CHO cells and the molecular weight of the protein was similar to what we expected. Its immunological activity was also established. CONCLUSION: The recombinant plasmid pcDNA/CD80-IgG was successfully constructed and it expressed the fusion protein CD80-IgG.
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To clone and construct eukaryotic expression plasmid containing mouse 4-IBB extra-membrane encoding region and human IgG Fc fusion gene, express 4-1BB-Fc fusion protein with high biological activity, and study its biological effect in vitro. Mouse full-length 4-1BB cDNA was cloned from total RNA of the mouse spleen with RT-PCR technique, ligated to the pGEM-T Easy vector and identified by sequence scanning. Its extra-membrane encoding region was cloned, digested by restrictive enzyme and inserted into the eukaryotic expression plasmid pcDNA3.1 together with human IgG1 Fc cDNA. The recombinant plasmid, pcDNA3.1-4-1BB-Fc, was transfected into COS-7 and CHO cells by using LipofectAMINE~(TM)2000, and the CHO cell lines stably expressing the fusion protein was obtained through G418 selection. The 4-1BB-Fc protein was purified by protein A affinity chromatography column. The expression of 4-1BB-Fc was identified by sandwich ELISA and Western blot. The binding of 4-1BB-Fc protein to 4-1BBL expressed in DC cell line DC2.4 was determined by flow cytometry (FACS). The suppression effect of 4-1BB-Fc on T lymphocyte proliferation in vitro was tested by MTT and CFSE-labeling method. The ORF and linking sequence of 4-1BB-Fc gene was coincident with what was expected. ELISA and Western blot confirmed protein expression in CHO cells and the purified 4-1BB-Fc protein was proved to suppress T lymphocyte proliferation in vitro. The 4-1BB-Fc eukaryotic expression vector was successfully constructed and a purified recombinant 4-1BB-Fc protein with biological activity was obtained. This lays the experimental foundation for further studies on the role of 4-1BB-Fc in transplant rejection and other biological functions.
Lipofectamine
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Objective To construct eukaryotic expression vector of attenuated Salmonella typhimurium containing hCTLA-4Ig cDNA and identify its expression in COS-7 cells for the further study of function in SLE models. Methods Touchdown PCR was used to amplify hCTLA-4Ig cDNA.The PCR product was ligated into the multiple clone site of eukaryotic expression vector pcDNA3.1(+) by gene recombination technique.Then the recombinated plasmid pcDNA3.1(+)-CTLA-4Ig was transfected into COS-7 cells using DOTAP.The expression of interest protein in the supernatant of the cell disruption was detected with SDS-PAGE and Western blotting.Results Restriction analysis and DNA sequence analysis showed that the CTLA-4Ig cDNA had been successfully inserted into pcDNA3.1(+) eukaryotic expression vector.The interest protein could be detected in the supernatant of cell disruption 48h after the transfection of pcDNA3.1(+)-CTLA-4Ig.This protein specifically bound with human CTLA-4 monoclonal antibody.Conclusion The eukaryotic expression vector containing hCTLA-4Ig gene was successfully constructed and bioactive interest protein could be successfully expressed in mammalian cells.
clone (Java method)
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Objective To construct eukaryotic expression vector containing the extracellular domain of the human AChRα1-subunit(Hα1-210) and Fc fragment of human IgG1,and to express human AChR-Fc fusion protein.Methods The extracellular domain of Hα1-210 was amplified and inserted into pAN1782 eukaryotic expression vector with a cytomegalovirus promoter,a murine immunoglobulin κ-chain leader sequence and the human genomic IgG γ1 constant region from the hinge to the end of CH3.Recombinant expression vector pAN-Hα1-210 was constructed and evaluated by restriction enzyme analysis and sequencing.The right pAN-Hα1-210 plasmid was transfected into CHO-K1 cells with lipofectin reagent and selected by G418.The positive clone with high expressing fusion protein were selected through ELISA detection and cultured.After purified by protein A affinity column chromatography,the AChR-Fc fusion protein was identified by SDS-PAGE and Western blot assay.Results Restriction enzyme and sequencing indicated Hα1-210 gene sequence was correct and pAN-Hα1-210 had been constructed successfully.AChR-Fc fusion protein could be detected by ELISA assay in the CHO-K1 culture supernatant.There was only one special band at the position of relative molecular mass 51 000 by SDS-PAGE assay,and it was equivalent to expected value.Western blot analysis also showed that fusion protein could react to mAb198.Conclusions We successfully constructed eukaryotic expression vector pAN-Hα1-210 and obtained AChR-Fc fusion protein with biological activity,thus lay a foundation for further works on the treatment of myasthenia gravis(MG) by targeting B cells.
Fragment crystallizable region
Protein A/G
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Objective: To construct the anti-human IL-33 scFv-Fc recombinant vector and transfection of Chinese hamster ovary cells( CHO),then express and identify the fusion protein. Methods: The IgG1 Fc was amplified and inserted into eukaryotic expression plasmid pcDNA3. 1 to construct recombinant vector pcDNA3. 1 / Fc. The signal peptide( SP) was synthesized and inserted into plasmid pcDNA3. 1 / Fc to construct recombinant vector pcDNA3. 1 / SP-Fc. Finally,the human anti-IL33 scFv which was selected in our previous work was inserted into plasmid pcDNA3. 1 / SP-Fc to construct recombinant vector pcDNA3. 1 / SP-scFv-Fc. After sequencing,the CHO cell was transfected by the correct recombinant plasmid pcDNA3. 1 / SP-scFv-Fc and the level of transcription and translation of target SP-scFv-Fc was identified by RT-PCR and Western blot. Results: The sequencing results showed that the recombinant vector pcDNA3. 1 / SP-scFv-Fc was successfully constructed and the size of inserted SP-scFv-Fc was about 1 560 bp. The RT-PCR results showed that the target SP-scFv-Fc was successfully transfected into CHO cells and the Western blot results indicated that the expressed protein had the specific binding reactions with goat anti human IgG1 Fc antibody. Conclusion: the SP-scFv-Fc eukaryotic expression vector was successfully constructed and could be used for the protein expression in CHO cells.
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Objective To construct a eukaryotic expression vector encoding human OX40-IgG1 fusion gene,express OX40Ig fusion protein with high biological activity,and make preparation for the gene therapy of induction of composite tissue allograft transplants immune tolerance.Methods The cDNA of extracellular domain of human OX40 gene and IgG1 Fc gene were synthesized and inserted into pUC57(+) vector,and then subcloned into eukaryotic expression vector pDC315(+) to construct the plasmid pDC315-OX40Ig.The constructed recombinant plasmid was identified by PCR,restriction enzymes and DNA sequence analysis.After these identifications,the recombinant plasmid was transfected into NIH/3T3 cells with Lipofectamine 2000.Expression and secretion of OX40Ig was confirmed by SDSPAGE and Western Blotting,and the inhibitory effect of OX40Ig on MLR in vitro was observed by MTT assay.Results A DNA fragment about 1320 bp was obtained.The OX40Ig gene sequence of pDC315-OX40Ig was consistent with GenBank.Restriction enzymes and PCR identification proved that the OX40Ig gene was correctly cloned into expression vector.SDS-PAGE and Western Blotting analysis showed that OX40Ig fusion protein was expressed in NIH/3T3 cells culture supernatant successfully.The relative molecular mass(Mr) of the expression protein was 48000,which wasaccorded with the predicted Mr value.It was confirmed that the OX40Ig protein can inhibit MLR in vitro.Conclusion The eukaryotic expression vector pDC315-OX40Ig was successfully constructed and OX40Ig fusion protein can inhibit lymphocytes proliferation.This study has laid the foundation for the intervention of composite tissue allograft rejective reaction.
Lipofectamine
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To construct a eukaryotic expression vector containing human complement receptor 2 (CR2)-Fc and express the CR2-Fc fusion protein in Chinese hamster ovary (CHO) cells.The extracellular domain of human CR2 and IgG1 Fc were respectively amplified, ligated and inserted into the eukaryotic expression vector PCI-neo. After verified by restriction enzyme digestion and sequencing, the recombinant plasmid was transfected into CHO K1 cells. The ones with stable expression of the fusion protein were obtained by means of G418 selection. The expression of the CR2-Fc fusion protein was detected and confirmed by SDS-PAGE and Western blotting.Restriction enzyme digestion and sequencing demonstrated that the recombinant plasmid was valid. SDS-PAGE showed that relative molecular mass (Mr;) of the purified product was consistent with the expected value. Western blotting further proved the single band at the same position.We constructed the eukaryotic expression vector of CR2-Fc/PCI-neo successfully. The obtained fusion protein was active and can be used for the further study of the role in HIV control.
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Cloning (programming)
clone (Java method)
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【Objective】The study constructed eukaryotic expression vector IL-32-IgG4(Fc)-pOptiVEC for expression of IL-32-IgG4(Fc) fusion protein in CHO cells.【Method】IL-32 gene was amplified by PCR from the cDNA library of human CD4+ T cell after LPS induction and inserted into IgG4(Fc)-pOptiVEC resulting in an eukaryotic expression vector IL-32-IgG4(Fc)-pOptiVEC.Then the eukaryotic expression vector was identified by enzyme digestion and sequencing.The CHO/DG44 cells were successfully transfected with the lined plasmid IL-32-IgG4(Fc)-pOptiVEC by Lipofectamine TM2000 and confirmed by detecting mRNA of the IL-32 in the transfected cells by RT-PCR.The positive clones were further screened in the presence of MTX at different concentrations.The IL-32-IgG4(Fc) fusion protein was purified from medium from the transfected CHO/DG44 clones by protein G affinity chromatography and then its expression and bioactivity were identified by SDS-PAGE and Western-blot.【Result】A 564 bp long cDNA sequence of human IL-32 gene was cloned by PCR.The sequence of IL-32 was consistent with that reported in GenBank.Eukaryotic expression vector IL-32-IgG4(Fc)-pOptiVEC was confirmed constructed successfully by restriction enzyme digestion,and was transfected into the CHO/DG44 cells which were further selected in presence of MTX.The immune activity of the fusion protein was verified by Western-blot,and its relative molecular weight was about 50 ku,which was very close to its expected value.The production of IL-32-IgG4(Fc) fusion protein was increased obviously by MTX selection.【Conclusion】An eukaryotic expression vector IL-32-IgG4(Fc)-pOptiVEC has been constructed successfully and the recombinant CHO/DG44 cell clone that can steadily express IL-32-IgG4(Fc) fusion protein with bioactivity has been obtained.
Lipofectamine
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The mCCL19 coding sequence without stop codon was amplified with specific primers,digested with enzymes,ligated and subcloned to reconstruct the recombinant eukaryotic expression vector pcDNA3.1-mCCl19Ig containing mouse chemokine 19 and human IgG1-Fc fragment fusion gene.This plasmid was then transfected into mammalian CHO cell line with LipofectAMINE reagent induction after sequencing.The expression of mCCL19Ig fusion protein in the conditioned tranfectants of CHO cells was assayed by RT-PCR and Western blotting,and the chemotactic activity of this protein was determined with Boyden chamber.It was demonstrated that this recombinant expression vector contained the coding sequences for mCCL19 and human IgG1-Fc fragment,and the inserted fragment was identifical with that of the published sequences in GenBank.The reading frame of the human IgG1-Fc fragment showed no any alteration.AS demonstrated by Western blotting,there was significant expression of the fusion protein with molecular mass of 39 Mr×10~2 in the culture supernatants of CHO cells transfected with vector pcDNA3.1-mCCL19-Ig.Also,definite chemotactic activity to the mouse spleen cells of this fusion protein could be demonstrated by in vitro chemotactic experiment with a dose-dependent relationship.
Lipofectamine
Coding region
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