[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:
Myc-tag
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To explore the feasibility of recombinant human interleukin 12 (IL-12) with biological activities by molecular biological techniques.Both p40 and p35 subunits cDNA of human IL-12 were cloned from mRNA extracted from NC-37 cell line by using RT-PCR, and the fusion gene (p40-linker-p35) of recombinant human single chain IL-12 (rhscIL-12) was constructed by using a polypeptide linker (Gly(4)Ser)(3). rhscIL-12 eukaryotic expressing vector pcDNA3.1 (+)-hscIL-12 was constructed by inserting the rhscIL-12 fusion gene into pcDNA3.1 (+) eukaryotic expressing plasmid. COS-7 cells were transfected with pcDNA3.1 (+)-hscIL-12 plasmid.A stable rhscIL-12 expressing cell line COS-rhscIL-12 was obtained by G418 selection. Western blot showed the presence of a 70 x 10(3) band of the fusion protein, which specifically bond to mouse-anti-human IL-12 monoclonal antibody. The assays of biological functions showed that the fusion protein had strong bioactivities in stimulating the lymphocyte proliferation, enhancing the NK cell cytotoxicity and increasing the IFN-gamma production.The constructed rhscIL-12 fusion gene could express biologically functional rhscIL-2 fusion protein.
Cell fusion
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Objective To construct eukaryotic expression plasmid of hOX40-Fc gene and stably express hOX40-Fc fusion protein with biological activity. Methods The extramembrane encoding region of OX40 molecule was cloned from a normal human activated T cells cDNA library by polymerase chain reaction. After sequencing, the extramembrane encoding region together with human IgG1-Fc cDNA was inserted into the eukaryotic expression plasmid pcDNA3. The right recombinant was transfected into COS-7 cells with lipofectamine reagent. OX40-Fc protein was purified by recombinated protein A affinity column chromatography. The molecular weight, purity, and antigenicity of OX40-Fc were identified by sandwich ELISA, SDS-PAGE, and Western blotting. Results The open-reading frame of OX40-Fc gene was coincident with what we had expected. OX40-Fc protein expression in COS-7 cells was confirmed, and the antigenicity of the purified OX40-Fc protein was analyzed. The purified OX40-Fc protein could inhibit the growth of activated Jurkat cells by proliferation assay. Conclusion The vector is constructed successfully and a purified recombinated OX40-Fc protein is obtained. This lays a foundation for further studies of OX40, such as its role in autoimmune diseases and immune homeostasis.
Lipofectamine
Antigenicity
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Objective:The immunogenicity of DNA vaccine immunogenicity can be improved by fusing antigenic genes to IgG Fc fragment.It is critical for enhancing immunogenicity of DNA vaccine to construct the secreting eukaryotic expressing vector.The purpose of this study is to construct a secreting eukaryotic expressing vector ligated with IgG Fc gene by amplifying human IgG Fc fragment and fusing the fragment with human CD5 signal peptide sequence for highly efficient expression.Methods:Human lymphocytes were isolated from the tonsil obtained by removal surgery.The total RNA of lymphocytes was extracted using Trizol reagent.Human IgG Fc cDNA was amplified by RT-PCR from lymphocytes and then inserted into pMD-18T vector.The Fc encoding sequence fused with CD5 signal peptide(sp) sequence was constructed by cross PCR using Fc fragment and CD5sp sequence and cloned in pcDNA-CD5 plasmid as the template.CD5sp-Fc fragment was inserted into pcDNA3.1 expressing vector to construct the pcDNA-CD5sp-Fc plasmid.The plasmids were transfected into HEK293T cells mediated by calcium phosphate.Western blot was used to detect expressed Fc protein in cultural supermatant of the cells.Results:The amplified sequence of human IgG Fc was consistent with that previously published.The secretory expression of Fc in HEK293T cells was achieved and the expressing level reached 50 μg/106 cells at 48 h culture after transfection.Conclusion:The human IgG Fc gene is amplified by RT-PCR methods and the secretory expression of Fc gene mediated by CD5 signal peptide in 293T cells is achieved.The results provide a experimental basis for further construction of antigen genes fused to IgG Fc fragment and investigation on DNA vaccines and Fc as a biological adjuvant.
Fragment crystallizable region
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Objective To clone human CD52 gene and construct a eukaryotic expression vector for stable expression in CHO cells.Methods The total RNA of human Hut-78 cells was extracted for amplification of CD52 gene by RT-PCR.The amplified gene was cloned into eukaryotic expression vector pcDNA3.1(+),and the constructed recombinant plasmid pcDNA3.1(+)/CD52 was transfected to CHO cells in mediation of liposome to establish a CHO-CD52 cell line for stable transfection.The transcription of target gene was determined by RT-PCR,and the expression of target protein by immunofluorescent histochemical assay and flow cytometry.Results The DNA fragment at a length of 186 bp was amplified by RT-PCR.PCR,restriction analysis and sequencing proved that recombinant plasmid pcDNA3.1(+)/CD52 was constructed correctly.The target gene band at a length of 186 bp was detected in CHO-CD52 cells by RT-PCR.Green fluorescence was observed in CHO-CD52 cells by immunofluorescent histochemical assay.Flow cytometry showed that the percentage of positive cells and mean fluorescence intensity of CHO-CD52 cells were 98.18 and 193.56 respectively.Conclusion Human CD52 gene was successfully cloned,and the CHO cell line for stable transfection was established,which laid a foundation of preparation of CD52 monoclonal antibody and development of antibody drugs.
CD52
clone (Java method)
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AIM: To construct the eukaryotic expression vector of DDR2-Fc fusion gene and to investigate its expression in HEK293 cells. METHODS: The two cDNAs was amplified by PCR respectively from the rat brain tissue and the plasmid con- tainingthe full-length cDNA of human IgG1 Fc,and cloned to the eukaryotic expression vector pcDNA3.1(-) by directional cloning.The resultant recombinant plamid pcDNA3.1(-)/DDR2-Fc was transfected into HEK293 cells with liposome transfection reagent. Then RT-PCR, Western Blot were used to detect the expression of the fusion protein. RESULTS: DNA sequencing and restriction enzyme digestion verified the correction of recombinantplasmid pcDNA3.1(-)/DDR2-Fc. The expressed fusion protein was detected in the transfected HEK293 cells and the molecular weight of the protein was the same as we expected. CONCLUSION: The recombinant plasmid pcDNA3.1(-)/DDR2-Fc was successfully constructed and the fusion protein DDR2/ Fc was expressed in HEK293 cells.
HEK 293 cells
Cloning (programming)
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To construct an eukaryotic expression vector of CD80-IgG1 Fc, and to express the fusion protein in CHO cells.The gene encoding the CD80-IgG1 Fc fusion protein were constructed in eukaryotic expression vector pcDNA3.1(+) by means of T-A cloning and subcloning techniques, then was transfected into CHO cells for stable expression. The expression of the fusion protein was detected by Western blot and ELISA.DNA sequencing and restriction endonuclease digestion analysis indicated that the eukaryotic expression vector CD80-IgG1 Fc/pcDNA3.1(+) was successfully constructed. After the recombinant plasmid was transfected into CHO cells, the stable expression of the fusion protein was demonstrated by Western blot and ELISA.The eukaryotic expression vector of CD80-IgG1 Fc/pcDNA3.1(+) was successfully constructed and stably expressed in CHO cells, providing basis for anti-tumor study.
Subcloning
CD80
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Objective To construct eukaryotic expression plasmid of human PD-1 extracellular region-hIgG1Fc-pcDNA3.1(+) eukaryotic expression vector, and express the functional fusion protein in mammalian cell. Methods Encoding sequence of human PD-1 extracellular region was amplified, and then inserted together with hIgG1Fc into pcDNA3.1(+) expression vector. The right recombinant was transfected into mammalian CHO cell by lipofectamine reagent. The supernatant of the cultured cell was collected and analyzed by the sandwich ELISA. The fusion protein was purified by HiTrap recombination protein A affinity chromatography. The molecular weight and immune activity of the fusion protein PD-1 were detected by Western blotting and SDS-PAGE. Results The extracellular region of hPD-1 about 727 bp was cloned from human T cell cDNA library, and then inserted together with hIgG1Fc into the eukaryotic expression vector pcDNA3.1(+). After the transfection of recombinant into mammalian CHO cell by lipofectamine reagent, the expression of PD-1 fusion protein was detected in the cultured CHO cell supernatant by the sandwich ELISA. The immune activity of the fusion protein was verified by Western blotting, and its relative molecular weight was about 42 000, which was very close to its expected value. Conclusion The hPD-1-Fc chimeric molecule is constructed and expressed successfully, which provides base for further investigation of the role of PD-1 in immune tolerance and autoimmune disease.
Lipofectamine
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Objective To construct eukaryotic expression plasmid of hBAFF-R-Fc gene and stably express hBAFF-R-Fc fusion protein possessing biological activity. Methods The extramembranous encoding region of hBAFF-R molecule was chemically synthesized by making 4 long oligos,followed by annealing and PCR to get the extracellular domain of coding sequence. The mutant hIgG4 was cloned by PCR with primers in order to change Leu235 to a Glu. The extramembranous encoding region of hBAFF-R fragment was digested with PciⅠ and XhoⅠ and cloned into the same sites in pSec/WG vector which harbored a mutant hIgG4 tail. The right recombinant was transfected into CHO cells with lipofectamine reagent. hBAFF-R-Fc protein was purified by recombinated protein A affinity column chromatography. The molecular weight,purity,and activity of hBAFF-R-Fc were identified by sandwich ELISA,SDS-PAGE,and Western blotting,respectively. Results The open reading frame of hBAFF-R-Fc gene was coincident with what we had expected. HBAFF-R-Fc protein expression in CHO cells was confirmed,and the activity of the purified hBAFF-R-Fc protein was analyzed. The purified hBAFF-R-Fc protein could inhibit the growth of activated B cells. Conclusion The vector is constructed successfully and a purified recombinated hBAFF-R-Fc protein is obtained,which lays a foundation for further studies of hBAFF-R-Fc,such as its role in lupus nephritis and other autoimmune diseases.
Lipofectamine
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Construction,expression and identification of human B7-H1-Fc chimeric molecule in mammalian CHO cell
Objective To construct eukaryotic expression plasmid of human B7-H1 extracellular region-hIgG1Fc-pCI-neo eukaryotic expression vector,and express the functional fusion protein in mammalian CHO cell.Methods Full-length human B7-H1 encoding sequence was amplified from human activated T cell cDNA library by PCR,fused with hIgG1Fc,then transformed into pCI-neo expression vector and verified by sequencing.The validated recombinant was transfected into mammalian CHO cell by lipofectamine reagent.The supernatant of the cultured cell was collected and analyzed by the sandwhich ELISA to detect if there was the fusion protein,and the fusion protein was purified by HiTrap recombination protein Protein A affinity chromatography.The concentrated supernatant or purified fusion protein were used for Western blotting after SDS-PAGE to identify the molecular weight and immune activity of the fusion protein of B7-H1.Results The extracellular region of hB7-H1 about 727 bp was cloned from human T cell cDNA library and was inserted with hIgG1Fc into the eukaryotic expression vector pCI-neo.After the transfection of recombinant into mammalian CHO cell by lipofectamine reagent,the expression of B7-H1 fusion protein was detected in the cultured CHO cell supernatant by the sandwhich ELISA.The immune activity of the fusion protein was verified by Western blotting,and its molecular weight was about 51.76×10~(3),very close to the expected value.Conclusion The hB7-H1-Fc chimeric molecule was successfully constructed and the expression of its functional fusion protein in mammalian CHO cells lays a foundation for further research on the role of B7-H1 in immune tolerance,autoimmune diseases.
Lipofectamine
Protein A/G
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AIM To construct an eukaryotic expression vector pEF-BOSneo-hdll1(ext)-Fc, and to express the fusion protein consisting of the extracellular region of delta-like1 and Fc fragment of human IgG1 in COS-7 cells. METHODS The extracellular region of human delta-like1 was amplified from a human brain cDNA library by PCR. The expression vector was constructed by DNA recombination. The recombinant plasmid was transfected into COS-7 cells via liposome mediation. The expression of the fusion protein was detected by RT-PCR, immunofluorescence assay and sandwich ELISA. RESULTS DNA sequencing and restriction endonuclease digestion analysis indicated that the eukaryotic expression vector pEF-BOSneo-hdll1(ext)-Fc had been constructed successfully. After recombinant plamid had been transfected into COS-7 cells, RT-PCR and DNA sequencing verified that the dll1(ext) gene and IgG1Fc gene were fused correctly. The results of immunofluorescence assay were positive and the fusion protein could be detected by sandwich ELISA in culture supernatant of transfected COS-7 cells. CONCLUSION hdll1(ext) was successfully cloned and expressed in the form of Fc fusion protein, which is helpful for further study of the function of Notch pathway.
Immunofluorescence
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