ABSTRACT
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.
Subject(s)
Antigens, Differentiation/metabolism , Antigens, Differentiation/pharmacology , Recombinant Fusion Proteins/pharmacology , Animals , Antigens, Differentiation/genetics , B-Lymphocytes/cytology , B-Lymphocytes/drug effects , CHO Cells , Cell Proliferation/drug effects , Cells, Cultured , Chromatography, Affinity , Cricetinae , Cricetulus , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Genetic Vectors/genetics , Humans , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/metabolism , Mice , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
AIM: To establish HUVECs line expressing mouse OX40(CD134) and to study its promotive effect on proliferation of B cells. METHODS: The cDNA fragment encoding mouse OX40 was obtained from the total RNA of ConA-activated lymphocytes of thymus by using RT-PCR and cloned into pUCm-T vector. The cDNA was then inserted into the eukaryotic expression vector pIRES2-EGFP. The recombinant vector was transfected into HUVECs with lipofectin reagent, and the positive cellular clones were selected by G418. Expression of mouse OX40 in the transfected cells was analyzed by FCM. The differentiation of B cells in vitro induced by OX40 signal was studied by means of (3)H-TdR method. RESULTS: The cDNA fragment in the recombinant plasmid was consistent with the reported mouse OX40 cDNA in GenBank, which was confirmed by DNA sequencing, PCR and enzyme digestion. The HUVECs stably expressing the mouse OX40 were successfully cloned. The transfected cells promoted the differentiation of B cells in vitro and there existed a synergic effect between OX40/OX40L and CD40/CD40L signals. CONCLUSION: Transfected cell line expressing the mouse OX40 gene is established successfully. OX40 enhances the proliferation of B cells.
Subject(s)
B-Lymphocytes/cytology , Cell Differentiation/physiology , Receptors, OX40/physiology , Animals , B-Lymphocytes/metabolism , Cell Differentiation/genetics , Cell Line , Cells, Cultured , DNA, Complementary/genetics , Female , Humans , Mice , Mice, Inbred BALB C , Receptors, OX40/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transfection/methodsABSTRACT
AIM: To explore the possibility of application of self-made anti-ssDNA mAb to detecting the apoptosis of tumor cells induced by chemotherapeutic drugs and evaluate the sensitivity of tumor cells to chemotherapeutic drugs from a apoptotic rate. METHODS: Apoptosis of both K562 and Hep-2 cells were induced by maximum plasma concentrations of 9 different chemotherapeutic drugs. The apoptosis of tumor cells was detected by immunohistochemical ( IHC) staining with anti-ssDNA mAb and their apoptotic rate were calculated. RESULTS: The apoptotic cells could be differentiated from non-apoptotic cells by the HIC staining with anti-ssDNAmAb. The apoptosis of tumor cells were induced by chemotherapeutic drugs, but apoptotic extent of different tumor cells, even the same one tumor cell, was distinct when they exposed to all kinds of anti-tumor drugs. CONCLUSION: The combination of anti-ssDNA mAb with IHC staining can be used to detect the sensitivity of tumor cells to chemotherapeutic drugs.
