[Expression of mOX40-Ig and its biological activity study].
OBJECTIVE
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.


CONCLUSIONS
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.
Liu
Yan-fei
YF
Laboratory of Molecular Immunology, Weifang Medical College, Weifang 261042, China.
Lin
Zhi-juan
ZJ
Feng
Yong-tang
YT
Xu
Chuan-wu
CW
Shi
Lin
L
Miao
Nai-fa
NF
D000818:Animals; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D001402:B-Lymphocytes / Q000166:cytology / Q000187:drug effects; D016466:CHO Cells; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000943:Antigens, Differentiation; D007141:Immunoglobulin Fc Fragments; C406562:OX40Ig; D011993:Recombinant Fusion Proteins