[Expression of mouse B7-H3-Fc fusion protein and characterization of its bioactivity].
OBJECTIVE
To obtain mouse B7-H3-Fc fusion protein and to investigate its biological function and effects on T lymphocyte activation.


METHODS
The genes coding extracellular domain of mouse B7-H3 and the Fc fragment of human IgG1 were amplified from pMD19-T/mouse B7-H3 and pMD19-T/human IgG1 vectors by PCR. The two genes were combined with mouse B7-H3-Fc fragment by overlap PCR. Then the resulting gene fragment was inserted into eukaryotic vector pIRES2-EGFP after digested with EcoR I and Bgl II to construct the recombinant vector pIRES2-EGFP/B7-H3-Fc. The recombinant vector was transfected into CHO cells with LipfectAMINE™ 2000, and the cells were further selected with G418. The collected supernatant of the transfected cell line cultured in serum-free media was ultrafiltrated and concentrated, then purified by Protein G column. The expression of mouse B7-H3-Fc fusion protein was confirmed by Western blot. Effects of fusion protein on T cells proliferation and cytokine production in vitro was studied by methods of CCK8 and ELISA.


RESULTS
The results showed that the transfected CHO cell line secreting mouse B7-H3-Fc fusion protein was constructed successfully. In vitro, mouse B7-H3-Fc fusion protein obviously promoted the proliferation of T cells in a dose-dependent manner.


CONCLUSIONS
A transfected CHO cell line stably expressing mouse B7-H3-Fc fusion protein has been obtained and the B7-H3-Fc fusion protein stimulates the proliferation of T cells and cytokines production in vitro.
Institute of Biotechnology, Clinical Diagnosis Laboratory of the Second Affiliated Hospital, Soochow University, Suzhou 215004, China.
Ru-hong
RH
Yan
Guang-bo
GB
Zhang
Jing
J
Sun
Feng-qing
FQ
Fu
Xue-guang
XG
Zhang
D000818:Animals; D060887:B7 Antigens; D018122:B7-1 Antigen / Q000096:biosynthesis / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000096:biosynthesis / Q000235:genetics*; D007371:Interferon-gamma / Q000096:biosynthesis; D007376:Interleukin-2 / Q000096:biosynthesis; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology
D060887:B7 Antigens; D018122:B7-1 Antigen; C486038:Cd276 protein, mouse; D007141:Immunoglobulin Fc Fragments; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D007371:Interferon-gamma