[Purification and characterization of the fusion protein TGF-betaR II/Fc].
OBJECTIVE
To express the fusion protein TGF-betaR II/Fc in large amounts by using recombinant Bac-TR II baculovirus expression system constructed by our laboratory and to purify and characterize it. Then, to verify whether the fusion protein TGF-betaR II/Fc can be able to block the biological activity of cytokine TGF-beta1.


METHODS
The viral titer was determined by plaque forming test. The recombinant baculovirus was amplified by infecting sf9 cells. The fusion protein was purified by FPLC using protein G column. The purified product was analyzed by SDS-PAGE and the amount of target protein calculated by gray scanning. Western blot and sandwich ELISA were used to affirm the expression of the fusion protein. MTT colorimetry was used to test whether the fusion protein can block the inhibition effect of cytokine TGF-beta1 on the growth of L929 cells. It was to verify whether the fusion protein can reduce the fibronectin production in L929 cells accelerated by TGF-beta1 by western blot.


RESULTS
The titer of recombinant Bac-TR II baculavirus in the primary culture fluid was 2x10(12) pfu/L. After electrophoresis, gray scanning analysis showed that the target protein accounted for 10 percent of the total protein. Western blot analysis and sandwich ELISA detection proved that the target protein has been expressed. The fusion protein could block the inhibitive effect of cytokine TGF-beta1 on the growth of L929 cells and fibronectin production in L929 cells.


CONCLUSIONS
The fusion protein TGF-betaR II/Fc can inhibit the biological activity of TGF-beta1 in-vitro. This study will be helpful to the mass production of the fusion protein, and will facilitate its further use in the therapy of pulmonary fibrosis.
Li
Sheng-Qing
SQ
Department of Respiratory Disease, Xijing Hospital,Fourth Military Medical University, Xi'an 710032, China.
Li
Huan-Zhang
HZ
Yang
Qiao-Xin
QX
Ni
Dian-Tao
DT
D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000276:immunology; D016212:Transforming Growth Factor beta / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000378:metabolism
D005353:Fibronectins; D011993:Recombinant Fusion Proteins; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1