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Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages
Published on: October 30, 2018
[Clone and expression of murine BTLA extracellular domain gene and its effect on the expression of B7 on dendritic
Jun-fa Xu1, Kun Zhao, Bao-jun Huang
1Department of Immunology, Tongji Medical College, Huazhong Universite of Science and Technology, Wuhan 430030, China.
Aim:
To study the regulatory effect of recombinant glutathione S-transferase (GST)-extracellular domain of murine B and T lymphocyte attenuator (mBTLAext) fusion protein GST-mBTLAext on the expression of B7 on murine dendritic cell (DC) line DC2.4.
Methods:
cDNA of mBTLA was amplified from total RNA of murine splenocytes by RT-PCR. The recombinant prokaryotic expression vector pGEX-4T-2/mBTLAext was constructed by cloning mBTLAext into pGEX-4T-2 and then transformed into E. coli BL21 (DE3). The fusion protein GST-mBTLAext was expressed under the induction of 1 mmol/L IPTG, and then was extracted from inclusion body and purified through Glutathione Sepharose 4B chromatography column. The fusion protein was added into the culture supernatant of DC2.4 and its effect on the expression of B7-1 and B7-2 on DC2.4 was analyzed by flow cytometry.
Results:
The recombinant prokaryotic expression vector pGEX-4T-2/mBTLAext was constructed and the fusion protein GST-mBTLAext was expressed successfully. The molecular weight of the fusion protein was 43.0 kDa, determined by SDS-PAGE, which was corresponding to expectation. GST-mBTLAext could up-regulate the expression of B7-1, but didn't alter the expression of B7-2, on DC2.4 in a dose dependent manner.
Conclusion:
BTLA had a regulatory effect on the expression of B7 on DC. It is significant to study the effect of BTLA on the biological behaviour of DC and its molecular mechanism.
