Construction and eukaryotic expression of human CD137-Fc chimeric molecule
- VernacularTitle:人重组CD137-Fc分子的构建与真核表达
- Author:
Gang HUANG
;
Wanling LI
;
Man JIANG
;
Yun BAI
;
- Publication Type:Journal Article
- Keywords:
CD137;
CD137 Fc;
eukaryotic expression
- From:Journal of Third Military Medical University
2003;0(24):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct eukaryotic expression plasmid of hCD137 Fc gene and express hCD137 Fc fusion protein with high biological activity. Methods PCR technique was employed to clone the human CD137 cDNA from a normal human activated T cells cDNA library, and then clone its extramembrane encoding region. The extramembrane sequence together with human IgG1 Fc cDNA were inserted into the eukaryotic expression plasmid pcDNA3. CD137 Fc gene was expressed transiently in 293T cells, and then CD137 Fc protein was purified by recombinated protein A affinity chromatography column. At last, the MW, purity and antigenicity of CD137 Fc were identified by sandwich ELISA, SDS PAGE and Western blotting, respectively. Results The ORF of CD137 Fc gene was coincident with what we expected. ELISA and SDS PAGE confirmed protein expression in 293T cells. Western blotting proved the antigenicity of the purified CD137 Fc protein. Conclusion We obtained a purified recombinated CD137 Fc protein with biological activity and the expected MW. This lays the foundation for further studies of CD137 such as its role in immune homeostasis and other biological functions.
