[Cloning, prokaryotic expression, and antigenicity identification of extracellular domain of mouse CD40].
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi
; 29(11): 1200-4, 2013 Nov.
Article
in Zh
| MEDLINE
| ID: mdl-24200070
ABSTRACT
OBJECTIVE:
To construct a prokaryotic expression plasmid for extracellular domain of mouse CD40 (mCD40), express the mCD40/GST recombinant protein in E.coli, purify the mCD40/GST recombinant protein and characterize its antigenicity.METHODS:
Extracellular domain of mouse CD40 was amplified by PCR from cell line DC2.4 and then was cloned into prokaryotic expression vector pGEX-6P-1 to construct the recombinant expression vector pGEX-6P-1-mCD40. The expression vector was transformed into E.coli BL21 (DE3) and the fusion protein mCD40/GST was induced by IPTG. The fusion protein was purified through sepharose 4B. Then antigenicity of the purified mCD40/GST protein was verified by Western blotting and ELISA.RESULTS:
The PCR product was verified by DNA sequencing to be consistent with the sequence of mouse CD40 on GenBank. The recombinant plasmid was identified by double digestion successfully. SDS-PAGE analysis showed the relative molecular mass of the fusion protein induced by IPTG was 45 000. Western blotting and ELISA demonstrated that the purified mCD40/GST protein had a good antigenicity.CONCLUSION:
The prokaryotic expression plasmid pGEX-6P-1-mCD40 was constructed successfully. In E.coli BL21 (DE3) transformed with the plasmid, the mCD40/GST fusion protein was expressed by IPTG induction. The purified mCD40/GST fusion protein had a high antigenicity, which provides a strong support for the future study of CD40.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
CD40 Antigens
/
Escherichia coli
/
Extracellular Space
Type of study:
Diagnostic_studies
/
Prognostic_studies
Limits:
Animals
Language:
Zh
Journal:
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi
Journal subject:
ALERGIA E IMUNOLOGIA
Year:
2013
Document type:
Article
Affiliation country:
China