Cloning and expression of the extracellular carbohydrate recognition domain of the fungal β-glucan receptor Dectin-1
Abstract：For further research on the biological functions of the extracellular carbohydrate recognition of β-glucan receptor Dectin-1, we cloned and expressed CRD gene of mouse peritoneal macrophages. The Dectin-1 CRD gene was amplified by RT-PCR from RNA of mouse peritoneal macrophages, and then inserted into prokaryotic expression vector pET28a (+). The size of the constructed recombinant plasmid digested by restricted enzymes of Nde I and Xho I were 552 bp and 5 369 bp, which were coincident with the expected sizes, while sequence analysis showed the plasma was completely correct, and the inserted target gene and its reading frames were coincidence with the expression vectors. Then the constructed recombinant plasmid was transformed into Escherichia coli BL21 (DE3) strain and induced by IPTG. The fusion protein had relative molecular mass M_r 22 000 by SDS-PAGE, and the specific positive signal was identified by Western blotting, β-glucans in the cell wall of fungi could be recognized by the fusion protein. All these results indicated that the recombinant expression plasmid pET28a-CRD was successfully constructed, and the expressed fusion protein in E.coli B121 may facilitate further functional study of the dectin-1 CRD gene.
Keywords：Fungusp-glucansProkaryotic expressionDectin-1
Publication Date：2010-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：4( 309-311,319 )