[Identification of functional sites of human IgA Fc receptor (CD89)]

To identify the domains and residues in IgA Fc receptor (FC alpha R) essential for interaction with IgA.

Various fragments of Fc alpha R cDNA were amplified by PCR or site-directed mutagenesis PCR. The amplified products were ligated into the EcoR I and Xho I sites of the pET30a plasmid, a prokaryotic expressing vector. Then the recombinant plasmids were transformed into Escherichia coli BL21. Plasmids with correct cDNA fragment inserts were identified by digestion with two restriction enzymes and by DNA sequencing. Expression of cDNA fragments was made by isopropyl-beta-D-thiogalactopyranoside (IPTG) induction. Western blotting was carried out to identify the functional site of Fc alpha R. Monoclonal antibodies against Fc alpha R were used as primary antibodies.

Five recombinant plasmids expressing various fragments of Fc alpha R were constructed. The monoclonal antibodies that can blot the IgA binding sites of Fc alpha R could only bind to the membrane-distal domains of Fc alpha R (EC1).

The functional site of Fc alpha R was located at EC1 domain.