Preparation and Identification of Polyclonal Antibody of Mouse Embryonic Cardiac Calcium Channel α1D

Objective To prepare high titer and specific polyclonal antibody of α1D subunit of L-type calcium channel encoded by Cav1.3 from mouse embryonic heart.Methods Prokaryotic expression plasmids pGEX-4T-α1DN/α1DC was obtained by recombination from mice embroynic heart.The recombinant of pGEX-4T-α1DN/α1DC was transformed into E.coli BL-21,induced and expressed by Isopropyl-β-D-thiogalactopyranoside(IPTG).Then glutathione S-transferase(GST) fusion proteins that recognize N terminus and C terminus of α1D respectively were highly expressed in E.coli and purified.Rabbits were immunized with purified GST-α1DN/α1DC.IgG type polyclonal antibody of α1D generated from gathered antiserum purified by protein G Agarose.The specificity of 2 kinds of antibody were tested by Western blot and the title of them were identified by enzyme linked immunosorbent assay (ELISA).Results The prokaryotic expression plasmid pGEX-4T-α1DN/α1DC were recombinated,the GST fusion protein GST-α1DN/α1DC were expressed and purifed and these 2 kinds of polyclonal antibody were prepared successfully.The titer of antibody by ELISA was 1:256 000 and the titer of anti-α1DN was higher than the titer of another.Western blot showed that α1D proteins extracted from mice heart were specifically recognize by either anti-α1DN or anti-α1DC.Conclusions Purified fusion GST-α1D proteins expressed by recombinants of pGEX-4T-α1D were used as antigen and the α1D polyclonal antibody with high titer and strong specificity has been successfully prepared.They will be beneficial to investigate the temporal and spatial expression pattern of α1D protein and to study the regulation of signal transduction concerning calcium ion during mouse embryonic heart development.