[Preparation and characterization of anti-Amelotin polyclonal antibody].

AIM

To construct a mouse Amelotin recombinant plasmid, express mouse Amelotin protein and prepare its polyclonal antibody.

METHODS

The cDNA sequence of mouse Amelotin gene was downloaded from GenBank. The coding region, which did not include signal peptide, was amplified by RT-PCR. Then it was recombined into prokaryotic expression vector pET32a and transformed into E.coli BL21(DE3). After induced by IPTG, the recombinant protein was expressed and purified using affinity purification. The polyclonal antibody was obtained from New Zealand rabbit immunized with the recombinant protein and the titer was identified by ELISA. The polyclonal antibody was identified by Western blot and immunohistochemistry assays.

RESULTS

The recombinant prokaryotic expression vector pET32a-Amelotin was constructed and the recombinant prorein was purified successfully. ELISA analysis showed the titer of the generated antiserum was 1:12,800. Western blot and immunohistochemistry analysis demonstrated this antibody bound specifically with Amelotin.

CONCLUSION

The anti-Amelotin antibody from the rabbit with high titer and specificity had been prepared with the purified recombinant Amelotin as immunogen, which is helpful for further research into the detection and function of Amelotin.