Preparation and identification of a polyclonal antibody against tooth development related gene-adam28 product / 实用口腔医学杂志

ABSTRACT

Objective:To prepare and identify a polyclonal antibody againstadam28 gene product.Methods:Theprotein coding region of ADAM28 was amplified by RT-PCR and cloned into pMD18-T Vector to produce the newconstruct, pMD18-T-adam28. The cloned ADAM28 segment was cut with two restriction enzymes and theadam28fregment was directed into the prokaryotic expression vector, pGEX-4T-1,to produce the expression vector pGEX-4T-adam28. The recombinant plasmid was transformed intoE. coliDH5?and GST-ADAM28 fusion protein was ob-tained after the inducement by IPTG. The fusion protein was extracted and purified by SDS-PAGE,and the newpro-tein band of 35 300 was isolated as antigen, the antigen was injected into rabbits to produce polyclonal antibody a-gainst ADAM28 product.Results:The expression vector pGEX-4T-adam28 was constructed successfully,and GST-ADAM28 fusion protein was obtained. The rabbit serum containing polyclonal antibody against ADAM28 productwas obtained and the antibody was purified by salting out method. Western blot analysis displayed that the antibodyhad high specificity. ELISA analysis confirmed that the titer for the antibody reached 1∶16 000.Conclusion:Thepolyclonal antibody against ADAM28 product with high titer is successfully prepared,it may be used for further studyof the role and expression of ADAM28 during tooth development.