Preparation and characterization of polyclonal antibodies against CD26

Objective To prepare polyclonal antibody against the catalytic center of CD26.Methods The coding gene of CD26 amplified using RT-PCR technology with mRNA template from human leucocyte was inserted into prokaryotic expressive vector PET-32a,and then the recombinant plasmids were transformed into BL21 and induced by IPTG.The fusion protein was purified by Ni+ affinity column chromatography,and then was used to immune two rabbits after being conformed by Western-blot.The 180 ml of the polyclonal antibody serum was then purified by protein A column and antibody affinity column.The titer and specificity were determined by ELISA,Western-blot and Immunocytochemistry.Results PET32a/CD26 prokaryotic expression plasmids were constructed successfully and highly expressed in BL21.Antigen concentration of the fusion protein was up to 2.3 mg/ml after being purified by Ni+ affinity column chromatography.The polyclonal antibody against CD26 was obtained with titer up to 1∶256000.Western-blot and Immunocytochemistry revealed that the final purified antibody was specific to the recombinant and native CD26.Conclusion The highly specific polyclonal antibody against the catalytic center of CD26 was obtained successfully.