Preparation and characterization of the rabbit polyclonal antibody against the alpha3 domain of human CD1d

AIM:To prepare the rabbit antibody against the α3 domain of the human CD1d(hCD1d-α3).METHODS:The gene fragment coding for hCD1d-α3 was amplified by PCR and cloned into prokaryotic expression vector pET28,then expressed in E.coli BL21(DE3).The recombinant protein hCD1d-α3 was purified with Ni2+-NTA agarose column and used as immunogen to immunize the rabbit.The titer and specificity of the anti-hCD1d-α3 antibody from the rabbit were analyzed by ELISA,Western blot and immunohistochemistry,respectively.RESULTS:The recombinant hCD1d-α3 was successfully expressed and purified,and the polyclonal anit-hCD1d-α3 antibody was successfully prepared.The titer of the antiserum was 1∶6400 by ELISA.Western blot analysis showed this antiboday reacted specifically with hCD1d.Immunohistochemistry analysis showed the antibody could recognize the native hCD1d in the human intestinal tissue.CONCLUSION:The anti-CD1d-α3 antibody from the rabbit with high titer and specificity has been prepared with purified recombinant hCD1d-α3 as immunogen,which lays a foundation for further research into detection and functional study of CD1d.