Prokaryotic expression of human mast cell chymase gene and preparation of its polyclonal antibody

AIM:To express the human mast cell chymase cDNA in E.coli and prepare the antibody against human mast cell chymase with recombinant chymase.METHODS:The human mast cell chymase cDNA was cloned by RT-PCR.The recombinant chymase was expressed in E.coli with L-Arabinose induction and purified by Ni-NTA agarose column.Then the purified chymase was used as immunogen to immunize the rabbit.The titer and specificity of the anti-chymase antibody from the rabbit were analyzed by indirect ELISA and Western blot,respectively.RESULTS:The recombinant chymase was successfully expressed in E.coli,and the polyclonal anit-chymase antibody was prepared by immunizing the rabbit with the purified recombinant chymase.The titer of the generated antiserum was detected to be 1:12 800 by ELISA.Western blot analysis showed this antiboday bound specifically with chymase.CONCLUSION:The anti-chymase antibody from the rabbit with high titer and specificity has been prepared with purified recombinant chymase as immunogen,which lays a foundation for further research into detection and function of chymase.