The testis-specific protein DMRT-like of the Chinese mitten crab:antibody preparation and immunological identification

Dmrt is a family of genes related to the sexual regulators Doublesex and Mab-3.Our previous studies showed that EsDmrt-like transcripts were detectable only in testis of Chinese mitten crab,Eriocheir sinensis.In order to verify whether EsDMRT-like protein is also specifically expressed in testis,the prokaryotic expression plasmid pET-32a-EsDmrt-like was constructed according to the sequence of EsDmrt-like.The recombinant plasmid was transferred into host bactria Escherichia coli BL21(DE3),and the recombinant protein Trx-EsDMRT was expressed under the induction of IPTG.SDS-PAGE analysis showed that the fusion protein was highly expressed as inclusion body with the molecular weight of 46 ku.The recombinant protein was purified by affinity for preparing EsDMRT-like antibody.The resultant antibody can specifically recognize the recombinant protein and endogenous EsDMRT-like protein in the testis as revealed by Western-blotting analysis.EsDMRT-like was specifically expressed in the testis,with molecular weight of 52 ku,which is twice as those of the monomer.After denatured treatment by urea and DTT,the protein displayed two forms of polypeptides:52 and 34 ku.Both of them can be recognized by antiserum raised against EsDMRT-like protein,indicating that the form of 34 ku was generated from 52 ku polypeptide and the EsDMRT-like protein exists as a dimer in testis.This result suggested that EsDmrt-like may regulate the male sex development/differentiation by dimerization in the crab.