Abstract The grass carp (Ctenopharyngodon idellus ) leptin gene was amplified by RT-PCR. PCR product was cloned into Pichia pastoris vector pPIC9K. The recombinant plasmid was transformed to competent cell GS115 by electroporation and expressed by methanol induction. Recombinant protein was analyzed with SDS-PAGE and Western blot. Results revealed that grass carp leptin gene had a length of 438 nucleotides which encoded a 146-amino peptide（GenBank Accession AY551335）. Its nucleotides sequence and deduced amino acid sequence homologues were compared with carp(Cyprinus carpio), humans, pigs and rats, which displayed a fairly high degree of conservation, and homologue of the nucleotides sequence were 99%, 84%, 86% and 95% respectively; homologue of the amino acid sequence were 99%, 84%, 82% and 96% respectively. There was significant difference between the grass carp and pupper(Takifugu rubripes） in amino acid sequence. The homologue was only 9%, which displayed that there was difference among the fish species. Grass carp leptin gene was expressed in Pichia pastoris. Molecular weight of expression product was estimated to be approximately 16 kD. Western blotting analysis showed that Leptin protein took on strong immunology activity.
DAI Han-chuan,WU Xiao-xiong,NIE Fen, et al. Identification of the leptin Gene from the Ctenopharyngodon idellus and Its Expression in Pichia pastoris[J]. , 2006, 14(5): 677-682.
