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Identification of the leptin Gene from the Ctenopharyngodon idellus
and Its Expression in Pichia pastoris |
| DAI Han-chuan;WU Xiao-xiong;NIE Fen;ZHAO Na;ZHANG Wei-min;ZENG Cui-ping;LONG Liang-qi |
| College of Animal Medicine ，Huazhong Agricultural University, Wuhan 430070 |
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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.
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Received: 18 October 2005
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Corresponding Authors:
LONG Liang-qi
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| [1] |
JIN Ya-Qi, YU Er-Meng, ZHANG Kai, LI Zhi-Fei, WANG Guang-Jun, XIE Jun, YU De-Guang, SUN Jin-Hui, WEI Dong, GONG Wang-Bao, TIAN Jing-Jing. Effects of Three Feeds on Serum Enzyme Activity, Intestinal Structure and Bacterial Flora of Ctenopharyngodon idellus[J]. 农业生物技术学报, 2019, 27(9): 1652-1663. |
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