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2025年4月4日 星期五
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草鱼leptin基因的分离鉴定及在巴斯德毕赤酵母中的表达

戴汉川;伍晓雄;聂芬;赵娜;张维民;曾翠平;龙良启
华中农业大学动物医学院,武汉430070
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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摘要 利用RT-PCR技术扩增出草鱼(Ctenopharyngodon idellus)的leptin基因,将leptin基因克隆至真核表达载体pPIC9K,电穿孔转化GS115菌株, 经G418筛选和甲醇诱导后,对表达产物进行SDS-PAGE琼脂糖凝胶电泳和Western blot分析。结果表明,草鱼leptin基因cDNA序列由438个核苷酸组成,编码146个氨基酸组成的多肽(GenBank登陆号AY551335),与鲤鱼(Cyprinus carpio)leptin基因相比, 核苷酸和氨基酸的同源性为99%; 与人、猪和鼠相比,核苷酸同源性分别为84%、86%和95%, 氯基酸的同源性分别为84%、82%和96%;与河豚(Takifugu rubripes)相比,氨基酸具有较大的差异,仅有9%的同源性,表明leptin在物种的进化上具有一定的差异;实现了草鱼leptin基因在毕赤酵母(Pichia pastoris)中的表达,表达蛋白的分子量约为16 kD, Western blot 分析表明,表达产物具有一定的免疫学活性。
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戴汉川
伍晓雄
聂芬
赵娜
张维民
曾翠平
龙良启
关键词 草鱼草鱼 leptin基因 毕赤酵母表达    
AbstractThe 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.
Key wordsCtenopharyngodon idellus    leptin gene    Pichia pastoris expression
收稿日期: 2005-10-18     
ZTFLH:  S188  
通讯作者: 龙良启   
引用本文:   
戴汉川;伍晓雄;聂芬;赵娜;张维民;曾翠平;龙良启.

草鱼leptin基因的分离鉴定及在巴斯德毕赤酵母中的表达

[J]. , 2006, 14(5): 677-682.
DAI Han-chuan;WU Xiao-xiong;NIE Fen;ZHAO Na;ZHANG Wei-min;ZENG Cui-ping;LONG Liang-qi. Identification of the leptin Gene from the Ctenopharyngodon idellus 
and Its Expression in Pichia pastoris. , 2006, 14(5): 677-682.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/      或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2006/V14/I5/677
 
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