草鱼leptin基因的分离鉴定及在巴斯德毕赤酵母中的表达

利用RT-PCR技术扩增出草鱼(Ctenopharyngodonidellus)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Westernblot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(Cyprinuscarpio)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(Takifugurubripes)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(Pichiapastoris)中的表达,表达蛋白的分子量约为16kD,Westernblot分析表明,表达产物具有一定的免疫学活性。

Identification of the leptin Gene from the Ctenopharyngodon idellus and Its Expression in Pichia pastoris

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.