草鱼IGF-ⅠcDNA的克隆和在原核生物中的表达  

CLONING AND EXPRESSION IN PROKARYOTE OF GRASS CARP IGF-Ⅰ cDNA

作　　者：华益民[1] 林浩然[1] 

机构地区：[1]中山大学生命科学学院水生经济动物研究所,广州510275

出　　处：《动物学报》2001年第3期274-279,共6页ACTA ZOOLOGICA SINICA

基　　金：国家自然科学基金资助项目 (No .3 95 70 0 99)&&

摘　　要：根据亲缘关系较近的鲤鱼胰岛素样生长因子 Ⅰ (IGF Ⅰ )cDNA设计一对引物 ,通过RT PCR从草鱼(Ctenopharyngodonidellus)肝组织首次克隆了草鱼 IGF ⅠcDNA开放阅读框 (ORF)片段。经序列分析表明克隆的草鱼IGF ⅠcDNA为Ea 2亚型 ,ORF与鲤鱼有 95 %的同源性 ,与人有 6 3%的同源性 ;草鱼IGF Ⅰ蛋白与鲤鱼IGF Ⅰ仅 2个氨基酸残基不同 ,与人IGF Ⅰ也仅有 13个残基不同。将表达成熟草鱼IGF Ⅰ (gcIGF Ⅰ)蛋白的cDNA片段亚克隆至谷胱甘肽S 转移酶 (GST)融合表达载体 pGEX 4T 3,再将构建的重组表达载体 pGEX T gcIGF Ⅰ转入大肠杆菌BL2 1。在IPTG的诱导下 ,GST gcIGF Ⅰ融合蛋白高效表达。兔抗鲑鱼IGF Ⅰ抗血清进行的WesternBlot检测显示重组草鱼IGF Ⅰ蛋白具有免疫活性。Fragment of open reading frame of Grass carp ( Ctenopharyngoden idellus ) cDNA was cloned for the first time from liver by RT PCR technique using a set of primers designed according to the closely related Common carp IGF Ⅰ cDNA. Sequence analysis showed that similar to Common carp IGF Ⅰ cDNA, the cloned Grass carp IGF Ⅰ cDNA was also Ea 2 subtype, and the homology of nucleotide sequence in open reading frame was up to 95% compared with the counterpart of Common carp, only nine amino acid were different between the two prepro IGF Ⅰ s. The homology of nucleotide sequence in ORF of Grass carp was 63% compared with the counterpart of human. Grass carp and Common carp mature protein differed in only two amino acids, while Grass carp and human mature protein differed in 13 amino acids. The fragment of Grass carp IGF Ⅰ DNA which expressed mature gcIGF Ⅰ protein, was subcloned into a GST fusion expression vector, pGEX 4T 3, to construct a recombinant expression vector, pGEX 4T 3 gcIGF Ⅰ. The pGEX 4T 3 gcIGF Ⅰ was then transformed into E. coli, BL21. When cultured and induced by IPTG, high amount of GST gcIGF Ⅰ fusion protein was expressed in the transformed BL21. The recombinant IGF Ⅰ protein had immune activity showed by Western Blotting technique.

关 键 词：草鱼 胰岛素样生长因子-Ⅰ 克隆 原核生物 表达 鱼类 IGF-Ⅰ 

分 类 号：S917[农业科学—水产科学]