Chen J Y, Chang C Y, Chen J C, Shen S C, Wu J L
Institute of Zoology, National Taiwan University, Taipei, R.O.C.
DNA Cell Biol. 1997 Jul;16(7):883-92. doi: 10.1089/dna.1997.16.883.
Insulin-like growth factor-II (IGF-II) is a fetal growth factor in humans, but has not been clearly identified in fish up to now. For a detailed understanding of the physiological response of fish IGF-II, the first step was to clone tilapia IGF-II cDNA from the brain cDNA library, coding the region of genomic DNA, and also expressing tilapia IGF-II polypeptides from Escherichia coli. Tilapia cDNA sequences total 1,977 bp, and predicted nucleotide sequences and amino acid sequences of tilapia share 77.9% and 90.7% homology identity with rainbow trout IGF-II, respectively. The genomic structure of the tilapia prepro-IGF-II coding region is very difficult to sequence in mammals and birds. The cloned tilapia IGF-II gene coding region appears much more complex than in other vertebrates. In tilapia IGF-II, the first coding exon I encoding part of the signal peptide sequence is 25 amino acids shorter than the first coding exon of mammals and birds. The other 23 amino acids of the signal peptide, and the first amino acids of the B domain and C domain are encoded by tilapia coding exon 2. The C, A, and D domains, and the first 20 amino acids of the E peptide are encoded by tilapia coding exon 3. The other E peptides and the 3' untranslated region (UTR) region are encoded by tilapia coding exon 4. These data show that the IGF-II genes have significantly differing structures in vertebrate evolution, and there are differences of interrupting introns in the IGF-I genomic structure compared with mammals. To obtain recombinant biologically active polypeptides, tilapia IGF-II B-C-A-D domains were amplified using the polymerase chain reaction (PCR), then ligated with glutathione S-transferase (GST, pGEX-2T vector). Tilapia recombinant IGF-II protein was purified and characterized in E. coli. The fusion protein was also digested with thrombin and appeared as a recombinant IGF-II polypeptide single band with a molecular mass of 7 kD. The recombinant tilapia IGF-II protein biological function was measured by stimulation of [3H]thymidine incorporation. The assay concentration was set up from 0 to 120 nM to stimulate tilapia ovary cell line (TO-2) significantly to uptake thymidine. The results suggest that the recombinant IGF-II protein was dose dependent.
胰岛素样生长因子-II（IGF-II）是人类的一种胎儿生长因子，但迄今为止在鱼类中尚未得到明确鉴定。为了详细了解鱼类IGF-II的生理反应，第一步是从大脑cDNA文库中克隆罗非鱼IGF-II cDNA，对基因组DNA的编码区域进行测序，并在大肠杆菌中表达罗非鱼IGF-II多肽。罗非鱼cDNA序列全长1977 bp，预测的罗非鱼核苷酸序列和氨基酸序列与虹鳟鱼IGF-II的同源性分别为77.9%和90.7%。罗非鱼前胰岛素原-IGF-II编码区的基因组结构在哺乳动物和鸟类中很难进行测序。克隆的罗非鱼IGF-II基因编码区似乎比其他脊椎动物的更为复杂。在罗非鱼IGF-II中，编码信号肽序列一部分的第一个编码外显子I比哺乳动物和鸟类的第一个编码外显子短25个氨基酸。信号肽的其他23个氨基酸以及B结构域和C结构域的第一个氨基酸由罗非鱼编码外显子2编码。C、A和D结构域以及E肽的前20个氨基酸由罗非鱼编码外显子3编码。其他E肽和3'非翻译区（UTR）区域由罗非鱼编码外显子4编码。这些数据表明，IGF-II基因在脊椎动物进化中具有显著不同的结构，并且与哺乳动物相比，IGF-I基因组结构中的内含子中断存在差异。为了获得重组生物活性多肽，使用聚合酶链反应（PCR）扩增罗非鱼IGF-II的B-C-A-D结构域，然后与谷胱甘肽S-转移酶（GST，pGEX-2T载体）连接。罗非鱼重组IGF-II蛋白在大肠杆菌中进行了纯化和鉴定。融合蛋白也用凝血酶消化，出现了一条分子量为7 kD的重组IGF-II多肽单条带。通过刺激[3H]胸腺嘧啶核苷掺入来测定重组罗非鱼IGF-II蛋白的生物学功能。测定浓度设置为0至120 nM，以显著刺激罗非鱼卵巢细胞系（TO-2）摄取胸腺嘧啶核苷。结果表明重组IGF-II蛋白具有剂量依赖性。
