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Isolation of a new chicken pancreatic hormone (glucagon like peptide) and its physiological functions

一种新型鸡胰激素（类胰高血糖素肽）的分离及其生理功能

基本信息

批准号：
05660328
负责人：
HASEGAWA Shin
金额：
$ 1.47万
依托单位：
Kobe university
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

cDNA clones coding for glucagon were isolated from a chicken pancreas cDNA library, and the nucleotide and amino acid sequences were determined. The amino acid sequence of chicken glucagon was HSQGTFTSDYSKYLDSR-RAQDFVQWLMST,which was contained in the 151-amino acid long precursor, being preceded by a signal sequence and an amino-terminal peptide(NH2-peptide)and followed by an intervening peptide and a GLP-I.Chicken preproglucagon, however, lacked GLP-II and intervening peptide II which have been shown to be contained in mammalian glucagon precursors. The DNA encoding chicken glucagon like peptide was synthesized by PCR method to construct a synthetic glucagon like peptide gene preceded by ATG coding for methionine and followed by TAA coding for the stop codon. The nucleotide sequence of glucagon like peptide was designed on the basis of preferential codon usage in Saccharomyces cerevisiae. Recombimant chicken glucagon lilke peptide was successfully produced with a high level of express … More ion in yeast as a fusion protein with MBP.The synthetic gene was designed to release glucagon like peptide, which does not contain methionine residues, from fusion protein with CNBr that specifically cleaves the peptide bond on the carboxyl side of the methionine residue. The resulting glucagon like peptide was purified to homogeneity by a combination of reverse-phase HPLC and ion-exchange HPLC.The yield of intact glucagon like peptide obtained from 11 of culture was approximately 1.5 mg. The structure of recombinant chicken glucagon like peptide was confirmed by HPLC and amino acid composition analysis. We produced monoclonal chicken glucagon like peptide antibodies, and tried to isolate chicken glucagon like peptide from chicken pancreas. Finally, we evaluated the effect of recombinant chicken glucagon like peptide on in vitro lipolysis in isolated adipocytes and on in vivo glycogenolysis in liver in chicks, by comparison to chicken glucagon and porcine glucagon, and indicated a definite role for chicken glucagon lilke peptide in regulating carbohydrate and lipid metabolism in chicks. Less

从鸡胰腺cDNA文库中分离出胰高血糖素的cDNA克隆，并测定其核苷酸和氨基酸序列。鸡胰高血糖素的氨基酸序列是HSQGTFTSDYSKYLDSR-RAQDFVQWLMST，其包含在151个氨基酸长的前体中，前面是信号序列和氨基末端肽（NH 2-肽），后面是插入肽和GLP-1。然而，鸡前胰高血糖素原缺乏GLP-II和插入肽II，这已显示包含在哺乳动物胰高血糖素前体中。用PCR方法合成了鸡胰高血糖素样肽基因，构建了以编码蛋氨酸的ATG和编码终止密码子的TAA为前导序列的合成胰高血糖素样肽基因。根据酿酒酵母的密码子偏好性设计胰高血糖素样肽的核苷酸序列。重组鸡胰高血糖素样肽在大肠杆菌中高效表达 ...更多信息该合成基因被设计为从具有CNBr的融合蛋白中释放胰高血糖素样肽，其不含甲硫氨酸残基，CNBr特异性地切割甲硫氨酸残基的羧基侧上的肽键。通过反相HPLC和离子交换HPLC的组合将所得胰高血糖素样肽纯化至均一，从11 μ l培养物中获得的完整胰高血糖素样肽的产量约为1.5mg。通过高效液相色谱和氨基酸组成分析，证实了重组鸡胰高血糖素样肽的结构。制备了鸡胰高血糖素样肽单克隆抗体，并尝试从鸡胰腺中分离鸡胰高血糖素样肽。最后，通过与鸡胰高血糖素和猪胰高血糖素的比较，评价了重组鸡胰高血糖素样肽对离体脂肪细胞脂解和鸡肝脏糖原分解的影响，表明鸡胰高血糖素样肽对鸡碳水化合物和脂质代谢有一定的调节作用。少