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

从鸡胰腺cDNA文库中分离编码胰高血糖素的cDNA克隆，测定其核苷酸和氨基酸序列。鸡胰高血糖素的氨基酸序列为HSQGTFTSDYSKYLDSR-RAQDFVQWLMST，包含在151个氨基酸长的前体中，前体为信号序列和氨基末端肽（nh2肽），后序为中间肽和glp - 1。然而，鸡胰高血糖素前体缺乏GLP-II和干预肽II，这已被证明是在哺乳动物胰高血糖素前体中含有的。采用PCR方法合成鸡胰高血糖素样肽编码DNA，构建了一个人工胰高血糖素样肽基因，其前导ATG编码蛋氨酸，后接TAA编码终止密码子。根据酿酒酵母的优先密码子使用规律，设计了胰高血糖素样肽的核苷酸序列。重组鸡胰高血糖素样肽作为MBP的融合蛋白，在酵母中高水平表达。合成的基因被设计为释放胰高血糖素样肽，不含蛋氨酸残基，由融合蛋白与CNBr特异性地切割蛋氨酸残基羧基侧的肽键。采用反相高效液相色谱和离子交换高效液相色谱相结合的方法纯化所得胰高血糖素样肽。从11个培养中获得的完整胰高血糖素样肽的产量约为1.5 mg。重组鸡胰高血糖素样肽的结构通过高效液相色谱法和氨基酸组成分析得到。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体外脂肪分解和鸡肝脏体内糖原分解的影响，表明鸡胰高血糖素样肽在调节鸡碳水化合物和脂质代谢方面具有一定的作用。少

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