Roles of vasoactive intestinal contractor/endothelin-2 in bovine ovary

内皮-2（Endothelia-2, ET2）是一种由21个氨基酸组成的生物活性肽，具有强烈的血管收缩和加压作用。本文报道了ET2的前体形式-牛内皮素-2（PPET2）的cDNA克隆和特征。牛cDNA编码PPET2多肽177个氨基酸，其中有21个氨基酸的成熟ET2肽、16个氨基酸的ET2样肽和23个氨基酸的推测信号肽。牛et2样肽序列在c端缺失双碱基氨基酸对，而人、小鼠和大鼠的et2样肽序列在n端和c端两侧都有双碱基氨基酸对。RT-PCR基因表达分析显示，该转录物在心、肺、肝、肾、胃肠道、子宫、卵巢等多个器官均有表达，但在脾脏中不表达。在胃肠道中，在反刍动物特有的消化器官瘤胃以及胃、十二指肠和结肠中检测到基因表达。基于获得的牛PPET2 cDNA全长序列，设计了基因特异性引物和TagMan探针，并利用实时荧光定量PCR系统建立了快速、精确的基因表达定量方法。

Endothelia-2(ET2), which was originally identified in human, is a bioactive peptide of 21 amino acids with strong vasoconstrictive and pressor effects. Here we report the cDNA cloning and characterization of bovine preproendothelin-2(PPET2), the precursor form of ET2. The bovine cDNA encodes 177 amino acids of the PPET2 polypeptide, in which a 21-amino acid mature ET2 peptide and a 16-amino acid ET2-like peptide as well as a 23-amino acid putative signal peptide were found. The bovine ET2-like peptide sequence was missing a dibasic amino acid pair at the C-terminal, in contrast to human, mouse and rat, for which the ET2-like sequence flanked dibasic pairs at both the N-and C-terminals. Gene expression analysis by RT-PCR showed that the transcript is expressed in various organs including heart, lung, liver, kidney, gastrointestinal tract, uterus and ovary, but not in spleen. Within the gastrointestinal tract, gene expression was detected in rumen, a ruminant-specific digestive organ, as well as stomach, duodenum and colon. Based on the full-length bovine PPET2 cDNA sequence obtained, we designed gene-specific primers and TagMan probe, and then established a rapid and precise quantitative method for the gene expression using a real-time PCR system.