垂体腺苷酸环化酶激活肽基因的合成及pIRES1-PACAP表达载体的构建

The Synthesis of PACAP and the Construction of Its Expression Vector

根据文献报道的垂体腺苷酸环化酶激活肽 ( pituitary adenylate cyclase activating polypeptide,PACAP)基因序列和信号肽基因序列 ,设计 10条单链寡核苷酸片段 ,通过 5次 PCR扩增获得 PACAP基因片段 ,同时两端设有 Sm a 和 Xba 酶切位点 ,酶切后将其克隆至 p IRES1- neo质粒中 ,经 PCR、酶切和测序鉴定 ,证明已获得 PACAP基因并构建了 p IRES1-

Pituitary adenylate cyclase activating polypeptide(PACAP) is a recently isolated bioactive polypeptide.In this research,PACAP gene was cloned in an attempt to express its recombinant peptide for experimantal and clinical application.We have synthesized 10 fregments according to sequence of PACAP gene.PACAP gene was amplified step by step for 5 times.It is 205 bp in length including signal peptide sequence and two restriction enzymic sites(SmaⅠ and XbaⅠ).Amplified PACAP DNA was ligated with prepared fragments of pIRES1-neo(SmaⅠ and XbaⅠ),then transformed to JM109.We selected and identified the positive clones by PCR and digestion with SmaⅠ and XbaⅠ.Sequencing result showed that sequence of PACAP gene has synthesized successfully.Which has the potential value in medical and veterinary clinical application.