大鼠降钙素基因相关肽重组腺病毒的构建与鉴定

Construction and detection of recombinant adenovirus of rat calcitonin gene-related peptide gene

目的利用AdMaxTM腺病毒载体系统,构建大鼠降钙素基因相关肽(CGRP)基因重组腺病毒载体,包装重组腺病毒并鉴定。方法根据Genbank提供的大鼠CGRP序列信息(NM_001033956.1),合成序列全长,PCR方法扩增目的基因,将CGRP/His基因片段亚克隆至穿梭质粒pDC316,形成重组质粒pDC316-CGRP/His,挑选测序正确的质粒与骨架质粒pBHGloxdelE13cre共转染293A细胞,包装出重组腺病毒Ad-CGRP/His,利用PCR和Western鉴定基因的表达。结果 (1)成功构建pDC316-rCGRP/His重组质粒。(2)包装出表达rCGRP/His的重组腺病毒。结论成功构建携带大鼠CGRP基因的重组腺病毒载体,并获得表达rCGRP/His的重组腺病毒,为深入研究CGRP基因并开展相关的基因治疗研究奠定基础。

Objective To construct a recombinant adenovirus vector of rat calcitonin gene -related peptide （CGRP） by AdMaxTM system, package recombinant adenovirus particles and identify it. Methods According to the information（ NM_001033956. 1 ）of gene order of rat CGRP from Genbank ,compose the whole length of CGRP. PCR technique amplify target gene. The full-length of CGRP/Hiss gene eDNA was cloned into the shuttle plasmid- pDC316,therefore the recombinant plasmid pDC316-CGRP/His was fol^ned. Choose the fight plasmid by sequencing and let it co-transfeet 293A cells with the skeleton plasmid-pBHGloxdelE13cre. Package recombinant adenovirus particles and identify it by PCR technique and Western blot technique. Results （1）Successfully constuct recombinant plasmid pDC316-rCGRP/His. （2）Recombinant adenovirus which express rCGRP/His was packaged. Conclusions We eonstruted a recombinant adenovirus vector which concludes the full -length of CGRP gene eDNA successfully and got recombinant adenovirus which express ICGRP/His. The results may provide a sound foundation for further study of CGRP and the research of related gene therapy in both laboratory and clinical trims .