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目的:应用基因同源重组技术,构建及鉴定携带小鼠betatrophin基因Gm6484(NM_001080940)的复制缺陷型重组腺病毒载体,为进一步研究betatrophin的功能奠定基础。方法:根据基因库中登录的小鼠betatrophin基因Gm6484(NM_001080940)序列,经化学合成得到含Bam HⅠ/AgeⅠ酶切位点的小鼠betatrophin基因全长c DNA质粒,将其酶切后插入到带有增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因的GV314载体CMV-MCS-3FLAG-SV40-EGFP中,得到重组病毒穿梭质粒p GV314-betatrophin;经Bam HⅠ/AgeⅠ双酶切后,与线性化的p DC315病毒基因组质粒体外同源重组,构建含有目的基因的腺病毒载体Ad-betatrophin,酶切线性化重组腺病毒质粒后转染HEK293T细胞包装成重组病毒颗粒,经在HEK293T细胞反复扩增数代后,利用聚合酶链式反应(PCR)、蛋白质印迹法及测序鉴定重组的腺病毒。结果:阳性克隆质粒Ad-betatrophin在HEK293T细胞中成功包装出重组病毒,经PCR、蛋白质印迹及测序检测表明重组腺病毒包装成功。结论:成功构建了携带小鼠betatrophin基因的复制缺陷型重组腺病毒过表达载体。
OBJECTIVE: To construct and identify the replication-deficient recombinant adenovirus vector carrying mouse betatrophin gene Gm6484 (NM_001080940) by gene homologous recombination technology, and lay a foundation for further study on the function of betatrophin. Methods: According to the sequence of mouse betatrophin gene Gm6484 (NM_001080940) registered in GenBank, the full length cDNA of mouse betatrophin gene containing BamHI / AgeI restriction site was obtained by chemical digestion and inserted into the band The GV314 vector CMV-MCS-3FLAG-SV40-EGFP with the enhanced green fluorescent protein (EGFP) gene was used to obtain the recombinant shuttle plasmid pGV314-betatrophin. After double digestion with Bam HI / Age I, Linearized p DC315 virus genome plasmid in vitro homologous recombination to construct the adenovirus vector containing the target gene Ad-betatrophin, linearized recombinant adenovirus plasmid was transfected HEK293T cells transfected with recombinant virus particles, repeatedly expanded in HEK293T cells After several generations, the recombinant adenovirus was identified by polymerase chain reaction (PCR), Western blot and sequencing. Results: Positive recombinant plasmid Ad-betatrophin successfully packaged recombinant virus in HEK293T cells. The recombinant adenovirus was successfully packaged by PCR, Western blot and sequencing. Conclusion: The replication-defective recombinant adenovirus overexpression vector carrying mouse betatrophin gene was successfully constructed.