表达胰高血糖素样肽1重组腺伴随病毒载体的构建

背景：胰高血糖素样肽1(glucagon-like peptide-1, GLP-1)在人体内半衰期过短限制了其应用。 目的：构建可表达GLP-1的重组腺伴随病毒。 方法：将NT4-GLP-1融合基因插入腺伴随病毒包装质粒pSSHG-CMV中,构建pSSHG/NT4-GLP-1重组腺伴随病毒包装质粒。采用磷酸钙共沉淀法将辅助质粒pAAV/Ad、腺病毒质粒pFG140及pSSHG/NT4-GLP-1转染至293细胞系,用其感染Hela细胞。 结果与结论：重组质粒pSSHG/NT4-GLP-1经限制性内切酶EcoRⅠ酶切鉴定可见342 bp的目的片段,说明NT4-GLP-1融合基因已经成功重组于腺伴随病毒包装质粒pSSHG-CMV内。免疫细胞化学结果显示,转染pSSHG/NT4-GLP-1重组腺伴随病毒包装质粒的Hela细胞内有大量棕黄色颗粒,阳性率达到70%以上,说明NT4-GLP-1重组腺伴随病毒在细胞中可以表达GLP-1。

Construction of a recombinant adeno-associated virus plasmid expressing glucagon-like peptide-1

BACKGROUND:Glucagon-like peptide-1 (GLP-1) is limited by half-life in human body. OBJECTIVE:To construct a recombinant adeno-associated virus vector expressing GLP-1. METHODS:The fusion gene NT4-GLP-1 was inserted into the adeno-associated virus packaging plasmid pSSHG-CMV to generate the adeno-associated virus packaging plasmid pSSHG/NT4-GLP-1. 80% of fused 293 cells were co-transfected by using the calcium phosphate precipitation method, with aid packaging plasmids pAAV/Ad, pFG140 and the recombinant adeno-associated virus packaging plasmid pSSHG/NT4-GLP-1. Virus effects were confirmed by immunohistochemistry at the same time. RESULTS AND CONCLUSION:Restriction enzyme EcoRⅠidentification showed that the recombinant plasmid pSSHG/NT4-GLP-1 contained a 342 bp target segment, indicating that the fusion gene NT4-GLP-1 has been successfully transfected into recombinant adeno-associated virus packaging plasmid pSSHG/NT4-GLP-1. Immunocytochemical staining results showed that Hela cells transfected by plasmid pSSHG/NT4-GLP-1 contained a large number of brown yellow particles, with the rate of positive cells more than 70%. These findings suggest that adeno-associated virus packaging plasmid pSSHG/NT4-GLP-1 expressed GLP-1.