小鼠PPA1重组腺病毒的构建及在胰岛β细胞中的表达
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Construction of recombinant adenovirus carrying mouse PPA1 gene and its expression in pancreatic β-cell
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    摘要:

    目的:构建小鼠PPA1重组腺病毒,观察重组腺病毒在小鼠胰岛和β-TC6细胞中的表达,以及对脂肪酸引起的胰岛β细胞凋亡的影响-方法:将目的基因PPA1插入到腺病毒穿梭质粒pAdtrack-CMV中,质粒经PCR鉴定正确后用PmeⅠ酶切线性化,转到含有腺病毒骨架pAdeasy-1的 BJ5183细菌中进行同源重组,重组成功的质粒经PacⅠ酶切线性化后转染QBI-293A细胞,经包装得到Ad-PPA1腺病毒-根据绿色荧光蛋白(GFP)检测病毒滴度和感染效率-用病毒感染小鼠胰岛和β-TC6细胞,以Western blot法检测PPA1蛋白表达水平-Hoechst染色法检测PPA1对细胞凋亡的影响-结果:重组腺病毒载体pAdeasy-PPA1经酶切鉴定确认构建成功,将pAdeasy-PPA1转染QBI-293A细胞,观察细胞病变效应提示病毒成功包装-重组腺病毒能够有效感染小鼠胰岛和β-TC6细胞并成功过表达 PPA1蛋白-Hoechst染色结果表明过表达PPA1可保护脂肪酸引起的胰岛β细胞凋亡-结论:成功构建携带小鼠PPA1基因的重组腺病毒,并证实过表达PPA1具有抗凋亡的作用,为进一步研究PPA1在胰岛β细胞中的功能奠定了基础-

    Abstract:

    Objective:To construct recombinant adenovirus carrying mouse PPA1 gene,and detect its expression in mouse islets and β-TC6 cells,and observe its effect on fatty acid-induced pancreatic β cell apoptosis. Methods:The target gene of PPA1 was cloned into the shuttle vector pAdtracd-CMV. After identified by PCR,the plasmid was linearized by PmeⅠ and recombined with backbone pAdeasy-1 in BJ5183 bacteria. The recombinant plasmid was linearized by PacⅠ enzymes digestion,transfected into QBI-293A cells,and packed for Ad-PPA1 adenovirus. Accoring to the green fluorescent protein (GFP),the titer and infection rate was detected. The mouse islets and β-TC6 cells were infected by recombinant adenovirus and the protein level of PPA1 was detected by Western blot. By Hoechst staining,the effect of PPA1 on cell apoptosis was detected. Results:The recombinant adenovirus vector pAdeasy-PPA1 was successfully constructed. After pAdeasy-PPA1 was transfected into QBI-293A cells,cytopathic effect showed that the adenovirus was successfully packed. The recombinant adenovirus containing mouse PPA1 gene effectively infected mouse islets and β-TC6 cells,and over-express the protein levels of PPA1. Hoechst staining showed that over-expression of PPA1 protected β-TC6 cells against fatty acid-induced apoptosis. Conclusion:The recombinant adenovirus expressing PPA1 gene has been successfully obtained and over-expression of PPA1 has anti-apoptosis effect,which provided a basis for further study of PPA1 on pancreatic β-cell function.

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郑 月,张 许,韩 晓.小鼠PPA1重组腺病毒的构建及在胰岛β细胞中的表达[J].南京医科大学学报(自然科学版),2014,(8):1019-1024

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  • 收稿日期:2014-04-17
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  • 在线发布日期: 2014-08-03
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