鸡miR-17-92基因簇重组腺病毒的制备及其细胞感染率分析
发布时间：2018-08-25 14:24
【摘要】：Mi R-17-92基因簇(mi R-17-92 cluster)在哺乳动物的细胞增殖、分化、凋亡、动物发育以及肿瘤发生等过程中发挥重要作用,但是目前mi R-17-92基因簇在鸡生长发育中的作用还不清楚。为了阐明mi R-17-92基因簇在鸡(Gallus gallus)生长发育中的作用和机制,本研究构建了鸡mi R-17-92基因簇的腺病毒表达载体。以鸡基因组为模板,通过PCR扩增出鸡mi R-17-92基因簇,将其克隆至腺病毒表达系统穿梭质粒pac Ad5 mi R-GFP Shuttle Vector,获得重组穿梭质粒pac Ad5-Mi R-17-92 cluster,进而将其与骨架质粒pac Ad5 9.2-100分别经PacⅠ酶切线性化后,共转染AD293细胞,经同源重组获得鸡重组腺病毒r Ad5-Mi R-17-92 cluster。重组病毒扩增后,采用半数细胞培养物感染量(50%tissue culture infective dose,TCID50)法测定病毒滴度,采用PCR鉴定和测序鉴定重组腺病毒r Ad5-mi R-17-92 cluster。结果显示重组腺病毒r Ad5-Mi R-17-92 cluster和空载体腺病毒r Ad-control的滴度均达到1×109 pfu/m L。PCR扩增出了879bp预期大小的特异产物;测序结果与预期序列完全一致。将重组腺病毒感染永生化鸡前脂肪细胞,实时stem-loop RT-PCR检测显示,在重组腺病毒感染细胞mi R-17-92基因簇中的mi R-19a和mi R-20a的表达量为对照组的2.2±0.2倍(P0.01)。将重组腺病毒分别感染4种人胚肾上皮细胞(human embryonic kidney epithelial cell,HEK)293A、293T、AD293和HEK293、永生化鸡前脂肪细胞(immortalized chicken preadipocytes,ICPA)、鸡成纤维细胞(chicken fibroblast cell,DF1)、人上皮细胞(human epithelial cells,Ha Ca T)和羊胚胎成纤维细胞(sheep embryonic fibroblasts,SEF),采用荧光显微镜观察判断病毒感染情况。结果发现,重组腺病毒对上述8种细胞的感染效率不同,其中,在4种人胚肾上皮细胞293A、293T、AD293和HEK293中的感染效率很高,在人上皮细胞及羊胚胎成纤维细胞中的感染效率中等,但是在永生化鸡前脂肪细胞和鸡成纤维细胞中的感染效率较低。本研究成功构建的鸡mi R-17-92基因簇重组腺病毒为进一步研究mi R-17-92基因簇在鸡生长发育中的作用及其机制提供研究工具。
[Abstract]:Mi R-17-92 gene cluster (mi R-17-92 cluster) plays an important role in mammalian cell proliferation, differentiation, apoptosis, animal development and tumorigenesis, but the role of mi R-17-92 gene cluster in chicken growth and development is unclear. In order to elucidate the role and mechanism of mi R-17-92 gene cluster in the growth and development of chicken (Gallus gallus), the adenovirus expression vector of chicken mi R-17-92 gene cluster was constructed. Using chicken genome as template, chicken mi R-17-92 gene cluster was amplified by PCR. The recombinant shuttle plasmid pac Ad5-Mi R-17-92 cluster, was cloned into the shuttle plasmid pac Ad5 mi R-GFP Shuttle Vector, of adenovirus expression system. The recombinant shuttle plasmid pac Ad5-Mi R-17-92 cluster, was then linearized with the skeleton plasmid pac Ad5 9.2-100 by restriction endonuclease digestion with Pac 鈪，
本文编号：2203144
[Abstract]:Mi R-17-92 gene cluster (mi R-17-92 cluster) plays an important role in mammalian cell proliferation, differentiation, apoptosis, animal development and tumorigenesis, but the role of mi R-17-92 gene cluster in chicken growth and development is unclear. In order to elucidate the role and mechanism of mi R-17-92 gene cluster in the growth and development of chicken (Gallus gallus), the adenovirus expression vector of chicken mi R-17-92 gene cluster was constructed. Using chicken genome as template, chicken mi R-17-92 gene cluster was amplified by PCR. The recombinant shuttle plasmid pac Ad5-Mi R-17-92 cluster, was cloned into the shuttle plasmid pac Ad5 mi R-GFP Shuttle Vector, of adenovirus expression system. The recombinant shuttle plasmid pac Ad5-Mi R-17-92 cluster, was then linearized with the skeleton plasmid pac Ad5 9.2-100 by restriction endonuclease digestion with Pac 鈪，
本文编号：2203144
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