【摘要】：Mi R-17-92基因簇(mi R-17-92 cluster)在哺乳動物的細胞增殖、分化、凋亡、動物發(fā)育以及腫瘤發(fā)生等過程中發(fā)揮重要作用,但是目前mi R-17-92基因簇在雞生長發(fā)育中的作用還不清楚。為了闡明mi R-17-92基因簇在雞(Gallus gallus)生長發(fā)育中的作用和機制,本研究構(gòu)建了雞mi R-17-92基因簇的腺病毒表達載體。以雞基因組為模板,通過PCR擴增出雞mi R-17-92基因簇,將其克隆至腺病毒表達系統(tǒng)穿梭質(zhì)粒pac Ad5 mi R-GFP Shuttle Vector,獲得重組穿梭質(zhì)粒pac Ad5-Mi R-17-92 cluster,進而將其與骨架質(zhì)粒pac Ad5 9.2-100分別經(jīng)PacⅠ酶切線性化后,共轉(zhuǎn)染AD293細胞,經(jīng)同源重組獲得雞重組腺病毒r Ad5-Mi R-17-92 cluster。重組病毒擴增后,采用半數(shù)細胞培養(yǎng)物感染量(50%tissue culture infective dose,TCID50)法測定病毒滴度,采用PCR鑒定和測序鑒定重組腺病毒r Ad5-mi R-17-92 cluster。結(jié)果顯示重組腺病毒r Ad5-Mi R-17-92 cluster和空載體腺病毒r Ad-control的滴度均達到1×109 pfu/m L。PCR擴增出了879bp預(yù)期大小的特異產(chǎn)物;測序結(jié)果與預(yù)期序列完全一致。將重組腺病毒感染永生化雞前脂肪細胞,實時stem-loop RT-PCR檢測顯示,在重組腺病毒感染細胞mi R-17-92基因簇中的mi R-19a和mi R-20a的表達量為對照組的2.2±0.2倍(P0.01)。將重組腺病毒分別感染4種人胚腎上皮細胞(human embryonic kidney epithelial cell,HEK)293A、293T、AD293和HEK293、永生化雞前脂肪細胞(immortalized chicken preadipocytes,ICPA)、雞成纖維細胞(chicken fibroblast cell,DF1)、人上皮細胞(human epithelial cells,Ha Ca T)和羊胚胎成纖維細胞(sheep embryonic fibroblasts,SEF),采用熒光顯微鏡觀察判斷病毒感染情況。結(jié)果發(fā)現(xiàn),重組腺病毒對上述8種細胞的感染效率不同,其中,在4種人胚腎上皮細胞293A、293T、AD293和HEK293中的感染效率很高,在人上皮細胞及羊胚胎成纖維細胞中的感染效率中等,但是在永生化雞前脂肪細胞和雞成纖維細胞中的感染效率較低。本研究成功構(gòu)建的雞mi R-17-92基因簇重組腺病毒為進一步研究mi R-17-92基因簇在雞生長發(fā)育中的作用及其機制提供研究工具。
[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，

本文編號：2203144