摘要
为了建立以及应用鸡细胞S期激酶相关蛋白2(S-phase kinase-associated protein 2,Skp2)的实时荧光定量PCR方法,以鸡Skp2基因为研究对象,根据GenBank中已发表的鸡Skp2和β-actin序列的高度保守区域设计引物,构建重组质粒作为标准品,成功建立了检测鸡胚成纤维细胞(Chicken embryo fibroblast,CEF)中Skp2 mRNA表达的实时荧光定量RT-PCR方法,并对其Tm值和引物浓度等进行了优化。该方法最优反应体系为20.0μL,包含Real-time PCRMaster mix(2×)10.0μL,上、下游引物(10.0μmol/L)各1.0μL,cDNA 2.0μL,ddH2O 6.0μL;最佳反应条件为95℃预变性10 s,95℃变性5 s,60℃退火/延伸34 s,30个循环。应用该方法对CEF中Skp2 mRNA的表达量进行检测,发现在CEF感染禽网状内皮组织增生病病毒(REV)后,其细胞中Skp2 mRNA表达量均高于对照组,且在24、48、72 h显著增加。研究首次建立了检测鸡Skp2基因的检测方法,该方法稳定性良好,精确度高,特异性强,为进一步定量研究鸡Skp2 mRNA表达及其与细胞周期的关系奠定了基础。
In order to establish and apply a real-time fluorescent quantitative PCR method for S-phase kinaseassociated protein 2(Skp2), the chicken Skp2 gene was used as a research object. Primers were designed based on highly conserved regions of chicken Skp2 and β-actin sequences published in GenBank, and a recombinant plasmid was constructed as a standard. A real-time quantitative RT-PCR method for detecting Skp2 mRNA expression in chicken embryo fibroblast(CEF) was established successfully, and its Tm was determined. The optimal reaction system of this method was 20.0 μL, which included real-time PCR Master mix(2×) 10.0 μL, upper and downstream primers(10.0 μmol/L)1.0 μL each, cDNA 2.0 μL, ddH2 O 6.0 μL;optimal reaction conditions were 95 ℃ pre-denaturation 10 s, denatured at 95 ℃ for 5 s, annealed/extended at 60 ℃ for 34 s with 30 cycles. The expression of Skp2 mRNA in CEF was detected by this method. The expression of Skp2 mRNA was higher than that of the control group after reticuloendotheliosis virus(REV) infected CEF, which was extremely significantly higher than that of the control group at 24 h, 48 h, 72 h post infection. This study firstly established a detection method for Skp2 gene in chicken. The method had good stability,high precision and strong specificity, which laid a technical foundation for further quantitative study on the expression of chicken Skp2 mRNA and its relationship with cell cycle.
作者
吴乐
高海婷
刘超男
高雪丽
张淑君
周放
吕晓萍
边洋
郑世民
WU Le;GAO Haiting;LIU Chaonan;GAO Xueli;ZHANG Shujun;ZHOU Fang;LV Xiaoping;BIAN Yang;ZHENG Shimin(Key Laboratory for Laboratory Animals and Comparative Medicine,College of Veterinary Medicine,Northeast Agricultural University,Harbin,Heilongjiang 150030)
出处
《中国家禽》
北大核心
2019年第24期17-22,共6页
China Poultry
基金
国家自然科学基金项目（31472169）
