荧光定量RT-PCR检测鸡CD4、CD8基因表达水平  

Development of Real-time RT-PCR for Detecting the Expression Level of CD4 and CD8 mRNA in Chicken

作　　者：岳华[1] 黄兴[1] 杨发龙[1] 李明义[2] 范根成[2] 马莉[1] 汤承[1] 

机构地区：[1]西南民族大学生命科学与技术学院,成都610041 [2]中国动物卫生与流行病学中心,青岛266032

出　　处：《畜牧兽医学报》2008年第6期784-790,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：“十一五”国家科技支撑计划重大项目资助(2006BAD06A11);四川省应用基础项目(05JY029-007-5)

摘　　要：白细胞分化抗原CD4和CD8在机体免疫应答及其信号传递过程中发挥着重要作用。本试验建立了定量检测鸡CD4和CD8 mRNA表达水平的SYBR Green I实时荧光定量RT-PCR（RRT-PCR）方法,并采用该方法对26-50日龄商品鸡外周血淋巴细胞（PBL）中CD4和CD8 mRNA表达水平进行了检测。结果显示：所建立的RRT-PCR对CD4和CD8 mRNA的扩增效率分别为93%和91%;线性范围分别在10^-4-10^-9和10^-3-10^-9;相关系数分别为0.998 0和0.999 9;最低分别能检测105和120拷贝;熔解曲线分别在78.2和86.7℃附近出现1个单特异峰;组内变异系数分别在1.13%-2.15%和1.17%-3.68%,组间变异系数分别在1.16%-3.25%和1.66%-2.86%。26-50日龄鸡PBL CD4和CD8的mRNA表达水平有小幅波动,与采用流式细胞术检测结果的报道一致。本试验建立的RRT-PCR方法敏感性高、稳定性和再现性好,为检测鸡CD4、CD8基因表达水平提供了精确定量的新方法。Leukocyte differentiation antigens CD4 and CD8 employed important role in avian immune response and signal transduction. In present study, a SYBR Green I based real-time reverse-transcription PCR （RRT-PCR） for quantitative analysis of chicken CD4 and CD8 mRNA was developed and the expression level of CD4 and CD8 mRNA were evaluated in 26-50 days old commercial chickens by using this method. The following results were obtained： The amplification efficiency of RRT-PCRs for CD4 and CD8 were 93% and 91%, respectively; The linear range of relative quantitative standard curve was 10^-4 to 10^-9 and 10^-3 to 10^-9 with good correlation coefficient of 0. 998 0（y=-3, 49x＋27.29） and 0. 999 9（y=-3.54x＋31.48）, respectively; The detection limit was 105 copies per reaction for CD4 and 120 copies per reaction for CD8; The melting curve presented a single peak at （78.05±0. 15）℃ and （86.50±0.20）℃ for CD4 and CD8 gene respectively. The intra-assay variability were 1.13%-2.15% for CD4 and 1.17%-3.68%for CDS, the inter-assay variability were 1.16% -3.25% and 1.66%-2.86%. CD4 and CD8 mRNA levels in PBL from 26-50 days old commercial chickens were slightly fluctuated, which is accord to the results obtained by using flow cytometry. The results in this study indicated that the RRT PCR have advantages of high sensitivity, repeatability and reproducibility, and provided a novel way for accurate quantification of CD4 and CD8 mRNA expression in chickens.

关 键 词：荧光定量RT-PCR 鸡 CD4 CD8 MRNA 

分 类 号：S852.4[农业科学—基础兽医学]