【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。更多还原

【Abstract】 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 CD8,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.更多还原