荧光定量RT-PCR检测雏鸡感染NDV强毒后胸腺和法氏囊中TLR2与TLR4 mRNA的表达

Development of a real-time RT-PCR for detection of Toll-like receptor2 and Toll-like receptor4 mRNAs in bursals and thymus of chicken infected with virulent NDV

Toll样受体2(TLR2)和Toll样受体4(TLR4)在识别病原微生物过程中发挥重要作用。为了定量检测TLR2、TLR4 mRNA表达水平,研究病原与机体的相互作用,本研究建立了检测鸡TLR2、TLR4 mRNA表达水平的SYBR GreenⅠ荧光定量RT-PCR(RRT-PCR)方法,检测了新城疫病毒强毒(vNDV)感染SPF雏鸡后36h、48h时胸腺、法氏囊中TLR2、TLR4 mRNA表达量变化。结果显示该方法特异性好,RRT-PCR产物分别在85.5℃、83.3℃出现单特异峰,对TLR2、TLR4的扩增效率分别为105.91%和95.30%,相关系数分别为0.9980、0.9996,最低检测限分别为108拷贝/反应和461拷贝/反应。感染后36h vNDV显著抑制TLR2、TLR4基因在法氏囊、胸腺中的表达;感染后48h时,法氏囊、胸腺中TLR2基因的表达水平显著升高,胸腺中TLR4基因的表达显著升高,而法氏囊中TLR4基因的表达仍处于抑制状态。本研究证明TLR2和TLR4参与了鸡体对NDV的感染应答。

Toll like receptor 2（TLR2） and Toll like receptor 4（TLR4） are crucial in pathogen recognition by the immune system. In present study,a real-time reverse-transcription PCR（RRT-PCR） based on SYBR GreenIwas developed for quantitative analysis of chicken TLR2 and TLR4 mRNA. SPF chickens were infected virulent Newcastle disease virus（vNDV）,and TLR2 and TLR4 mRNA levels in the bursals and thymus was detected at 36 and 48 h post infection（PI） . The results showed that the RRT-PCR assay was highly specific,and had a sensitivity for detection of 108 copies TLR2 and 461 copies TLR4 mRNAs,repectively. The expression levels of TLR2 and TLR4 mRNA were down-regulation in bursals and thymus 36 h PI. However,the expression level of TLR2 was up-regulated in both bursals and thymus 48 h PI. The TLR4 expression level in thymus was also up-regulated at 48 h PI,but down-regulated in bursals. Our results indicated that TLR2 and TLR4 may play an important role in responsing to NDV infection.