禽传染性喉气管炎病毒体内外感染模型中鸡Toll样受体21 mRNA转录水平的研究

mRNA transcription profile of chicken toll-like receptor 21 in vitro and in vivo under avian infectious laryngotracheitis virus infection

为研究病毒与机体的相互作用,本研究参考GenBank中鸡Toll样受体21(ChTLR21)的基因序列设计实时定量PCR特异性引物,以鸡核糖体蛋白L4(RPL4)为内参基因,建立检测ChTLR21 mRNA相对转录水平的实时定量PCR方法,分析ChTLR21在禽传染性喉气管炎病毒(ILTV)感染的鸡胚成纤维细胞(CEF)中和感染SPF雏鸡免疫器官脾脏、法氏囊和胸腺组织中的转录水平。结果显示:ILTV感染CEF后2 h、4 h、8 h和18 h时间点ChTLR21 mRNA转录水平分别为未感染对照细胞的1 540.53、0.98、1.19和3.70倍。但仅在ILTV感染2 h时引起ChTLR21转录水平升高,与对照组相比差异显著(p<0.05)。ILTV感染SPF雏鸡后6 h、24 h和30 h脾脏中ChTLR21 mRNA转录水平分别为未感染对照的56.34、59.85和3.61倍;法氏囊中分别为0.03、25.98和3.08倍;胸腺中分别为2.52、50和7.32倍。在感染初期,脾脏中ChTLR21转录量显著升高,随后有所降低,但均高于未感染对照(p<0.05);法氏囊中仅在感染6 h时呈显著抑制(p<0.01);胸腺中呈波动性转录水平升高,但与对照组无统计学差异(p>0.05)。本研究证明ChTLR21参与了鸡体对ILTV感染的应答,并在体内外感染模型中呈现不同的表达规律。

To investigate the effect of avian infectious laryngotracheitis virus （ILTV） infection on chicken Toll-like receptor 21 （ChTLR21） expression, a SYBR Green I real-time PCR was established for detecting ChTLR21 mRNA transcription with Ribosomal protein L4 （RPIA） as housekeeping gene. Both of chicken embryonic fibroblasts （CEF） and two-week-old SPF chickens were infected with ILTV and the mRNA levels in infected CEF and thymus, bursal and spleen from infected SPF chickens were detected at different time points post infection. The results indicated that transcription profiles of ChTLR21 mRNA in infected CEFs were 1,540.53, 0.98, 1.19 and 3.70 folds of control cells at 2, 4, 8 and 18 hours post virus infection, respectively. ChTLR21 mRNA levels in ILTV infected CEFs was significantly upregnlated only at 2 hours （p〈0.05）. Furthermore, SPF chickens were detected at 6, 24 and 30 hours post ILTV infection and the transcription profiles of ChTLR21 mRNA were 56.34, 59.85 and 3.61 times in the spleen, 0.03, 25.98 and 3.08 times in the bursal and 2.52, 50 and 7.32 times higher in the thymus than that of control, respectively. The mRNA levels of ChTLR21 in the spleen were significantly upregulated （p〈0.05）, although it was lower at 30 hour, but ChTLR21 mRNA in the bursal was significantly downregulated at 6 hours （p〈0.05）. However, no significant change of the mRNA was found in thymus even it was higher than that in control group at all the time points. Those results indicated that ChTLR21 participated in response to ILTV infection in different levels both in vivo and in vivo models.