禽腺病毒血清4型感染鸡组织中NLRP3基因转录水平的实时荧光定量PCR分析

Analysis of the Transcription Level of NLRP 3 Gene in Fowl Adenovirus Serotype 4 Infected Chicken Tissues by a Real-time Fluorescence Quantitative PCR

旨在分析禽腺病毒血清4型(FAdV-4)感染鸡组织中NLRP3基因的转录水平,本研究设计鸡NLRP3特异性引物,利用RT-PCR扩增NLRP3基因180 bp片段并克隆至pMD-18T载体,制备重组质粒pMD-18T-NLRP3。以pMD-18T-NLRP3质粒作为标准品进行荧光定量PCR并建立标准曲线。通过反应条件优化,成功建立了检测NLRP3基因的实时荧光定量PCR方法,并利用该方法对致病性FAdV-4感染鸡组织中NLRP3基因的转录水平进行了分析。结果显示,所设计的NLRP3引物可特异性扩增鸡NLRP3基因,建立的实时荧光定量PCR对鸡NLRP3标准质粒的扩增曲线良好,标准品的拷贝数与Cq值呈现良好的线性关系。与对照组相比,NLRP3分子在FAdV-4感染鸡肝和脾中的转录水平极显著高于对照组(P<0.001),在盲肠扁桃体和法氏囊的表达显著高于对照组(P<0.01)。本研究所建立的鸡NLRP3基因SYBR GreenⅠ实时荧光定量PCR可以检测FAdV-4感染鸡不同组织中NLRP3的转录水平;致病性FAdV-4感染所造成的组织炎症损伤与NLRP3分子密切相关。

To analyze the transcription level of NLRP 3 gene in chickens infected with FAdV-4, a pair of NLRP 3 specific primers was designed, and the 180 bp NLRP 3 gene was amplified by RT-PCR and cloned into the pMD-18T vector to create a recombinant plasmid pMD-18T-NLRP3. Using the constructed pMD-18T-NLRP3 plasmid as standards, a standard curve assay for SYBR Green I real-time PCR was established. After optimization of reaction conditions, a real-time fluorescent quantitative PCR was successfully established to detect NLRP 3, and it was applied to measure the transcription level of NLRP 3 gene in the organs of chickens infected with pathogenic FAdV-4. The results indicated that the primers designed for this assay were specific to chicken NLRP 3 gene;the established assay was able to provide a well-defined quantification standard curve for the chicken NLRP 3 standard plasmid, and a linear correlation was observed between the copy numbers of the standard plasmid pMD-18T-NLRP3 and the Cq values. Comparing to the control group, the transcription level of NLRP 3 was significantly higher in the liver and spleen ( P <0.001) and higher in the cecal tonsils and bursa of Fabricius ( P <0.01) of the infected chickens. The chicken NLRP 3 SYBR Green I real-time PCR established in this study was able to detect the transcription level of NLRP3 in different organs of chickens infected with FAdV-4, and the inflammation induced by FAdV-4 infection is highly related to NLRP3.