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

doi:10.11843/j.issn.0366-6964.2021.020

摘要/Abstract

摘要： 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 Green Ⅰ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。

关键词: 禽腺病毒血清4型, 鸡NLRP3基因, 实时荧光定量PCR, 组织炎症, 转录水平

Abstract: To analyze the transcription level of NLRP3 gene in chickens infected with FAdV-4, a pair of NLRP3 specific primers was designed, and the 180 bp NLRP3 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 NLRP3, and it was applied to measure the transcription level of NLRP3 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 NLRP3 gene; the established assay was able to provide a well-defined quantification standard curve for the chicken NLRP3 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 NLRP3 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 NLRP3 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.

Key words: fowl adenovirus serotype 4, chicken NLRP3 gene, real-time fluorescence quantitative PCR, tissue inflammation, transcription level

中图分类号:

S855.3

郭慧芳, 李宁, 王白玉, 乔麒龙, 黄庆, 李永涛, 王增, 赵军. 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[J]. 畜牧兽医学报, 2021, 52(1): 195-201.

GUO Huifang, LI Ning, WANG Baiyu, QIAO Qilong, HUANG Qing, LI Yongtao, WANG Zeng, ZHAO Jun. Analysis of the Transcription Level of NLRP3 Gene in Fowl Adenovirus Serotype 4 Infected Chicken Tissues by a Real-time Fluorescence Quantitative PCR[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(1): 195-201.

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