猪NF-kappaB p65/p50基因克隆、序列鉴定及实时荧光定量PCR检测方法的建立

doi:10.11843/j.issn.0366-6964.2013.08.017

摘要/Abstract

摘要：

NF-κB信号通路在病毒感染、免疫损伤性疾病、肿瘤疾病中发挥重要作用，其活化水平常作为机体免疫应答水平、疾病发展进程的检测指标。为获得猪NF-κB p65/p50基因序列特征并建立体外定量检测其表达水平的实时荧光定量PCR方法，作者对猪NF-κB p65 ORF和成熟p50编码区进行RT-PCR扩增、克隆、测序及生物信息学分析；设计特异性荧光定量引物，建立检测p65、p50实时荧光定量PCR方法。结果显示：所扩增的猪NF-κB p65 ORF长1 662 bp，编码553 aa，成熟p50编码区长1 653 bp，编码551 aa；与不同动物的p65、p50序列相似性均较高；56.5% p65位于细胞核内，78.3% p50存在于细胞质中，p65、p50均无信号肽及跨膜区。荧光定量结果表明：起始模板与Ct值之间线性关系好，相关系数达到0.999，扩增效率均在95%左右；敏感性高，初始模板的检出下限达到10¹copies·μL^－1；特异性强，扩增产物形成单一的特异性熔解峰；重复性好，组内变异系数均小于5‰。本试验为进一步研究猪NF-κB p65/p50生物学功能及其在猪相关疾病发生发展中表达量变化奠定了基础。

Abstract:

The signaling pathway of Nuclear factor-kappaB (NF-κB) plays an important role in the viral infection, immune injury diseases and tumor diseases. Its activation level is often used as a detection indicator for immune response level and disease development. In order to obtain porcine NF-κB p65/p50 sequence characteristics and establish a real-time fluorescence quantitative PCR (real-time FQ-PCR) method to detect its mRNA expression in vitro, porcine NF-κB p65 ORF and mature p50 coding region were amplified, cloned and analyzed. Specific primers were designed to develop the real-time FQ-PCR assay to detect p65, p50 mRNA expression. Sequence analysis showed that porcine p65 ORF contained 1 662 nucleotides in full length encoding a protein of 533 amino acid residues, the p50 mature protein coding region contained 1 653 nucleotides encoding 551 aa. The nucleotide sequence of p65 and p50 genes shared a high similarity with other animals. 56.5% p65 located in the nucleus, 78.3% p50 existed in the cytoplasm, no signal peptide and transmembrane region had found in both p65 and p50. The real-time FQ-PCR results showed a good liner relationship between template copy number and circulation number, the correlation coefficient (R²) of the standard curves were all 0.999, the amplification efficiency at about 95%. These assays were highly specific and there are single specific melting peak. The assays were highly sensitive and have a detection limit of 10¹copies·μL^－1, and it was highly repeatable and had a coefficient of variation less than 5‰. This study laid a foundation for researching biological function of NF-κB p65/p50 and its differential expression in pig diseases.

中图分类号:

S828
Q781

王潇娣，朱玲，廖春燕，黄仆，徐志文，郭万柱. 猪NF-kappaB p65/p50基因克隆、序列鉴定及实时荧光定量PCR检测方法的建立[J]. 畜牧兽医学报, 2013, 44(8): 1288-1296.

WANG Xiao-di, ZHU Ling, LIAO Chun-yan, HUANG Pu, XU Zhi-wen, GUO Wan-zhu. Clone,Sequence Identification and Development of Real-time PCR Assays for Detection of Porcine Nuclear Transcription Factor-kappaB p65/p50 Genes[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(8): 1288-1296.

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