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

Abstract

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.

CLC Number:

S828
Q781

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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Clone,Sequence Identification and Development of Real-time PCR Assays for Detection of Porcine Nuclear Transcription Factor-kappaB p65/p50 Genes

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