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

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

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 p65ORF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 p65ORF长1 662bp,编码553aa,成熟p50编码区长1 653bp,编码551aa;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101 copies.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。

The signaling pathway of Nuclear factor-kappaB （NF-~B） 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 oth- er 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 pS0. The real-time FQ-PCR results showed a good liner relationship between template copy number and circulation number, the cor- relation 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 as- says were highly sensitive and have a detection limit of 101 copies ·μL^-1 , and it was highly re- peatable and had a coefficient of variation less than 5‰. This study laid a foundation for researc- hing biological function of NF-κB p65/p50 and its differential expression in pig diseases.