鸡NLRX1表达的细胞定位及其siRNA干扰效果的研究

Localization and siRNA of chicken NLRX1 expression in cells

NLRX1 (NLR family member X1)是细胞内模式识别受体NLRs家族中重要成员之一,在炎症反应和抗病毒等天然免疫中主要发挥负调控作用。为了解鸡NLRX1 (chNLRX1)分子的特征及其生物学功能,本研究采用cDNA末端快速克隆(RACE)技术,从鸡肺脏组织中克隆了chNLRX1基因的cDNA序列,其最长开放阅读框(ORF)为2 970 bp,编码989个氨基酸,含有较为保守的结构域,分子构象分析显示chNLRX1在空间结构上更接近于鱼类NLRX1;将chNLRX1真核表达质粒转染HEK293T细胞和鸡DF-1细胞,显示chNLRX1主要定位于细胞质内的线粒体区;以腺病毒介导RNAi干扰chNLRX1表达,采用荧光显微镜观察和western blot方法筛选出干扰效率相对较高的si RNA (sh-NLRX1-2)。上述结果为进一步研究chNLRX1的生物学功能奠定基础。

NLRX1(NLR family member X1)is an important member of the NLRs family of intracellular pattern recognition receptors,which plays a negative regulatory role in innate immunity such as inflammation and antiviral immune response.In this study,in order to investigate the molecular characteristics and biological functions of chicken NLRX1(chNLRX1),chNLRX1gene was cloned from chicken lung tissue by RACE technology.The longest ORF of chNLRX1 gene was 2,970bp,encoding 989 amino acids,containing a conserved structural domain.Molecular conformational analysis showed that chNLRX1 is structurally closer to fish NLRX1.After transfection with the chNLRX1 eukaryotic expression plasmid into HEK293T cells and chicken DF-1 cells,chNLRX1 protein was dominantly expressed in the mitochondrial region of the cytoplasm.With adenovirus-mediated RNA interference(siRNA)for target on chNLRX1,an effective siRNA sequence(named sh-NLRX1-2)was identified by fluorescence observation and western blot.The above results lay a foundation for further study on the biological function of chNLRX1.