兔NLRC3基因克隆及其表达特性研究

本研究旨在探究兔NOD样受体家族蛋白3（NOD-like receptor family CARD domain containing 3，NLRC3）基因序列、分子结构和体内外表达特性。根据GenBank中公布的预测序列（登录号：XM_017338739.1）设计引物，PCR扩增并克隆兔NLRC3基因，利用生物信息学方法对其分子结构进行预测分析。构建真核表达载体pcDNA3.1-rNLRC3-HA，通过间接免疫荧光试验探究兔NLRC3的亚细胞定位。通过实时荧光定量PCR检测NLRC3基因在兔各组织中的分布情况及肠出血性大肠杆菌感染后表达水平的变化。结果表明，兔NLRC3基因编码区长3 192 bp，相似性比对及系统进化树显示，兔NLRC3与其他哺乳动物相似性较高，并在进化树中处于同一分支。兔NLRC3由N-端、NACHT及LRR结构域组成，三级结构模型呈单曲率马蹄形，凸面由α-螺旋组成，凹面由β-折叠组成。间接免疫荧光试验结果显示，兔NLRC3位于细胞浆中，且不与线粒体共定位。兔NLRC3基因在所有被检组织中均有表达，且在脾脏中的表达量最高，其次是肠系膜淋巴结、淋巴滤泡。肠出血性大肠杆菌感染机体后，在脾脏、肝脏、肾脏中兔NLRC3基因mRNA表达量均上调，表明兔NLRC3基因参与了肠出血性大肠杆菌感染后的免疫应答。综上，本研究成功克隆了兔NLRC3基因并进行了生物信息学分析，明确其为胞内受体，在各组织中广泛分布，并参与了细菌感染后的免疫应答，为进一步探究兔NLRC3在炎症反应中的调控机制提供了基础材料。

Study on Cloning and Expression Characteristic of NLRC3 Gene in Rabbit

This study was aimed to investigate the sequence, molecular structure, and expression characteristics of Oryctolagus cuniculus NOD-like receptor family CARD domain containing 3 (NLRC3) in vivo and in vitro. The primers were designed according to the predicted sequence published by GenBank (accession No. :XM_017338739.1), NLRC3 gene in rabbit was amplified by PCR and cloned, and its molecular structure was predicted and analyzed by bioinformatics method. The pcDNA3.1-rNLRC3-HA vector was constructed, and the subcellular localization of NLRC3 gene in rabbit was carried out by indirect immunofluorescent assay. The tissue distribution of NLRC3 gene in rabbit was detected by Real-time quantitative PCR. After challenged with enterohemorrhagic Escherichia coli (EHEC), the expression level of NLRC3 gene in rabbit was further analyzed. The results showed that the CDS sequence of NLRC3 gene in rabbit was 3 192 bp. The similarity and phylogenetic tree analysis showed that NLRC3 gene in rabbit had highly similarity with other mammalian species in the same branch. NLRC3 protein in rabbit consisted of N-terminal domain, NACHT domain and LRR domain. Tertiary structural model of NLRC3 protein in rabbit formed a typical horseshoe-like structure in a single curvature, with alpha helix forming the convex surface and beta sheet in the concave faces. Indirect immunofluorescence assay showed that NLRC3 protein in rabbit was located in the intracytoplasm, and did not colocate with mitochondria. NLRC3 protein was detected in all tested tissues of rabbit, with the highest level in spleen, followed by mesenteric lymphnodes and lymphoid follicles. The mRNA expression of NLRC3 gene in rabbit was upregulated in spleen, liver and kidney after EHEC infection. The results showed that NLRC3 gene in rabbit was involved in the immune response after EHEC infection. In summary, NLRC3 gene in rabbit was successfully cloned and analyzed by bioinformatics, it was an intracellular receptor and widely distributed in various tissues, it participated in the immune response after bacterial infection. This study provided basic materials for further exploring the regulatory mechanism of NLRC3 in rabbit in inflammatory response.