斜带石斑鱼核苷酸结合结构域及亮氨酸重复序列受体对病原类似物的识别——通过核因子κB信号途径

Pathogenic Analogues Recognization of NLRs Receptors of Orange-spotted Grouper(Epinephelus coioides) Through NF-κB Signal Pathway

构建斜带石斑鱼(Epinephelus coioides)核苷酸结合结构域(Nucleotide-binding domain,NOD)1及2基因的表达载体pEGFP-Nod1和pEGFP-Nod2,与报告基因质粒pNFκB-Luc及内参质粒pRL-TK共转染HEK293T细胞,以脂多糖(LPS)、肽聚糖(PGN)、胞壁酰二肽(MDP)、及二氨基庚二酸(DAP)为细菌类似物代表,以多聚肌苷酸-胞苷酸(Poly I:C)、多聚尿苷酸(PolyU)、多聚脱氧腺苷酸-脱氧胸腺苷酸[Poly(da:dt)]为病毒类似物代表,研究病原类似物刺激后核因子κB(NF-κB)的活化程度。结果表明,斜带石斑鱼Nod2基因可通过NF-κB信号途径识别多种病原成分;Nod1基因可识别DAP,对其他配体呈抑制作用或未通过NF-κB信号通路发挥作用。斜带石斑鱼Nod1及Nod2基因可不同程度地通过NF-κB信号途径识别病原成分。

Expression vectors of pEGFP-Nod1 and pEGFP-Nod2 were constructed from Orange-spotted grouper, Epinephelus coioides, followed by co-transfection into HEK293T cells with pNFκB-Luc reporter plasmid and pRL-TK reference plasmid. NF-κB activity was monitored after HEK293T cells were stimulated by virus analogs, such as Polyinosinic: polycytidylic (Poly, I: C), Poly-uridine (poly U),Poly (deoxyadenylic-deoxythymidylic) (Poly (da:dt)) and bacteria analogues as Lipopolysaccharide(LPS), Peptidoglycan (PGN), Muramyl dipeptide (MDP) and L-Ala-γ-D-Glu-mesu-diaminopimelic acid(DAP). The results showed that the grouper Nod2 gene discriminated on various pathogenic components through NF-κB signal pathway, while Nod1 gene could recognize DAP. For other ligands, Nod1 gene showed inhibitory effect on some or no effect on the other by the NF- kappa B signaling pathway. These results indicated that, the grouper Nod1 and Nod2 were able to make identification in different extent of pathogenic components through NF-κB signal pathway.