摘要
为分析鸭NOD样受体蛋白3(NOD-like receptor protein 3,NLRP3)基因和蛋白特性,根据鸭NLRP3基因组序列(Gene ID:101795933)设计引物,经PCR扩增、测序后获得长2 805 bp基因序列,可编码934个氨基酸,包含PYRIN、NACHT和LRR结构域。构建pCAGGS-duNLRP3-Flag真核表达质粒,转染细胞后,蛋白印迹试验检测到约104 ku蛋白,间接免疫荧光试验发现该蛋白在转染后12 h开始表达,弥散分布在细胞质,但经病毒刺激后可在核周发生斑点样聚集。上述结果表明,研究获得了鸭NLRP3基因全长CDS序列,构建的重组质粒p CAGGSduNLRP3-Flag转染DEF细胞后可表达有活性的NLRP3蛋白,为后续鸭NLRP3基因和蛋白功能研究提供参考。
In order to characterize duck NOD-like receptor protein 3(NLRP3) gene and protein,primers based on duck NLRP3 genome sequence(Gene ID: 101795933) were designed to amplify the CDS sequence of this gene by PCR. After sequencing, a 2 805 bp long gene sequence, encoding 934amino acids that contained PYRIN, NACHT, and LRR structural domains was obtained. The p CAGGSduNLRP3-Flag eukaryotic expression plasmid was then constructed and transfected into DEF. A protein that was approximately 104 ku and expressed diffusely in the cytoplasm at 12 h after transfection was detected by Western-blot and indirect immunofluorescence assay. Further, it was found that the protein could form puncta around the perinuclear region after virus stimulation. These results suggested that the full-length CDS sequence of duck NLRP3 was obtained and the recombinant plasmid pCAGGSduNLRP3-Flag was able to express functional NLRP3 protein after transfection with DEF, which provided a reference for subsequent studies on the function of duck NLRP3 gene and protein.
作者
伍单丹
孙迪
汪铭书
程安春
毛赛
WU Dandan;SUN Di;WANG Mingshu;CHENG Anchun;MAO Sai(Avian Disease Research Center,College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China;Institute of Preventive Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China;Key Laboratory of Animal Disease and Human Health of Sichuan Province,Sichuan Agricultural University,Chengdu 611130,China)
出处
《东北农业大学学报》
CAS
CSCD
北大核心
2022年第12期46-56,共11页
Journal of Northeast Agricultural University
基金
四川省科技计划项目(2020YJ0397)
财政部和农业农村部:国家现代农业产业技术体系资助(CARS-42-17)。
作者简介
伍单丹(1996-),女,硕士研究生,研究方向为鸭甲肝病毒致病机制。E-mail:wudandan1123@163.com;通讯作者:毛赛,讲师,博士,研究方向为鸭肝炎病毒与宿主相互作用。E-mail:sarrawin@163.com。
