【摘要】 为了研究赤羽病毒（AKAV）N基因的遗传变异情况，并利用原核表达技术制备检测抗原。参照GenBank上已发布的赤羽病毒N基因组序列，设计引物，将AKAV的RNA反转录后通过PCR方法对目的基因进行了扩增，将目的片段测序之后进行蛋白理化性质、基因序列分析以及原核表达。结果表明，成功扩增出大小为717bp的赤羽病毒N基因，编码的蛋白属于稳定蛋白，无跨膜区、无信号肽，偏碱性的亲水蛋白，核苷酸序列分析显示该毒株的N基因序列与NCBI上已经公布的基因序列同源性为82.7%～97.9%。构建pET28a-N的原核表达质粒并转化BL21菌株，成功表达出27ku重组N蛋白，Western blot鉴定表明该蛋白能与阳性血清发生结合。试验结果为进一步研制赤羽病毒疫苗及ELISA诊断试剂盒奠定了基础。更多还原

【Abstract】 To investigate the genetic variation of the N gene of the Akabane virus(AKAV)and to prepare detection antigens using prokaryotic expression technology,primers were designed based on the N genome sequence of AKAV available in GenBank.After reverse transcription of AKAV RNA,the target gene was amplified using PCR.Following the sequencing of the target fragment,comprehensive analyses were conducted regarding the physicochemical properties of the protein,gene sequence,and prokaryotic expression.The results indicated that the 717 bp N gene of AKAV was successfully amplified.The encoded protein was stable,lacking transmembrane region and signal peptides,and exhibited hydrophilic and alkaline properties.Nucleotide sequence analysis revealed that the homology of the N gene sequence of this strain,compared to those published in NCBI,ranged from 82.7%to 97.9%.The prokaryotic expression plasmid pET28a-N was constructed and subsequently transformed into the BL21 strain,resulting in the successful expression of the 27 ku recombinant N protein.Western blot analysis confirmed that this protein could bind to positive serum.This experiment laid the foundation for the further development of AKAV vaccines and ELISA diagnostic kits.更多还原