摘要
目的利用PiggyBac转座系统构建稳定表达NLRC5的BEAS-2B细胞系,为后续针对病毒抗原提呈功能的研究提供有力的工具。方法根据NLRC5基因信息扩增目的基因蛋白编码序列,构建目的基因NLRC5的转座子质粒,将辅助质粒和转座子质粒共转染至BEAS-2B细胞。嘌呤霉素筛选稳定转导的细胞后进行单克隆化,最后对获取的单克隆细胞在分子水平上进行过表达验证。结果成功构建一株NLRC5过表达的BEAS-2B细胞,且该细胞系可维持正常细胞活性与增殖能力。结论通过PiggyBac转座子技术实现了NLRC5的过表达,进而提高了MHC-Ⅰ分子基因的表达。通过对该基因的过表达从而研究NLRC5对MHC-Ⅰ抗原肽提呈途径的调控机制,为理解抗病毒免疫的复杂机制以及开发新的治疗策略提供有力支持。
Objective To construct a BEAS-2B cell line stably overexpressing NLRC5 by using PiggyBac transposon system,so as to provide a powerful tool for further exploration of viral antigen presentation mechanisms.Methods Based on NLRC5 gene data,the protein-coding sequence of NLRC5 was amplified,and a transposon plasmid containing NLRC5 was constructed.The helper plasmid and transposon plasmid were co-transfected into BEAS-2B cells.Following puromycin screening for stably transduced cells,monoclonalization was conducted to validate molecular-level overexpression in the obtained monoclonal cells.Results A BEAS-2B cell line,which overexpressed NLRC5,was successfully constructed while maintaining regular cell viability and proliferation capacity.Conclusion The overexpression of NLRC5 is accomplished using PiggyBac transposon,resulting in heightened expression of MHC-Ⅰmolecule genes.Investigation into the regulatory role of NLRC5 in the MHC-Ⅰantigen peptide presentation pathway via its overexpression offers substantial insights into the intricate mechanisms of antiviral immunity,thus facilitating the development of novel therapeutic approaches.
作者
孙丹妮
刘欣悦
饶子凡
杜岚
张湘琳
刘梓谕
侯诗源
沈星
刘蓉蓉
吴兴安
SUN Danni;LIU Xinyue;RAO Zifan;DU Lan;ZHANG Xianglin;LIU Ziyu;HOU Shiyuan;SHEN Xing;LIU Rongrong;WU Xingan(Department of Microbiology and Pathogen Biology,School of Basic Medical Sciences,Air Force Medical University,Xian 710032,China;No.5 Cadet Regiment,School of Basic Medical Sciences,Air Force Medical University,Xian 710032,China)
出处
《空军军医大学学报》
2025年第4期526-532,共7页
Journal of Air Force Medical University
基金
国家自然科学基金(82272330)
陕西省自然科学基金(2024JC-ZDXM-42)。
