利用PiggyBac转座子技术构建NLRC5基因过表达BEAS-2B细胞系及其功能研究  

Construction of BEAS-2B cell line with NLRC5 overexpression through PiggyBac transposon and its function study

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)

机构地区：[1]空军军医大学基础医学院微生物与病原生物学教研室,陕西西安710032 [2]空军军医大学基础医学院学员五大队,陕西西安710032

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)。

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.

关 键 词：NLRC5 PIGGYBAC转座子 基因过表达 稳定转染细胞株 

分 类 号：R392[医药卫生—免疫学] Q78[医药卫生—基础医学]