利用昆虫细胞表达猪RANKL蛋白及其生物学活性分析
Expression of porcine RANKL protein using insect cells and analysis of its biological activity
周亚伟 1蒋华正 1杨宁 1付钰广 1李宝玉 1杨彬 1刘光亮1
作者信息
- 1. 中国农业科学院兰州兽医研究所动物疫病防控全国重点实验室,甘肃兰州 730046
- 折叠
摘要
将猪RANKL基因克隆至杆状病毒供体质粒pFastBacHTA中,经酶切鉴定及测序筛选出阳性重组供体载体pFastBacHTA-RANKL,将其转化至含有杆状病毒穿梭载体Bacmid的DH10Bac感受态细胞,构建杆状病毒表达载体,获得重组转座子Bacmid-RANKL,在脂质体介导下转染sf9昆虫细胞,获得含RANKL基因的重组杆状病毒.经SDS-PAGE分析、Western-blot分析以及IFA试验,证明RANKL基因在sf9细胞中成功表达.利用表达的RANKL蛋白刺激IPEC-J2细胞,采用qPCR方法检测处理后细胞中M细胞相关标志基因的表达.结果显示,与阴性对照组相比,猪RANKL处理后的细胞中M细胞特异性的标记基因CK-18、Marcksll、Sgenel、Spib、CCL20、UBD、NCF4、TRAF6表达量都显著上调.这表明本研究表达的猪RANKL具有良好的生物学活性,可成功诱导IPEC-J2细胞向M细胞分化.本研究结果为后续进一步研究猪肠道M细胞的分化,及研制增强猪肠道黏膜免疫应答的M细胞靶向疫苗打下了良好的物质基础.
Abstract
The porcine RANKL gene was cloned into the baculovirus donor plasmid pFastBacHTA,and the positive recombinant donor vector pFastbachTA-RANKL was identified by restriction enzyme diges-tion and sequencing.The recombinant vector pFastbachTA-RANKL was transformed into DH10Bac competent cells containing Bacmid to construct baculovirus expression vector.The recombinant baculovirus con-taining porcine RANKL gene were obtained via sf9 insect cells transfected by recombinant transposon Bacmid-RANKL using Liposome.The identification results of SDS-PAGE,Western-blot and IFA showed that RANKL protein was expressed successfully by recombinant baculovirus in sf9 insect cells.IPEC-J2 cells were treated with expressed RANKL protein,and M cell-associated gene were tested by PCR assay.The re-sults showed that the expression of M cell-specific marker genes CK-18,Marcksll,Sgenel,Spib,CCL20,UBD,NCF4,and TRAF6 were significantly up-regulated in cells treated with porcine RANKL,indicating the expressed porcine RANKL protein has biological activity and could induce IPEC-J2 cells differentia-tion towards M cells.In conclusion,the successfully expressed porcine RANKL protein provides the ma-terial basis for further study of M cell differentiation in porcine intestinal tract and development of M cell targeted vaccines.
关键词
RANKL/M细胞/杆状病毒表达系统Key words
RANKL/M cell/BEVS引用本文复制引用
出版年
2024
国家科技期刊平台
NSTL
万方数据