天然免疫分子LILRB5慢病毒稳定转染THP-1细胞系的构建  

Creation of a THP-1 cell line via stable transfection of a lentivirus vector containing LILRB5

作　　者：王希娣 张之勇[2] 任霞[2] 宋冠华[2] 禹林昌 史美艳[2] 袁晓芬[2] 郭强[2] 李莲莲[2] 张晓瑜[2] 姜国胜[1,2] 

机构地区：[1]济南大学医学与生命科学学院,山东济南250022 [2]山东省医学科学院基础医学研究所,山东济南250062

出　　处：《中国病原生物学杂志》2014年第10期865-868,共4页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81101605;81172792);山东省自然科学基金项目(No.ZR2010HQ034;ZR2009CL014);山东省科技发展计划项目(No.2010GSF10210);山东省医药卫生科技发展计划项目(No.2011QZ021);山东省优秀中青年科学家科研奖励基金项目(No.BS2009YY008)

摘　　要：目的构建天然免疫分子LILRB5的慢病毒表达载体,获得稳定转染LILRB5的单核细胞系。方法将LILRB5构建入真核表达载体pEGFP-flag,瞬时转染293T细胞系,通过免疫荧光和流式细胞术检测LILRB5-GFP和LILRB5-flag在293T细胞中的表达。构建慢病毒表达载体pHR-LILRB5,与p8.91和pMD共同转入293T细胞,产生标记有绿色荧光的LILRB5慢病毒,感染单核细胞系THP-1后通过流式细胞术检测LILRB5的表达。结果测序显示真核表达载体pEGFP-LILRB5-flag的序列与预期相符,瞬时转染pEGFP-LILRB5-flag的293T细胞可检测到绿色荧光蛋白的表达,流式细胞术检测显示LILRB5-flag在细胞膜表面有表达;构建亚克隆慢病毒载体pHR-LILRB5,经测序验证后,转染293T细胞,产生LILRB5的慢病毒,并感染THP-1细胞,流式细胞术检测LILRB5在THP-1细胞稳定表达。结论构建LILRB5的慢病毒载体,通过LILRB5慢病毒感染建立LILRB5的稳转THP-1细胞系。Objectives To construct a lentivirus vector containing the innate immune receptor LILRB5 and to obtain monocytes stably transfected with LILRB5. Methods LILRB5 was subcloned into the expression vector pEGFP-flag,which was then transiently transfected into 293 Tcells.The expression of LILRB5-GFP and LILRB5-flag was detected in293 Tcells transiently transfected with pEGFP-LILRB5-flag using immunofluorescence and flow cytometry（FCM）.The lentivirus vector pHR-LILRB5 was constructed and co-transfected into 293 Tcells with p8.91 and pMD to produce a medium containing LILRB5-GFP lentiviral particles.The lentivirus vector was then transfected into THP-1cells,and the expression of LILRB5 in transfected THP-1cells was detected using FCM. Results The expression vector pEGFPLILRB5-flag was verified via sequencing.Immunofluorescence revealed the expression of LILRB5-GFP in the cytoplasm of 293 Tcells,and FCM revealed LILRB5-flag in the membrane of 293 Tcells.The lentivirus vector pHR-LILRB5 was subcloned and identified by sequencing.The lentivirus vector containing LILRB5 was transfected in 293 Tcells and THP-1cells.FCM revealed that LILRB5 was stably expressed in transfected THP-1cells. Conclusion The lentivirus vector pHR-LILRB5 was constructed and a THP-1cell line stably transfected with LILRB5 was established using the lentivirus vector containing LILRB5.

关 键 词：绿色荧光蛋白 LILRB5 单核细胞 慢病毒 转染 

分 类 号：R37[医药卫生—病原生物学]