中文摘要：

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。

英文摘要：

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.