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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。
Objective To construct lentiviral expression vector of natural immune molecule LILRB5 and obtain monocyte cell line stably transfected with LILRB5. Methods LILRB5 was constructed into eukaryotic expression vector pEGFP-flag and transiently transfected into 293T cell line. The expression of LILRB5-GFP and LILRB5-flag in 293T cells was detected by immunofluorescence and flow cytometry. The lentiviral expression vector pHR-LILRB5 was constructed and transfected into 293T cells with p8.91 and pMD to generate LILRB5 lentivirus with green fluorescence. The expression of LILRB5 was detected by flow cytometry after infected with monocytic cell line THP-1. Results Sequencing showed that the sequence of eukaryotic expression vector pEGFP-LILRB5-flag was in accordance with the expectation. The expression of green fluorescent protein (GFP) was detected in 293T cells transiently transfected with pEGFP-LILRB5-flag. The results of flow cytometry showed that the expression of LILRB5- LILRB5 was constructed and transfected into 293T cells. The lentiviral vector LILRB5 was constructed and infected with THP-1 cells. The expression of LILRB5 in THP-1 cells was detected by flow cytometry. Conclusion The lentiviral vector of LILRB5 was constructed and the THP-1 cell line was established by LILRB5 lentivirus infection.