关键词: 白细胞免疫球蛋白样受体B2, 悬浮细胞, 慢病毒载体, 稳定转染株

Abstract: Objective To construct stable human myeloid leukemia mononuclear cells (THP-1) overexpressing leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2) cDNA by viral packaging with a lentiviral vector overexpressing LILRB2 and infecting THP-1 cells using puromycin selection, and to successfully validate the overexpression effects at both mRNA and protein levels, we will provide prerequisites to study the role of LILRB2 in monocytes. Methods The LILRB2 overexpression lentiviral vector was constructed by inserting polymerase chain reaction (PCR) amplified LILRB2 gene fragments into Xba I and BamH I followed by the lentiviral backbone vector pLV-SFFV-MCS-EF1-ZsGreen1-T2A-Puro. After positive clones were screened and identified by sequencing, the multiplicity of infection (MOI) value was determined experimentally before virus infection. Virus was packaged in 293T cells, and the stock obtained after concentration was subjected to virus titer. To confirm the setting of the overexpression (LV-OE-LILRB2) group and control (LV-OE-NC) group, the green fluorescence of virus-infected THP-1 in each group was observed by fluorescence microscope. Puromycin selection of THP-1 cell lines stably expressing LILRB2. The mRNA and protein expression levels of LILRB2 in THP-1 cells were determined by quantitative real-time PCR (qPCR) and Western blotting (WB), respectively. Results The base sequences in the vector were confirmed to be correct by PCR and sequencing to confirm the completion of the LILRB2 overexpression lentiviral vector construction. Virus infection pre experiments were performed to determine the monocyte cell line THP-1 infected for 72 h at MOI=5. Viral titer measurements yielded LV-OE-LILRB2 group titers of 1 × 10⁸ TU / mL, while the titer in the LV-OE-NC group was 3.3 × 10⁸TU/mL. A concentration of 2 μ g / mL puromycin selected stable cell lines subjected to qPCR showed that the mRNA expression level of LILRB2 in the LV-OE-LILRB2 group was significantly upregulated compared with that in the LV-OE-NC group (P<0.001). Moreover, WB assay showed that the protein expression level of LILRB2 in LV-OE-LILRB2 group was significantly increased compared with that in LV-OE-NC group (P <0.05). Conclusion The successful establishment of THP-1 cell lines stably overexpressing LILRB2 was achieved, providing a cell model for studying the mechanism of LILRB2 in monocytes.

Key words: leukocyte immunoglobulin-like receptor subfamily B member 2(LILRB2), suspended cells, lentiviral vector, stable transformation