LILRB2过表达慢病毒载体以及THP-1-LILRB2稳转细胞株的构建

目的 采用慢病毒载体(lentivirus, LV)感染并探究其在人髓系白血病单核细胞(human myeloid leukemia mononuclear cells, THP-1)中的最佳感染条件，之后病毒包装白细胞免疫球蛋白样受体B2(leukocyte immunoglobulin-like receptor subfamily B member 2, LILRB2)的过表达病毒载体并感染THP-1细胞，最终嘌呤霉素筛选出经mRNA和蛋白水平均成功验证LILRB2过表达效果的THP-1稳定转染细胞株，为研究LILRB2在单核细胞中的作用提供前提条件。方法 LILRB2过表达慢病毒载体以pLV-SFFV-MCS-EF1-ZsGreen1-T2A-Puro为慢病毒骨架载体，通过Xba I与BamH I位点插入经聚合酶链式反应(polymerase chain reaction, PCR)扩增的LILRB2基因片段构建而成。待筛选阳性克隆与测序鉴定后，病毒感染预实验确定感染复数(multiplicity of infection, MOI);以293T细胞包装病毒，浓缩后获得的原液进...

Construction of LILRB2 overexpression lentivirus vector and THP-1-LILRB2 stable transformation cell

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.