关键词: TLR2, 慢病毒载体, B淋巴细胞, PI3K-AKT信号通路, 细胞增殖

Abstract: To construct a B lymphocyte model stably expressing human TLR2, the coding sequences of humanTLR1,TLR2andTLR6were cloned and inserted into the pCDH-CMV-MCS-EF1-GFP-puro lentiviral vector. After verification of correct insertion by sequencing, these plasmids were packaged into complete infectious virus particles by transfection into 293T cells and transduced into Nalm-6 cells (TLR2－), and puromycin was used to select Nalm-6 cells that stably expressed TLR2 (TLR2+). An inverted fluorescence microscope was used to observe the cell status and green fluorescent protein expression. Western blott was used to measure the expression levels of related proteins. CCK-8 and flow cytometry assays were used to determine the cell viability and evaluate cell proliferation. The results of inverted fluorescence microscopy showed that Nalm-6 cells were successfully infected with lentiviruses with green fluorescent protein expression and puromycin resistance. Western Blot analysis showed that TLR2 was successfully expressed in Nalm-6 cells and that activation of TLR2 signaling significantly increased the levels of phosphorylated PI3K-AKT signaling axis proteins. The CCK-8 and flow cytometry assays showed that TLR2 activation could increase the cell viability and promote cell proliferation. In summary, in this study, a B lymphocyte line model overexpressing TLR2 was successfully constructed and was used to verify that TLR2 activation can upregulate the PI3K-AKT signaling pathway in B cells and promote their proliferation. This model lays the foundation for further exploring the impact of the TLR2 pathway on the anti-infective immune function of B cells.

Key words: LR2, lentiviral vector, B lymphocyte, PI3K-AKT pathway, cell proliferation