Establishment of B lymphocyte model overexpressing human TLR2/1 and TLR2/6
To construct a B lymphocyte model stably expressing human TLR2,the coding sequences of human TLR1,TLR2 and TLR6 were cloned and inserted into the pCDH-CMV-MCS-EF1-GFP-puro lentiviral vector.After verification of correct insertion by sequen-cing,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 micro-scope 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 fluo-rescent 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 foun-dation for further exploring the impact of the TLR2 pathway on the anti-infective immune function of B cells.
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维普
