Establishment Of Overexpression Model And Functional Study Of SP110 In THP1 Macrophages

Objective:To construct a human mononuclear macrophage THP-1 cell model with overexpression of SP110 gene,to infect mycobacterium tuberculosis H37Ra in vitro,and preliminarily investigate the mechanism of SP110 gene in macrophage against Mtb infection.Methods:(1)The SP110 gene sequence was obtained by PCR,the lentiviral vector LV5-SP110 was constructed,and the recombinant plasmid and the lentivirus-assisted plasmid system(pGag/Pol pRev pvsv-g)were packaged into recombinant lentivirus particles by RNAi-Mate after double enzyme digestion and sequencing identification.And the lentivirus venom was obtained and the titer was calculated by co-transfection of 293T cells;(2)THP-1 cells were transfected with lentivirus venom with known titer,the empty plasmid LV5-NC group was used as control,and the transfection efficiency was calculated,the expression of SP110 gene was identified by rt-pcr and Western blotting;(3)MTT method was used to detect the effect of SP110 gene overexpression on the growth status of macrophages,Purinomycin was used to screen THP-1 cells with stable expression of SP110 gene and stable expression of eGFP(green fluorescence),respectively,as the experimental group and the control group;After induction and differentiation with a final concentration of 200ng/mlPMA,in vitro infection with H37Ra and cell lysate bacterial culture colony count(CFU)after 24h and 96h of infection were used to observe the anti-h37ra infection activity of cells in the experimental group and control group.Results:1.Double enzyme digestion identification and DNA sequencing comparison showed that the gene fragment(1650bp)was completely consistent with the expected SP110gene sequence,LV5-SP110 and LV5-NC vectors were successfully constructed;Recombinant lentiviruses LV5-SP110 and LV5-NC with virus titer of 1x10~8 TU/ml were successfully obtained after 293T cell packaging;2.Thp-1 cells were successfully transfected with a transfection efficiency of 50%,Rt-pcr and Western blotting were used to identify that SP110gene was highly expressed in the transcription and translation levels of cells in the experimental group;3.MTT method did not find that LV5-SP110 could inhibit the growth of thp-1 macrophages;After screening by purinomycin,thp-1 cells with high expression of SP110 gene and cells with stable expression of eGFP(green fluorescent protein)were obtained;In vitro anti-mycobacterium H37Ra experiment showed that the bacterial culture colony count(CFU)of macrophage lysates of the experimental group with overexpression of SP110 gene was lower than that of the control group,with statistically significant difference(p<0.05).Conclusions:The overexpression model of SP110 gene was successfully constructed,and the overexpression of SP110 gene has no inhibitory effect on the proliferation of macrophages,In vitro antibacterial experiments showed that the overexpression of SP110gene enhanced the ability of macrophages to kill intracellular phagocytic mycobacterium tuberculosis H37Ra.