The anti-infective effect and application of hnrnpa2b1
A technology of A2B1 and DNA virus, which is applied in the field of biotechnology and medicine, and can solve problems such as the unclear role of hnRNPA2B1 protein
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Embodiment 1
[0109] Example 1: hnRNPA2B1 directly recognizes DNA of pathogen origin in the nucleus
[0110] Obtain mouse primary peritoneal macrophages: use C57BL / 6 mice (6-8 weeks old, female, purchased from Shanghai Bikai Experimental Animal Co., Ltd.), intraperitoneally inject 2ml of 3% thioglycolate (purchased from Sigma -Aldrich) solution once, three days later, the mice were killed by cervical dislocation, the peritoneal cavity was washed with serum-free medium, and cells were obtained by centrifugation after aspiration.
[0111] Primary peritoneal macrophage cells were cultured in DMEM medium. After 2 hours of infection with HSV-1 (F strain, purchased from ATCC), the nuclear protein was isolated and passed through anti-hnRNPA2B1 antibody (purchased from Santa Cruz) or control antibody (IgG, purchased from Santa Cruz) for immunoprecipitation and detection of HSV-1 DNA by PCR.
[0112] hnRNPA2B1 binds to HSV-1 DNA as figure 1 shown.
[0113] The results showed that the hnRNPA2B1 pr...
Embodiment 2
[0115] Example 2: Interfering with the expression of hnRNPA2B1 has an inhibitory effect on the type I interferon induced by HSV-1 infection
[0116] Primary peritoneal macrophage cells were cultured in DMEM medium, and human THP1 cells (purchased from ATCC) were cultured in 1640 medium. The cells were transfected with small interfering RNA against hnRNPA2B1 (hnRNPA2B1 siRNA) and mock control (control siRNA) (transfection reagent INTERFERin was purchased from Polyplus).
[0117] Small interfering RNA against hnRNPA2B1 (hnRNPA2B1 siRNA) and mock control (control siRNA) were purchased from Genephama. The sequence of mouse hnRNPA2B1 siRNA is shown in SEQ ID NO: 5 and SEQ ID NO: 6, the sequence of human hnRNPA2B1 siRNA is shown in SEQ ID NO: 7 and SEQ ID NO: 8, and the sequence of control siRNA is shown in SEQ ID NO: 9 and SEQ ID NO: As shown in ID NO: 10, 2 dTs were added at the 3' during synthesis to make the sequence more stable. The specific sequence is as follows:
[0118]h...
Embodiment 3
[0132] Example 3: Overexpression of hnRNPA2B1 inhibits IFN-β secretion in cells
[0133] First, the cDNA of hnRNPA2B1 (NM_182650) was introduced into the eukaryotic expression vector pcDNA3.1 plasmid (purchased from Addgene) according to conventional methods to construct the hnRNPA2B1 expression vector.
[0134] The hnRNPA2B1 expression vector was transfected into mouse peritoneal macrophages at a density of 1 μg / ml, and the fresh DMEM medium was replaced 48 hours later.
[0135] to 4×10 5 cells / ml, HSV-1 (MOI, 10) infected cells, collected the cell culture supernatant after 12 hours, and detected the IFN-β level in the cell culture supernatant with an ELISA kit.
[0136] The secretion of IFN-β is as follows Figure 5 shown.
[0137] The results showed that: hnRNPA2B1 plasmid transfected cells can promote the production of IFN-β induced by HSV-1.
[0138] The results indicated that overexpression of hnRNPA2B1 could promote the production of IFN-β induced by HSV-1 infection...
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