Application of lambda interferon in the preparation of anti-hantaan virus drugs
A Hantaan virus and drug technology, which is applied in the application field of the new interferon-λ interferon in the preparation of anti-Hantaan virus drugs, can solve the problems of lowering the fatality rate, not reducing complications, and long antipyretic time, achieving Obvious therapeutic effect, reduced nucleic acid expression level, fast action time
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Embodiment 1
[0032] Embodiment 1MTT method detects the cytotoxic effect of IFN-λ1 / λ2
[0033] A549 cells cultured in DMEM (containing 10% fetal bovine serum, V / V) were digested with trypsin and diluted to 1~3×10 5 cells / mL, 100 μL per well was inoculated in a 96-well plate. After the cells grew into a single layer, different concentrations (1ng / mL, 10ng / mL, 100ng / mL, 1000ng / mL) of IFN-λ1, IFN- λ2, cultured for 72 hours, and the cytotoxicity of IFN-λ1 and IFN-λ2 was detected by MTT method. The results showed that the cytotoxicity of IFN-λ1 and IFN-λ2 from 1ng / mL to 1000ng / mL was less than 10% on A549 cells, and statistics There was no significant difference in the analysis, so it can be considered that IFN-λ1 and IFN-λ2 have no toxicity to A549 cells ( figure 1 ).
Embodiment 2I
[0034] Example 2 IFN-λ anti-HTNV infection in vitro has time effect and dose effect
[0035] (1) IFN-λ has a time effect on HTNV infection in vitro
[0036] A549 cells cultured in DMEM (containing 10% fetal bovine serum, V / V) were digested with trypsin and diluted to 1~3×10 5 cells / mL, 400 μL per well was inoculated in a 24-well plate. After the cells grow into a single layer, add IFN-λ1 and IFN-λ2 (100ng / mL) diluted with DMEM (containing 2% fetal bovine serum, V / V) to pretreat A549 cells for 24h, and HTNV 76-118 strain infection for 2h ( MOI=5), and washed three times with PBS, then added DMEM (containing 2% fetal bovine serum, V / V) medium, 37 ° C 5% CO 2 (V / V) culture; cells were collected on the 1st day, 4th day, 7th day and 10th day of infection, and real-time quantitative RT-PCR was used to detect the expression of HTNV S gene, GAPDH was used as an internal reference, and IFN-λ was not added treated as the control group. Primers are as follows:
[0037] HTNV S gene u...
Embodiment 3I
[0044] Example 3 Antiviral Factors Induced by IFN-λ
[0045] A549 cells cultured in DMEM (containing 10% fetal bovine serum, V / V) were digested with trypsin and diluted to 1~3×10 5 cells / mL, 400 μL per well was inoculated in a 24-well plate. After the cells grow into a single layer, add IFN-λ1 and IFN-λ2 (100ng / mL) diluted with DMEM (containing 2% fetal bovine serum, V / V) to pretreat A549 cells, and then treat them 3h, 6h, and 12h after treatment, respectively. and 24h, the cells were collected, and the expression of antiviral factors OAS-1, ISG-56, MxA and MxB was detected by real-time quantitative RT-PCR. GAPDH was used as an internal reference, and those without interferon treatment were used as a control group. Primers are as follows:
[0046] OAS-1 upstream primer: 5'-AGAAGGCAGCTCACGAAACC-3',
[0047] OAS-1 downstream primer: 5'-CCACCACCCAAGTTTCCTGTA-3';
[0048] ISG-56 upstream primer: 5'-GCTGAAGTGTGGAGGAAAGA-3',
[0049] ISG-56 downstream primer: 5'-AGCAAAGAAAATGGC...
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