Application of Compound mln4924 in the Preparation of Bunyaviridae Phlebovirus Inhibitors
A bunya virus, virus inhibitor technology, used in antiviral agents, resistance to vector-borne diseases, medical preparations containing active ingredients, etc.
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Embodiment 1
[0019] Embodiment 1, plasmid construction
[0020] Plasmid construction by a method comprising the following steps
[0021] Synthesis of Nonstructural Protein cDNA of Fever with Thrombocytopenia Syndrome Virus (SFTSV) and Sicilian Sandfly Fever Virus (SFSV)
[0022] According to GENBANK gene sequence numbers SFTSV (GI: 747024328) and SFSV (GI: 334035), the gene sequence was retrieved, and the cDNA sequences of SFTSV and SFSV non-structural protein NSs were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.
[0023] Amplification of target gene
[0024] According to the sequence of SFTSV and SFSV non-structural protein NSs genes, NSs-specific primers were designed, the restriction site of the upstream primer was Sal1, the restriction site of the downstream primer was BamH1, and an HA tag was added at the end of the downstream primer. Using cDNA as a template, the target gene was amplified according to the PCR instruction system.
[0025]
[0026] PCR product gel reco...
Embodiment 2
[0038] Embodiment 2, cell culture, transfection, activity detection of interferon promoter
[0039] cell culture
[0040] 1) 293T cells ( CRL-3216 TM ) cultivation
[0041] 293T cells were cultured in DMEM liquid medium containing 10% FBS, 1% penicillin-streptomycin, placed in an incubator containing 5% CO2 at 37°C, and incubated in an incubator containing 0.25% EDTA after overgrowth. Enzyme digestion, after about 2-3min, add 10% FBS fresh medium to terminate, centrifuge at room temperature at 1200rpm for 3min, discard the supernatant, take an appropriate amount of fresh medium to resuspend, return one-third of the culture medium to the culture bottle, and the remaining Transfer to 24-well cell culture plates.
[0042] 2) HeLa cells ( CCL-2 TM ) cultivation
[0043] HeLa cells were cultured in DMEM liquid medium containing 10% FBS and 1% penicillin-streptomycin, and incubated at 37°C in an incubator containing 5% CO2. After about 2-3 days, digest with trypsin contain...
Embodiment 3
[0059] Example 3, SFTSV SFSV NSs inhibit IFN-β promoter activity
[0060] Perform experiments such as cell transfection, fluorescent dual reporter analysis, and Western blot according to the specified steps (the method is the same as in Example 2)
[0061] 200ng RIG-I-Flag plasmid (Addgene) was co-transfected into 293T cells with 200ng VR1012 vector, 50ng SFTSV NSs and 500ng SFSV or NSs respectively, and 500ng IFN-β-Luc and 50ng pRL-TK plasmids were transfected at the same time. Forty-eight hours after transfection, the expression of the reporter gene was detected by a dual reporter gene detection method. NSs protein expression level by Western-Blot results as follows figure 1 show. The results of dual reporter gene detection showed that the NSs protein of the virus significantly inhibited the activation of the RIG-I-induced interferon beta promoter, which also indicated that the NSs protein inhibited the production of host type I interferon.
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