Ebola virus entry inhibitor composition and application thereof
An Ebola virus and entry inhibitor technology, applied in the field of medicine, can solve the problems of large dosage of antibody drugs, high cost, poor anti-Ebola virus activity, etc.
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Embodiment 1
[0033] Embodiment 1: EBOVZairePsV system screening principle
[0034] The entry of Ebola virus into the host cell is the first step of virus infection, and the inhibition of virus entry can effectively block the virus infection; EBOV envelope surface glycoprotein (Glycoprotein.GP) is the mediator of Ebola virus adsorption and entry into the target cell the key protein;
[0035]The present invention synthesized the envelope GP gene of Zaire type Ebola virus (EBV-ZaireGP, GeneAccesionNo.KM034549.1). By co-transfecting EBOV-GP and co-transfecting cells with pNL4-3.Luc-, obtained EBV-GP is the EBOV recombinant virus EBOV-ZairePsV of the HIV core wrapped in the shell; the virus particle has the following characteristics: 1) the selectivity of the virus to the host cell depends on the characteristics of EBOV-GP; 2) due to the env on the HIV vector, The nef and vpr genes are deleted, so the virus can only enter the host cell once and cannot replicate, so the virus is safe; 3) The HI...
Embodiment 2
[0036] Embodiment 2: Utilize EBOV-ZairePsV pseudovirus system to evaluate the antiviral effect of tamoxifen and antibody complex, virus inhibition experiment
[0037] In the present invention, EBV-Zaire (Gene Accession No. KM034549.1) is used to evaluate the pharmacological activity of the drug against Ebola virus invasion and infection.
[0038] Recombinant virus preparation: co-transfect 2ugpcDNA3.1 / EBV-GP plasmid and 20ugpNL4-3.Luc.R-E-plasmid into 293T cells, collect the supernatant after 48 hours of transfection, and filter the supernatant through a 0.45μM filter membrane. Contains EBV-GP / HIV virus particles, the recombinant virus can be used for infection;
[0039] Infection and detection: the day before infection, 10 per well 4 Huh-7 cells were inoculated onto a 96-well plate at a density of 100 cells, and the positive control compound was dissolved in DMEM (tamoxifen in DMEM) as a culture medium, and added to the cell culture medium 15 minutes before infection, and DM...
Embodiment 3
[0040] Embodiment 3: Use pseudovirus evaluation system to evaluate whether drugs and drug combinations have synergistic effects
[0041] 10 per well the day before infection 4 Inoculate Huh-7 cells on a 96-well plate at a density of one cell, and the specific infection and detection steps are the same as above; the evaluation of drug synergy requires the virus inhibition experiments of drug A, drug B, and the same amount of A+B. Do two repetitions, take the blank cells plus the same virus solution as the positive control, and carry out the calculation according to the virus inhibition effect (inhibition rate) into the relevant formula (the calculation formula has patent protection). This calculation scheme can calculate the concentration of the drug in the composition Activity and drug-antibody synergy coefficient, the degree of drug synergy is determined by the synergy coefficient; the synergy coefficient (CI) value indicates the interaction between the two drugs, which are >...
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