Function inhibitor for EBV (Epstein-Barr virus) LMP (latent membrane protein)1 and application
A protein function and inhibitor technology, which can be applied to medical preparations containing active ingredients, antiviral agents, organic active ingredients, etc., and can solve the problem of less research
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Embodiment 1
[0018] Example 1 Fluorescence spectroscopy detects the interaction between EGCG and LMP1.
[0019]Protein molecules contain strong endogenous fluorescence due to amino acids such as tryptophan, tyrosine, and phenylalanine. The phenomenon that the fluorescent quencher interacts with the fluorescent substance and causes the fluorescence intensity to decrease is called fluorescence quenching. Fluorescence quenching can be divided into dynamic fluorescence quenching and static fluorescence quenching due to different quenching mechanisms. Fluorescence spectroscopy based on this principle is an important means to study the interaction between biological macromolecules, small molecules and ions.
[0020] The experimental method is as follows: under the conditions of 25°C and 37°C, add a certain molar concentration gradient (0-75 μM) of EGCG and purified LMP1 protein solution to the cuvette respectively (the volume of EGCG added does not exceed 1.5% of the total volume of the solutio...
Embodiment 2
[0022] Example 2 Pull-down affinity chromatography experiments confirmed that EGCG binds to LMP1 protein.
[0023] The operation steps are as follows:
[0024] Put the prepared total protein (including LMP1) 500μg (or purified LMP1 protein monomer) and 50μl EGCG-Sepharose 4B (or 50μl Sepharose 4B, as a negative control) in the reaction buffer (50mmol / LTris (pH 7.5), 5mmol / L EDTA, 150mmol / L NaCl, 1mmol / L DTT, 0.01% Ninidet P-40, 0.02mmol / L Phenylmethylsulfonyl fluoride, 4Ag / mL bovine serum albumin, 1Xprotease inhibitor cocktail), shake gently at 4℃, Incubate overnight. The next day, the beads were washed 5 times with WashingBuffer ([50mmol / L Tris (pH 7.5), 5mmol / L EDTA, 150mmol / L NaCl, 1mmol / L DTT, 0.01% NP40, 0.02mmol / L phenylmethylsulfonyl fluoride]) to remove untreated bound protein. Add an appropriate amount of SDS loading buffer, boil in water for 5 minutes, centrifuge at 13,000 rpm for 5 minutes, take the supernatant and load it, separate it in a discontinuous SDS dena...
Embodiment 3
[0026] Example 3 The ITC curve of the combination of EGCG and LMP1, and the nonlinear fitting curve of the binding reaction heat to the molar ratio of EGCG and protein obtained from the integration of the measured ITC curve.
[0027] Isothermal titration calorimetry is an important method to study biothermodynamics and biokinetics in situ, online and without damage. It uses a method similar to chemical titration to record the change of reaction heat flow with time. It has the characteristics of high sensitivity, good reproducibility, and in-situ online without interfering with the research reaction. It is currently one of the most accurate and widely used thermodynamic methods. It is often used in protein-protein interactions, protein-small molecule interactions, and enzymatic reaction kinetics.
[0028] The operation steps are as follows:
[0029] At 25 °C, the determination of the thermodynamic properties of the interaction between EGCG and LMP1 was carried out on an isothe...
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