Method for screening SARS corona virus major protease inhibitor from traditional Chinese medicine and screened SARS corona virus major protease inhibitor
A technology of coronavirus and main protease, applied in antiviral agents, pharmaceutical formulations, biochemical equipment and methods, etc.
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Embodiment 1
[0038] Expression, purification of embodiment 1.SARS coronavirus main protease
[0039] According to literature (Yang H.Proc Natl Acad Sci., 2003,100,13190-13195), carry out the expression of SARS coronavirus main protease, purify, the aminoacid sequence of this SARS coronavirus main protease is as follows:
[0040] Ser Gly Phe Arg Lys Met Ala Phe Pro Ser Gly Lys Val Glu Gly Cys
[0041] 1 5 10 15
[0042] Met Val Gln Val Thr Cys Gly Thr Thr Thr Leu Asn Gly Leu Trp Leu
[0043] 20 25 30
[0044] Asp Asp Thr Val Tyr Cys Pro Arg His Val Ile Cys Thr Ala Glu Asp
[0045] 35 40 45
[0046] Met Leu Asn Pro Asn Tyr Glu Asp Leu Leu Ile Arg Lys Ser Asn His
[0047] 50 55 60
[0048] Ser Phe Leu Val Gln Ala Gly Asn Val Gln Leu Arg Val Ile Gly His
[0049] 65 70 75 80
[0050]Ser Met Gln Asn Cys Leu Leu Arg Leu Lys Val Asp Thr Ser Asn Pro
[0051] 85 90 95
[0052] Lys Thr Pro Lys Tyr Lys Phe Val Arg Ile Gln Pro Gly Gln Thr Phe
[0053] ...
Embodiment 2
[0098] Embodiment 2. preparation croton extract
[0099] Get 500 grams of dry and pulverized croton medicinal material, reflux extraction with 4500 milliliters of 95% ethanol, extract 3 times, each time for 2 hours, combine the extracts, and concentrate to obtain extract (i.e. the total extract of croton 95% ethanol);
[0100] Weigh 10 grams of this extract, suspend it with 150 milliliters of double distilled water, pour it into a 1000 milliliter separatory funnel, extract it with ethyl acetate, use 650 milliliters of ethyl acetate for each extraction, shake it well, and let it stand for 6 hours. layer, extracted 3 times in total, separated the ethyl acetate phase and the water phase, combined the water phase, and set aside, combined each ethyl acetate extract, and evaporated the ethyl acetate extract to remove the solvent with an EYELAN1001 rotary evaporator to obtain a thick paste Croton ethyl acetate extract sample (i.e. croton ethyl acetate extract), standby;
[0101]The ...
Embodiment 3
[0102] Embodiment 3. Screen SARS coronavirus main protease inhibitor from Croton extract
[0103] The above-mentioned ethyl acetate extraction sample, aqueous phase macroporous resin 95% ethanol elution sample and croton 95% ethanol total extract are carried out in vitro inhibitory activity test of SARS coronavirus main protease, and the inhibition curve is shown in figure 1 . Assay method specifically comprises as follows: (1). Add SARS coronavirus main protease in the buffer solution containing 50mM Tris-HCl, pH 7.3, 1mM EDTA, final concentration is 0.5 μ M, then in different buffer solutions containing this protein Add croton ethyl acetate extraction sample, aqueous phase macroporous resin 95% ethanol elution sample and 95% ethanol total extract respectively, and their concentration is 100 μ g / mL; (2). Add substrate MCA-AVLQSGFRL (DNP) L-NH 2 In the buffer solution, the final concentration is 16 μ M; and (3). measure the in vitro inhibitory activity of the SARS coronaviru...
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