CN1449822A - New use of gene recombinant human lysozyme in pharmacy for curing coronal virus of SARS - Google Patents
New use of gene recombinant human lysozyme in pharmacy for curing coronal virus of SARS Download PDFInfo
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- CN1449822A CN1449822A CN 03127014 CN03127014A CN1449822A CN 1449822 A CN1449822 A CN 1449822A CN 03127014 CN03127014 CN 03127014 CN 03127014 A CN03127014 A CN 03127014A CN 1449822 A CN1449822 A CN 1449822A
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- human lysozyme
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Abstract
The present invention relates to a new application of gene recombinant human lysozyme, in particula is relates to new application of gene recombinant human lysozyme for preventing and inhibiting coronavirus SARS. It can be made into injection, tablet, capsule, granules, aerosol and drops, and it is simple in preparation process, and has no toxic side effect.
Description
Technical field:
The present invention relates to a kind of new purposes of gene recombinant human lysozyme, particularly relate in the pharmaceutical field gene recombinant human lysozyme in new purposes at coronavirus SARS.
Background technology:
Antibiotic has been created many medical science miracles, makes the no track of numerous disease disappearance, as pneumonia, meningitis, lochiopyra, septicemia, tuberculosis etc.Today of 21 century, coronavirus sars is the variant viral of a kind of RNA, has extremely strong infectiousness, has all brought very big influence for people's live and work, does not also find the method for radical cure at present.
Summary of the invention:
The object of the present invention is to provide a kind of gene recombinant human lysozyme at the new purposes of coronavirus SARS in pharmacy.
In fact, the present invention relates to gene recombinant human lysozyme as the application in the pharmacy of preparation prevention and inhibition coronavirus SARS.
In order to understand essence of the present invention better, will its new purposes in pharmaceutical field be described with the pharmacological testing and the result of gene recombinant human lysozyme below.
Gene recombinant human lysozyme is as the criterion to prepare 200 milliliters of culture medium, with 6 milliliters of phosphoric acid, magnesium sulfate 3 grams, potassium sulfate 4 grams, potassium hydroxide 1 gram, calcium sulfate 1.5 grams, adding distil water to 200 milliliter, inoculation glycerol pipe seed behind the autoclaving, the shaking table revolution is that per minute 250 changes, and cultivation temperature is 20-35 ℃, cultivates 36-48 hour on the constant temperature bed.Carry out seed tank culture, produce a jar cultivation at last.With lyophilized powder 2mg, the injection that makes soluble in water is that every bottle of 30000U/mg is standby; Make aerosol, the every ml10000U of every bottle of 5ml of drop is standby; It is standby to make powder 2mg.
The drug effect of the external prevention of gene recombinant human lysozyme medicine, inhibition coronavirus: in the HELA cell culture, adopt viral CPE method, the recombinant human lysozyme medicine of observing variable concentrations is to the prevention of coronavirus, the effect of inhibition.
Experimental example: one, test material
1. checking medicine: the recombinant human lysozyme protein content: 4.3845mg is provided by ChangChun QiLong Biology Technology Research Institute
2. positive control medicine: injection ganciclovir lot number: 020802 is provided by Hubei KeYi Pharmacentic Co., Ltd..
3. cell: heLa cell (HELA) is provided by virus resource center of China Sickness Prevention Control Center Virus Disease Prevention Control Institute.
4. viral: No. 04 separated strain of coronavirus (No. 04 specimen of SARS patients serum) is provided by You An hospital.
5.CO
2Incubator NUAIR US AUTO FLOW provides
6. (XSZ-D2) produced by the optical instrument factory, Chongqing
7. other reagent, equipment etc. provide by virus resource center of China Sickness Prevention Control Center Virus Disease Prevention Control Institute.Two, test method pilot study: 1. recombinant human lysozyme is to the toxicity test of HELA cell
The HELA cell is inoculated 96 well culture plates with 400,000/ml concentration, 37 ℃, 5%CO
2Cultivated 2 hours, and added the checking medicine, the recombinant human lysozyme doubling dilution is 6000ug/ml～11.7ug/ml, the dilution of positive control medicine ganciclovir is 6000ug/ml～11.7ug/ml, and every concentration is inoculated 3 holes, every hole 100 μ l, establish the normal cell contrast simultaneously, 37 ℃ of 5%CO
2Incubator was cultivated 6 days, every day is observation of cell metamorphosis (CPE) under inverted microscope, with 25% following metamorphosis is "+", 26%～50% metamorphosis is " ++ ", 51%～75% metamorphosis is " +++", 76%～100% metamorphosis is " ++ ++ ", uses the Reed-Muench method, calculates medicine median toxic concentration (TD
50) and maximal non-toxic concentration (TD
0).In the HELA cell culture to the separation (SARS patients serum separation) of the mensuration coronavirus of the virulence of coronavirus No. 04 No. 04:
The HELA cell is with every milliliter 400,000 concentration inoculation test tube, 37 ℃ of 5%CO
2Cultivated 24 hours, and discarded culture fluid, every pipe adds SARS patients serum 0.2ml, and 33 ℃ of rotary drums were cultivated after 5 hours, added and kept liquid 1ml, established the normal cell contrast simultaneously, and 33 ℃ of rotary drums were cultivated 5～7 days.After the CPE variation appears in cell, adopt the method for PCR to detect coronavirus, No. 04 specimen PCR positive is defined as the coronavirus separated strain.With whole last dilution method purified virus 2 times, it is still positive that PCR detects coronavirus, through immunofluorescence assay double SARS patients serum, and the IgM positive, IgG4 doubly raises, and is defined as coronavirus.Adopting viral CPE method to measure it tires.Virus CPE method:
The HELA cell is inoculated 96 well culture plates with every milliliter 400,000 concentration, 37 ℃ of 5%CO
2Cultivated 24 hours, and added viral liquid, viral dilution 10
-1～10
-5, 5 concentration, every concentration 3 holes, every hole 100 μ l establish the normal cell contrast, 37 ℃ of 5%CO
2Cultivated 5～7 days, observed and recorded cellular morphology variation (CPE) under inverted microscope in per 24 hours: to be changed to "+" below 25%, 26%～50% is changed to " ++ ", 5 1%～75% be " +++", 76%～100% be changed to " ++ ++ ", use the Reed-Muench method, calculate viral half and infect concentration TCID
50Formal test: recombinant human lysozyme in the HELA cell culture to the inhibitory action test objective of coronavirus No. 04:
To the inhibitory action of coronavirus No. 04, virocyte (CPE) method is adopted in test to the recombinant human lysozyme of observing variable concentrations, calculates the medium effective concentration (IC of medicine in the HELA cell culture
50) and minimum effective drug concentration (MIC) and therapeutic index TI, judge drug effect.
The HELA cell is inoculated 96 well culture plates with 400,000/ml concentration, 37 ℃ of 5%CO
2Incubator was cultivated 24 hours, and cell culture discards culture fluid to monolayer, adds 100 TCID
50Coronavirus liquid, put 37 ℃ of 5%CO
2Adsorb after 2 hours, discard viral liquid, add the recombinant human lysozyme medicinal liquid of variable concentrations, the maximal non-toxic concentration (TD of choice of drug pair cell
0) the i.e. 750 μ g/ml～1.46 μ g/ml of 10 concentration of 2 times of dilutions, positive control medicine ganciclovir is the i.e. 6000 μ g/ml～1.46 μ g/ml of 10 concentration of 2 times of dilutions, add the medicine of dilution in the cell hole respectively, every concentration 3 holes, establish normal cell contrast and virus control simultaneously, put 37 ℃ of 5%CO
2Incubator was cultivated 5～7 days, observed viral CPE day by day under inverted microscope, occurred with virus control +++-++ ++ in time, finish to test, and uses the Reed-Muench method, calculates the medium effective concentration (IC of medicine
50) and minimum effective drug concentration (MIC) and therapeutic index (TI) judgement drug effect, the test triplicate.Recombinant human lysozyme is to the preventive effect of coronavirus No. 04: test objective:
To the preventive effect of coronavirus No. 04, virocyte (CPE) method is adopted in test to the recombinant human lysozyme of observing variable concentrations, calculates the medium effective concentration (IC of medicine in the HELA cell culture
50) and minimum effective drug concentration (MIC) and therapeutic index TI, judge drug effect.
The HELA cell is inoculated 96 well culture plates with 400,000/ml concentration, 37 ℃ of 5%CO
2Incubator was cultivated 24 hours, and cell culture discards culture fluid to monolayer, added the recombinant human lysozyme medicinal liquid of variable concentrations, the maximal non-toxic concentration (TD of choice of drug pair cell
0) the i.e. 750 μ g/ml～1.46 μ g/ml of 10 concentration of 2 times of dilutions, positive control medicine ganciclovir is the i.e. 6000 μ g/ml～1.46 μ g/ml of 13 concentration of 2 times of dilutions, and the medicine of dilution is added respectively in the cell hole, 37 ℃ of 5%CO are put in every concentration 3 holes
2Adsorb after 2.5 hours, add 100 TCID
50Coronavirus liquid, establish normal cell contrast and virus control simultaneously, put 37 ℃ of 5%CO
2Incubator was cultivated 5～7 days, observed viral CPE day by day under inverted microscope, occurred with virus control +++-++ ++ in time, finish to test, and calculates the medium effective concentration (IC of medicine with the Reed-Muench method
50) and minimum effective drug concentration (MIC) and therapeutic index (TI) judgement drug effect, the test triplicate.Three, result of the test pilot study result: recombinant human lysozyme is to the toxic action of HELA cell:
Recombinant human lysozyme that is provided by ChangChun QiLong Biology Technology Research Institute and positive control medicine injection ganciclovir adopt cellular morphology to change (CPE) method in the HELA cell culture.Calculate medicine maximal non-toxic concentration (TD
0) and median toxic concentration (TD
50), following result of the test is the average of three result of the tests.1. recombinant human lysozyme is to the toxicity test result verification medicine of HELA cell: recombinant human lysozyme: maximal non-toxic concentration (TD
0) be 750 ± 0 μ g/ml, median toxic concentration (TD
50) be 1500 ± 0 μ g/ml positive control medicines: injection ganciclovir: maximal non-toxic concentration (TD
0) be＞6000 ± 0 μ g/ml, median toxic concentration (TD
50) be＞6000 ± 0 μ g/ml.In the HELA cell culture to No. 04 toxicity test result of coronavirus (TCID
50) coronavirus No. 04: median infective dose (TCID
50) be 10
-3, see Fig. 1.The formal test result sees Fig. 1.Recombinant human lysozyme in the HELA cell culture to inhibitory action (following test data is the average of three result of the tests) the checking medicine of coronavirus No. 04:
Recombinant human lysozyme: CPE method, medicine medium effective concentration (IC
50) be 23.4 ± 0 μ g/ml, minimum effective drug concentration (MIC) is 46.8 ± 0 μ g/ml, therapeutic index (TI) is 16.The positive control medicine:
Injection ganciclovir: CPE method, medicine medium effective concentration (IC
50) be 11.7 μ g/ml ± 0, minimum effective drug concentration (MIC) is 23.44 μ g/ml ± 0, therapeutic index (TI) is 256.Recombinant human lysozyme in the HELA cell culture to preventive effect (following test data is the average of three result of the tests) the checking medicine of coronavirus No. 04:
Recombinant human lysozyme: CPE method, medicine medium effective concentration (IC
50) be 5.9 ± 0 μ g/ml, minimum effective drug concentration (MIC) is 11.7 ± 0 μ g/ml, therapeutic index (TI) is 64.The positive control medicine:
Injection ganciclovir: CPE method, medicine medium effective concentration (IC
50) be 11.7 μ g/ml ± 0, minimum effective drug concentration (MIC) is 23.44 μ g/ml ± 0, therapeutic index (TI) is 256.
Above result can draw and the invention has the advantages that: gene recombinant human lysozyme has been excavated new medical application, has opened up a new application.Gene recombinant human lysozyme is safe in utilization to have no side effect, and good prospect in medicine is arranged.The gene recombinant human lysozyme source is abundant, preparation technology is simple, and can make various injections, tablet, granule, aerosol, drop etc., easy to use, usage and consumption: intravenous injection: adult every day 1～3 time, each 1,000,000 U～3,000,000 U, children's every day 1～3 time, each 500,000 U～2,000,000 U.Aerosol: directly nebulization is in the focus topical, and every day 1～6 time, each 1500u～30000u adult is identical with children's.Drop: directly act on focus topical every day 1～6 time, each 1500u～30000u.Tablet, capsule, granule: adult every day 1～3 time, each 1,000,000 u～3,000,000 u.Children's every day 1～3 time, each 500,000 u～1,500,000 u.
Description of drawings:
Fig. 1 coronavirus No. 04.
No. 04 median infective dose (TCID of Fig. 2 coronavirus
50).
The specific embodiment:
The present invention will be further described below in conjunction with embodiment:
Embodiment 1:
Behind the culture medium autoclaving, inoculation human lysozyme bacterial strain, shaking table revolution are that per minute 250 changes, and cultivation temperature is 20 ℃, cultivates 36 hours on the constant temperature bed.Carry out seed tank culture, produce a jar cultivation at last.And the culture fluid that fermentation expression is finished extracted purification, and the protein concentrated solution that extracts purification is carried out lyophilizing, preserve after recording protein active.Get human lysozyme lyophilized powder 1%, phosphate buffer 5mM (pH5.0) 90%; Make injection, be mainly used in coronavirus SARS (severe acute respiratory syndrome) disease.
Embodiment 2:
Behind the culture medium autoclaving, inoculate the human lysozyme bacterial strain, again culture fluid carried out purification and make the aerosol that contains human lysozyme lyophilized powder 0.1%, phosphate buffer 20mM (pH7.0) 80%, 25% glycerol, 5/0,000 Tween 80s after 40 hours in cultivation on the constant temperature bed.
Embodiment 3:
Behind the culture medium autoclaving, inoculate the human lysozyme bacterial strain, again culture fluid carried out purification and make the drop that contains human lysozyme lyophilized powder 0.005%, phosphate buffer 10 (pH6.0) 80%, 20% glycerol, 6%2-pyrrolidone-5-carboxylic acid sodium, 0.9% water solublity azone, 3/0,000 Tween 80s after 36 hours in cultivation on the constant temperature bed.
Embodiment 4:
Behind the culture medium autoclaving, inoculate the human lysozyme bacterial strain, again culture fluid carried out purification and make capsule, tablet, granule after 40 hours in cultivation on the constant temperature bed.It contains human lysozyme lyophilized powder 10%, medical starch 50, medicinal dextrin 25%, phosphate buffer 1 0mM (pH6.0), sucrose 5%.
Claims (3)
1, gene recombinant human lysozyme relates to the application for preparing in the medicine that prevents coronavirus SARS as a kind of.
2, gene recombinant human lysozyme relates to the application for preparing in the inhibition coronavirus SARS medicine as a kind of.
3, gene recombinant human lysozyme relates to as a kind of pharmaceutical pack for preparing prevention and suppress coronavirus SARS and draws together injection, tablet, capsule, granule, aerosol, drop.
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| CN 03127014 CN1449822A (en) | 2003-05-04 | 2003-05-04 | New use of gene recombinant human lysozyme in pharmacy for curing coronal virus of SARS |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005120556A1 (en) * | 2004-06-10 | 2005-12-22 | Mi An | Human lysozyme medicine, its manufacturing method and application thereof |
| CN112135625A (en) * | 2020-04-27 | 2020-12-25 | 广州新创忆药物临床研究有限公司 | Medicine and food for preventing or treating COVID-19 new coronary pneumonia and application thereof |
| IT202100016037A1 (en) * | 2021-06-18 | 2021-09-18 | Antonio Bianchi | SPRAY FORMULATION FOR NASAL/ORAL USE WITH ACTIVITY AGAINST RESPIRATORY CORONAVIRUSES |
| WO2021218154A1 (en) * | 2020-04-27 | 2021-11-04 | 广州新创忆药物临床研究有限公司 | Drug, food and application of anti-coronavirus infection |
| IT202000025207A1 (en) * | 2020-10-23 | 2022-04-23 | Antonio Bianchi | SPRAY FORMULATION FOR NASAL/ORAL USE WITH ACTIVITY AGAINST RESPIRATORY CORONAVIRUSES |
| WO2022084931A1 (en) * | 2020-10-23 | 2022-04-28 | Antonio Bianchi | Spray formulation for nasal/oral use with activity against respiratory coronaviruses and in particular the sars-cov-2 virus |
-
2003
- 2003-05-04 CN CN 03127014 patent/CN1449822A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005120556A1 (en) * | 2004-06-10 | 2005-12-22 | Mi An | Human lysozyme medicine, its manufacturing method and application thereof |
| JP2008501734A (en) * | 2004-06-10 | 2008-01-24 | 安米 | Production and application of human lysozyme |
| CN112135625A (en) * | 2020-04-27 | 2020-12-25 | 广州新创忆药物临床研究有限公司 | Medicine and food for preventing or treating COVID-19 new coronary pneumonia and application thereof |
| CN112135625B (en) * | 2020-04-27 | 2021-07-06 | 广州新创忆药物临床研究有限公司 | Medicine for preventing or treating COVID-19 new coronary pneumonia and application thereof |
| WO2021218154A1 (en) * | 2020-04-27 | 2021-11-04 | 广州新创忆药物临床研究有限公司 | Drug, food and application of anti-coronavirus infection |
| WO2021217702A1 (en) * | 2020-04-27 | 2021-11-04 | 广州新创忆药物临床研究有限公司 | Medicine and food for preventing or treating covid-19, and application thereof |
| IT202000025207A1 (en) * | 2020-10-23 | 2022-04-23 | Antonio Bianchi | SPRAY FORMULATION FOR NASAL/ORAL USE WITH ACTIVITY AGAINST RESPIRATORY CORONAVIRUSES |
| WO2022084931A1 (en) * | 2020-10-23 | 2022-04-28 | Antonio Bianchi | Spray formulation for nasal/oral use with activity against respiratory coronaviruses and in particular the sars-cov-2 virus |
| IT202100016037A1 (en) * | 2021-06-18 | 2021-09-18 | Antonio Bianchi | SPRAY FORMULATION FOR NASAL/ORAL USE WITH ACTIVITY AGAINST RESPIRATORY CORONAVIRUSES |
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