CN1557485A - Application of gene recombined human lysozyme in eliminating pathogenic microorganism infection - Google Patents
Application of gene recombined human lysozyme in eliminating pathogenic microorganism infection Download PDFInfo
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- CN1557485A CN1557485A CNA2004100211009A CN200410021100A CN1557485A CN 1557485 A CN1557485 A CN 1557485A CN A2004100211009 A CNA2004100211009 A CN A2004100211009A CN 200410021100 A CN200410021100 A CN 200410021100A CN 1557485 A CN1557485 A CN 1557485A
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Abstract
The present invention provides the application of gene recombinant human lysozyme extracted in industrial saccharomycete production and with purity up to 99.8 % and activity as high as 70,000 u/mg in preparing medicine for various inflammations caused by bacteria, fungi and viruses to infect skin and mucous membrane; externally applied and orally taken medicines with bactericidal and antiphlogistic gene recombinant human lysozyme for treating various kinds of inflammation clinically; and the method of applying the gene recombinant human lysozyme in treating various kinds of inflammation alone or in combination with antibiotic.
Description
Technical field
The present invention relates to utilize in the biomedicine gene recombinant human lysozyme (HLZ) to cause the application of various inflammation medicines as preparation treatment cause pathogeny imcrobe infection.
Background technology
Lysozyme is found (Proc.R.Soc.Lond.Biol.Sci.1922 by Fleming in nineteen twenty-two; 93:306).It is a kind of effective antibacterial, and full name is: 1, and 4-β-N-lysozyme or title: mucopeptide N-acetyl group muramyl hydrolytic enzyme.The connection that it can cut off β-1,4 glycosidic bond between the N-acetylglucosamine and-acetylmuramic acid in the Peptidoglycan of bacterial cell wall destroys the Peptidoglycan support, and antibacterial cell spalling under the effect that internal penetration is pressed is opened, and causes the antibacterial cracking.The acellular wall construction of humans and animals cell does not also have Peptidoglycan, so lysozyme is to the human body cell free of toxic effects.Lysozyme is present in human multiple tissue and the secretions, also is present in the multiple animal and plant.Except the effect of lysozyme aspect the body prevention infection, it also has other biological action.
The antibacterial action that a large amount of bibliographical information lysozyme is arranged.Lysozyme is dense in a lot of birds Ovum Gallus domesticus album.Although these vertebratess have the immunoglobulin system, ovum and developmental embryo do not produce immunoglobulin, and 7 talentes before hatching can produce (Jolles P, et al.Mol.Cell.Biol.Chem.1984; 63:165).Therefore, play defense reaction at lysozyme during this period of time.And in invertebrates owing to do not produce immunoglobulin, lysozyme plays rudimentary protection system effect in the vertebrates.Someone thinks that lysozyme is a kind of primary non-special defense mechanism of mononuclear phagocyte system, kind is being early than more special lymphocyte-plasma cell-immunoglobulin system in the growth.But think that now the lysozyme and the immunoglobulin function first line of a couplet fastens close.Lysozyme is similar with the S-IgA tissue distribution, IgA and lysozyme all be present in some exocrine secretions as tear, saliva, tracheal bronchus secretions and gastric juice etc., has only the small part lysozyme to be present in endocrine liquid as blood, meninges liquid and cerebrospinal fluid.Lysozyme extensively is present in nature animal, plant and the microorganism.
Lysozyme removes directly cracking antibacterial, can also regulate the effect of neutrophilic granulocyte as immunomodulator, and the normal structure of protection inflammation part plays negative feedback (Leo IG, et al.J.Clin.Invist.1979 in regulating inflammatory reaction; 64:222).It may combine by the polysaccharide part with polymorphonuclear granulocyte film surface; suppressing polymorphonuclear granulocyte moves to inflammation part; and the inflammation part of can decaying is by the effect of the super oxidation that polymorphonuclear granulocyte produced, the tissue injury that the protection body takes place owing to the immunne response transition when inflammatory reaction.Also report in monocytic training liquid, to add the high concentration lysozyme, can improve mononuclear cell and engulf the ability of tubercule bacillus (Kokoshis PL, etal.Science.1978; 199:1340).Lysozyme also can with the combined effect of other biological activated protein, give full play to antibacterial action, as complement, Lactotransferrin etc.When the bacterial endotoxin (LPS) of research such as beam Aiwa lysozyme on ceftazidime-induced discharges inhibitory action, find that lysozyme can suppress the inductive bacterial endotoxin of ceftazidime and discharge.Recent research finds that lysozyme except that bacteriolysis, also has anti HIV-1 virus effect (Lee-Huang S.Proc Natl Acad Sci USA1999; 96 (6): 2678), its effect may be because lysozyme structure and histone are similar, makes lysozyme combine (Steinrauf LK.Biochem Biophys Res Commun 1999 with DNA and RNA; 266 (2): 366).
At present, people mainly produce lysozyme of chicken from Ovum Gallus domesticus album.Yet lysozyme of chicken can cause allergy and can't be used for the mankind that the price of natural human lysozyme too costliness also can't be used for clinical as foreign protein.
The objective of the invention is to utilize biology gene engineering technology to realize suitability for industrialized production in application Chinese patent 00110463.2 in 2000 from yeast or escherichia coli with my company, the product gene recombinant human lysozyme purity of extracting purification is up to more than 99.8%, active in 70,000u/mg has the bactericidal activity of natural human lysozyme simultaneously.On this basis, for the invention provides the new purposes of gene recombinant human lysozyme, the new application in the medicine that causes inflammation as preparation treatment cause pathogeny imcrobe infection.
Summary of the invention
The present invention at first uses gene recombinant human lysozyme (HLZ) to treat pathogenic microorganism by test tube method (seeing accompanying drawing 1) and flat band method (seeing accompanying drawing 2) as preparation: the medicine that antibacterial, fungus, viral infection cause inflammation carries out activity, sterilization and antiinflammatory to be identified.Then, with purity 99.8% or more, active 70, the gene recombinant human lysozyme of 000u/mg is treated antibacterial, fungus and viral infection cause upper respiratory tract infection, colpitis, scytitis and oral inflammation medicine on the skin mucosa surface application as preparation; The gene recombinant human lysozyme that will have sterilization and antiinflammatory action is made body innerlich anwenden oral tablet, atomizing suction spray and external medication drop, unguentum, suppository; And the application that causes inflammation medication combined medication in inside and outside with 1: 1 compositions of gene recombinant human lysozyme and antibiotic as preparation treatment cause pathogeny imcrobe infection.
The antibacterial that relates in the pathogenic microorganism of the present invention is meant Gram-positive, negative bacteria, anaerobe, drug tolerant bacteria and L type antibacterial; The virus that relates to is meant DNA viruses and RNA viruses; Upper respiratory tract infection is to infect tracheitis, bronchitis, pneumonia, the pharyngitis that causes by the drug resistance staphylococcus aureus; Scytitis is eczema, allergic dermatitis, comedo, rubella, psoriasis or the urticaria that is caused by Gram-positive, negative bacterial infections; Oral inflammation is to infect gingivitis or the oral ulcer that causes by the anaerobic coccus.
The invention still further relates to and use gene recombinant human lysozyme and antibiotic, but antibiotic is meant clarithromycin, Roxithromycin, vancomycin or moral mycin by combinatorial association medication in 1: 1.
The invention still further relates to the medicine for the treatment of upper respiratory tract infection, colpitis and scytitis as preparation with gene recombinant human lysozyme is the injection that is mixed with by 1% gene recombinant human lysozyme lyophilized powder and 5mM phosphate PH5 buffer, the intravenous injection dosage is 30, the 000u/ml/20g Mus, every day three times; Medicine as preparation treatment oral inflammation is the spray that is mixed with by 0.1% gene recombinant human lysozyme lyophilized powder, 20mM phosphate PH7 buffer, 25% glycerol and 5/0,000 Tween 80s, and dosage is 1500 μ l/20g Mus, every day six times; As the cause inflammation oral tablet of medication of bacterial infection in the preparation treatment body is formulated by 10% gene recombinant human lysozyme lyophilized powder, 50% medical starch, 25% medicinal dextrin, 10mM phosphate PH6 buffer and 5% sucrose; Atomizing sucks the concentration 0.025～0.4mg/ml of gene recombinant human lysozyme, inhaled medication 2～5 days in the body; Drop as the external medication of preparation treatment is formulated by 0.005% gene recombinant human lysozyme lyophilized powder, 10mM phosphate PH6 buffer, 20% glycerol, 6%2-ketopyrrolidine-5 carboxylic acid sodium, 0.9% water solublity azone and 3/0,000 Tween 80s; The agent of external use ointment is formulated by 0.1% gene recombinant human lysozyme lyophilized powder, 20mM phosphate PH7 buffer, 20% glycerol, 1% carbomer and 0.3% essence.
Essence for a better understanding of the present invention, at first, we use as active component with gene recombinant human lysozyme by test tube method (seeing accompanying drawing 1) fastbacteria are carried out the bacteriolyze activity identification, and its method is:
With PH7.2 Tris-HCl buffer golden Portugal bacterium or escherichia coli are made into 10
7～10
8The bacterium liquid of CFU/ml concentration quantitatively is added in the different magnificent test tubes, the gene recombinant human lysozyme that adds 100 μ g～1mg various dose simultaneously respectively, 37 ℃ are spent the night, every pipe is got a certain amount of with 10 times of dilutions, and be applied to the LB culture medium, 37 ℃ of overnight incubation, observed result is calculated the total number of bacteria (embodiment 1 is seen in concrete operations) in the original pipe.
Total number of bacteria * 100% in the bacteriolyze effect of gene recombinant human lysozyme=(in the control tube in total number of bacteria-experiment tube total number of bacteria)/control tube
Flat band method (seeing accompanying drawing 2) the active evaluation of gene recombinant human lysozyme bacteriolyze, its method is:
Behind PH7.2 Tris-HCl buffer preparation agarose autoclaving, cooling adds the antibacterial pour plate, punching, add 0～100mg/ml variable concentrations gene recombinant human lysozyme solution in the hole, hatched 4～6 hours for 37 ℃, LB Nutrient agar autoclaving, cooling, be covered on the agarose plate, uviol lamp was hatched overnight incubation for 37 ℃ according to 20 minutes, observe bacterial growth situation between agarose and the Nutrient agar layer, measure variable concentrations lysozyme bacteriostatic diameter.Antibacterial circle diameter well diameter 4mm person is minimum effective bacteriocidal concentration (embodiment 2 is seen in concrete operations).
By above-mentioned lysozyme activity authentication method, respectively antibacterial, fungus, virus are carried out activity and use mensuration, division is as follows as a result:
1, the application of active component in the medicine that causes inflammation as the treatment bacterial infection with gene recombinant human lysozyme: respectively to Gram-positive, negative bacteria, anaerobe, drug tolerant bacteria and L type antibacterial have carried out activity identification, promptly to 8 strain staphylococcus aureuses, 6 Klebsiella pneumoniaes, the green dense false pseudomonas bacillus of 3 strains, 7 strain escherichia coli, 1 strain Bao eel bacillus, 1 strain serratia marcescens, 3 strain Bacillus proteuss and 1 strain alpha streptococcus carry out the bacteriolyze activity test, this 30 strain antibacterial is all to the gene recombinant human lysozyme sensitivity, and effectively the Mlc scope is between 50 μ g～1.0mg/ml.Gene recombinant human lysozyme mainly is present between the different strains of identical bacterium the difference between the minimum inhibitory concentration of different bacterium.
2, the application of active component in the medicine that causes as the treatment fungal infection with gene recombinant human lysozyme: respectively to 8 fungal strains, be that Candida albicans, Oidium tropicale and gram Rou Shi candidiasis have carried out bacteriostatic test, they all have tangible fungistatic effect (seeing accompanying drawing 3a and 3b) human lysozyme to have an activity of significantly killing fungus external.Its bacteriostatic test method is identical with the bacteriostatic experiment of said gene recombinant human lysozyme effect drug-resistant cell.
3, the application of active component in the medicament that causes inflammation as the treatment viral infection with the gene recombinant human lyase, the inhibitory action that the coronavirus that gene recombinant human lysozyme is cultivated heLa cell (HELA) is No. 04, adopt the gene recombinant human lysozyme of variable concentrations, with virocyte CPE method, calculate the medium effective concentration (IC of medicine
50) and minimum effective drug concentration (MIC) and therapeutic index TI judge drug effect.Concrete grammar is:
HeLa cell HELA inoculates 96 well culture plates with 400,000/ml concentration, at 37 ℃ of 5%CO
2Incubator was cultivated 24 hours, and cell culture discards culture fluid to monolayer, added 100TCID
50Coronavirus liquid, put 37 ℃ of 5%CO
2Adsorb after 2 hours, discard viral liquid, add the gene recombinant human lysozyme medicinal liquid of variable concentrations, the maximal non-toxic concentration (TD of choice of drug pair cell
0) 10 concentration of 2 times of dilutions are 6000 μ g/ml～1.46 μ g/ml, and the medicine of dilution is added respectively in the cell hole, every concentration 3 holes are established 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.
(1) gene recombinant human lysozyme is made comparisons the meansigma methods of three result of the tests to the toxic action of HELA cell with positive control medicine ganciclovir:
Checking medicine: the maximal non-toxic concentration (TD of gene recombinant human lysozyme
0) be 750 ± 0mg/ml, median toxic concentration (TD
50) be 1500 ± 0 μ g/ml.
Positive control medicine: the maximal non-toxic concentration of injection ganciclovir＞60000 ± 0 μ g/ml, median toxic concentration (TD
50) be＞60000 ± 0 μ g/ml
In the HELA cell culture to No. 04 toxicity test result of coronavirus, median infective dose (TCID
50) be 10
-3(seeing accompanying drawing 4).
(2) gene recombinant human lysozyme in the HELA cell culture to the meansigma methods of three result of the tests of No. 04 inhibitory action of coronavirus (seeing accompanying drawing 4):
The checking medicine is a gene 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 is the injection ganciclovir, CPE method, medicine medium effective concentration (IC
50) be 11.7 ± 0 μ g/ml, minimum effective drug concentration (MIC) is 23.44 ± 0 μ g/ml, therapeutic index (TI) is 256.
(3) gene recombinant human lysozyme in the HELA cell culture to the meansigma methods of three result of the tests of No. 04 preventive effect of coronavirus:
The checking medicine is a gene 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 is the injection ganciclovir, CPE method, medicine medium effective concentration (IC
50) be 11.7 ± 0 μ g/ml, minimum effective drug concentration (MIC) is 23.44 ± 0 μ g/ml, therapeutic index (TI) is 256.
One, gene recombinant human lysozyme is as the application of preparation treatment upper respiratory tract infection, colpitis, oral inflammation and scytitis medicine.
Upper respiratory tract tracheitis, the bronchitis that division is caused by the drug resistance infection of staphylococcus aureus below; The vaginitis that causes by drug resistance streptococcal infection; Infect the stomatitis that causes by the anaerobic coccus; Preparation, medication and the therapeutic effect of the eczema that causes by Gram-positive, negative bacterial infections, allergic dermatitis, comedo, rubella, urticaria medicine:
(1) medicine preparation
A, with behind the culture medium autoclaving, inoculation gene recombinant human lysozyme bacterial strain, shaking table revolution are that per minute 250 changes, 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 albumen that extracts purification is concentrated carry out lyophilizing, preserve after recording protein active.Get gene recombinant human lysozyme lyophilized powder 1%, phosphate buffer 5mM (PH=5.0) 90%: make injection, be mainly used in the disease of the anti-drug resistance that gram positive bacteria and gram negative bacteria are caused.As diseases such as tracheitis, pneumonia.
B, with behind the culture medium autoclaving, inoculation gene recombinant human lysozyme bacterial strain, after cultivating 40 hours on the constant temperature bed, again culture fluid is carried out purification and make the spray that contains human lysozyme lyophilized powder 0.1%, phosphate buffer 20mM (PH=7.0) 80%, 25% glycerol, 5/0,000 Tween 80s.
C, with behind the culture medium autoclaving, inoculate the gene recombinant human lysozyme bacterial strain, again culture fluid carried out purification and make the drop that contains human lysozyme lyophilized powder 0.005%, phosphate buffer 1 0mM (PH=6.0) 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 shaking table.
D, with behind the culture medium autoclaving, inoculation gene recombinant human lysozyme bacterial strain, after cultivating 48 hours on the constant temperature shaking table, again culture fluid is carried out purification and makes and contain human lysozyme lyophilized powder 0.1%, phosphate buffer 20mM (PH=7.0) 85%, 20% glycerol, carbomer 1%, essence 0.3% unguentum.
E, with behind the culture medium autoclaving, inoculation gene recombinant human lysozyme bacterial strain, after cultivating 40 hours on the constant temperature shaking table carries out purification with culture fluid again and makes capsule, tablet.It contains human lysozyme lyophilized powder 10%, medical starch 50%, medicinal dextrin 25%, phosphate buffer 1 0mM (PH=6.0), sucrose 5%.
(2) to the model test of mice
A, drug resistance staphylococcus aureus upper respiratory tract infection induced mice tracheitis, bronchitis Preparation of model:
Select 20 of healthy Kunming mouses for use, male and female half and half, body weight is the 18-22 gram, divide two groups, 10 every group, choose a certain amount of drug resistance staphylococcus aureus and cause 10 of mice tracheitiies, 10 of bronchitis with spray pattern infecting mouse upper respiratory tract, intravenous injection at once is subjected to reagent 30000u/ml/20g Mus heavy after the infection, observe a week, three administrations every day, record mice effective percentage.Experimental result shows that gene recombinant human lysozyme has obvious curative effects to drug resistance staphylococcus aureus upper respiratory tract infection induced mice tracheitis, bronchitis.Result such as following table:
| The sick kind | Animal subject (only) | Recovery from illness | Produce effects | Invalid | Effective percentage % |
| Tracheitis | ?10 | ?7 | ????3 | ????0 | ????100 |
| Bronchitis | ?10 | ?7 | ????3 | ????0 | ????100 |
B, drug resistance streptococcal infection induced mice vaginitis Preparation of model:
Select 10 of healthy Kunming mouses for use, male and female half and half, body weight is the 18-22 gram, a certain amount of drug resistance streptococcus of picking causes scorching 10 of mouse vagina with smearing mode infecting mouse vagina, be that intravenous injection is subjected to reagent 30000u/ml/20g Mus heavy after the infection, observe a week, three administrations every day, record mice effective percentage.Experimental result shows that gene recombinant human lysozyme has obvious curative effects to drug resistance streptococcal infection induced mice vaginitis.Result such as following table:
| The sick kind | Animal subject (only) | Recovery from illness | Produce effects | Invalid | Effective percentage % |
| Vaginitis | ????10 | ????7 | ????3 | ????0 | ????100 |
C, anaerobic coccus upper respiratory tract infection induced mice stomatitis Preparation of model:
Select 10 of healthy Kunming mouses for use, male and female half and half, body weight is the 18-22 gram, a certain amount of anaerobic coccus of picking causes 10 of mice stomatitis with spray pattern infecting mouse upper respiratory tract, be that mouthspray is subjected to reagent 1500u/ml/20g Mus heavy after the infection, observe a week, six administrations every day, record mice effective percentage.Experimental result shows that gene recombinant human lysozyme has obvious curative effects to anaerobic coccus upper respiratory tract infection induced mice stomatitis.Result such as following table:
| The sick kind | Animal subject (only) | Recovery from illness | Produce effects | Invalid | Effective percentage % |
| Stomatitis | ????10 | ???8 | ????2 | ????0 | ????100 |
Curative effect such as following table that skin sexuality such as D, the eczema to being caused by gram positive bacteria and part negative bacterium, allergic dermatitis, comedo, rubella, psoriasis, urticaria are dyed:
| The sick kind | Animal subject (only) | Recovery from illness | Produce effects | Invalid | Effective percentage % |
| Eczema | ????10 | ????6 | ????3 | ????1 | ????90 |
| Allergic dermatitis | ????10 | ????4 | ????4 | ????2 | ????80 |
| Comedo | ????10 | ????9 | ????1 | ????0 | ????100 |
| Rubella | ????10 | ????7 | ????1 | ????2 | ????80 |
| Psoriasis | ????10 | ????4 | ????4 | ????2 | ????80 |
| Urticaria | ????10 | ????6 | ????2 | ????2 | ????80 |
Two, gene recombinant human lysozyme causes the application of respiratory inflammation (tracheitis, bronchitis, pneumonia, pharyngitis etc.) medicine as bacterial infection in the preparation treatment body.
Adopt mouse peritoneal infection septicemia or throat infection antibacterial model to observe the therapeutical effect of HLZ to inflammation in the mice body.
Test strain: staphylococcus aureus 01193, staphylococcus aureus MSSA022, staphylococcus aureus MRSA028, table staphylococcus MRSE0210, streptococcus pneumoniae 01182, enterococcus ATCC700802 are used for the infection experiment of mice septicemia.Method is that a certain amount of lawn of picking is inoculated in the 2mlM-H fluid medium, and after 35 ℃ of cultivations 6 as a child, taking-up adapts to dilution (10 with sterile saline
-1, 10
-2, 10
-3, 10
-4), every group of 5 Mus, respectively the different bacterium amounts of abdominal cavity infection tried bacterium liquid, measure the minimum that the causes mice 100% death bacterium amount (MLD) that causes death, measure as infection dosage in the body with this bacterium.
The intravenous injection of gene recombinant human lysozyme medicament is to the therapeutic test of bacterial infection mice septicemia, evenly divided into groups by the body weight sex trying mice, every group of 10 Mus, take out the bacterium liquid of cultivating above-mentioned 6 kinds of strains of 6 hours through 35 ℃ then, be diluted to 1MLD with physiological saline solution, inject respectively in the mouse peritoneal body, every Mus 0.5ml, preparation mice septicemia model, to respectively be subjected to the reagent thing to establish 5-6 dosage group again with 1: 0.5 dose of spacing, respectively at after infecting at once the intravenous injection variable concentrations be subjected to reagent liquid, 0.5ml/20g Mus is heavy, establishes simultaneously and infects matched group (not administration), observes and record dead mouse number, observed 7-14 days continuously, press the Bliss method and calculate median effective dose ED
50And 95% fiducial limit.
Bactericidal assay result shows: intravenous administration has certain interior curative effect to bacterial infection, its median effective dose ED
50Be respectively: staphylococcus aureus 01193 is 41.47mg/kg, staphylococcus aureus MSSA022 is 38.35mg/kg, staphylococcus aureus MRSA028 is 153.40mg/kg, staphylococcus epidermidis MRSE0210 is 18.57mg/kg, streptococcus pneumoniae 01182 is 14.08mg/kg, and enterococcus ATCC700802 is 150.2mg/kg.The interior curative effect of the staphylococcus epidermidis MRSE0210 of gene recombinant human lysozyme intravenously administrable, streptococcus pneumoniae 01182 infecting mouse is better than clarithromycin respectively more than 4 times and 9 times significantly.The result of the test data see attached list 1,2.
Three, gene recombinant human lysozyme and clarithromycin coupling can strengthen the clarithromycin list respectively and use 19.6,18.7 times of the interior curative effects of the staphylococcus aureus MRSA028 that is tried, suppurative hammer 01181D.
Test strain: micrococcus scarlatinae 01-18-1, staphylococcus aureus MRSA BAA-42 are used for through the therapeutical effect of atomizing suction to mice bacterial respiratory road infection inflammation.Method is that mice is put into ultrasound atomizer, the above-mentioned test organisms liquid of interior dress 30ml, and concentration is 10
8CFU/ml, sucked 20 minutes to the mice atomizing, 10 minutes at interval, atomizing sucked 20 minutes again, and atomizing sucks 3 times continuously, and atomizing at last sucked back 6 hours, be applied to the sticking fat planar surface of no medicine with the pharyngeal mucosa of aseptic cotton carrier picking respectively, cultivated 18-20 hour for 35 ℃, observation has or not bacterial growth, carries out Bacteria Identification and count plate.Antibacterial culturing is positive, and viable count 〉=10
3CFU/ml shows the infection model success, and this infection model can be used for therapeutic test.
The gene recombinant human lysozyme atomizing sucks the therapeutic test to mice bacterial respiratory tract infection inflammation:
Get the mice of respiratory tract infection antibacterial success, every group of 10 Mus, pack into and carry out atomization inspiration treatment in the glass jar that is subjected to reagent liquid that fills variable concentrations, each atomizing sucked 20 minutes, 10 minutes at interval, atomizing sucked 20 minutes again, and atomizing sucks three times altogether, was applied to no medicine agar plate dash board with sterilization cotton swab picking bottleneck throat mucosa respectively in first day, second day, the 3rd day, the 4th day, the 5th day after atomizing sucks medicinal liquid.Cultivated 18-20 hour at 35 ℃, carry out count plate.Bacterial respiratory tract infection matched group (not administration) is established in test.Pharyngeal viable count of administration group mice and the pharyngeal viable count of infection control group mice are carried out the Student verification, be subjected to the reagent thing through the therapeutical effect of atomizing suction mice bacterial respiratory tract infection inflammation with evaluation.Result of the test shows: concentration is 0.4,0.2,0.1,0.05, the gene recombinant human lysozyme of 0.025mg/ml sucks through atomizing, mouse breathing road infection suppurates streptococcus 01-18-1 and staphylococcus aureus MRSA BAA-42 had obvious curative effects, administration second day, pharyngeal bacterial infection number is reduced, and infect matched group and relatively have significant difference (P＜0.05) and curative effect to be better than clarithromycin, Roxithromycin administration group (P＜0.05).The data of result of the test see attached list 3.
Gene recombinant human lysozyme and clarithromycin (1: 1) drug combination sucks the curative effect of mice bacterial respiratory tract infection staphylococcus aureus BAA-42, micrococcus scarlatinae 01181 and clarithromycin list with significant difference (P＜0.05) is relatively arranged through atomizing.The optium concentration of gene recombinant human lysozyme and clarithromycin coupling is (1.5+0.1) mg/ml, and under this concentration, it is better than other each administration concentration group evident in efficacyly.
Four, gene recombinant human lysozyme to antibacterial 1040 strain antibacterial activity in vitro and with the potentiation of clarithromycin, Roxithromycin coupling.
The clinical isolates kind that adopts in this test has 1040 strain bacterium altogether such as staphylococcus aureus MRSA 211 strains, staphylococcus aureus MSSA 92 strains, the coccus MRSE of epidermis Portugal 123 strains, the coccus MSSA of epidermis Portugal 69 strains, streptococcus pneumoniae 40 strains, escherichia coli 123 strains, Klebsiella Pneumoniae 92 strains, Enterococcus 55 strains, acinetobacter calcoaceticus 55 strains, citrobacter 57 strains, pseudomonas aeruginosa 29 strains, Serratieae 44 strains, enterobacter cloacae 12 strains.
But contrast antibiotics is clarithromycin, Roxithromycin, amoxicillin, vancomycin, moral mycin.
Test method: dissolved with the sterilization distilled water by the reagent thing, suitably dilution.Get the 1ml medicinal liquid respectively and add the Tris-HCl agarose solid medium mixing that 9ml melts, doubling dilution prepares serial pastille plate.Every ware contained drug final concentration is respectively 4,2,1,0.5,0.25,0.125,0.06,0.03-0.001mg/ml; To be diluted to 10 with multiple spot inoculation instrument
5The test organisms liquid of CFU/ml is inoculated in each pastille plate surface, putting 35 ℃ cultivated 8-10 hour, take out, draw the above-mentioned serial plate of M-H culture medium (50 ℃) the covering surface that 6ml melts again respectively, place 35 ℃ to cultivate taking-up in 10 hours again, observed result is the minimum inhibitory concentration (MIC) of this bacterium with contained lowest concentration of drug in the no bacterial growth plate.Count MIC with the MIC value of accumulation inhibition 50%, 90% bacterial strain that tried respectively
50, MIC
90MIC value minima to its maximum of the bacterial strain that tries is counted MIC
Range(scope).The contrast lysozyme of test is a lysozyme of chicken.
Result of the test shows: gene recombinant human lysozyme all has antibacterial action to examination Gram-positive, negative bacterium.To clarithromycin, the drug-fast staphylococcus aureus MRSA of Roxithromycin, the coccus MRSE of epidermis Portugal, have antibacterial action, the effect of his-and-hers watches Portugal coccus is better than the antibacterial action to staphylococcus aureus.Escherichia coli in the gram negative bacteria, Klebsiella Pneumoniae, citrobacter, husky thunder bacterium all had certain antibacterial action.The present invention sees attached list 4 to the result of the test data of above-mentioned 58 strain bacterium.
Heavy group of human lysozyme of gene is to the MIC of staphylococcus aureus MRSA, MSSA bacterial strain
50, be 4000 μ g/ml, MIC
RangeAt 0.5-〉4000 μ g/ml; The MIC of the coccus MRSE of his-and-hers watches Portugal, MSSE
50, be respectively 6.25 μ g/ml and 2000 μ g/ml (table 5).To streptococcus pneumoniae, Streptococcus and enterococcal antibacterial action a little less than, the MIC value of most bacterial strains is at 〉=4000 μ g/ml.
Though gene recombinant human lysozyme has antibacterial action to the escherichia coli in the gram negative bacteria, Klebsiella Pneumoniae, acinetobacter calcoaceticus, pseudomonas aeruginosa and husky thunder bacterium, citrobacter, enterobacter cloacae etc., but a little less than its antibacterial action, the MIC value of most bacterial strains 〉=4000 μ g/ml.
Gene recombinant human lysozyme and clarithromycin, Roxithromycin to treated in vitro (seeing attached list 5), have the notable synergistic effect with coupling in 1: 1, can make more than antibacterial activity potentiation 2-16 times of clarithromycin, Roxithromycin, but indivedual bacterial strain potentiation is more than 1000 times.Clarithromycin, Roxithromycin are increased to 39.8,55.1% and 30.7,38.5% by 25.3,24.4% and 25.9,25.6% of single usefulness respectively to the responsive rate of drug resistance staphylococcus aureus and table Portugal coccus; Make clarithromycin, Roxithromycin be increased to 23.1% and 23.1% (seeing attached list 6) by 11.5%, 15.4% of single usefulness respectively to the responsive rate of streptococcus pneumoniae.Coupling and single with having significant difference (P＜0.05), especially remarkable with the potentiation of human lysozyme and clarithromycin use in conjunction.But but gene recombinant human lysozyme and vancomycin, the coupling of moral mycin can reduce vancomycin, the moral mycin MIC to staphylococcus aureus MRSA, the table coccus MRSE of Portugal
50, MIC
90Value is more than 2-4 times.
More than all application results show: the high-purity, the highly active gene recombinant human lysozyme that utilize our company's biology gene engineering technology to develop, not only have and cut off the β-1 between the N-acetyl-glucosamine and-acetylmuramic acid in the Peptidoglycan, the 4-glycosidic bond, destroy thalline Peptidoglycan support, directly the normal structure of cracking antibacterial, protection inflammation part, raising mononuclear cell are engulfed the ability of tubercule bacillus, the release action of inhibition bacterial endotoxin; And gene recombinant human lysozyme can decompose sticking thick mucin, eliminates the mucosa inflammation, decomposes mucosa liquid, impels mucopolysaccharide metabolism and the effect of cleaning upper respiratory tract.
Application test also shows: gene recombinant human lysozyme not only can be used as preparation treatment antibacterial, fungus, the viral medicine that causes various inflammation at the skin mucosa surface infection; With gene recombinant human lysozyme and antibiotic drug combination, can also strengthen the inside and outside bacterial infection greatly and cause various inflammation therapeutic effect at double.This just provides new development direction for biotech medicine product, also preparation and the Gospel that causes inflammation and provide new for the clinical cure bacterial infection.
Description of drawings
Fig. 1 is with the activity test of gene recombinant human lysozyme test tube method evaluation to antibacterial;
Fig. 2 is with the activity test of gene recombinant human lysozyme flat band method evaluation to antibacterial;
Fig. 3 a handles back fungistatic effect figure with gene recombinant human lysozyme to fungus;
Fig. 3 b is contrast figure before with gene recombinant human lysozyme fungus being handled;
Fig. 4-A is normal HELA cell photo;
Fig. 4-B is No. 04 coronavirus infection HELA cell photo;
Fig. 4-C handles the minimum effective drug concentration photo with gene recombinant human lysozyme to coronavirus.
The specific embodiment
Embodiment 1
Present embodiment is for to carry out bacteriolyze activity identification method with the gene recombinant human lysozyme test tube method to antibacterial.
10mMTris-HCl buffer with PH7.2 is made into 10 with staphylococcus aureus (or escherichia coli)
7-10
8The bacterium liquid of CFU/ml left and right sides concentration, join respectively in the different magnificent test tubes with every pipe 2ml, then, add the gene recombinant human lysozyme of 100 μ g-1mg various dose in the magnificent test tube of difference respectively, 37 ℃ are spent the night, and every pipe is got 20 μ l and diluted with 10 times of dilution methods, get 100 μ l again and be applied to the LB culture medium, in 37 ℃ of overnight incubation, observed result is calculated total number of bacteria in the original pipe.
Total number of bacteria * 100% in the bacteriolyze effect of gene recombinant human lysozyme=(total number of bacteria in the total number of bacteria-experiment tube in the control tube)/control tube.
Embodiment 2
Present embodiment is for to carry out the bactericidal activity authentication method with the gene recombinant human lysozyme flat band method to antibacterial.(material is with embodiment 1)
After preparing 1% agarose autoclaving with the 10mMTris-HCl buffer of PH7.2, be cooled to 40 ℃, add antibacterial to 106 pour plate, punch on flat board with card punch, the gene recombinant human lysozyme solution that in the hole, adds the 0-100mg/ml variable concentrations, hatched 4-6 hour in 37 ℃ of incubators, then with behind the LB Nutrient agar autoclaving, be cooled to 40 ℃, be covered on the above-mentioned agarose plate, this flat board placed under the uviol lamp shone 20 minutes, 37 ℃ of incubator overnight incubation, observe bacterial growth situation between agarose and the Nutrient agar layer, measure the diameter of the inhibition zone of variable concentrations lysozyme formation.The diameter well diameter 4mm person of inhibition zone is minimum effective bacteriocidal concentration.
Embodiment 3
Present embodiment is with the bacteriostatic experiment method of gene recombinant human lysozyme to fungus.
Bacterial strain: the 8 strain Candida albicans, Oidium tropicale, the gram Rou Shi candidiasis that provide by verification section of Xijing hospital of The Fourth Military Medical University.
The 10mMTris-HCl buffer of buffer: PH=7.2
10％SDS
The LB culture medium
Implementation method is identical with embodiment 1 bacteriostatic experiment of gene recombinant human lysozyme effect fastbacteria.
Embodiment 4
Present embodiment is to suck mice bacterial respiratory tract infection therapeutical effect through atomizing with gene recombinant human lysozyme.
The model preparation:
Before the test mice is put into the seal glass cylinder, glass jar is connected to ultrasound atomizer (model: 402, make a concerted effort medical apparatus and instruments factory production of Shanghai), interior dress 30ml test organisms liquid, and bacterial concentration is 10
8CFU/ml starts ultrasound atomizer, sucks 20 minutes to the mice atomizing, and 10 minutes at interval, atomizing needed to suck 20 minutes again, and mist sucks 3 times continuously.The last atomizing sucked back 6 hours, was applied to the dull and stereotyped table of the sticking fat of no medicine with the pharyngeal mucosa of aseptic cotton carrier picking respectively and existed, and cultivated 18-20 hour for 35 ℃, observed to have or not bacterial growth, carried out Bacteria Identification and count plate.Positive bacterial culture, and viable count 〉=10
3CFU/ml shows the infection model success.Point out this infection model to can be used for therapeutic test.
Gene recombinant human lysozyme (HLZ) atomizing sucks the therapeutic test of mice bacterial respiratory tract.
Get the mice of respiratory tract infection antibacterial success, random packet, every group of 10 Mus, pack into fill concentration be subjected to carry out atomization inspiration treatment in the reagent liquid glass jar of (having connected ultrasound atomizer), each atomizing sucked 20 minutes, and 10 minutes at interval, atomizing sucked 20 minutes again, atomizing sucks 3 times altogether, is applied to no medicine agar plate dash board with sterilization cotton swab picking bottleneck throat mucosa respectively in first day, second day, the 3rd day, the 4th day, the 5th day after atomizing sucks medicinal liquid.Cultivated 18-20 hour, and carried out count plate for 35 ℃.Bacterial respiratory tract infection matched group (not administration) is established in experiment.Pharyngeal viable count of administration group mice and the pharyngeal viable count of infection control group mice are carried out the Student check, be subjected to the reagent thing through the therapeutical effect of atomizing suction the mice bacterial respiratory tract infection with evaluation.
Embodiment 5
This example execute into gene recombinant human lysozyme to No. 04 antibacterial virulence experiment of coronavirus.
Test material
1. checking medicine: gene recombinant human lysozyme protein content: 4.3845mg
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.
Test method
Pilot study:
1. gene 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 gene 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 "+", the 26%-50% metamorphosis is " ++ ", the 51%-75% metamorphosis is " +++", the 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 mensuration of the virulence of coronavirus No. 04
The separation that coronavirus is No. 04 (SARS patients serum separation):
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 " ++ ", 51%-75% be " +++; 76%-100% is changed to " ++ ++ "; use the Reed-Muench method, calculates viral half and infects concentration TCID
50
Formal test
Gene recombinant human lysozyme in the HELA cell culture to the inhibitory action of coronavirus No. 04
Test objective: to the inhibitory action of coronavirus No. 04, virocyte pathological changes (CPE) method is adopted in test to the gene 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 gene 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, and the medicine that dilutes is added in the cell hole every concentration 3 holes respectively, 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.
Result of the test
The pilot study result:
Gene recombinant human lysozyme is to the toxic action of HELA cell:
Gene recombinant human lysozyme 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 meansigma methods of three result of the tests.
1. gene recombinant human lysozyme is to the toxicity test result of HELA cell
The checking medicine:
Gene 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
The positive control medicine:
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(seeing accompanying drawing 4)
Formal test result (seeing accompanying drawing 4)
Gene recombinant human lysozyme in the HELA cell culture to the inhibitory action (following test data is the meansigma methods of three result of the tests) of coronavirus No. 04
The checking medicine:
Gene 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 ± 0 μ g/ml, minimum effective drug concentration (MIC) is 23.44 ± 0 μ g/ml, therapeutic index (TI) is 256.
Gene recombinant human lysozyme in the HELA cell culture to the preventive effect (following test data is the meansigma methods of three result of the tests) of coronavirus No. 04
The checking medicine:
Gene 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 ± 0 μ g/ml, minimum effective drug concentration (MIC) is 23.44 ± 0 μ g/ml, therapeutic index (TI) is 256.
Embodiment 6
Present embodiment is the preparation method of gene recombinant human lysozyme lyophilized powder.
Behind the culture medium autoclaving, inoculation gene recombinant human lysozyme bacterial strain, the shaking table revolution is that per minute 250 changes, cultivation temperature is 20, cultivates 36-40 hour on the constant temperature shaking table, carries 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.
Embodiment 7
Present embodiment is with the therapeutic test of gene recombinant human lysozyme to injection medicament in the bacterial infection body.
Being tried mice by body weight, sex is evenly divided into groups, every group of 10 Mus, take out the bacterium liquid of cultivating above-mentioned 6 kinds of strains of 6 hours through 35 then, be diluted to 1MLD with physiological saline solution, inject respectively in the mouse peritoneal body, every Mus 0.5ml, preparation mice septicemia model, to respectively be subjected to the reagent thing to establish 5-6 dosage group again with 1: 0.5 dose of spacing, respectively at after infecting at once the intravenous injection variable concentrations be subjected to reagent liquid, the 0.5ml/20g Mus is heavy, establish simultaneously and infect matched group (not administration), observe and record dead mouse number, observed continuously 7-14 days, press the Bliss method and calculate median effective dose ED
50And 95% fiducial limit.(seeing Table 1,2)
Embodiment 8
Present embodiment is with gene recombinant human lysozyme and the antibiotic interior drug combination treatment of composition in 1: 1 inflammation effect.
Mice is put into ultrasound atomizer, interior dress 30ml staphylococcus aureus MRSA028 or streptococcus pyogenes 01181 test organisms liquid, concentration is 10
8CFU/ml, sucked 20 minutes to the mice atomizing, 10 minutes at interval, atomizing sucked 20 minutes again, and atomizing sucks 3 times continuously, and atomizing at last sucked 6 hours, be applied to the sticking fat planar surface of no medicine with the pharyngeal mucosa of aseptic cotton carrier picking respectively, cultivated 18-20 hour for 35 ℃, observation has or not bacterial growth, carries out Bacteria Identification and count plate.Be positive viable count 〉=10
3CFU/ml shows and infects successfully.
Get respiratory tract infection antibacterial success mice, every group 10, be incorporated with the gene recombinant human lysozyme of variable concentrations and 1: 1 atomizing of clarithromycin and sucked behind the medicinal liquid the 1st, 2,3,4,5 day, be applied to no medicine agar plate dash board with the cotton swab picking bottleneck throat mucosa of sterilizing respectively.Cultivated 18-20 hour at 35 ℃, carry out count plate.Bacterial respiratory tract infection matched group (not administration) is established in test.Pharyngeal viable count of administration group mice and the pharyngeal viable count of infection control group mice are carried out the Student check, estimate therapeutical effect.
The result shows: above-mentioned bacterial infection inflammation has significant difference (P＜0.05) than single with clarithromycin to the optium concentration of gene recombinant human lysozyme and clarithromycin coupling in 1: 1 for (1.5+0.1) mg/ml treats.Body internal respiration road is respectively strengthened 19.6 times and 18.7 times than single with clarithromycin by the curative effect of staphylococcus aureus, streptococcus pyogenes infection inflammation.
Embodiment 9
Present embodiment be with gene recombinant human lysozyme to the antibacterial antibacterial activity in vitro and with the potentiation of antibiotic coupling.
With gene recombinant human lysozyme with sterilization distilled water dissolving, suitably dilution, get respectively 1ml respectively medicinal liquid add the Tris-HCl agarose solid medium mixing that 9ml melts, doubling dilution prepares serial pastille plate.The contained gene recombinant human lysozyme final concentration of every ware is respectively 4,2,1,0.5,0.25,0.125,0.06,0.03-0.001mg/ml; To be diluted to 10 with multiple spot inoculation instrument
5The test organisms of CFU/ml (staphylococcus aureus, table Portugal coccus, streptococcus pneumoniae, enterococcus etc.) is inoculated in each pastille plate surface, putting 35 ℃ cultivated 8-10 hour, take out, draw the above-mentioned serial plate of M-H culture medium (50 ℃) the covering surface that 6ml melts again respectively, place 35 ℃ to cultivate taking-up in 10 hours, observed result again.With contained lowest concentration of drug in the asepsis growth plate is the minimum inhibitory concentration MIC of this bacterium.Can count MIC with the MIC value of accumulation inhibition 50%, 90% bacterial strain that tried respectively
50, MIC
90
The gene recombinant human lysozyme list is used all has antibacterial action to Gram-positive, negative bacterium; Clarithromycin, the drug-fast staphylococcus aureus of Roxithromycin, epidermis Portugal coccus are presented antibacterial action; The effect of his-and-hers watches Portugal coccus is better than the antibacterial action to staphylococcus aureus.If will be subjected to the reagent thing to change gene recombinant human lysozyme and antibiotic coupling into, result of the test is as follows:
Heavy group of human lysozyme of gene and clarithromycin, Roxithromycin coupling in 1: 1 can make more than antibacterial activity potentiation 2-16 times of clarithromycin, Roxithromycin; Clarithromycin, Roxithromycin are increased to 39.8,55.1% and 30.7,38.5% by 25.3,24.4% and 25.9,25.6% of single usefulness respectively to the responsive rate of drug resistance staphylococcus aureus and table Portugal coccus; Make clarithromycin, Roxithromycin be increased to 23.1,23.1% by 11.5,15.4% of single usefulness respectively to the responsive rate of streptococcus pneumoniae; More remarkable with heavy group of human lysozyme of gene and clarithromycin coupling potentiation; But but can reduce vancomycin, moral mycin MIC to staphylococcus aureus, table Portugal coccus with vancomycin, the coupling of moral mycin
50, MIC
90Value is more than 2-4 times.
Table 1 gene recombinant human bacteriolyze alcohol infects the interior curative effect result of the test of induced mice septicemia to staphylococcus aureus MRSA028
Bacterial infection (bacterium number) infects concentration number of animals death toll death rate ED50(mg/kg)
Tested drug administration approach dosage (mg/kg)
Bacterium amount (CFU/ML) is (only) (only) (%) (95% fiducial limit) (mg/ml)
Staphylococcus aureus MRSA028 human lysozyme i.v 400 (1,200,000 units) 16 10 10 100
1.9×10
5(HLZ) 200 (600,000 units) 8 10 10 100
100 (300,000 units) 4 10 10 100
50 (150,000 units) 2 10 10 100
CLA i.v 200 8 10 3 30
(CLA) 100 4 10 6 60 122.8
25 1 10 10 100 (82.5-246.7)
CLA/molten i.v 12.5 0.5/0.5 10 3 30
Bacterium enzyme 6.25 0.25/0.25 10 4 40 6.29
3.13 0.125/0.125 10 8 80 (2.21-18.03)
Infect control group (1MLD) ip* - - 10 10 100
Infect control group (1/10MLD) ip--10 8 80
*Ip: abdominal cavity infection bacterium.
Table 2 gene recombinant human lysozyme infects the interior curative effect result of the test of induced mice septicemia to streptococcus pneumonia 01181
Bacterial infection
(bacterium number) concentration number of animals death toll ED50(mg/kg)
Tested drug administration approach dosage (mg/kg) death rate (%)
Infection dosage (mg/ml) (only) (only) (95% fiducial limit)
(CFU/ML)
25 1.00 10 8 80
12.5 0.50 10 7 70
Streptococcus pneumonia 01181 HLZ i.v 6.25 0.25 10 6 60 2.09
3.12 0.13 10 5 50 0.00-1000.00
1.56 0.06 10 3 30
200 8.0 10 3 30
i.v 100 4.0 10 5 50 98.2
CLA 50 2.0 10 7 70 60.1-233.1
25 1.0 10 8 80
12.5 0.5 10 10 100
25/25 1.00/1.00 10 10 100
i.v 12.5/12.5 0.50/0.50 10 7 70 2.521
Carat+lysozyme 6.25/6.25 0.25/0.25 10 4 40 0-1000.0
3.12/3.12 0.13/0.13 10 2 20
1.56/1.56 0.06/0.06 10 8 80
Infect control group-10 10 100
The clinical trial result of table 3 gene recombinant human lysozyme atomizing inhalation in mice respiratory tract infection staphylococcus aureus
Colony counting (CFU) after dosage (ul) administration
The bacterium administration
The infection dosage drug concentration *
(cfu/ml) (mg/ml) administration number of times the 1st day the 2nd day the 3rd day the 4th day the 5th day
(times/day)
Staphylococcus aureus 0.2 100 * 4 253.80 ± 22.88 222.10 ± 18.46 165.80 ± 16.08 116.20 ± 13.97 76.70 ± 11.60
MRSA 0.1 100×4 254.20±25.42 221.30±18.12 172.70±17.62 116.70±15.59 78.30±17.86
BAA-42 human lysozyme 0.05 100 * 4 253.90 ± 15.91 218.80 ± 18.70 174.50 ± 26.28 119.80 ± 17.64 80.80 ± 14.28
0.025 100×4 256.40±14.31 227.20±17.02 176.90±11.14 121.50±12.41 84.20±20.90
0.0125 100×4 258.50±46.60 230.50±12.99 182.80±20.67 121.90±14.69 86.50±15.71
6.0 100×4 241.90±16.62 211.50±16.85 134.60±13.58 82.00±12.09 57.60±15.37
3.0 100×4 246.30±19.03 217.50±24.42 141.00±18.54 83.20±13.45 60.70±13.98
CLA 1.5 100 * 4 247.10 ± 21.31 218.30 ± 23.98 146.30 ± 13.55 83.00 ± 13.02 64.40 ± 15.69
0.75 100×4 248.50±26.32 219.50±30.00 156.10±20.87 92.00±11.08 68.00±14.02
0.375 100×4 250.80±19.69 221.00±16.21 165.00±16.63 93.60±13.12 69.20±13.80
6+0.4 100×4 232.10±34.94 204.10±18.16 126.20±17.74 78.80±12.05 50.40±16.93
CLA 3+0.2 100 * 4 232.60 ± 24.76 206.80 ± 21.70 134.00 ± 20.96 79.70 ± 13.00 52.60 ± 19.20
+ people bacteriolyze 1.5+0.1 100 * 4 224.00 ± 18.65 195.10 ± 10.69 118.00 ± 15.13 69.30 ± 16.23 46.50 ± 19.29
Enzyme 0.75+0.05 100 * 4 239.00 ± 25.11 206.60 ± 15.76 137.00 ± 18.87 88.20 ± 14.31 52.70 ± 17.78
0.375+0.025 100×4 239.30±20.22 209.40±34.95 138.40±18.65 90.40±12.23 64.30±10.82
Bacterium contrast--275.60 ± 21.60 250.30 ± 29.12 194.30 ± 17.63 130.10 ± 19.34 86.70 ± 12.88
Table 4 gene recombinant human lysozyme, lysozyme of chicken, clarithromycin and Roxithromycin in-vitro antibacterial spectrum
MIC
Antibacterial
HLZ????????????????CLZ???????????CLA???????ROX
μ g/ml (the μ g/ml of ten thousand μ/ml) (the μ g/ml μ g/ml of ten thousand μ/ml)
Staphylococcus aureus MRSA02-22 250 (0.8) 8 (0.04) 4,000 4000
Staphylococcus aureus MRSA02-23 250 (0.8) 8 (0.04) 4,000 4000
Staphylococcus aureus MRSA02-26 250 (0.8) 4 (0.02) 4,000 4000
Staphylococcus aureus MRSA02-28 500 (1.5) 15.6 (0.08) 4,000 4000
Staphylococcus aureus 02-19-5 31.3 (0.1) 2 (0.001) 4,000 4000
The table coccus MssE25 of Portugal 15.6 (0.05) 4 (0.02) 0.5 0.5
The table coccus MRSE02-29 of Portugal 62.5 (0.2) 500 (2.5) 31.3 500
The table coccus MRSE02-5 of Portugal 0.5 (0.0015) 0.5 (0.0025) 0.5 0.5
The table coccus MRSE02-6 of Portugal 0.5 (0.0015) 0.5 (0.0025) 0.5 0.5
The table coccus MRSE02-20-2 of Portugal 0.5 (0.0015) 0.5 (0.0025) 4,000 1000
The table coccus MRSE02-20-3 of Portugal 0.5 (0.0015) 4 (0.02) 4,000 1000
The table coccus MRSE02-20-4 of Portugal 0.5 (0.0015) 0.5 (0.0025) 4,000 4000
The table coccus MRSE02-20-5 of Portugal 4 (0.012) 0.5 (0.0025) 4,000 4000
The table coccus MRSE02-20-6 of Portugal 4 (0.012) 0.5 (0.0025) 4,000 4000
The table coccus MRSE02-20-7 of Portugal 0.5 (0.0015) 0.5 (0.0025) 11
The table coccus MRSE02-20-8 of Portugal 0.5 (0.0015) 0.5 (0.0025) 0.5 0.5
The table coccus MRSE02-20-9 of Portugal 0.5 (0.0015) 0.5 (0.0025) 0.5 0.5
The table coccus MRSE02-20-1 of Portugal 0.5 (0.0015) 0.5 (0.0025) 0.5 0.5
The table coccus MRSE02-3 of Portugal 1000 (3) 15.6 (0.08) 4,000 4000
The table coccus MRSE02-4 of Portugal 4 (0.012) 8 (0.04) 500 4000
Continuous table 4
MIC
Antibacterial
HLZ???????????????CLZ????????????CLA?????????ROX
μ g/ml (the μ g/ml of ten thousand μ/ml) (the μ g/ml μ g/ml of ten thousand μ/ml)
The table coccus MRSE02-10 of Portugal 4 (0.012) 250 (1.25) 1,000 1000
The table coccus MRSE02-12 of Portugal 0.5 (0.0015) 0.5 (0.0025) 8 125
The table coccus MRSE02-11 of Portugal 0.5 (0.0015) 0.5 (0.0025) 0.5 0.5
The table coccus MRSE02-15 of Portugal 8 (0.024) 2 (0.01) 1,000 4000
The table coccus MRSE02-17 of Portugal 4 (0.012) 0.5 (0.0025) 250 4000
The table coccus MRSE02-18 of Portugal 0.5 (0.0015) 0.5 (0.0025) 0.5 0.5
The table coccus MRSE02-20 of Portugal 8 (0.024) 0.5 (0.0025) 4,000 4000
The table coccus MRSE02-21 of Portugal 15.6 (0.05) 250 (1.25) 0.5 0.5
The table coccus MRSE02-22 of Portugal 4 (0.012) 0.5 (0.0025) 0.5 0.5
Citrobacter 02-7-4 8 (0.02) 4000 (20) 22
Citrobacter 02-7-5 8 (0.02) 250 (0.63) 0.5 0.5
Citrobacter 02-7-6 8 (0.02) 62.5 (0.31) 8 250
Citrobacter 02-7-9 62.5 (0.2) 125 (0.63) 8 15.6
Citrobacter 02-7-7 125 (0.4) 15.6 (0.08) 31.3 250
Escherichia coli 01-2-22 31.3 (0.1) 500 (2.5) 1,000 1000
Escherichia coli 01-2-30 62.5 (0.2) 4000 (10) 4,000 4000
Escherichia coli 01-2-32 15.6 (0.05) 250 (1.25) 8 500
Escherichia coli 01-2-33 31.3 (0.1) 2000 (10) 4,000 4000
Escherichia coli 01-2-36 62.5 (0.2) 4000 (20) 250 500
Escherichia coli 01-2-37 31.3 (0.1)＞4000 (20) 4,000 4000
Escherichia coli 01-2-39 31.3 (0.1) 500 (2.5) 500 1000
Continuous table 4
MIC
Antibacterial
HLZ?????????????????CLZ??????????CLA???????ROX
μ g/ml (the μ g/ml of ten thousand μ/ml) (the μ g/ml μ g/ml of ten thousand μ/ml)
Escherichia coli 01-2-41 31.3 (0.1) 500 (2.5) 4,000 4000
Escherichia coli 01-2-42 15.6 (0.05) 500 (2.5) 62.5 4000
Escherichia coli 01-2-43 0.5 (0.0015)＞4000 (20) 15.6 250
Escherichia coli 01-2-45 31.3 (0.1) 2000 (10) 250 500
Escherichia coli 01-2-51 31.3 (0.1)＞4000 (20) 4,000 4000
Escherichia coli 01-2-52 31.3 (0.1)＞4000 (20) 4,000 250
Escherichia coli 01-2-53 8 (0.024) 4000 (20) 31.3 250
Escherichia coli 01-2-54 31.3 (0.1) 1000 (5) 4,000 1000
Escherichia coli 01-2-56 31.3 (0.1)＞4000 (20) 15.6 250
Escherichia coli 01-2-57 0.5 (0.0015) 200 (10) 31.3 250
Escherichia coli 01-2-58 0.5 (0.0015) 400 (20) 4,000 500
Escherichia coli 01-2-59 31.3 (0.1) 400 (20) 125 250
Serratieae 2-31-8 8 (0.02) 500 (0.25) 8 15.6
Serratieae 2-31-8 8 (0.02) 1000 (5) 8 15.6
Serratieae 2-31-8 8 (0.02) 125 (0.63) 15.6 15.6
Klebsiella Pneumoniae 02-6-14 15.6 (0.05) 1000 (5) 62.5 4000
Klebsiella Pneumoniae 02-6-18 4 (0.012)＞4000 (20) 500 1000
Annotate: HLZ refers to human lysozyme, and CLZ refers to contrast lysozyme (lysozyme of chicken), and CLA refers to clarithromycin, and ROX refers to Roxithromycin
Table 5 gene recombinant human lysozyme and clarithromycin, Roxithromycin list are used and (1: 1) use in conjunction interaction in vitro compares
MIC(μg/ml)
Bacteria name
HLZ?????????CLZ???????CLA?????????CLA/HLE????????ROX???????ROX/HLZ
The gold MRSA02-20 of Portugal＞4,000 1,000 4000 62.5/62.5,4000 62.5/62.5
Staphylococcus aureus MRSA02-22 250 8 4000 62.5/62.5 4000 62.5/62.5
Staphylococcus aureus MRSA02-26 250 4 4000 0.5/0.5 4000 0.5/0.5
Staphylococcus aureus 02-19-1＞4,000 62.5 4,000 5,00/,500 4,000 2000/2000
Staphylococcus aureus 02-19-2＞4,000 62.5 4,000 5,00/,500 4,000 2000/2000
Staphylococcus aureus 02-19-5 31.3 0.5 4,000 8/8 4,000 250/250
Staphylococcus aureus 02-19-25＞4,000 0.5 1 0.5/0.5,4000 0.5/0.5
Staphylococcus aureus 02-19-28＞4,000 8 250 1,25/,125 1,000 500/500
Staphylococcus aureus 02-19-39＞4,000 0.5 8 0.5/0.5,62.5 0.5/0.5
The table coccus 02-20-2 of Portugal 22 4000 0.5/0.5 4000 0.5/0.5
The table coccus 02-20-3 of Portugal 24 4,000 1/1 1,000 8/8
The table coccus 02-20-4 of Portugal 22 4000 0.5/0.5 4,000 8/8
The table coccus 02-20-6 of Portugal 42 4000 0.5/0.5 4000 0.5/0.5
The table coccus 02-20-7 of Portugal 22 1000 0.5/0.5 1000 0.5/0.5
Continuous table 5
Bacteria name MIC (μ g/ml)
HLZ?????????CLZ???????CLA???????CLA//HLZ???????ROX???????ROX/HLZ
The table coccus MRSE02-4 of Portugal 48 500 62.5/62.5 4000 15.6/15.6
The table coccus MRSE02-5 of Portugal 88 4,000 200,0/2,000 4,000 8/8
The table coccus MRSE02-9 of Portugal＞4,000 15.6 4000 0.5/0.5 4,000 1/1
The table coccus MRSE02-10 of Portugal 4 250 1000 0.5/0.5 1000 0.5/0.5
The table coccus MRSE02-12 of Portugal 0.5 0.5 8 0.5/0.5 125 0.5/0.5
The table coccus MRSE02-13 of Portugal 15.6 62.5 15.6 2/2 500 15.6/15.6
The table coccus MRSE02-15 of Portugal 82 1000 0.5/0.5 4000 0.5/0.5
The table coccus MRSE02-17 of Portugal 4 0.5 250 2/2 4,000 8
The table coccus MRSE02-20 of Portugal 8 0.5 4000 0.5/0.5 4000 0.5/0.5
The table coccus MRSE02-23 of Portugal＞4,000 0.5 1000 0.5/0.5,4000 0.5/0.5
Escherichia coli 02-1-20＞4,000 250 500 15.6/15.6,4000 62.5/62.5
Escherichia coli 02-1-24＞4000＞4,000 4,000 1,25/,125 4,000 4/4
Escherichia coli 02-1-3＞4000＞4,000 500 2,50/,250 250 62.5/62.5
Escherichia coli 02-1-4＞4000＞4,000 125 62.5/62.5,125 31.3/31.3
Continuous table 5
MIC(μg/ml)
Bacteria name
HLZ?????????CLZ???????CLA???????CLA/HLZ????????ROX???????ROX/HLZ
Escherichia coli 02-1-5 31.3 15.6 125 31.3/31.3 250 31.3/31.3
Escherichia coli 02-1-7＞4,000 2 62.5 62.5/62.5,125 62.5/62.5
Escherichia coli 02-1-8＞4,000 2 125 15.6/15.6,500 31.3/31.3
Escherichia coli 02-1-13＞4000＞4,000 62.5 31.3/31.3,250 31.3/31.3
Escherichia coli 02-1-14＞4000＞4,000 500 31.3/31.3 500 125/125
Citrobacter 02-7-7＞4,000 1 4000 0.5/0.5 4,000 4/4
Citrobacter 02-7-8 21 1000 15.6/15.6 4000 0.5/0.5
Citrobacter 02-7-10＞4000＞4,000 1000 15.6/15.6 1,000 500/500
Citrobacter 02-7-11＞4,000 1 62.5 4/4 4,000 125/125
Citrobacter 02-7-12 125 2 31.3 0.5/0.5 250 4/4
Klebsiella Pneumoniae 02-6-1＞4,000 4 125 0.5/0.5,0125 15.6/15.6
Klebsiella Pneumoniae 02-6-2＞4,000 2 500 31.3/31.3,1000 31.3/31.3
Klebsiella Pneumoniae 02-6-3＞4000＞4,000 62.5 31.3/31.3,125 31.3/31.3
Klebsiella Pneumoniae 02-6-4＞4,000 4 500 15.6/15.6 1,000 500/500
Continuous table 5
MIC(μg/ml)
Bacteria name
HLZ?????????CLZ???????CLA???????CLA/HLZ????????ROX???????ROX/HLZ
Klebsiella Pneumoniae 02-6-12＞4000＞4,000 4,000 100,0/1,000 4,000 1000/1000
Klebsiella Pneumoniae 02-6-14 250 1,000 62.5 62.5/62.5 4,000 125/125
Klebsiella Pneumoniae 02-6-16＞4000＞4,000 1,000 1,25/,125 4000 62.5/62.5
Klebsiella Pneumoniae 02-6-19＞4,000 1,000 62.5 4/4 1000 62.5/62.5
Enterobacter cloacae 02-9-1＞4,000 4,000 1000 31.3/31.3,250 31.3/31.3
Enterobacter cloacae 02-9-2＞4,000 4,000 125 31.3/31.3 500 125/125
Enterobacter cloacae 02-9-3＞4,000 1,000 125 31.3/31.3 250 125/125
Enterobacter cloacae 02-9-5 31.3 15.6 4000 0.5/0.5 4 0.5/0.5
Enterobacter cloacae 02-9-6＞4,000 250 125 0.5/0.5,125 0.5/0.5
Acinetobacter calcoaceticus 02-24-4＞4,000 500 125 15.6/15.6,8 0.5/0.5
Annotate: HLZ refers to human lysozyme, and CLZ refers to contrast lysozyme (lysozyme of chicken), and CLA refers to clarithromycin, and ROX refers to Roxithromycin.
But table 6 human lysozyme and clarithromycin, Roxithromycin, vancomycin, moral mycin
Single with and unite the responsive rate (%) of use to 500 strain clinical isolates
| Antibacterial (strain number) | Medicine | Responsive (strain number) | Medium sensitivity (strain number) | Drug resistance (strain number) | Responsive rate (%) |
| Staphylococcus aureus (166) | ????HLZZ * | ????53 | ????0 | ????113 | ????31.9 |
| ????CLZ | ????160 | ????0 | ????6 | ????96.4 | |
| ????CLA | ????42 | ????1 | ????123 | ????25.3 | |
| ????CLA/HLZ | ????66 | ????1 | ????99 | ????39.8 | |
| ????ROX | ????43 | ????5 | ????118 | ????25.9 | |
| ????ROX/HLZ | ????51 | ????8 | ????107 | ????30.7 | |
| ????VAN | ????141 | ????20 | ????5 | ????84.9 | |
| ????VAN/HLZ | ????147 | ????0 | ????19 | ????88.6 | |
| ????TEC | ????123 | ????35 | ????8 | ????74.1 | |
| ????TEC/HLZ | ????148 | ????0 | ????18 | ????89.2 | |
| Table Portugal coccus (78) | ????HLZ | ????53 | ????0 | ????25 | ????67.9 |
| ????CLZ | ????78 | ????0 | ????0 | ????100 | |
| ????CLA | ????19 | ????1 | ????58 | ????24.4 | |
| ????CLA/HLZ | ????43 | ????1 | ????34 | ????55.1 | |
| ????ROX | ????20 | ????0 | ????58 | ????25.6 | |
| ????ROX/HLZ | ????30 | ????4 | ????44 | ????38.5 | |
| ????VAN | ????76 | ????0 | ????2 | ????97.4 | |
| ????VAN/HLZ | ????74 | ????0 | ????4 | ????94.9 | |
| ????TEC | ????53 | ????23 | ????2 | ????67.9 | |
| ????TEC/HLZ | ????73 | ????0 | ????5 | ????93.6 | |
| Escherichia coli (48) | ????HLZ | ????13 | ????0 | ????35 | ????27.1 |
| ????CLZ | ????19 | ????0 | ????29 | ????65.5 | |
| ????CLA | ????1 | ????0 | ????47 | ????2.1 | |
| ????CLA/HLZ | ????3 | ????1 | ????44 | ????6.3 | |
| ????ROX | ????0 | ????1 | ????47 | ????0 | |
| ????ROX/HLZ | ????2 | ????1 | ????45 | ????4.2 | |
| ????VAN | ????2 | ????2 | ????44 | ????4.2 | |
| ????VAN/HLZ | ????14 | ????10 | ????24 | ????29.2 | |
| ????TEC | ????3 | ????0 | ????45 | ????6.3 | |
| ????TEC/HLZ | ????2 | ????4 | ????42 | ????4.2 | |
| Klebsiella Pneumoniae (72) | ????HLZ | ????12 | ????0 | ????60 | ????16.7 |
| ????CLZ | ????23 | ????0 | ????49 | ????31.9 | |
| ????CLA | ????0 | ????0 | ????72 | ????0 | |
| ????CLA/HLZ | ????2 | ????0 | ????70 | ????2.8 | |
| ????ROX | ????0 | ????0 | ????72 | ????0 | |
| ????ROX/HLZ | ????2 | ????0 | ????70 | ????2.8 | |
| ????VAN | ????2 | ????0 | ????70 | ????2.8 | |
| ????VAN/HLZ | ????14 | ????15 | ????43 | ????19.4 | |
| ????TEC | ????1 | ????0 | ????71 | ????1.4 | |
| ????TEC/HLZ | ????4 | ????1 | ????67 | ????5.6 | |
| Pseudomonas aeruginosa (13) | ????HLZ | ????10 | ????0 | ????3 | ????76.9 |
| ????CLZ | ????11 | ????0 | ????2 | ????84.6 | |
| ????CLA | ????0 | ????0 | ????13 | ????0 | |
| ????CLA/HLZ | ????0 | ????0 | ????13 | ????0 | |
| ????ROX | ????0 | ????0 | ????13 | ????0 | |
| ????ROX/HLZ | ????0 | ????0 | ????13 | ????0 | |
| ????VAN | ????0 | ????0 | ????13 | ????0 | |
| ????VAN/HLZ | ????9 | ????0 | ????2 | ????69.2 | |
| ????TEC | ????0 | ????0 | ????13 | ????0 | |
| ????TEC/HLZ | ????10 | ????1 | ????2 | ????76.9 |
Continuous table 6
| Antibacterial (strain number) | Medicine | Responsive (strain number) | Medium sensitivity (strain number) | Drug resistance (strain number) | Responsive rate (%) |
| Acinetobacter calcoaceticus (19) | ????HLZ | ????13 | ????0 | ????6 | ????68.4 |
| ????CLZ | ????13 | ????0 | ????6 | ????68.4 | |
| ????CLA | ????0 | ????1 | ????18 | ????0 | |
| ????CLA/HLZ | ????0 | ????1 | ????18 | ????0 | |
| ????ROX | ????0 | ????0 | ????19 | ????0 | |
| ????ROX/HLZ | ????1 | ????0 | ????18 | ????5.3 | |
| ????VAN | ????0 | ????5 | ????14 | ????0 | |
| ????VAN/HLZ | ????8 | ????4 | ????7 | ????42.1 | |
| ????TEC | ????0 | ????0 | ????19 | ????0 | |
| ????TEC/HLZ | ????12 | ????0 | ????7 | ????63.2 | |
| Enterococcus (33) | ????HLZ | ????5 | ????0 | ????28 | ????15.2 |
| ????CLZ | ????33 | ????0 | ????0 | ????100 | |
| ????CLA | ????2 | ????1 | ????30 | ????6.1 | |
| ????CLA/HLZ | ????10 | ????1 | ????22 | ????30.3 | |
| ????ROX | ????2 | ????0 | ????31 | ????6.1 | |
| ????ROX/HLZ | ????13 | ????0 | ????20 | ????39.4 | |
| ????VAN | ????31 | ????2 | ????0 | ????93.9 | |
| ????VAN/HLZ | ????13 | ????0 | ????20 | ????39.4 | |
| ????TEC | ????33 | ????0 | ????0 | ????100 | |
| ????TEC/HLZ | ????13 | ????0 | ????20 | ????39.4 | |
| Streptococcus pneumoniae (26) | ????HLZ | ????2 | ????0 | ????24 | ????7.7 |
| ????CLZ | ????26 | ????0 | ????0 | ????100 | |
| ????CLA | ????3 | ????1 | ????22 | ????11.5 | |
| ????CLA/HLZ | ????6 | ????0 | ????20 | ????23.1 | |
| ????ROX | ????4 | ????0 | ????22 | ????15.4 | |
| ????ROX/HLZ | ????6 | ????0 | ????20 | ????23.1 | |
| ????VAN | ????24 | ????2 | ????0 | ????92.3 | |
| ????VAN/HLZ | ????6 | ????0 | ????20 | ????23.1 | |
| ????TEC | ????26 | ????0 | ????0 | ????100 | |
| ????TEC/HLZ | ????6 | ????0 | ????20 | ????23.1 | |
| Serratieae (15) | ????HLZ | ????4 | ????0 | ????11 | ????26.7 |
| ????CLZ | ????3 | ????0 | ????12 | ????25.0 | |
| ????CLA | ????0 | ????0 | ????15 | ????0 | |
| ????CLA/HLZ | ????0 | ????0 | ????15 | ????0 | |
| ????ROX | ????0 | ????0 | ????15 | ????0 | |
| ????ROX/HLZ | ????0 | ????0 | ????15 | ????0 | |
| ????VAN | ????0 | ????0 | ????15 | ????0 | |
| ????VAN/HLZ | ????0 | ????2 | ????13 | ????0 | |
| ????TEC | ????0 | ????0 | ????15 | ????0 | |
| ????TEC/HLZ | ????0 | ????4 | ????11 | ????0 | |
| Citrobacter (30) | ????HLZ | ????0 | ????0 | ????30 | ????0 |
| ????CLZ | ????11 | ????0 | ????19 | ????0 | |
| ????CLA | ????0 | ????0 | ????30 | ????36.7 | |
| ????CLA/HLZ | ????0 | ????0 | ????30 | ????0 | |
| ????ROX | ????0 | ????0 | ????30 | ????0 | |
| ????ROX/HLZ | ????1 | ????0 | ????29 | ????0 | |
| ????VAN | ????0 | ????0 | ????30 | ????3.4 | |
| ????VAN/HLZ | ????0 | ????5 | ????25 | ????0 | |
| ????TEC | ????0 | ????0 | ????30 | ????0 | |
| ????TEC/HLZ | ????2 | ????5 | ????25 | ????6.7 |
*HLZ, CLZ, CLZ, ROX, VAN, TEC represent human lysozyme, lysozyme of chicken, clarithromycin, Roxithromycin, vancomycin and De Ke mycin respectively.
Claims (14)
1, a kind of with human lysozyme as the cause inflammation application of medicine of preparation treatment cause pathogeny imcrobe infection.It is characterized in that the present invention utilizes by purity 99.8% or more, active 70, the gene recombinant human lysozyme of 000u/mg is treated antibacterial, fungus and viral infection cause upper respiratory tract infection, colpitis, scytitis and oral inflammation medicine on the skin mucosa surface application as preparation; Cause that with gene recombinant human lysozyme preparation treatment bacterial infection inflammation drug oral tablet in the body, atomizing suck the application of spray and external inflammation medicine drop, unguentum and suppository with sterilization and antiinflammatory performance; And the drug combination that causes the inside and outside medicine with 1: 1 compositions of gene recombinant human lysozyme and antibiotic as preparation treatment cause pathogeny imcrobe infection.
2, according to the described gene recombinant human lysozyme of claim 1 as the cause inflammation application of medicine of preparation treatment cause pathogeny imcrobe infection, it is characterized in that the antibacterial of indication is Gram-positive, negative bacteria, anaerobe, drug tolerant bacteria and L type antibacterial in the pathogenic microorganism; Gene recombinant human lysozyme to the responsive effectively Mlc of antibacterial between 50 μ g-1.0mg/ml.
3, according to the described gene recombinant human lysozyme of claim 1 as the cause inflammation application of medicine of preparation treatment cause pathogeny imcrobe infection, it is characterized in that the virus of indication is DNA viruses and RNA viruses in the pathogenic microorganism.
4, according to the described gene recombinant human lysozyme of claim 1 as the cause inflammation application of medicine of preparation treatment cause pathogeny imcrobe infection, it is characterized in that upper respiratory tract infection is meant by drug-resistant staphylococcus aureus infects tracheobronchitis, pneumonia and the pharyngitis that causes.
5, according to the described gene recombinant human lysozyme of claim 1 as the cause inflammation application of medicine of preparation treatment cause pathogeny imcrobe infection, it is characterized in that scytitis is meant eczema, allergic dermatitis, comedo, rubella, the urticaria that is caused by Gram-positive, negative bacterial infections.
6, according to the described gene recombinant human lysozyme of claim 1 as the cause inflammation application of medicine of preparation treatment cause pathogeny imcrobe infection, it is characterized in that oral inflammation is meant by the anaerobic coccus infects gingivitis or the oral ulcer that causes.
7, according to the described gene recombinant human lysozyme of claim 1 as the cause inflammation application of medicine of preparation treatment cause pathogeny imcrobe infection, the medicine that it is characterized in that treating upper respiratory tract infection, colpitis and scytitis is the injection that is mixed with by 1% gene recombinant human lysozyme lyophilized powder and 5mM phosphate PH5 buffer, intravenous injection dosage 30, the 000u/ml/20g Mus is heavy, every day three times, effective cure rate 100% of upper respiratory tract and colpitis, effective cure rate 80-100% of scytitis; The curative effect of staphylococcus, streptococcus pneumoniae infection inflammation being shown in body internal respiration road with gene recombinant human lysozyme is better than 4 times of clarithromycins and 9 times.
8, according to the described gene recombinant human lysozyme of claim 1 as the cause inflammation application of medicine of preparation treatment cause pathogeny imcrobe infection, the medicine that it is characterized in that treating oral inflammation is the spray that is mixed with by 0.1% gene recombinant human lysozyme lyophilized powder, 20mM phosphate PH7 buffer, 25% glycerol and 5/0,000 Tween 80s, dosage 1500 μ l/20g Mus are heavy, every day six times, effectively cure rate 100%.
9, according to the described gene recombinant human lysozyme of claim 1 as the cause inflammation application of medicine of preparation treatment cause pathogeny imcrobe infection, the oral tablet that it is characterized in that the body innerlich anwenden is the tablet that is mixed with by 10% gene recombinant human lysozyme lyophilized powder, 50% medical starch, 25% medicinal dextrin, 10mM phosphate PH6 buffer and 5% sucrose.
10, according to the described gene recombinant human lysozyme of claim 1 as the cause inflammation application of medicine of preparation treatment cause pathogeny imcrobe infection, the concentration that it is characterized in that the preparation of body innerlich anwenden atomizing suction gene recombinant human lysozyme is 0.025-0.4mg/ml, inhalation 2-5 days.
11, according to the described gene recombinant human lysozyme of claim 1 as the cause inflammation application of medicine of preparation treatment cause pathogeny imcrobe infection, it is characterized in that external medication drop is formulated by 0.005% gene recombinant human lysozyme lyophilized powder, 10mM phosphate PH6 buffer, 20% glycerol, 6%2-pyrrolidone-5-carboxylic acid sodium, 0.9% water solublity azone and 3/0,000 Tween 80s.
12, according to the described gene recombinant human lysozyme of claim 1 as the cause inflammation application of medicine of preparation treatment cause pathogeny imcrobe infection, it is characterized in that the agent of external use ointment is formulated by 0.1% gene recombinant human lysozyme lyophilized powder, 20mM phosphate PH7 buffer, 20% glycerol, 1% carbomer and 0.3% essence.
13, according to the described gene recombinant human lysozyme of claim 1 as the application of medicine that causes inflammation of preparation treatment cause pathogeny imcrobe infection, but it is characterized in that the antibiotic in gene recombinant human lysozyme and the antibiotic drug combination compositions is clarithromycin, Roxithromycin, vancomycin or moral mycin; With gene recombinant human lysozyme and 1: 1 compositions drug combination of clarithromycin optium concentration is (1.5+0.1) mg/ml, and body internal respiration road is respectively strengthened 19.6 and 18.7 times than single with clarithromycin by the curative effect of staphylococcus aureus, watch chain coccus infection inflammation.
14, according to the described gene recombinant human lysozyme of claim 1 as the cause inflammation application of medicine of preparation treatment cause pathogeny imcrobe infection, it is characterized in that gene recombinant human lysozyme to external individually dosed treatment Gram-positive, negative bacterial infections causes inflammation that antibacterial action is all arranged; 1: 1 drug combination of gene recombinant human lysozyme and clarithromycin or Roxithromycin, the antibacterial activity potentiation 2-16 that can make clarithromycin, Roxithromycin doubly, responsive rate is significantly increased; But can reduce their MIC to staphylococcus aureus, table Portugal coccus with vancomycin, the coupling of moral mycin
50And MIC
90Value 2-4 doubly.
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