CN103357011A - Pharmaceutical composition and composite burn cream for treating burns and scalds and preparation method thereof - Google Patents
Pharmaceutical composition and composite burn cream for treating burns and scalds and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a pharmaceutical composition and composite burn cream for treating burns and scalds and a preparation method thereof. The pharmaceutical composition or the composite burn cream comprises ultra-pure yolk oil and immunoglobulin of yolk with effective treatment dosages, as well as optional pharmaceutically acceptable auxiliary materials, wherein the antigen of the immunoglobulin of yolk is the mycoprotein of one or at least two of staphylococcus aureus, pseudomonas aeruginosa, escherichia coli and hemolytic streptococcus. The composite burn cream can be used for effectively promoting skin regeneration, inhibiting one or at least two of staphylococcus aureus, pseudomonas aeruginosa, escherichia coli and hemolytic streptococcus, and preventing occurrence of infection.
Description
Technical field
The present invention relates to treat the drug world of empyrosis, be specifically related to a kind of pharmaceutical composition for the treatment of empyrosis, burn cream and preparation method thereof.
Background technology
Empyrosis is people's incident a kind of universality unexpected injuries in life, under comparatively serious empyrosis situation, scalds such as third degree burn, and skin even muscle all can be subject to heavy damage.In the process for the treatment of, to impel on the one hand the regeneration of new skin as far as possible, also to prevent on the other hand the infection of pathogenic microorganism, the generation that alleviates even stop inflammation.Cause easily infection, typical pathogenic microorganism comprises: staphylococcus aureus (Staphyloccocusaureus), bacillus pyocyaneus (Pseudomonas aeruginosa), escherichia coli (Escherichia coli), Hemolytic streptococcus (Hemolytic streptococcus) etc.
Burn and scald ointment is main mainly with the Chinese medicine product on the domestic market, and the doctor also can exhort and be aided with the antibiotics product usually in clinical treatment.On the one hand, existing Chinese medicine product great majority there is no especially significantly effect aspect the short granulation promoting, and are mainly reflected in anti-inflammatory analgetic.On the other hand, antibiotic abuse has more and more produced drug resistance so that cause inflammation bacterioid (such as staphylococcus aureus, bacillus pyocyaneus, escherichia coli, Hemolytic streptococcus etc.) for a long time, this also so that fastbacteria a thorny difficult problem in the empyrosis therapeutic process appears becoming in succession.
Egg oil claims again egg-yolk lipids, it is the yolk extract that the bright ovum separation and Extraction from egg-laying bird (mainly being laying hen) obtains, contain abundant lecithin and unsaturated fatty acid, be rich in simultaneously the multiple medicinal and nutritional labelings such as folic acid, carotene, calcium, phosphorus, ferrum, be substantially free of the range protein that comprises antibody.Egg oil has clearing heat, releasing toxin, removing swelling, nourishing YIN and benefiting blood is moisturized, expelling pus and promoting granulation is put on flesh effect.The method that the external egg oil is cured the disease is burnt skin ulcer side first appeared in controlling the Northern Zhou Dynasty " collection proved recipe " to scald; The therapy of the diseases such as external or egg oil for oral administration treatment skin ulcer, fistula, dysentery, on the books repeatly in the successive dynasties monographs such as " Japan hanako materia medica ", Compendium of Material Medica, " the medical center bun is wanted ", " Bencao Pinhui Jingyao ".
Egg oil promotes the good medicine among the people of union of wounded skin to be widely used as clinical adjuvant drug in many hospitals as having.In life, thereby widely used with the yellow method of obtaining egg oil of pan dried fried egg in without oily situation.Through modern medicine clinical experiment checking, egg oil can also effectively be alleviated gastroenteritis disease except having the function that promotes union of wounded skin, and also quite effective aspect other treatment of inflammatory diseases such as face.In Germany, egg oil also is beneficial to skin new life, increases the skin defying age as the main effective ingredient in the cosmetics, and can also increase skin elasticity.
Yolk immunoglobulin (Immunoglobulin of egg yolk; be called for short IgY) be the immunoglobulin that is present in the yolk of egg-laying bird (mainly being laying hen); claim again chicken yolk antibody; being that immunoglobulin in the hen blood is passed to Ovum Gallus domesticus Flavus and hatches the immunoglobulin that after the process neutralization is hatched chicken is shielded in chicken, is immunoglobulin unique in the yolk.The IgY molecular weight of normal chicken is about 180KD, is comprised of two heavy chains and two light chains.Research finds that the IgY hydrophobic group more than IgG, has good stability and anti-hypertonicity, and is heat-resisting, acidproof, alkaline-resisting better.Research shows that also IgY can not activate mammiferous complement system, not with serum in the rheumatoid factor (RF) that exists react, do not react with a-protein and G, do not affect mammal IgG and play a role, and do not react with mammiferous Fc receptor.
The production of Yolk immunoglobulin and application are defined as the Yolk immunoglobulin technology.Nowadays, the Yolk immunoglobulin technology has become an international standardized technique.This technology has satisfied the zoopery 3R principle that proposes in the human experimentation engineering philosophy well, namely in zoopery, adopt and reduce (Reduction), substitute (Replacement) and optimize (Refinement) method, to safeguard Laboratory Animal Welfare.Based on the advantage of this technology at aspects such as animal protections, the European trials method is replaced center recommendation Yolk immunoglobulin (IgY) and is substituted mammalian immune globulin (IgG), to reduce owing to the antibody collection causes injury to animal.
Because Yolk immunoglobulin has stable chemical nature, output is high, cost is low, satisfy the animal welfare needs, and the advantage with animal phylogenetics distance, therefore is more suitable for the production specific antibody.Its passive immunity function can be used for research opposing virus and bacterial disease, has the potential of development functionality food and new drug.Simultaneously, U.S. FDA is listed it in " general generally recognized as safe material category ", and this is indicating that also Yolk immunoglobulin will welcome a new development opportunity at the human product scope.
Defective in view of present burn and scald ointment, and consider that above-mentioned egg oil is in the function that promotes aspect the skin regeneration, the function of Yolk immunoglobulin aspect opposing virus and bacterial disease, research and development had not only had the medicine that promotes skin regeneration but also can resist bacterial disease targetedly, with the treatment empyrosis, significant.
Summary of the invention
The invention provides a kind of pharmaceutical composition for the treatment of empyrosis, burn cream and preparation method thereof, not only to promote skin regeneration but also can resist bacterial disease in the treatment that is implemented in empyrosis.
For realizing purpose of the present invention, the invention provides a kind of pharmaceutical composition for the treatment of empyrosis, described pharmaceutical composition comprises obtaining high-purity egg oil and the Yolk immunoglobulin of dose therapeutically effective, and optional pharmaceutically acceptable adjuvant, the antigen of described Yolk immunoglobulin is staphylococcus aureus (Staphyloccocusaureus), bacillus pyocyaneus (Pseudomonas aeruginosa), the tropina of a kind of or at least two kinds of thalline in escherichia coli (Escherichia coli) and the Hemolytic streptococcus (Hemolytic streptococcus).
Described obtaining high-purity egg oil promotes skin regeneration in the treatment of empyrosis, described Yolk immunoglobulin can suppress a kind of or at least two kinds of antibacterials in staphylococcus aureus, bacillus pyocyaneus, escherichia coli and the Hemolytic streptococcus, the generation of protecting from infection.
Preferably, in the pharmaceutical composition of the present invention, the antigen of described Yolk immunoglobulin is the tropina of staphylococcus aureus, bacillus pyocyaneus, escherichia coli and 4 kinds of thalline of Hemolytic streptococcus.
Preferably, in the pharmaceutical composition of the present invention, take the quality of described pharmaceutical composition as 100%, the mass percent of described obtaining high-purity egg oil is 52～70%; Take the volume of described pharmaceutical composition as 100%, the content of described Yolk immunoglobulin is 0.2～5mg/ml.
Preferably, in the pharmaceutical composition of the present invention, described pharmaceutically acceptable adjuvant is lanoline and Borneolum Syntheticum, and take the quality of described pharmaceutical composition as 100%, the mass percent of described lanoline is 26～46%, and the mass percent of described Borneolum Syntheticum is 1～5%.
The present invention also provides a kind of burn cream, and it is the described pharmaceutical composition of above-mentioned arbitrary scheme.
The present invention provides again the preparation method of the described pharmaceutical composition of a kind of above-mentioned arbitrary scheme, and described method comprises:
(1) prepares described obtaining high-purity egg oil;
(2) prepare described Yolk immunoglobulin;
(3) with described obtaining high-purity egg oil and the Yolk immunoglobulin of dose therapeutically effective, and optional pharmaceutically acceptable adjuvant is mixed to get described pharmaceutical composition.
Particularly, described step (2) comprising:
(a) cultivate respectively described staphylococcus aureus, bacillus pyocyaneus, escherichia coli and Hemolytic streptococcus, and centrifugal, fragmentation obtains corresponding tropina antigen;
(b) the tropina antigen with described staphylococcus aureus, bacillus pyocyaneus, escherichia coli and Hemolytic streptococcus is mixed to get hybrid antigen according to concentration ratio 1-10:1-10:1-10:1-10;
(c) utilize described hybrid antigen to adopt for five steps contained the multipath immunization method immunity egg-laying bird of adjuvant, get the immune egg that immune birds produces;
(d) from described immune egg, extract described Yolk immunoglobulin.
Further, described step (b) specifically comprises:
The tropina antigen of described staphylococcus aureus, bacillus pyocyaneus, escherichia coli and Hemolytic streptococcus is mixed to get hybrid antigen according to concentration ratio 1:1:1:1, and the final concentration of described hybrid antigen is 50～500 μ g/ml.In the described step (b), the concentration ratio of the tropina antigen of staphylococcus aureus, bacillus pyocyaneus, escherichia coli and Hemolytic streptococcus is not limited to 1:1:1:1, such as being 1:5:10:1 or 2:10:5:10 etc.
Further, immune egg-laying bird specifically comprises in the described step (c):
(c1) the 1st day: with chest muscle 4 points of a laying hen of injection behind the described hybrid antigen of 0.5ml and the abundant mixing and emulsifying of Freund's complete adjuvant;
(c2) the 15th day: the described hybrid antigen of 1ml and incomplete Freunds adjuvant are injected chest muscle 4 points of described laying hen;
(c3) the 31st day: with chest muscle 4 points of the described laying hen of injection behind the described hybrid antigen of 1.5ml and the abundant mixing and emulsifying of incomplete Freunds adjuvant;
(c4) the 60th day: with the ear vein of the described laying hen of injection behind the described hybrid antigen of 1.5ml and the abundant mixing and emulsifying of incomplete Freunds adjuvant;
(c5) the 100th day: with the ear vein of the described laying hen of injection behind the described hybrid antigen of 1.5ml and the abundant mixing and emulsifying of incomplete Freunds adjuvant.
Further, described step (d) specifically comprises:
(d1) described immune egg is broken egg and process, separate obtaining egg yolk liquid, add the distilled water of 9 times of volumes, place 6h in 4 ℃, then under 4 ℃, 10000g, centrifugal 25min gets supernatant;
(d2) 50% saturated ammonium sulfate solution is slowly joined in the supernatant that step (d1) obtains, the volume ratio that makes described ammonium sulfate and described supernatant is 3:1, ambient temperature overnight after mixing;
(d3) mixed liquor that step (d2) is obtained under 4 ℃, 10000g, centrifugal 25min abandons supernatant, keeps precipitation;
(d4) then the precipitation dissolved in distilled water that step (d3) is obtained filters by the 100KD ultrafilter membrane, obtains Yolk immunoglobulin liquid, then prepares the Yolk immunoglobulin sterling by lyophilization;
(d5) detect the purity of described Yolk immunoglobulin sterling by SDS-PAGE method and ELISA method respectively and tire, the purity of described Yolk immunoglobulin sterling 〉=90% is to the antibody titers of four kinds of pathogen 〉=12800.
Term of the present invention " dose therapeutically effective " refers to that the corresponding component of this term is at the dosage for the treatment of, alleviate and/or alleviate disease or the disease of empyrosis of the present invention.
Term of the present invention " pharmaceutical composition " refers to can be used for treating, alleviating and/or alleviate the material of disease or the disease of empyrosis of the present invention.
Term of the present invention " burn cream " refers to can be used for treating, alleviating and/or alleviate the ointment of disease or the disease of empyrosis of the present invention, and it is interpreted as is a kind of in " pharmaceutical composition ".
Term of the present invention " optional " refers to contain or does not contain the corresponding component of this term.
Obtaining high-purity egg oil of the present invention promotes skin regeneration in the treatment of empyrosis, described Yolk immunoglobulin can suppress a kind of or at least two kinds of antibacterials in staphylococcus aureus, bacillus pyocyaneus, escherichia coli and the Hemolytic streptococcus, the generation of protecting from infection has reached in the treatment of empyrosis and not only to have promoted skin regeneration but also the purpose that can resist bacterial disease.
Description of drawings
Fig. 1 is implementation flow chart of the present invention.
The specific embodiment
The invention discloses a kind of pharmaceutical composition for the treatment of empyrosis and preparation method thereof and burn cream.Those skilled in the art can use for reference this paper content, suitably improve technological parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, the related personnel obviously can change or suitably change and combination methods and applications as herein described within not breaking away from content of the present invention, spirit and scope, realizes and use the technology of the present invention.The below is not marked concrete experimental procedure, method and condition in the example, all is well known to those skilled in the art, carries out according to conventional steps, method and condition.
Embodiment 1: the preparation obtaining high-purity egg oil
Select Fresh Egg, isolate egg yolk stock solution after the broken shell; Dried under 45～65 ℃ of conditions of temperature; Be ground into the yolk powder of 15～45 order granularities; Yolk powder is dropped in the extraction kettle, pass into the extractant carbon dioxide, the flow velocity of carbon dioxide is 15～25kg/h, and extracting pressure is 30～50MPa, and extraction temperature is 35 ℃, and extraction time is 4～10h; Mixed material after the extraction passes into the decompression separation device, and its bottom is that the sedimentation sub-argument goes out obtaining high-purity egg oil.
At present, the preparation technology of obtaining high-purity egg oil is comparative maturity, the method of a lot of similar above-described embodiments can both realize that the present invention prepares the purpose of obtaining high-purity egg oil, the present invention is not particularly limited the method for preparing obtaining high-purity egg oil, and those skilled in the art can select suitable method according to prior art.
Embodiment 2: the immunity preparation of tropina antigen
(1) cultivation of thalline:
Adopt broth medium solid plate line activation staphylococcus aureus (ATCC26031), the single colony inoculation after the picking activation is in broth medium, 37 ℃ of shaken cultivation 6～12 hours; Adopt Sabouraud culture medium solid plate line activation bacillus pyocyaneus (ATCC27853), the single colony inoculation after the picking activation is in the Sabouraud culture medium, 37 ℃ of shaken cultivation 6～12 hours; Adopt serum broth solid plate line activation escherichia coli (ATCC25922), the single colony inoculation after the picking activation is in serum broth, 37 ℃ of shaken cultivation 6～12 hours; Adopt hexadecane trimethyl amine bromide culture medium solid plate line activation beta hemolytic streptococcus (CMCC32210), the single colony inoculation after the picking activation is in hexadecane trimethyl amine bromide culture medium, 37 ℃ of shaken cultivation 6～12 hours.(illustrate: strain of the present invention derives from the U.S. typical case ATCC of DSMZ and Chinese medicine microorganism fungus kind preservation management center C MCC, the preserving number of described strain marks out, and those skilled in the art can ask for or buy by legitimate channels).
(2) separation of tropina antigen:
The thalline of the above-mentioned cultivation of centrifugal collection,, washing thalline resuspended with the PBS solution of pH7.2, then centrifugal and resuspended with the PBS solution of pH7.2 after, adopt the mode fragmentation thalline of multigelation or ultrasonication, obtain the tropina antigen of each bacterial strain.
Embodiment 3: Immune Laying Hens
(1) preparation hybrid antigen:
The tropina antigen of above-mentioned staphylococcus aureus, bacillus pyocyaneus, escherichia coli and Hemolytic streptococcus is mixed to get hybrid antigen according to concentration ratio 1:1:1:1, and the final concentration of described hybrid antigen is 50～500 μ g/ml, and-80 ℃ save backup.
Need to prove: the present invention can use in staphylococcus aureus, bacillus pyocyaneus, escherichia coli and the Hemolytic streptococcus combination of any one or at least two kinds as the antigen immune egg-laying bird.But in order to obtain better fungistatic effect, preferably adopt the mixture of above-mentioned four kinds of antibacterials as hybrid antigen, immune egg-laying bird.The mixed proportion of above-mentioned four kinds of antibacterials can rule of thumb be determined, but the present invention preferably mixes according to concentration ratio 1:1:1:1, and in fact, concentration ratio 1-10:1-10:1-10:1-10 mixing all is fine; The final concentration of described hybrid antigen is that 50～500 μ g/ml are better, and further, the final concentration of described hybrid antigen is that 200 μ g/ml are better.
(2) injecting immune:
Polytype egg-laying bird (such as chicken, duck or goose etc.) can both be fit to needs of the present invention, but the present invention preferably adopts laying hen as immune object, especially selects SPF(Specific Pathogen Free, specific pathogen free) the level laying hen.SPF level laying hen does not almost have cause pathogeny imcrobe infection, and the purity of the Yolk immunoglobulin that produces and specificity are high.The method of immunity egg-laying bird can have multiple, through my company's long-term experiment research, can obtain preferably immune effect according to following laying hen immune programme for children (table 1), the resulting Yolk immunoglobulin of this immune programme for children exceeds more than 30% than routine immunization method output, and average energy reaches the 90mg/ egg.And the method can continue to produce the high-titer Yolk immunoglobulin up to 260 days, compares 200 days stable tiring of the resulting Yolk immunoglobulin of common immunization method, and significant progress is arranged.
Table 1 laying hen immune programme for children
Embodiment 4: Yolk immunoglobulin extracts and purification
(1) give birth to immune egg through the laying hen behind the above-mentioned immune programme for children, collect immune egg and break egg and process, separate obtaining egg yolk liquid, add the distilled water of 9 times of volumes, in 4 ℃ of placement 6h, then under 4 ℃, 10000g, centrifugal 25min gets supernatant;
(2) 50% saturated ammonium sulfate solution is slowly joined in the supernatant that step (1) obtains, the volume ratio that makes described ammonium sulfate and described supernatant is 3:1, ambient temperature overnight after mixing;
(3) mixed liquor that step (2) is obtained under 4 ℃, 10000g, centrifugal 25min abandons supernatant, keeps precipitation;
(4) then the precipitation dissolved in distilled water that step (3) is obtained filters by the 100KD ultrafilter membrane, obtains Yolk immunoglobulin liquid, then prepares the Yolk immunoglobulin sterling by lyophilization.
Embodiment 5: Yolk immunoglobulin purity and bioactivity
Detect the purity of the Yolk immunoglobulin sterling of preparation by the SDS-PAGE method, determine its purity 〉=90%; Measure the tiring of Yolk immunoglobulin sterling of preparation by the ELISA method, determine its tire 〉=12800, satisfy the pharmaceutical composition of preparation treatment empyrosis and the needs of burn cream.
After obtaining obtaining high-purity egg oil and Yolk immunoglobulin by said method, obtaining high-purity egg oil and Yolk immunoglobulin can be mixed to get the pharmaceutical composition for the treatment of empyrosis according to proper ratio, described obtaining high-purity egg oil and Yolk immunoglobulin and pharmaceutically acceptable substrate can also be mixed making burn cream.Specifically be illustrated by following embodiment.
Embodiment 6: the preparation of burn cream
Get obtaining high-purity egg oil 52g, Yolk immunoglobulin 20mg adds lanoline 46g, and Borneolum Syntheticum 2g, mix homogeneously make about 100ml burn cream.
Embodiment 7: the preparation of burn cream
Get obtaining high-purity egg oil 70g, Yolk immunoglobulin 500mg adds lanoline 26g, and Borneolum Syntheticum 4g, mix homogeneously make about 100ml burn cream.
Embodiment 8: the preparation of burn cream
Get obtaining high-purity egg oil 60g, Yolk immunoglobulin 100mg adds lanoline 35g, and Borneolum Syntheticum 5g, mix homogeneously make about 100ml burn cream.
In above-described embodiment 6～8, the content of each component is some more suitable proportionings of the present invention, and those skilled in the art can rule of thumb adjust corresponding proportioning, and all do not break away from the adjustment of spirit of the present invention, fall into protection scope of the present invention without exception.Need to prove: the present invention's " burn cream " is a kind of of sensu lato " pharmaceutical composition ", so above-described embodiment 6～8 also can be described as the preparation method of " pharmaceutical composition ".
Embodiment 9: burn plaster effect research (In Vitro Bacteriostasis experiment)
(1) prepares culture medium: dispose respectively broth medium, Sabouraud culture medium, serum broth and hexadecane trimethyl amine bromide culture medium, behind high pressure steam sterilization, be cooled to about 50 ℃, after shaking up, under aseptic condition, be poured in the sterilized culture dish, the about 8～12ml culture medium of each culture dish naturally cools to and solidifies;
(2) be coated with bacterium: with cultured staphylococcus aureus (ATCC26031), bacillus pyocyaneus (ATCC27853), escherichia coli (ATCC25922) and hemolytic hammer (CMCC32210) are diluted to respectively 10 with sterilized water in advance
8The bacterial suspension of CFU/mL is got respectively the suspension of the above-mentioned four kinds of antibacterials of 100 μ L, is coated on respectively successively on above-mentioned four kinds of culture medium flat plates, about 37 ℃ of oven dry 20min;
(3) paster: the burn cream of (about 10 μ L) embodiment 6 preparations that take a morsel, be uniformly coated on diameter and be (the circle scraps of paper are high temperature sterilize in advance) on the round scraps of paper about 0.5cm, the one side that the above-mentioned round scraps of paper are contained burn cream is attached on the flat board of the above-mentioned culture medium that is coated with bacterium (with the round scraps of paper of sterilization as blank), pastes at a certain distance round scraps of paper;
(4) cultivate: place 37 ℃ incubator to cultivate at least 24h above-mentioned flat board;
(5) diameter of survey inhibition zone: take out flat board, the size of testing respectively antibacterial circle diameter.
The result shows: the burn cream of above-described embodiment 6 preparations all has antibacterial phenomenon to above-mentioned 4 kinds of antibacterials, and the diameter of its inhibition zone does not wait from 10～20mm, illustrates that the burn cream of Yolk immunoglobulin content 0.2mg/mL has stronger fungistatic effect.
Applicant's statement, the present invention illustrates detailed features of the present invention and method by above-described embodiment, but the present invention is not limited to above-mentioned detailed features and method, does not mean that namely the present invention must rely on above-mentioned detailed features and method could be implemented.The person of ordinary skill in the field should understand, any improvement in the present invention to the increase of the equivalence replacement of the selected condition of the present invention and subsidiary conditions, the selection of concrete mode etc., all drops within protection scope of the present invention and the open scope.
Claims (10)
1. pharmaceutical composition for the treatment of empyrosis, it is characterized in that, described pharmaceutical composition comprises obtaining high-purity egg oil and the Yolk immunoglobulin of dose therapeutically effective, and optional pharmaceutically acceptable adjuvant, the antigen of described Yolk immunoglobulin is the tropina of a kind of or at least two kinds of thalline in staphylococcus aureus (Staphyloccocus aureus), bacillus pyocyaneus (Pseudomonas aeruginosa), escherichia coli (Escherichia coli) and the Hemolytic streptococcus (Hemolytic streptococcus).
2. pharmaceutical composition according to claim 1 is characterized in that, the antigen of described Yolk immunoglobulin is the tropina of staphylococcus aureus, bacillus pyocyaneus, escherichia coli and 4 kinds of thalline of Hemolytic streptococcus.
3. pharmaceutical composition according to claim 1 and 2 is characterized in that, take the quality of described pharmaceutical composition as 100%, the mass percent of described obtaining high-purity egg oil is 52～70%; Take the volume of described pharmaceutical composition as 100%, the content of described Yolk immunoglobulin is 0.2～5mg/ml.
4. pharmaceutical composition according to claim 1 and 2, it is characterized in that described pharmaceutically acceptable adjuvant is lanoline and Borneolum Syntheticum, take the quality of described pharmaceutical composition as 100%, the mass percent of described lanoline is 26～46%, and the mass percent of described Borneolum Syntheticum is 1～5%.
5. a burn cream is characterized in that, described burn cream is each described pharmaceutical composition of claim 1 to 4.
6. the preparation method of each described pharmaceutical composition of claim 1 to 4 is characterized in that, described method comprises:
(1) prepares described obtaining high-purity egg oil;
(2) prepare described Yolk immunoglobulin;
(3) with described obtaining high-purity egg oil and the Yolk immunoglobulin of dose therapeutically effective, and optional pharmaceutically acceptable adjuvant is mixed to get described pharmaceutical composition.
7. preparation method according to claim 6 is characterized in that, described step (2) comprising:
(a) cultivate respectively described staphylococcus aureus, bacillus pyocyaneus, escherichia coli and Hemolytic streptococcus, and centrifugal, fragmentation obtains corresponding tropina antigen;
(b) the tropina antigen with described staphylococcus aureus, bacillus pyocyaneus, escherichia coli and Hemolytic streptococcus is mixed to get hybrid antigen according to concentration ratio 1-10:1-10:1-10:1-10;
(c) utilize described hybrid antigen to adopt for five steps contained the multipath immunization method immunity egg-laying bird of adjuvant, get the immune egg that immune birds produces;
(d) from described immune egg, extract described Yolk immunoglobulin.
8. preparation method according to claim 7, it is characterized in that, the tropina antigen of the described staphylococcus aureus of described step (b), bacillus pyocyaneus, escherichia coli and Hemolytic streptococcus is mixed to get hybrid antigen according to concentration ratio 1:1:1:1, and the final concentration of described hybrid antigen is 50～500 μ g/ml.
9. preparation method according to claim 7 is characterized in that, immune egg-laying bird specifically comprises in the described step (c):
(c1) the 1st day: with chest muscle 4 points of a laying hen of injection behind the described hybrid antigen of 0.5ml and the abundant mixing and emulsifying of Freund's complete adjuvant;
(c2) the 15th day: the described hybrid antigen of 1ml and incomplete Freunds adjuvant are injected chest muscle 4 points of described laying hen;
(c3) the 31st day: with chest muscle 4 points of the described laying hen of injection behind the described hybrid antigen of 1.5ml and the abundant mixing and emulsifying of incomplete Freunds adjuvant;
(c4) the 60th day: with the ear vein of the described laying hen of injection behind the described hybrid antigen of 1.5ml and the abundant mixing and emulsifying of incomplete Freunds adjuvant;
(c5) the 100th day: with the ear vein of the described laying hen of injection behind the described hybrid antigen of 1.5ml and the abundant mixing and emulsifying of incomplete Freunds adjuvant.
10. preparation method according to claim 7 is characterized in that, described step (d) specifically comprises:
(d1) described immune egg is broken egg and process, separate obtaining egg yolk liquid, add the distilled water of 9 times of volumes, place 6h in 4 ℃, then under 4 ℃, 10000g, centrifugal 25min gets supernatant;
(d2) 50% saturated ammonium sulfate solution is slowly joined in the supernatant that step (d1) obtains, the volume ratio that makes described ammonium sulfate and described supernatant is 3:1, ambient temperature overnight after mixing;
(d3) mixed liquor that step (d2) is obtained under 4 ℃, 10000g, centrifugal 25min abandons supernatant, keeps precipitation;
(d4) then the precipitation dissolved in distilled water that step (d3) is obtained filters by the 100KD ultrafilter membrane, obtains Yolk immunoglobulin liquid, then prepares the Yolk immunoglobulin sterling by lyophilization;
(d5) detect the purity of described Yolk immunoglobulin sterling by SDS-PAGE method and ELISA method respectively and tire, the purity of described Yolk immunoglobulin sterling 〉=90% is to the antibody titers of four kinds of pathogen 〉=12800.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105688210A (en) * | 2016-03-10 | 2016-06-22 | 陕西瑞凯生物科技有限公司 | Yolk biological protein skin repair dressing and preparation method thereof |
| CN105769918A (en) * | 2016-05-20 | 2016-07-20 | 四川师范大学 | Collagen peptide granulation-promoting film and preparation method thereof |
| CN108640988A (en) * | 2018-05-23 | 2018-10-12 | 成都安蒂康生物科技有限公司 | A kind of rapid extracting method of Yolk antibody, the anti-burn and scald infection product of preparation and its application |
| CN113288931A (en) * | 2021-05-25 | 2021-08-24 | 卢云龙 | Skin injury repairing preparation and preparation method and application thereof |
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| CN105688210A (en) * | 2016-03-10 | 2016-06-22 | 陕西瑞凯生物科技有限公司 | Yolk biological protein skin repair dressing and preparation method thereof |
| CN105769918A (en) * | 2016-05-20 | 2016-07-20 | 四川师范大学 | Collagen peptide granulation-promoting film and preparation method thereof |
| CN108640988A (en) * | 2018-05-23 | 2018-10-12 | 成都安蒂康生物科技有限公司 | A kind of rapid extracting method of Yolk antibody, the anti-burn and scald infection product of preparation and its application |
| CN113288931A (en) * | 2021-05-25 | 2021-08-24 | 卢云龙 | Skin injury repairing preparation and preparation method and application thereof |
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