CN104844709A - Preparation for specific aspergillus fumigatus infection resisting chicken egg yolk antibody and application - Google Patents
Preparation for specific aspergillus fumigatus infection resisting chicken egg yolk antibody and application Download PDFInfo
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- CN104844709A CN104844709A CN201510275294.3A CN201510275294A CN104844709A CN 104844709 A CN104844709 A CN 104844709A CN 201510275294 A CN201510275294 A CN 201510275294A CN 104844709 A CN104844709 A CN 104844709A
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- aspergillus fumigatus
- yolk antibody
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- egg yolk
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Abstract
The invention relates to the technical field of immunity, in particular to a specific aspergillus fumigatus infection resisting chicken egg yolk antibody and application thereof. The specific aspergillus fumigatus infection resisting chicken egg yolk antibody is prepared by the following steps: 1, preparing an aspergillus fumigatus antigen; 2, performing immune injection on laying hens; 3, preparing specific chicken egg yolk antibody freeze-dried powder by utilizing an ammonium sulfate salting-out method. The valence of the prepared specific aspergillus fumigatus infection resisting chicken egg yolk antibody is higher than 1:10,000; the prepared specific aspergillus fumigatus infection resisting chicken egg yolk antibody is a biological agent, the side effects generated to the human body are small, the irritation to human body conjunctivas, corneas, mucosa, skin and the like is small, no adverse reaction exists when the antibody is externally used or orally taken, and the physicochemical property is stable; the antibody can be saved for a long time and is particularly suitable for being locally applied to treating aspergillus fumigatus infection of ocular surface, oral cavity, skin and the like.
Description
Technical field
The present invention relates to immunological technique field, the Synthesis and applications of the chicken yolk antibody of a particularly species specificity anti-aspergillus fumigatus infection.
Background technology
In recent years along with the widespread use of antimicrobial drug, immunosuppressor, chemotherapeutics and radiotherapy, and the utilization of invasive diagnosis and treatment technology, opportunistic fungi infection rate obviously raises.Eurotium is a kind of important opportunistic fungi, and Aspergillus fumigatus is modal Eurotium.The Common drugs of China's treatment fungal disease comprises natamycin, amphotericin, Novel triazole medicine etc.According to WHO Report, in pulmonary infection worldwide and corneal infection, fungi infestation to be infectious keratopathy be one of main causes of blindness, the aspergillin infection of lung is one of Main Pathogenic Bacteria of fungoid pulmonary infection.
Common antifungal drug exist in varying degrees water-soluble not good, be that toxic side effect is large, some patients also there will be the shortcomings such as anaphylaxis entirely.Many patients stop treatment owing to tolerating, curative effect is often not good, and conditions of patients often repeatedly, treats fungal disease at present, not a kind of medicine of control fungi infestation that can be permanently effective.
Summary of the invention
The present invention is directed to deficiency of the prior art, the Synthesis and applications of the chicken yolk antibody providing a species specificity anti-aspergillus fumigatus to infect.
The said aspergillus fumigatus of the present invention, this bacterial strain is deposited in China General Microbiological culture presevation administrative center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and preservation name is called: aspergillus fumigatus, preservation date: 2011. 10.05, deposit number is: NO.3.0772.
Technical scheme of the present invention is:
The chicken yolk antibody that one species specificity anti-aspergillus fumigatus infects, described chicken yolk antibody preparation process comprises the following steps:
Step 1: preparation deactivation Aspergillus fumigatus mycelia suspension: inoculation aspergillus fumigatus is cultivated 5 days in Sharpe liquid nutrient medium, and take out Aspergillus fumigatus hypha body and grind in mill, filter spore, mycelial concentration is adjusted to 2 × 10 the most at last
9cFU/ml;
Step 2: animal immune: by mycelial concentration 2 × 10
9after the bacteria suspension of CFU/mL is fully emulsified after mixing by 1:1 with Freund's complete adjuvant, every chicken only injects 1 mL/, is changed to Freund's incomplete adjuvant booster immunization 3 times after 3 weeks, 5 weeks, 8 weeks;
Step 3: adopt ammonium sulfate salting-out process to prepare the chicken yolk antibody albumen of specificity anti-aspergillus fumigatus infection: beating eggs removes egg white and membrane of yolk, egg yolk liquid and ultrapure water 1:8 is mixed, adjust pH to 5.1, hold over night; Centrifuging and taking supernatant liquor; Add according to the solubleness of ammonium sulfate and liquid volume the amount can separating out at most IgY albumen afterwards, leave standstill 2h; The centrifugal Supernatant protein that removes is separated out again, adds phosphate buffered saline buffer soluble protein, again adds ammonium sulfate and leave standstill 2h; The centrifugal supernatant liquor that removes stays albumen, again adds minimum solvable protelytic phosphate buffered liquid measure redissolution albumen, loads dialysis tubing dialysis 48h; Put into Freeze Drying Equipment after the complete liquid of dialysis is frozen into solid and be made into lyophilized powder.4 DEG C of preservations, still can keep higher immunocompetence after 6 months, have characteristic with long preservation period.
On the basis of above scheme, the method for animal immune described in step 2 injection is that chicken used is Lay henry laying hen in the neck of chicken, the subcutaneous loose injection of dipteron and chest muscle many places.
Above-mentioned chicken yolk antibody is applied in the medicine of preparation control fungal keratitis.
Different pharmaceutical dosage forms can be made into, as pulvis, tablet, ointment, mouth spray, collyrium, spongaion etc.
The invention has the beneficial effects as follows:
1) manufacture craft is uncomplicated, cost is not high, be easy to preservation, can produce in a large number;
2) the chicken yolk antibody stable in physicochemical property of specificity anti-aspergillus fumigatus infection of the present invention, shows higher thermostability and pH stability, all can keep most immune affinities lower than 60 DEG C and pH3-9.
3) chicken yolk antibody good water solubility compared with at present conventional antifungal drug of infecting of specificity anti-aspergillus fumigatus, be easy to absorb, systemic side effects be little, anaphylaxis is few, pungency is little, pH close to neutral, be a kind of natural biological bacteriostatic agent.
Accompanying drawing explanation
Accompanying drawing 1 is the fungistatic effect of the chicken yolk antibody group that the specificity anti-aspergillus fumigatus of the specific embodiment of the invention infects.
Embodiment
The specific embodiment of the present invention is as follows:
Embodiment 1
The preparation of the chicken yolk antibody that specificity anti-aspergillus fumigatus infects:
(1) prepare deactivation Aspergillus fumigatus mycelia suspension: inoculation aspergillus fumigatus is cultivated 5 days in Sharpe liquid nutrient medium, take out Aspergillus fumigatus hypha body and grind in mill, filter spore, mycelial concentration is adjusted to 2 × 10 the most at last
9cFU/ml;
(2) by mycelial concentration 2 × 10
9after the bacteria suspension of CFU/mL is fully emulsified after mixing by 1:1 with Freund's complete adjuvant, every chicken injection (1 mL/ only), is changed to freund 's incomplete adjuvant booster immunization 3 times after 3 weeks, 5 weeks, 8 weeks.
(3) adopt ammonium sulfate salting-out process to prepare the chicken yolk antibody albumen of specificity anti-aspergillus fumigatus infection, beating eggs removes egg white and membrane of yolk, egg yolk liquid and ultrapure water 1:8 is mixed, and adjusts pH to 5.1, hold over night; Centrifuging and taking supernatant liquor; After add 50 % saturation ratios ammonium sulfate can separate out at most the amount of IgY albumen, leave standstill 2h; The centrifugal Supernatant protein that removes is separated out again, adds phosphate buffered saline buffer soluble protein, again adds ammonium sulfate and leave standstill 2h; The centrifugal supernatant liquor that removes stays albumen, again adds, and pBS, 0.01mol/ L, pH 7.4 loads dialysis tubing dialysis 48h; The maximum protein concentration of separating out aspergillus fumigatus Igy is 20mg/ml, and most high-titer is 1:20000, puts into Freeze Drying Equipment and be made into lyophilized powder after the complete liquid of dialysis is frozen into solid.
(4) be dissolved in physiological saline by lyophilized powder, being adjusted to concentration is 10mg/ml, and indirect Elisa method measures weekly it and tires, for subsequent use as effective antibacterials when reaching 1:10000 when tiring.
Embodiment 2
The chicken yolk antibody antibacterial experiment in vitro that specificity anti-aspergillus fumigatus infects:
Aspergillus fumigatus, in the medium after shaking culture 96 h, adopts blood cell counting to calculate the concentration of Aspergillus fumigatus mycelia in nutrient solution, the total amount being scaled light absorption value with this with ultraviolet spectrophotometer and then calculating sample mycelia.Rear 1 × 10 will be ground
9cFU/ml mycelia Sharpe liquid nutrient medium carries out gradient dilution, measures each dilution light absorption value in 600 nm places, draws the typical curve between mycelial concentration and light absorption value.By 1 × 10
5cFU/ml bacteria suspension and tiring of obtaining in embodiment 1 are configured to the chicken yolk antibody of different concentration higher than the antibody of 1:10000, survey light absorption value and count after mixed culture 24h, 48h, 72h.As shown in Figure 1, the fungistatic effect of the chicken yolk antibody group that specificity anti-aspergillus fumigatus infects is apparently higher than blank group and negative chicken yolk antibody group, and within the specific limits, the higher bacteriostatic action of concentration is stronger.
Embodiment 3
The test of the chicken yolk antibody physico-chemical property that specificity anti-aspergillus fumigatus infects:
By IgY solution dilution to certain concentration (protein concentration is 0.02mg/mL), hatch 30 min respectively in 40,50,60,70,80,90 DEG C of water-baths after, indirect ELISA measures antibody activity; Be critical temperature place of IgY solution in its loss of activity of 0.02 mg/mL simultaneously by protein concentration, hatch 0.5,1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5 h respectively, indirect ELISA measures the activity of antibody.Use respectively when investigating the ph stability of antibody pH l, 2,3,4,5,6,7,8,9, lO, 11,12,13, IgY solution dilution to protein concentration is 0.02mg/ml by the PBS solution of 14, after placing 1h at 4 DEG C, indirect ELISA measures antibody activity.
Test result shows the antibody titer that obtained by embodiment 1 higher than the specific IgY of 1:10000, below 60 DEG C, have good thermostability, and at 60 DEG C heat treated 4 more than h, immunocompetence only have dropped less than about 1/3,60 DEG C does not have considerable change; At pH 4 ~ 6, there is the highest activity, can more than 70% be remained in the immunocompetence of pH 3 ~ 9; Along with further reduction or the rising of pH value, its active reduction rapidly.As can be seen here, the antibody titer obtained by embodiment 1 is higher than the specific IgY of 1:10000, there is good thermostability and ph stability, stable in physicochemical property, as various kinds of drug, as oral medicine, external-applied ointment, collyrium etc. can resist the decomposition of human body temperature and acid and alkali substance, keep the bacteriostatic action that its activation plays is lasting preferably.
Embodiment 4
The test of room temperature preservation and 4 DEG C of stability of preserving:
By the IgY obtained in room temperature and 4 DEG C of embodiments 1 of preserving, at being kept at room temperature and 4 DEG C respectively, indirect Elisa method measures 1,2,3,4,5,6,7,8,9 month tiring.Result shows, and in room temperature preservation within 2 months, the IgY obtained in embodiment 1 tires and do not have considerable change, within 2 months, starts to decline gradually later; And tire within half a year there is no considerable change at 4 DEG C of IgY preserved, start to have a declining tendency after 6 months.It can thus be appreciated that the IgY obtained in embodiment 1 can preserve for a long time, this just for a large amount of productions of IgY preserve, transport and widespread use etc. provide favourable condition.
Embodiment 5
The application of the chicken yolk antibody that specificity anti-aspergillus fumigatus infects in treatment rat fungal keratitis:
Wistar rat 18, right eye is all the IgY treatment group of gained in embodiment 1,1-9 left eye is blank group, and No. 10-18 is negative chicken yolk antibody control group (the yolk antibody group namely not having aspergillus fumigatus to stimulate is negative), after aspergillus fumigatus modeling 24h, with Aspergillus fumigatus yolk antibody made in the embodiment 1 of 200mg/ml, be configured to solution with physiological saline, adjust pH to be 7, carry out eye drip, right eye every two hours drips once, and the same 2h of left eye physiological saline drips once.Observe under 8h, 24h, 48h, 72h, 96h slit lamp, mark according to O ' Day standard row keratohelcosis.Result shows, treatment group keratohelcosis is commented lower than blank group and negative group, and blank group and the scoring of negative group do not have significant difference.
Claims (4)
1. the chicken yolk antibody of a species specificity anti-aspergillus fumigatus infection, it is characterized in that, described chicken yolk antibody preparation process comprises the following steps:
Step 1: preparation deactivation Aspergillus fumigatus mycelia suspension: inoculation aspergillus fumigatus is cultivated 5 days in Sharpe liquid nutrient medium, and take out Aspergillus fumigatus hypha body and grind in mill, filter spore, mycelial concentration is adjusted to 2 × 10 the most at last
9cFU/ml;
Step 2: animal immune: by mycelial concentration 2 × 10
9after the bacteria suspension of CFU/mL is fully emulsified after mixing by 1:1 with Freund's complete adjuvant, every chicken only injects 1 mL/, is changed to Freund's incomplete adjuvant booster immunization 3 times after 3 weeks, 5 weeks, 8 weeks;
Step 3: adopt ammonium sulfate salting-out process to prepare the chicken yolk antibody albumen of specificity anti-aspergillus fumigatus infection: beating eggs removes egg white and membrane of yolk, egg yolk liquid and ultrapure water 1:8 is mixed, adjust pH to 5.1, hold over night; Centrifuging and taking supernatant liquor; Add according to the solubleness of ammonium sulfate and liquid volume the amount can separating out at most IgY albumen afterwards, leave standstill 2h; The centrifugal Supernatant protein that removes is separated out again, adds phosphate buffered saline buffer soluble protein, again adds ammonium sulfate and leave standstill 2h; The centrifugal supernatant liquor that removes stays albumen, again adds minimum solvable protelytic phosphate buffered liquid measure redissolution albumen, loads dialysis tubing dialysis 48h; Put into Freeze Drying Equipment after the complete liquid of dialysis is frozen into solid and be made into lyophilized powder.
2. the chicken yolk antibody that infects of specificity anti-aspergillus fumigatus according to claim 1, is characterized in that, the method for the animal immune injection described in step 2 is that chicken used is Lay henry laying hen in the neck of chicken, the subcutaneous loose injection of dipteron and chest muscle many places.
3. apply in the medicine of preparation control fungal keratitis according to chicken yolk antibody according to claim 1.
4. chicken yolk antibody according to claim 3 is applied in the medicine of preparation control fungal keratitis, it is characterized in that described medicine is prepared into pulvis, tablet, ointment, mouth spray, collyrium, spongaion.
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| CN105106953A (en) * | 2015-08-21 | 2015-12-02 | 天津耀宇生物技术有限公司 | Antibody-based drug for treating psoriasis |
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| CN102977208A (en) * | 2012-12-12 | 2013-03-20 | 大连医科大学 | Preparation method, application and medicine composition and preparation of specific egg yolk immunoglobulin (IgY) and acinetobacter baumannii, as well as preparation and kit |
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| CN101343320B (en) * | 2008-07-01 | 2012-06-27 | 华南农业大学 | Anti-vibrio parahaemolyticus chicken yolk antibody, preparation method and application thereof |
| BR112013012594A2 (en) * | 2010-11-23 | 2018-10-16 | Pantheryx Inc | compositions and methods for treatment in broad spectrum undifferentiated or mixed clinical applications |
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| CN102977208A (en) * | 2012-12-12 | 2013-03-20 | 大连医科大学 | Preparation method, application and medicine composition and preparation of specific egg yolk immunoglobulin (IgY) and acinetobacter baumannii, as well as preparation and kit |
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| MARITA CHAKHTOURA 等: "Preparation of anti-Aspergillus fumigatus antibodies in egg laying hens and their protective efficacy in BALB/c mice", 《THE JOURNAL OF APPLIED RESEARCH 》 * |
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| CN105106953A (en) * | 2015-08-21 | 2015-12-02 | 天津耀宇生物技术有限公司 | Antibody-based drug for treating psoriasis |
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