CN103550286B - A kind of infectious bursal disease live-vaccine effervescent tablet immunological adjuvant - Google Patents
A kind of infectious bursal disease live-vaccine effervescent tablet immunological adjuvant Download PDFInfo
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Abstract
A kind of infectious bursal disease live-vaccine effervescent tablet immunological adjuvant, belongs to veterinary biologics technical field.Containing herbal polysaccharide 30 ~ 80g, Chinese medicine flavone 20 ~ 50g, vitamin 6 ~ 10g, gelatin 4 ~ 6g and sucrose 40 ~ 70g in the every 1000ml phosphate buffer of this immunological adjuvant.Immunological adjuvant of the present invention has the following advantages: 1. the immunological adjuvant prepared by the present invention, has heat-resisting effect, can improve the conveniences such as vaccine transport, storage.2. adopt the vaccine prepared by immunological adjuvant of the present invention can reduce stress when inoculation, reduce chicken group disease infection risk.3. adopt immunological adjuvant of the present invention to prepare vaccine, greatly can improve the result of use of live vaccine, produce neutralizing antibody fast.
Description
Technical field
The invention belongs to veterinary biologics technical field, be specifically related to a kind of infectious bursal disease live-vaccine effervescent tablet immunological adjuvant.
Background technology
Along with the rise of intensive aviculture and the increase of international trade, fowl diseases is popular to be on the rise, and causes the high mortality of poultry, seriously governs the development of poultry husbandry, causes the loss that world's poultry husbandry is serious, jeopardizes economic development, food safety and social stability.Infectious bursal disease (Infection bursal disease, IBD) be by infectious bursa of Fabricius virus (Infection bursal disease virus, a kind of high degree in contact sexually transmitted disease of the chicken IBDV) caused and turkey, main infection 3-12 week age young chicken, this virus is mainly in the lymphocyte internal breeding of fabricius bursa, and damage is in various degree caused to other immune organ, finally causes the fabricius bursa atrophy of chicken and cause the immunosuppressant of chicken.This disease is without significantly seasonal, all can occur throughout the year, propagate usually through approach such as contaminated feedstuff, drinking-water, feces, hen house apparatus, staff garments, incubation period is 1-5 days, and after there is symptom the 3rd day starts dead, within 4-6 days, be peak mortality phases, stop death gradually later, sickness rate can reach more than 90%, and mortality rate not etc., is not generally 10%-50%.Primary disease can cause losing weight clinically, poor growth and the symptom such as skeletal muscle is hemorrhage, often occur together when sanitary condition is poor other diseases, mortality rate improves further, and chickling individuality is little, and premunition is poor, once generation epidemic disease, then infect fast, mortality rate is high, and mortality rate reaches more than 80%, loss is larger, has carried out huge economic loss to poultry industrial belt.
Vaccine is the goods be made up after breeding and process of the good pathogenic microorganism of immunogenicity, after inoculation animal body, body is stimulated to produce specific antibody, after the antibody titer in body reaches certain numerical value, just can resist invasion and attack, the infection of this pathogenic microorganism, play the effect preventing this disease.Along with the fast development of China's aviculture, more and more important to the control of infectious bursal disease.
Polysaccharide is one of active ingredient of Chinese herbs, and large quantity research shows, polysaccharide and saccharide complex are not only in vivo as energy resource and constituent material, the more important thing is that it is present in all membrane structures, participates in the various activities of cell in biosis.Polysaccharose substance is the important component part of all Living organisms, has scavenging free radicals, improves the ability of activities of antioxidant enzymes and anti-lipid peroxidation.In addition, herbal polysaccharide has special protective effect to the production of unicellular lower eukaryote and storage.Herbal polysaccharide is rich in hydroxyl, can with low isoreactivity bioconjugation, build multidimensional network space hydrogen bond structure, form protecting film at active biological surface, realize the protective effect to active bio.
Vitamin is requisite organic compound in organism metabolism.The chemical plant that body is very complicated just as one, constantly carries out various biochemical reaction.Its reaction has substantial connection with the catalytic action of enzyme.Enzyme will produce activity, and coenzyme must be had to participate in.Known many vitamin are the coenzyme of enzyme or the ingredient of coenzyme.Therefore, vitamin is the important substance maintaining and regulate body homergy.Vitamin is one of indispensable nutrient substance in growth of animal process, and especially when occurring, the effect of vitamin is more obvious.Suitable edible vitamin can reduce Animal stress, reduces disease incident, improves animal disease resistant ability.
Flavone compound is the polyatomic phenol material that occurring in nature exists, and is also one of main active in nature medicinal plants.It refers to have 15 carbon atoms and with the three ring natural organic matters that the mode of C6-C3-C6 is formed, be the secondary metabolism product that plant produces in long-term natural selection, now isolation identification have kind more than 4000.It is extensively present in fruit and vegerable, Chinese herbal medicine, have no side effect, it has the medicine healthy sofa functions such as significant scavenger interior free yl, aging resistance, mutation, Adjust-blood lipid blood pressure lowering, it is the natural organic oxidation-resistant agent that a class has DEVELOPMENT PROSPECT, these anti-oxidation active substances can reduce and scavenger interior free yl, have prophylactic effect.
Summary of the invention
For prior art Problems existing, the object of the invention is to design the technical scheme that a kind of infectious bursal disease live-vaccine effervescent tablet immunological adjuvant is provided, this immunological adjuvant can increase substantially the immunity of body, stimulate body to produce efficient neutralizing antibody, improve the prophylactic ability of body.
Described a kind of infectious bursal disease live-vaccine effervescent tablet immunological adjuvant, it is characterized in that described herbal polysaccharide and Chinese medicine flavone extract and obtain from following Chinese medicine composition: Herba Andrographis, Radix Achyranthis Bidentatae, Poria, Radix Isatidis, Radix Glycyrrhizae, Herba Urticae Cannabinae, Herba Equiseti Arvinsis and the Radix Astragali containing herbal polysaccharide 30 ~ 80g, Chinese medicine flavone 20 ~ 50g, vitamin 6 ~ 10g, gelatin 4 ~ 6g and sucrose 40 ~ 70g in the every 1000ml phosphate buffer of this immunological adjuvant.
Described a kind of infectious bursal disease live-vaccine effervescent tablet immunological adjuvant, is characterized in that in the every 1000ml phosphate buffer of this immunological adjuvant containing herbal polysaccharide 40 ~ 60g, Chinese medicine flavone 30 ~ 40g, vitamin 6 ~ 8g, gelatin 4.5 ~ 5.5g and sucrose 50 ~ 60g.
Described a kind of infectious bursal disease live-vaccine effervescent tablet immunological adjuvant, is characterized in that in described vitamin containing vitamin C 20 ~ 40%, vitamin E 10 ~ 30%, vitamin D3 5 ~ 15%, vitamin B1 15 ~ 35%, vitamin K 10 ~ 20% and vitamin B2 5 ~ 20%.
Described a kind of infectious bursal disease live-vaccine effervescent tablet immunological adjuvant, is characterized in that in described Chinese medicine composition containing Herba Andrographis 10 ~ 30 parts, Radix Achyranthis Bidentatae 20 ~ 40 parts, 10 ~ 20 parts, Poria, Radix Isatidis 10 ~ 30 parts, 10 ~ 30 parts, Radix Glycyrrhizae, Herba Urticae Cannabinae 10 ~ 30 parts, Herba Equiseti Arvinsis 10 ~ 20 parts and the Radix Astragali 10 ~ 30 parts.
Described a kind of infectious bursal disease live-vaccine effervescent tablet immunological adjuvant, is characterized in that in described Chinese medicine composition containing Herba Andrographis 15 ~ 25 parts, Radix Achyranthis Bidentatae 25 ~ 35 parts, 12 ~ 18 parts, Poria, Radix Isatidis 15 ~ 25 parts, 15 ~ 25 parts, Radix Glycyrrhizae, Herba Urticae Cannabinae 15 ~ 25 parts, Herba Equiseti Arvinsis 12 ~ 18 parts and the Radix Astragali 15 ~ 25 parts.
Described a kind of infectious bursal disease live-vaccine effervescent tablet immunological adjuvant, is characterized in that described herbal polysaccharide and Chinese medicine flavone are obtained by following steps:
A, take Herba Andrographis, Radix Achyranthis Bidentatae, Poria, Radix Isatidis, Radix Glycyrrhizae, Herba Urticae Cannabinae, Herba Equiseti Arvinsis and the Radix Astragali by described weight portion, chopping, clean after spend the night by cold water soak, add the purified water of raw material weight 15 times again, water-bath to 90 DEG C, and remain on 90 DEG C, boil 2 hours, boil in process every 10 minutes and stir once;
B, discard a Chinese medicine slag, collect medicinal liquid, after room temperature cooling through 10000rpm centrifugal 10 minutes, discard precipitation, collect supernatant;
C, supernatant in b is carried out precipitate with ethanol, precipitation is rough herbal polysaccharide, and supernatant is rough Chinese medicine flavone;
D, with rough herbal polysaccharide in sterilizing purified water washing c, dissolves with 42 DEG C of sterilizing purified water of 20 times amount after washing, after dissolving, add active carbon 4 DEG C of attach overnight in 0.5% ratio, after absorption through 10000rpm centrifugal 10 minutes, discard precipitation, collection supernatant;
E, supernatant in d carried out 10 times and concentrate, obtained herbal polysaccharide concentrated solution;
F, herbal polysaccharide concentrated solution in e is carried out vacuum lyophilization obtain herbal polysaccharide;
G, Chinese medicine flavone rough in c is carried out reduced vacuum concentrate, concentrate time sterilized water for injection dilution cleaning, again carries out reduced vacuum and concentrates, make Chinese medicine flavone after three times like this through lyophilisation.
Above-mentioned a kind of infectious bursal disease live-vaccine effervescent tablet immunological adjuvant, reasonable in design, it has the following advantages compared with traditional immunological adjuvant:
1. the immunological adjuvant prepared by the present invention, has heat-resisting effect, can improve the conveniences such as vaccine transport, storage.
2. adopt the vaccine prepared by immunological adjuvant of the present invention can reduce stress when inoculation, reduce chicken group disease infection risk.
3. adopt immunological adjuvant of the present invention to prepare vaccine, greatly can improve the result of use of live vaccine, produce neutralizing antibody fast.
Detailed description of the invention
In order to make the present invention easier to understand, below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention and place restrictions on scope of the present invention, NM specific experiment method in the following example, experimental technique carries out routinely usually.
Embodiment 1: the preparation of herbal polysaccharide and Chinese medicine flavone
A, by Herba Andrographis 20 parts, Radix Achyranthis Bidentatae 30 parts, 20 parts, Poria, Radix Isatidis 10 parts, 10 parts, Radix Glycyrrhizae, Herba Urticae Cannabinae 30 parts, Herba Equiseti Arvinsis 20 parts, the Radix Astragali 30 parts take each raw material of Chinese medicine, chopping, clean after spend the night by cold water soak, add the purified water of raw material weight 15 times again, water-bath to 90 DEG C, and remain on 90 DEG C, boil 2 hours, boil in process every 10 minutes and stir once;
B, discard a Chinese medicine slag, collect medicinal liquid, after room temperature cooling through 10000rpm centrifugal 10 minutes, discard precipitation, collect supernatant;
C, supernatant in b is carried out precipitate with ethanol, precipitation is rough herbal polysaccharide, and supernatant is rough Chinese medicine flavone;
D, with rough herbal polysaccharide in sterilizing purified water washing c, dissolves with 42 DEG C of sterilizing purified water of 20 times amount after washing, after dissolving, add active carbon 4 DEG C of attach overnight in 0.5% ratio, after absorption through 10000rpm centrifugal 10 minutes, discard precipitation, collection supernatant;
E, by supernatant in d after 0.22um membrane filtration is degerming, carry out 10 times and concentrate, obtained herbal polysaccharide concentrated solution;
F, herbal polysaccharide concentrated solution in e is carried out vacuum lyophilization namely obtain herbal polysaccharide;
G, Chinese medicine flavone rough in c is carried out reduced vacuum concentrate, concentrate time sterilized water for injection dilution cleaning, again carries out reduced vacuum and concentrates, after three times like this, more degerming through 0.22um membrane filtration, then namely obtain Chinese medicine flavone through lyophilisation.
Embodiment 2: the preparation of herbal polysaccharide and Chinese medicine flavone
The Chinese medicine composition of the step a of embodiment 1 is changed into Herba Andrographis 10 parts, Radix Achyranthis Bidentatae 20 parts, 15 parts, Poria, Radix Isatidis 25 parts, 25 parts, Radix Glycyrrhizae, Herba Urticae Cannabinae 10 parts, Herba Equiseti Arvinsis 15 parts and the Radix Astragali 25 parts, other step condition is all identical with embodiment 1.
Embodiment 3:
The Chinese medicine composition of the step a of embodiment 1 is changed into Herba Andrographis 30 parts, Radix Achyranthis Bidentatae 40 parts, 10 parts, Poria, Radix Isatidis 30 parts, 30 parts, Radix Glycyrrhizae, Herba Urticae Cannabinae 20 parts, Herba Equiseti Arvinsis 10 parts and the Radix Astragali 15 parts, other step condition is all identical with embodiment 1.
Embodiment 4: a kind of preparation of infectious bursal disease live-vaccine effervescent tablet immunological adjuvant
In every 1000ml phosphate buffer, add herbal polysaccharide 80g, Chinese medicine flavone 50g, vitamin 8g(vitamin C 1.8g, vitamin E 1g, vitamin D3 1g, vitamin B1 2g, vitamin K 1.2g, vitamin B2 1g), gelatin 5g and sucrose 50g, namely obtain infectious bursal disease live-vaccine effervescent tablet immunological adjuvant.Wherein herbal polysaccharide and Chinese medicine flavone are obtained by embodiment 1.Phosphate buffer by sodium chloride 6 ~ 10g, potassium chloride 0.05 ~ 0.5g, sodium hydrogen phosphate 1 ~ 1.2g, potassium dihydrogen phosphate 0.05 ~ 0.5g, calcium chloride 0.05 ~ 0.2g, the magnesium chloride 0.05 ~ 0.2g containing 6 water of crystallization is dissolved in 1000ml distilled water, through 116 DEG C, autoclaving obtains for 30 minutes.
Embodiment 5: a kind of preparation of infectious bursal disease live-vaccine effervescent tablet immunological adjuvant
In every 1000ml phosphate buffer, add herbal polysaccharide 30g, Chinese medicine flavone 20g, vitamin 6g(vitamin C 1.2g, vitamin E 1.8g, vitamin D3 0.9g, vitamin B1 1.2g, vitamin K 0.6g, vitamin B2 0.3g), gelatin 4g and sucrose 40g, namely obtain Porcine reproductive and respiratory syndrome composite live vaccine immunological adjuvant.Wherein herbal polysaccharide and Chinese medicine flavone are obtained by embodiment 1.Phosphate buffer by sodium chloride 6 ~ 10g, potassium chloride 0.05 ~ 0.5g, sodium hydrogen phosphate 1 ~ 1.2g, potassium dihydrogen phosphate 0.05 ~ 0.5g, calcium chloride 0.05 ~ 0.2g, the magnesium chloride 0.05 ~ 0.2g containing 6 water of crystallization is dissolved in 1000ml distilled water, through 116 DEG C, autoclaving obtains for 30 minutes.
Embodiment 6: the preparation of effervescent materials
The preparation of a, acid material
Take citric acid 15 parts, lactose 10 parts, EDTA 15 parts, light blue indicator 0.5 part by weight respectively, fully mixed by mentioned component, cross 8 ~ 12 mesh sieves after 40 ~ 65 DEG C of oven dry, obtained acid particles is for subsequent use;
The preparation of b, basic matterial
Take sodium bicarbonate 15 parts, lactose 10 parts, light blue indicator 1 part by weight respectively, fully mixed by mentioned component, cross 8 ~ 12 mesh sieves after 40 ~ 65 DEG C of oven dry, obtained alkali grain is for subsequent use;
C, above-mentioned basic matterial to be mixed homogeneously by 1:1 with acid material, and add the levamisole of 0.5 ~ 1 weight portion, obtain effervescent materials through Co-60 radiation sterilization.
Embodiment 7: the using method of immunological adjuvant
1. infections chicken cloacal bursa virus suspension preparation
(1) prepared by production seed culture of viruses:
To grow up to the DF-1 cell of monolayer, and discard cell growth medium, by cell growth medium 1% amount virus inoculation, 37 DEG C adsorb 1 hour, add cell maintenance medium, put 37 DEG C and continue to cultivate, start after 40 hours to observe, when cytopathy reaches 80%, harvesting culture, puts-20 DEG C of preservations.
(2) selection of seedling material: the DF-1 cell monolayer that growth selection is good.
(3) the seedling breeding of cell venom:
To grow up to the DF-1 cell of good monolayer, discard growth-promoting media, by the DMF12 maintenance medium of pH value 7.2-7.6, production seed culture of viruses be diluted 3-4 doubly, by the virus after 1% inoculation dilution of growth liquid measure, 37 DEG C adsorb 1 hour, add cell maintenance medium, put 37 DEG C and continue rotating and culturing.After connecing poison, every day observation of cell pathological changes situation, when sick cell reaches 80%, can harvesting culture, multigelation 3 times, puts less than-20 DEG C Cryopreservations.
2. lyophilized formulations preparation
Steriling test and viral level are detected qualified cell venom, be mixed in same sterilization container, in 1:1(V:V) ratio to add in embodiment 4 or 5 immunological adjuvant of preparation, and fully quantitative separating after mixing, lyophilisation makes infectious bursal disease lyophilized formulations.
3. effervescent tablet preparation
Effervescent materials infectious bursal disease lyophilized formulations and embodiment 6 by above-mentioned preparation obtained, by after mixing homogeneously by weight 2:1 ~ 3, is pressed into tablet with tablet machine, quantitative separating.
Above-mentioned cell growth medium contains 90%DMEM/F12,10% hyclone, 90 ~ 110U/ml antibiotics, pH 7.2 ~ 7.3, DO 30 ~ 60%; Cell maintenance medium contains 98%DMEM/F12,2% hyclone, 90 ~ 100U/ml antibiotics, pH 7.2 ~ 7.3, DO 30 ~ 50%.
The application of test example 1 infectious bursal disease live-vaccine effervescent tablet of the present invention immunological adjuvant
1 material
1.1 infectious bursal disease live-vaccine effervescent tablets
Obtained by infections chicken cloacal bursa virus lyophilized formulations and infectious bursal disease live-vaccine effervescent tablet immunological adjuvant.
1.2 tests chicken 200 age in days sea blue laying breed 2000; 10 age in days Sanhuang broilers 2000.
2 methods
2000 200 blue laying breed in age in days sea are divided into two groups by 2.1, first group 1000, as experimental group, the infectious bursal disease live-vaccine effervescent tablet 10 of feeding prepared by the present invention while complete feed of feeding (with reference to fowl raising standard NY/33-2004); Second group 1000, as a control group, complete feed of only feeding.The indexs such as laying rate, rate of fertilization, death rate, infections chicken cloacal bursa antibody horizontal respectively organized in test implementation period record, and the observation period is surrounding.
2000 10 age in days Sanhuang broilers are divided into two groups, first group 1000 by 2.2, as experimental group, and the infectious bursal disease live-vaccine effervescent tablet 10 of feeding prepared by the present invention while complete feed of feeding (with reference to fowl raising standard NY/33-2004); Second group 1000, as a control group, complete feed of only feeding.The indexs such as average daily gain, death rate and infections chicken cloacal bursa antibody horizontal respectively organized in test implementation period record, and the observation period is surrounding.
3 results
3.1 disintegration result
Get 1 effervescent tablet, put and fill in the 250ml beaker of 200ml water, water temperature 15-25 DEG C, observe and record the consoluet time, the results are shown in Table 1.
Table 1 assay disintegration
The 3.2 200 blue laying breed result of the tests in age in days sea, in table 2.
The blue laying breed result of the test in table 2 200 age in days sea
3.3 10 age in days Sanhuang broiler result of the tests, in table 3.
Table 3 10 age in days Sanhuang broiler result of the test
4 conclusions
In sum, the infectious bursal disease live-vaccine effervescent tablet prepared by adjuvant of the present invention, can improve infections chicken cloacal bursa antibody titer when chicken group uses, and improves laying rate, improves fertility rate of hatching egg, improves broiler daily gain, reduces chicken group death rate.
Claims (4)
1. an infectious bursal disease live-vaccine effervescent tablet immunological adjuvant, it is characterized in that described herbal polysaccharide and Chinese medicine flavone extract and obtain from following Chinese medicine composition: Herba Andrographis 10 ~ 30 parts, Radix Achyranthis Bidentatae 20 ~ 40 parts, 10 ~ 20 parts, Poria, Radix Isatidis 10 ~ 30 parts, 10 ~ 30 parts, Radix Glycyrrhizae, Herba Urticae Cannabinae 10 ~ 30 parts, Herba Equiseti Arvinsis 10 ~ 20 parts and the Radix Astragali 10 ~ 30 parts containing herbal polysaccharide 30 ~ 80g, Chinese medicine flavone 20 ~ 50g, vitamin 6 ~ 10g, gelatin 4 ~ 6g and sucrose 40 ~ 70g in the every 1000ml phosphate buffer of this immunological adjuvant;
Described herbal polysaccharide and Chinese medicine flavone are obtained by following steps:
A, take Herba Andrographis, Radix Achyranthis Bidentatae, Poria, Radix Isatidis, Radix Glycyrrhizae, Herba Urticae Cannabinae, Herba Equiseti Arvinsis and the Radix Astragali by described weight portion, chopping, clean after spend the night by cold water soak, add the purified water of raw material weight 15 times again, water-bath to 90 DEG C, and remain on 90 DEG C, boil 2 hours, boil in process every 10 minutes and stir once;
B, discard a Chinese medicine slag, collect medicinal liquid, after room temperature cooling through 10000rpm centrifugal 10 minutes, discard precipitation, collect supernatant;
C, supernatant in b is carried out precipitate with ethanol, precipitation is rough herbal polysaccharide, and supernatant is rough Chinese medicine flavone;
D, with rough herbal polysaccharide in sterilizing purified water washing c, dissolves with 42 DEG C of sterilizing purified water of 20 times amount after washing, after dissolving, add active carbon 4 DEG C of attach overnight in 0.5% ratio, after absorption through 10000rpm centrifugal 10 minutes, discard precipitation, collection supernatant;
E, supernatant in d carried out 10 times and concentrate, obtained herbal polysaccharide concentrated solution;
F, herbal polysaccharide concentrated solution in e is carried out vacuum lyophilization obtain herbal polysaccharide;
G, Chinese medicine flavone rough in c is carried out reduced vacuum concentrate, concentrate time sterilized water for injection dilution cleaning, again carries out reduced vacuum and concentrates, make Chinese medicine flavone after three times like this through lyophilisation.
2. a kind of infectious bursal disease live-vaccine effervescent tablet immunological adjuvant as claimed in claim 1, is characterized in that in the every 1000ml phosphate buffer of this immunological adjuvant containing herbal polysaccharide 40 ~ 60g, Chinese medicine flavone 30 ~ 40g, vitamin 6 ~ 8g, gelatin 4.5 ~ 5.5g and sucrose 50 ~ 60g.
3. a kind of infectious bursal disease live-vaccine effervescent tablet immunological adjuvant as claimed in claim 1 or 2, is characterized in that in described vitamin containing vitamin C 20 ~ 40%, vitamin E 10 ~ 30%, vitamin D3 5 ~ 15%, vitamin B1 15 ~ 35%, vitamin K 10 ~ 20% and vitamin B2 5 ~ 20%.
4. a kind of infectious bursal disease live-vaccine effervescent tablet immunological adjuvant as claimed in claim 1, is characterized in that in described Chinese medicine composition containing Herba Andrographis 15 ~ 25 parts, Radix Achyranthis Bidentatae 25 ~ 35 parts, 12 ~ 18 parts, Poria, Radix Isatidis 15 ~ 25 parts, 15 ~ 25 parts, Radix Glycyrrhizae, Herba Urticae Cannabinae 15 ~ 25 parts, Herba Equiseti Arvinsis 12 ~ 18 parts and the Radix Astragali 15 ~ 25 parts.
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Denomination of invention: A live infectious bursal disease vaccine Effervescent tablet Immunologic adjuvant Effective date of registration: 20230626 Granted publication date: 20150930 Pledgee: Agricultural Bank of China Limited by Share Ltd. Jinhua Economic Development Zone Branch Pledgor: ZHEJIANG MIBOLERONE BIOLOGICAL TECHNOLOGY Co.,Ltd. Registration number: Y2023980045679 |
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