Chicken mycoplasma synoviae genetic engineering subunit vaccine

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a chicken mycoplasma synoviae genetic engineering subunit vaccine, which comprises the steps of culture medium preparation, antigen liquid preparation, vaccine preparation, vaccine inspection and adjuvant preparation, and the culture medium comprises the following main components: PPLO broth powder, yeast extract powder, nicotinamide, lactalbumin hydrolysate, 1% acyl red white and water; in the culture medium, the mass of PPLO broth powder is 30 g, the mass of yeast extract powder is 10 g, the mass of nicotinamide is 5 ml, the mass of whey hydrolysate is 10 g, and the mass of water is 1 L; the adjuvant is a water-in-oil-in-water type adjuvant; by arranging the water-in-oil-in-water type adjuvant, the immunogenicity and immune effect of the vaccine can be improved, meanwhile, the oil content is low, stimulation to animal bodies is small, safety is high, the organism can be effectively induced to generate immune response, the efficiency of uptake and presentation of antigens is improved, and the vaccine can be used for preparing the vaccine. Therefore, the genetic engineering subunit vaccine is high in safety, simple in culture condition and capable of effectively preventing chicken mycoplasma synoviae from prevalence.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a genetically engineered subunit vaccine of Mycoplasma synoviae. Background Art

[0002] Mycoplasma gallisepticum is a highly contagious pathogen that mainly causes chronic respiratory diseases, air sacitis and sinusitis in chickens. There are two main transmission routes of Mycoplasma gallisepticum: horizontal transmission and vertical transmission. Horizontal transmission mainly occurs through respiratory droplets, hatchery transmission, direct contact with infected birds or indirect modes. Vertical transmission may occur in eggs and embryos. Since the abdominal air sacs are adjacent to the oviduct, they may also be transmitted to offspring through respiratory infections in hens. The clinical symptoms of Mycoplasma gallisepticum in infected chickens usually include tracheal gong sounds, coughing, runny nose, tears, swelling or adhesion of one or both eyelids, and chicks show growth retardation, poor development, an increase in sick and weak chicks, and an increased culling rate; laying hens often show a decrease in egg production.

[0003] Mycoplasma has no cell wall and is insensitive to many antibiotics. Antibiotic treatment cannot eliminate MS infection in chickens. How to control the spread of MS? Vaccine research has become the key to preventing and controlling chicken synoviae Mycoplasma disease. In terms of vaccine research, the main focus is on developing new vaccines that are more effective and safer. Summary of the Invention

[0004] To solve the problems raised in the above background technology, the present invention provides a genetically engineered subunit vaccine of Mycoplasma synoviae, which has the characteristics of high safety, high immunogenicity and high stability, and can effectively prevent the epidemic of Mycoplasma synoviae.

[0005] To achieve the above object, the present invention provides the following technical solution: a genetically engineered subunit vaccine of Mycoplasma synoviae, comprising culture medium preparation, antigen solution preparation, vaccine preparation, vaccine testing, and adjuvant preparation, wherein the main components of the culture medium include PPLO broth powder, yeast extract powder, nicotinamide, hydrolyzed lactic acid, 1% acyl red and white, and water;

[0006] The culture medium contains 30 g of PPLO broth powder, 10 g of yeast extract powder, 5 ml of nicotinamide, 10 g of lactolyl hydrolyzate, and 1 L of water.

[0007] The adjuvant is a water-in-oil-in-water type adjuvant.

[0008] Preferably, the steps of preparing the culture medium are as follows:

[0009] Step 1: Prepare a container and pour water into it. Then add PPLO bouillon powder, yeast extract powder, niacinamide, lactolysate, and 1% acyl red and white into the container in the order of mass proportion. Stir while adding each component until completely dissolved. The stirring temperature is 25-35℃. Be careful not to affect its activity at high temperature.

[0010] Step 2: Sterilize the prepared basal culture medium by high-pressure steam sterilization at a temperature of 115°C for 25 minutes. After sterilization, allow the culture medium to cool to room temperature.

[0011] Step 3: After the base solution has cooled, aseptically add 200 ml of inactivated horse serum and 1.2 ml of 800,000 units / ml penicillin solution, adjust the pH to 7.6-7.8 with 1 mol / L sodium hydroxide solution, and store at 2-8°C.

[0012] Preferably, the steps of preparing the antigen solution are as follows:

[0013] Step 1: Inoculate Mycoplasma synoviae into the finished culture medium at a ratio of 1:10, culture at 37°C for 24-48 hours, and harvest as the first-level seed liquid. Use the same method to inoculate the first-level seed liquid again at a ratio of 1:10 and harvest as the second-level seed liquid.

[0014] Step 2: Fermentation is carried out using a stirred fermenter. The culture medium in the fermenter is first sterilized. When the temperature drops to 37°C, the secondary seed liquid is inoculated at a ratio of 1:5-1:10, and then the pH is adjusted to 7.6-7.8. Feeding is performed using a continuous enrichment culture method, controlling the rubbing pressure at 0.03-0.05 Pa, the dissolved oxygen content at 40%, and culturing at a constant temperature of 37°C. When the pH drops to about 6.5, 30% of the original culture volume of the culture medium is added, and the pH is adjusted to 7.6-7.8 again. This process is repeated for two enrichment cultures until the pH of the second enrichment culture drops to 6.4, and the antigen liquid is harvested;

[0015] Step 3: Inactivation: Use 10% formaldehyde solution prepared from sterile saline as an inactivator. Add the formaldehyde solution to the antigen solution at 0.3% of the total bacterial solution. After fully mixing the antigen solution with formaldehyde, place it at a specific temperature, such as 37°C, and let it stand for a period of 6-24 hours. During this period, shake or stir it every 2 hours to ensure that the inactivator is in full contact with the antigen solution to improve the inactivation effect.

[0016] Preferably, the vaccine preparation steps are as follows:

[0017] Step 1: Preparation of oil phase: Take 95 parts of injection adjuvant and 1 part of aluminum stearate, mix them evenly in an oil phase preparation tank and heat to 80°C, then add 5 parts of Siben-80 and maintain the temperature at 115°C for 40 minutes, then cool and set aside;

[0018] Step 2: Preparation of aqueous phase: Take 95 parts of the inactivated antigen solution and add 5 parts of Tween-80 to it, and mix thoroughly to obtain the aqueous phase;

[0019] Step 3: Emulsification: Take 3 parts of the oil phase and add it to an appropriate sterile container. Start the mixer at 10,000 r / min and stir for 10 minutes. Then slowly add 3 parts of the water phase and stir at 15,000 r / min for 5 minutes. After stirring is completed, the vaccine can be obtained.

[0020] Preferably, the vaccine inspection includes sample reception and registration, safety inspection and storage stability inspection;

[0021] The sample reception and registration includes: receiving vaccine samples for registration, recording basic sample information such as batch number, production date, production unit, etc., and checking the physical properties of the vaccine such as appearance, color, transparency, etc.;

[0022] The safety test is carried out in accordance with the Chinese Veterinary Pharmacopoeia or other relevant standards, by inoculating the vaccine sample into a specific culture medium to observe whether there is microbial growth to ensure the effectiveness of the vaccine;

[0023] The storage stability test is as follows: the vaccine sample is stored under specified storage conditions for a certain period of time, which is 3-12 months. During this period, the changes in the vaccine's physical properties, sterility, immunogenicity and other indicators are regularly tested to ensure the quality stability of the vaccine during storage.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention uses a fermenter for a continuous proliferation and enrichment process, which not only increases yield but also reduces production costs. By providing a water-in-oil-in-water adjuvant, the immunogenicity and immune effect of the vaccine can be improved. At the same time, the oil content is low, the stimulation to the animal body is small, the safety is high, and the efficiency of antigen uptake and presentation is improved. Therefore, the genetically engineered subunit vaccine of the present invention is highly safe, the culture conditions are simple, and it can effectively prevent the epidemic of Mycoplasma synoviae. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0027] Figure 1 This is the production process flow chart of this vaccine;

[0028] Figure 2 This is a production process flow chart for preparing the culture medium of the present invention;

[0029] Figure 3 This is a production process flow chart for preparing the antigen solution of the present invention;

[0030] Figure 4 The figure is a flow chart of the production process for preparing the vaccine of the present invention. DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] Example 1

[0033] See also Figure 1-4 The present invention provides the following technical solution: a genetically engineered subunit vaccine for Mycoplasma synoviae, comprising culture medium preparation, antigen liquid preparation, vaccine preparation, and vaccine testing, characterized in that the culture medium comprises mainly PPLO broth powder, yeast extract powder, nicotinamide, hydrolyzed lactic acid, 1% acyl red and white lactic acid, and water; the culture medium comprises 30 g of PPLO broth powder, 10 g of yeast extract powder, 5 ml of nicotinamide, 10 g of hydrolyzed lactic acid, and 1 L of water; and the adjuvant is an oil-in-water (O / W) adjuvant. The O / W adjuvant improves the immunogenicity and immune efficacy of the vaccine, has a low oil content, causes less irritation to the animal body, is highly safe, can effectively induce an immune response, and improves the efficiency of antigen uptake and presentation. Thus, the genetically engineered subunit vaccine of the present invention is highly safe, requires simple culture conditions, and can effectively prevent the spread of Mycoplasma synoviae.

[0034] See also Figure 2The steps for preparing the culture medium are as follows: Step 1: Prepare a container, pour water into the container, and then pour PPLO broth powder, yeast extract powder, nicotinamide, hydrolyzed lactose, and 1% acyl red and white into the container in order according to the mass ratio, and add each component while stirring until it is completely dissolved. The stirring temperature is 25-35°C, and care should be taken to avoid high temperature affecting its activity; Step 2: Sterilize the prepared basal culture medium by high-pressure steam sterilization. The temperature of the high-pressure steam is 115°C and the time is 25 minutes. After sterilization, wait for the culture medium to cool to room temperature; Step 3: After the basal liquid is cooled, aseptically add 200ml of inactivated horse serum and 1.2ml of 800,000 units / ml penicillin solution, and adjust the pH value to 7.6-7.8 with 1mol / L sodium hydroxide solution, and store it at 2-8°C.

[0035] See also Figure 3 The steps for preparing the antigen liquid are as follows: Step 1: inoculate the chicken synoviae Mycoplasma into the finished culture medium at a ratio of 1:10, culture at 37°C for 24-48 hours, harvest as the first-level seed liquid, and inoculate the first-level seed liquid again at a ratio of 1:10 in the same way, and harvest as the second-level seed liquid; Step 2: fermentation, using a stirred fermentation tank, first sterilize the culture medium in the fermentation tank, when the temperature drops to 37°C, inoculate the second-level seed liquid at a ratio of 1:5-1:10, and then adjust the pH to 7.6-7.8, feed, adopt continuous bacterial culture method, control the rubbing pressure at 0.03-0.05Pa, the dissolved oxygen content is 40%, and culture at a constant temperature of 37°C. When the pH drops to about 6.5, add culture The method comprises the following steps: the first step is to add 10% formaldehyde solution (made of sterile physiological saline) as an inactivator, and adding the formaldehyde solution to the antigen liquid according to 0.3% of the total amount of the bacterial liquid. After the antigen liquid to which formaldehyde is added is fully mixed, the antigen liquid is placed at a specific temperature such as 37°C and allowed to stand for a period of time for 6-24 hours. During this period, the antigen liquid is fully shaken or stirred every 2 hours to ensure that the inactivator is in full contact with the antigen liquid. This improves the inactivation effect and can reduce production costs by adopting a fermentation tank to carry out a continuous proliferation and cultivation process.

[0036] See also Figure 4The steps for preparing the vaccine are as follows: Step 1: Preparation of oil phase: Take 95 parts of injection adjuvant and 1 part of aluminum stearate, mix them evenly in an oil phase preparation tank and heat them to 80°C, then add 5 parts of Siben-80 and maintain the temperature at 115°C for 40 minutes, cool and set aside; Step 2: Preparation of aqueous phase: Take 95 parts of inactivated antigen solution and add 5 parts of Tween-80 thereto, mix thoroughly to form the aqueous phase; Step 3: Emulsification: Take 3 parts of the oil phase and add it to an appropriate sterile container, start the mixer at 10,000 r / min and stir for 10 minutes, then slowly add 3 parts of the aqueous phase, and then stir at 15,000 r / min for 5 minutes. After stirring is completed, the vaccine can be obtained.

[0037] Specifically, vaccine inspection includes sample reception and registration, safety inspection and storage stability inspection; sample reception and registration include: receiving vaccine samples for registration, recording basic information of the samples, such as batch number, production date, production unit, etc., and checking the physical properties of the vaccine such as appearance, color, transparency, etc.; safety inspection includes: in accordance with the "Chinese Pharmacopoeia of Veterinary Medicine" or other relevant standards, inoculating vaccine samples in a specific culture medium, observing whether there is microbial growth, and ensuring whether the vaccine is effective; storage stability inspection includes: keeping vaccine samples under prescribed storage conditions for a certain period of time, which is 3-12 months. During this period, the changes in the physical properties, sterility, immunogenicity and other indicators of the vaccine are regularly inspected to ensure the quality stability of the vaccine during storage.

[0038] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A genetically engineered subunit vaccine of Mycoplasma synoviae, comprising culture medium preparation, antigen solution preparation, vaccine preparation, vaccine testing, inactivation agent, and adjuvant, characterized in that: The main components of the culture medium include PPLO broth powder, yeast extract powder, nicotinamide, hydrolyzed lactose, 1% acyl red and white, and water; The culture medium contains 30 g of PPLO broth powder, 10 g of yeast extract powder, 5 ml of nicotinamide, 10 g of lactolyl hydrolyzate, and 1 L of water. The inactivator is a 10% formaldehyde solution (prepared with sterile saline); The adjuvant is a water-in-oil-in-water type adjuvant.

2. The genetically engineered subunit vaccine of Mycoplasma synoviae according to claim 1, characterized in that: The steps of preparing the culture medium are as follows: Step 1: Prepare a container and pour water into it. Then add PPLO bouillon powder, yeast extract powder, niacinamide, lactolysate, and 1% acyl red and white into the container in the order of mass proportion. Stir while adding each component until completely dissolved. The stirring temperature is 25-35℃. Be careful not to affect its activity at high temperature. Step 2: Sterilize the prepared culture medium by high-pressure steam sterilization at a temperature of 115°C for 25 minutes. After sterilization, allow the culture medium to cool to room temperature. Step 3: After the culture medium has cooled, aseptically add 200 ml of inactivated horse serum and 1.2 ml of 800,000 units / ml penicillin solution, adjust the pH to 7.6-7.8 with 1 mol / L sodium hydroxide solution, and store at 2-8°C.

3. The genetically engineered subunit vaccine of Mycoplasma synoviae according to claim 1, characterized in that: The steps of preparing the antigen solution are as follows: Step 1: Inoculate Mycoplasma synoviae into culture medium at a ratio of 1:10, culture at 37°C for 24-48 hours, and harvest as the first-level seed solution. Use the same method to inoculate the first-level seed solution again at a ratio of 1:10 and harvest as the second-level seed solution. Step 2: Fermentation is carried out using a stirred fermenter. The culture medium in the fermenter is first sterilized. When the temperature drops to 37°C, the secondary seed liquid is inoculated at a ratio of 1:5-1:10, and then the pH is adjusted to 7.6-7.

8. Feeding is performed using a continuous enrichment culture method, controlling the rubbing pressure at 0.03-0.05 Pa, the dissolved oxygen content at 40%, and culturing at a constant temperature of 37°C. When the pH drops to about 6.5, 30% of the original culture volume of the culture medium is added, and the pH is adjusted to 7.6-7.8 again. This process is repeated for two enrichment cultures until the pH of the second enrichment culture drops to 6.4, and the antigen liquid is harvested; Step 3: Inactivation: Add the inactivator to the antigen solution at 0.3% of the total bacterial solution. After fully mixing the antigen solution with the inactivator, place it at 37°C for a period of time, which is 6-24 hours. During this period, shake or stir it every 2 hours to ensure that the inactivator is in full contact with the antigen solution to improve the inactivation effect.

4. The genetically engineered subunit vaccine of Mycoplasma synoviae according to claim 1, characterized in that: The steps of vaccine preparation are as follows: Step 1: Preparation of oil phase: Take 95 parts of injection adjuvant and 1 part of aluminum stearate, mix them evenly in an oil phase preparation tank and heat to 80°C, then add 5 parts of Siben-80 and maintain the temperature at 115°C for 40 minutes, then cool and set aside; Step 2: Preparation of aqueous phase: Take 95 parts of the inactivated antigen solution and add 5 parts of Tween-80 to it, and mix thoroughly to obtain the aqueous phase; Step 3: Emulsification: Take 3 parts of the oil phase and add it to an appropriate sterile container. Start the mixer at 10,000 r / min and stir for 10 minutes. Then slowly add 3 parts of the water phase and stir at 15,000 r / min for 5 minutes. After stirring is completed, the vaccine can be obtained.

5. The genetically engineered subunit vaccine of Mycoplasma synoviae according to claim 1, characterized in that: The vaccine inspection includes sample reception and registration, safety inspection and storage stability inspection; The sample reception and registration includes: receiving vaccine samples for registration, recording basic sample information such as batch number, production date, production unit, etc., and checking the physical properties of the vaccine such as appearance, color, transparency, etc.; The safety test is carried out in accordance with the Chinese Veterinary Pharmacopoeia or other relevant standards, by inoculating the vaccine sample into a specific culture medium to observe whether there is microbial growth to ensure the effectiveness of the vaccine; The storage stability test is as follows: the vaccine sample is stored under specified storage conditions for a certain period of time, which is 3-12 months. During this period, the changes in the vaccine's physical properties, sterility, immunogenicity and other indicators are regularly tested to ensure the quality stability of the vaccine during storage.

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