CN109385385B - Preparation method and application of mycoplasma avium culture medium and mycoplasma avium bacterial liquid - Google Patents
Preparation method and application of mycoplasma avium culture medium and mycoplasma avium bacterial liquid Download PDFInfo
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Abstract
The invention relates to a mycoplasma avium culture medium, a preparation method of mycoplasma avium bacterial liquid and application of the mycoplasma avium bacterial liquid, and belongs to the technical field of biology. The avian mycoplasma culture medium consists of a basic culture medium and auxiliary components, wherein the basic culture medium is a conventional avian mycoplasma liquid culture medium, and the auxiliary components are gelatin, tween 80 and calcium chloride; the strain for production is mycoplasma gallisepticum or mycoplasma synoviae, and the mycoplasma gallisepticum bacterial solution is produced through bacterial solution inoculation, culture and harvesting to obtain the mycoplasma gallisepticum vaccine. The preparation method of the invention improves the antigen content of the mycoplasma avian bacterial liquid, simplifies the production process, reduces the production cost, reduces the occurrence probability of vaccine immune side reactions and improves the vaccine immune effect.
Description
Technical Field
The invention relates to the technical field of biomedicine, and particularly relates to a mycoplasma avium culture medium, a preparation method of mycoplasma avium bacterial liquid and application of the mycoplasma avium bacterial liquid.
Background
Avian Mycoplasmosis (AM) is an infectious disease of poultry caused by Avian Mycoplasma, and clinically common pathogens are Mycoplasma Gallisepticum (MG) and Mycoplasma Synoviae (MS). Mycoplasma gallisepticum infection can cause Chronic Respiratory Disease (CRD), bursitis and sinusitis in birds; mycoplasma synoviae infection can cause symptoms such as arthrocele, inflammation of bursa and tendon, and enlargement of parenchymal organ; effective vaccine immunization is the primary means for preventing and controlling the disease.
At present, the production of the avian mycoplasma vaccine mainly uses an improved Frey culture medium, a CM2 culture medium and the like for culture, the serum concentration of the culture medium is higher (generally more than 10 percent), and the concentration of a culture solution is not high (generally 10 percent)8-10CCU/ml), strains need to be reproduced again in each production, the production operation is complicated, the cost is high, the quality of vaccine products is unstable (the content and the purity of antigens are not high), and side reactions are easy to occur. Therefore, the development of an efficient, low-cost and convenient avian mycoplasma culture medium, a preparation method of an avian mycoplasma bacterial liquid and an application of the avian mycoplasma bacterial liquid are needed.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a preparation method and application of a high-efficiency, convenient and low-cost mycoplasma avium culture medium and mycoplasma avium bacterial liquid.
In order to solve the technical scheme, the invention provides the following technical scheme: the avian mycoplasma culture medium is composed of an avian mycoplasma basic culture medium and auxiliary components, wherein the basic culture medium is a conventional avian mycoplasma liquid culture medium, and the auxiliary components are composed of gelatin, tween 80 and calcium chloride.
Further, the basic culture medium is any one of a conventional CM2 liquid culture medium, a CM culture medium or a modified Frey's culture medium.
Furthermore, the dosage of the auxiliary components is the following components in the mass-volume ratio (m: V) of the finished product culture medium:
0.2 to 0.4 percent of gelatin,
tween 800.5-1%,
0.2 to 0.4 percent of calcium chloride.
The invention relates to a preparation method of mycoplasma avian bacterial liquid, wherein the strain for production is mycoplasma gallisepticum or mycoplasma synoviae, and the bacterial liquid comprises the following steps:
(1) inoculating qualified mycoplasma avium production seeds into a mycoplasma avium culture medium according to the volume ratio (V: V) of 10%, fully and uniformly mixing, standing and culturing at 36-37 ℃, and culturing for 6-8 hours;
(2) raising the culture temperature to 38-39 ℃, stirring and culturing for 8-12 hours at 50-100r/min, stopping stirring when the pH value of the culture medium is reduced to 7.2, reducing the culture temperature to 22 ℃, standing for 2-4 hours, and harvesting the culture solution at the lower layer, wherein the harvesting amount is 1/3-1/2 of the total volume of the culture solution;
(3) adding 2-3 times of concentrated mycoplasma avium culture medium into the rest culture solution, repeating the step (2) to continue subculture, and harvesting the lower layer culture solution with the total volume of 1/3-1/2; continuously feeding and culturing for 4-6 generations according to the method to obtain the mycoplasma avian bacterial liquid.
The invention relates to an application of an avian mycoplasma culture medium in preparation of avian mycoplasma vaccines.
The invention relates to application of mycoplasma avium bacterial liquid in preparation of mycoplasma avium vaccines.
Has the advantages that: the preparation method of the invention improves the antigen content of the mycoplasma avian bacterial liquid, simplifies the production process, reduces the production cost, reduces the occurrence probability of vaccine immune side reactions and improves the vaccine immune effect.
Compared with the prior art, the invention has the following advantages:
(1) the invention adopts a novel culture medium, adjusts the culture temperature in the culture process, obviously improves the antigen content of the mycoplasma avian bacterial liquid, and the titer of the bacterial liquid reaches up to 1013-14CCU/ml is higher than the quality standard of the prior vaccine semi-finished bacterial liquid.
(2) The method adopts a fed-batch culture mode, only prepares the seeds for producing the avian mycoplasma before the first culture, omits the preparation of the seeds for producing each generation in the later period, simplifies the production process, saves the labor and shortens the production period by more than 20 days.
(3) The mycoplasma avian bacterial liquid can be diluted to prepare a vaccine, so that the production cost is reduced; and the purity of the antigen of the finished vaccine is high, impurities such as serum protein and metabolites are greatly reduced, the occurrence probability of immune side reactions is reduced, and the immune effect of the vaccine is improved.
Detailed Description
The present invention is further illustrated in detail by the following examples, but it should be noted that the scope of the present invention is not limited by these examples at all.
Example 1
The avian mycoplasma culture medium is composed of an avian mycoplasma basic culture medium and auxiliary components, wherein the basic culture medium is a conventional avian mycoplasma liquid culture medium, and the auxiliary components are composed of gelatin, tween 80 and calcium chloride.
The basic culture medium is an improved Frey's culture medium.
The dosage of the auxiliary components is the following components in the mass volume ratio (m: V) of the finished product culture medium:
0.2 to 0.4 percent of gelatin,
tween 800.5-1%,
0.2 to 0.4 percent of calcium chloride.
The invention relates to a preparation method of mycoplasma avian bacterial liquid, wherein the strain for production is mycoplasma gallisepticum or mycoplasma synoviae, and the bacterial liquid comprises the following steps:
(1) inoculating qualified mycoplasma avium production seeds into a mycoplasma avium culture medium according to the volume ratio (V: V) of 10%, fully and uniformly mixing, standing and culturing at 36 ℃, and culturing for 8 hours;
(2) raising the culture temperature to 39 ℃, stirring and culturing for 8-12 hours at 100r/min, stopping stirring when the pH value of the culture medium is reduced to 7.2, reducing the culture temperature to 22 ℃, standing for 4 hours, and harvesting 1/2 percent of the culture solution at the lower layer, wherein the harvesting amount is the total volume of the culture solution;
(3) adding 2 times of concentrated mycoplasma avium culture medium into the residual culture solution, repeating the step (2) to continue subculture, and harvesting the lower-layer culture solution with the total volume of 1/2; continuously feeding and culturing for 4 generations according to the method to obtain the mycoplasma avian bacterial liquid.
The invention relates to an application of an avian mycoplasma culture medium in preparation of avian mycoplasma vaccines.
The invention relates to application of mycoplasma avium bacterial liquid in preparation of mycoplasma avium vaccines.
Example 2
Example 2 differs from example 1 in that: the avian mycoplasma culture medium is composed of an avian mycoplasma basic culture medium and auxiliary components, wherein the basic culture medium is a conventional avian mycoplasma liquid culture medium, and the auxiliary components are composed of gelatin, tween 80 and calcium chloride.
The basic culture medium is a CM culture medium.
The dosage of the auxiliary components is the following components in the mass volume ratio (m: V) of the finished product culture medium:
0.2 to 0.4 percent of gelatin,
tween 800.5-1%,
0.2 to 0.4 percent of calcium chloride.
The invention relates to a preparation method of mycoplasma avian bacterial liquid, wherein the strain for production is mycoplasma gallisepticum or mycoplasma synoviae, and the bacterial liquid comprises the following steps:
(1) inoculating qualified mycoplasma avium production seeds into a mycoplasma avium culture medium according to the volume ratio (V: V) of 10%, fully and uniformly mixing, standing and culturing at 36.5 ℃, and culturing for 6 hours;
(2) raising the culture temperature to 38.5 ℃, stirring and culturing for 8-12 hours at 50r/min, stopping stirring when the pH value of the culture medium is reduced to 7.2, reducing the culture temperature to 22 ℃, standing for 2 hours, and harvesting the culture solution at the lower layer, wherein the harvesting amount is 1/3 of the total volume of the culture solution;
(3) adding 3 times of concentrated mycoplasma avium culture medium into the residual culture solution, repeating the step (2) to continue subculture, and harvesting the lower-layer culture solution with the total volume of 1/3; according to the method, continuous feeding culture is carried out for 6 generations, and mycoplasma avian bacterial liquid is prepared.
Example 3
Example 3 differs from example 1 in that: the avian mycoplasma culture medium is composed of an avian mycoplasma basic culture medium and auxiliary components, wherein the basic culture medium is a conventional avian mycoplasma liquid culture medium, and the auxiliary components are composed of gelatin, tween 80 and calcium chloride.
The basic culture medium is a conventional CM2 liquid culture medium.
The dosage of the auxiliary components is the following components in the mass volume ratio (m: V) of the finished product culture medium:
0.2 to 0.4 percent of gelatin,
tween 800.5-1%,
0.2 to 0.4 percent of calcium chloride.
The invention relates to a preparation method of mycoplasma avian bacterial liquid, wherein the strain for production is mycoplasma gallisepticum or mycoplasma synoviae, and the bacterial liquid comprises the following steps:
(1) inoculating qualified mycoplasma avium production seeds into a mycoplasma avium culture medium according to the volume ratio (V: V) of 10%, fully and uniformly mixing, standing and culturing at 37 ℃, and culturing for 7 hours;
(2) raising the culture temperature to 38 ℃, carrying out stirring culture at the speed of 80r/min for 8-12 hours, stopping stirring when the pH value of the culture medium is reduced to 7.2, reducing the culture temperature to 22 ℃, standing for 3 hours, and harvesting 1/3 percent of the culture solution at the lower layer, wherein the harvesting amount is the total volume of the culture solution;
(3) adding 3 times of concentrated mycoplasma avium culture medium into the residual culture solution, repeating the step (2) to continue subculture, and harvesting the lower-layer culture solution with the total volume of 1/3; continuously feeding and culturing for 5 generations according to the method to obtain the mycoplasma avian bacterial liquid.
Test 1
1. Preparation of avian mycoplasma culture medium
The raw materials for preparing the culture medium are commercially available; strain: the mycoplasma gallisepticum F36 strain, the mycoplasma gallisepticum CR strain, the mycoplasma synoviae (CVCC385 strain) and the mycoplasma gallisepticum R strain are all purchased from Chinese veterinary medicine inspection institute, and the mycoplasma synoviae MS-H strain is obtained from a market vaccine strain and is only used for experimental verification of the method.
(1) Preparing avian mycoplasma culture medium 1
The basic culture medium is CM2 liquid culture medium, and the culture medium is prepared according to 'farming-grazing letters (1998) No. 38'; when preparing the basic components of the CM2 liquid culture medium, adding auxiliary components at the same time, adding 0.2-0.4% of gelatin, 800.5-1% of tween and 0.2-0.4% of calcium chloride according to the mass volume ratio (m: V) of the finished culture medium, performing high pressure treatment at 115 ℃ for 20min, cooling, aseptically adding pig serum, aseptic 25% of yeast extract and penicillin, adjusting the pH value to 7.6-8.0 by using 1.0mol/L NaOH, and preparing the mycoplasma avium culture medium 1. And (5) performing sterile inspection for later use.
(2) Preparing avian mycoplasma culture medium 2
The basic culture medium is CM liquid culture medium, and the culture medium is prepared according to ' Ministry of agriculture ' No. 297 '; when preparing the basic components of the CM liquid culture medium, auxiliary components are added at the same time, 0.2-0.4 percent of gelatin, 0.2-1 percent of tween 800.5 and 0.2-0.4 percent of calcium chloride are added according to the mass volume ratio (m: V) of the finished product culture medium, the mixture is pressurized at 115 ℃ for 20min, pig serum, 25 percent of yeast extract and penicillin are added aseptically after cooling, the PH value is adjusted to 7.6-8.0 by sodium hydroxide, and the avian mycoplasma culture medium 2 is prepared. And (5) performing sterile inspection for later use.
(3) Preparing poultry mycoplasma culture medium 3
The basic culture medium is an improved Frey liquid culture medium, and is prepared according to the appendix of the pharmacopoeia of the people's republic of China (2015 edition); when preparing the basic components of the improved Frey liquid culture medium, auxiliary components are added at the same time, 0.2-0.4 percent of gelatin, 0.2-1 percent of Tween 800.5 and 0.2-0.4 percent of calcium chloride are added according to the mass volume ratio (m: V) of the finished product culture medium, the mixture is pressurized at 115 ℃ for 20min, pig serum, 2 percent of coenzyme I, 1 percent of L-cysteine solution and penicillin are added aseptically after being cooled, the pH value is adjusted to 7.6-8.0 by sodium hydroxide, and the avian mycoplasma culture medium 3 is prepared. And (5) performing sterile inspection for later use.
2. Culture of Mycoplasma gallisepticum (strain F36) with bacterial solution
(1) Inoculating 10% of seeds for producing mycoplasma gallisepticum (F36 strain) in a mycoplasma gallisepticum culture medium 1 according to the volume ratio (V: V), fully mixing uniformly, standing at 37 ℃, culturing for 7 hours, then increasing the culture temperature to 39 ℃, stirring at 80r/min for 10.5 hours, reducing the pH of the culture medium to 7.2, stopping stirring, reducing the culture temperature to 22 ℃, standing for 3 hours, harvesting a lower-layer culture solution, wherein the harvesting amount is 1/2 of the total volume of the culture solution, and measuring the concentration CCU of a bacterial solution. Meanwhile, a control group is set up and produced according to the existing culture process (farming-grazing letter (1998) No. 38).
(2) Adding 2 times of concentrated mycoplasma avium culture medium into the residual culture solution, culturing at 39 ℃ for 8-12 hours under stirring at 80r/min, stopping stirring when the pH of the culture medium is reduced to 7.2, reducing the culture temperature to 22 ℃, standing for 3 hours, harvesting the culture solution at the lower layer, wherein the harvesting amount is 1/2 of the total volume of the culture solution, and determining the concentration CCU of the bacterial liquid; continuously feeding and culturing for 5 generations according to the method, and harvesting the bacterial liquid to be used as a semi-finished product for preparing the vaccine.
(3) Viable bacteria count of the above 1, 2, 4, 6 generations of bacterial liquid and control group bacterial liquid was measured, and the concentration was 4.0 × 1014CCU/ml、3.0×1014CCU/ml、6.0×1013CCU/ml、8.0×1014CCU/ml and 4.0X 109CCU/ml。
3. Culture of mycoplasma gallisepticum (CR strain) bacterial liquid
Mycoplasma gallisepticum CR strain was cultured in Mycoplasma gallisepticum medium 2 according to the method of example 2, and a control group (produced by the conventional method, published by the Ministry of agriculture No. 297) was set up, and viable cell counts of bacterial solutions of generations 1, 2, 4 and 6 and the control group were measured at concentrations of 1.0X 1013CCU/ml、6.0×1013CCU/ml、5.0×1014CCU/ml、7.0×1014CCU/ml and 3.0X 109CCU/ml。
4. Preparation of mycoplasma gallisepticum vaccine
Preparation of live vaccine: the semi-finished bacterial solutions of the strains Mycoplasma gallisepticum F36 of 1, 2, 4 and 6 generations in example 1 were diluted to 4.0X 10 with 0.1M PBS (pH7.2PBS)9CCU/ml, and control bacteria, were prepared according to the protocol draft for the manufacture and testing of Mycoplasma gallisepticum live vaccine (farming letters (1998) No. 38). Spraying the prepared 5 groups of vaccines with SPF chicken of 20 days old, 10 chickens/group and 1 feather/chicken respectively, and setting blank control to avoidAnd observing for 14 days after epidemic, performing challenge with mycoplasma gallisepticum virulent R strain 28 days after immunization, observing for 14 days after challenge, performing dissection to observe pathological changes, and comparing the safety and efficacy of mycoplasma gallisepticum live vaccines by different culture processes as shown in Table 1.
TABLE 1
As can be seen from Table 1, the spray immunization of the mycoplasma gallisepticum live vaccine product prepared by the original production process has slight immune side reaction, the immune protection rate after challenge is 9/10, the spray immunization of the vaccine prepared by the process of the invention has no immune side reaction, and the immunized group of chickens are well protected.
Preparation of inactivated vaccine: the semi-finished bacterial liquid of the Mycoplasma gallisepticum CR strains 1, 2, 4 and 6 generations in example 1 was diluted to 3.0X 10 with 0.1M PBS (pH7.2PBS)9CCU/ml and control bacterial liquid were prepared according to the protocol draft (Ministry of agriculture, No. 297) for the manufacture and testing of Mycoplasma gallisepticum inactivated vaccine. The prepared 5 groups of vaccines are injected intramuscularly to immunize SPF chickens of 40 days old, 10 vaccines/group and 1 feather vaccine/vaccine respectively, a blank control is set, after immunization, the vaccines are observed for 14 days, the vaccines are attacked by mycoplasma gallisepticum virulent R strain 28 days after immunization, after attacking, the vaccines are observed for 14 days, the pathological changes are observed by dissection, and the safety and efficacy comparison results of the mycoplasma gallisepticum inactivated vaccines of different culture processes are shown in Table 2.
TABLE 2
As can be seen from Table 2, the mycoplasma gallisepticum inactivated vaccine product prepared by the original production process has slight immune side reaction, the immune protection rate after challenge is 9/10, the vaccine prepared by the process of the invention has no immune side reaction, and the immunized group chickens are well protected.
5. Mycoplasma synoviae bacterial liquid culture and vaccine preparation
(1) Inoculating 10% of seeds for producing mycoplasma synoviae (MS-H strain) into a mycoplasma gallisepticum culture medium 3 according to the volume ratio (V: V), fully and uniformly mixing, standing and culturing at 36 ℃, culturing for 8 hours, then increasing the culture temperature to 38 ℃, stirring and culturing at 100r/min for 9 hours, reducing the pH of the culture medium to 7.2, stopping stirring, reducing the culture temperature to 22 ℃, standing for 4 hours, harvesting a lower-layer culture solution, wherein the harvesting amount is 1/3 of the total volume of the culture solution, and measuring the concentration CCU of a bacterial solution.
(2) Adding 3 times of concentrated mycoplasma avium culture medium into the residual culture solution to the original volume, then stirring and culturing at 38 ℃ for 8-12 hours at 100r/min, stopping stirring when the pH of the culture medium is reduced to 7.2, reducing the culture temperature to 22 ℃, standing for 4 hours, harvesting the culture solution at the lower layer, wherein the harvesting amount is 1/3 of the total volume of the culture solution, and determining the concentration CCU of the bacterial liquid; continuously feeding and culturing for 5 generations according to the method, and harvesting the bacterial liquid to be used as a semi-finished product for preparing the vaccine.
(3) The viable count of the obtained bacteria liquid of 1, 2, 4 and 6 generations was measured, and the concentration was 3.0X 1014CCU/ml、5.0×1014CCU/ml、7.0×1013CCU/ml and 8.0X 1014CCU/ml。
(4) Diluting the semi-finished bacterial liquid of the strains F36 of the 1, 2, 4 and 6 generations of mycoplasma synoviae to 4.0 × 10 by 0.1M PBS (pH7.2PBS)9CCU/ml, refer to farming letter (1998) No. 38 for the preparation of live vaccine of M.synoviae. The prepared 4 groups of vaccines are respectively sprayed to immunize SPF (specific pathogen free) chickens of 40 days old, 10 vaccines/group and 1 feather vaccine/group, an imported vaccine control group and a blank control group are set, 14 days are observed after immunization, the mycoplasma synoviae is used for virus challenge 28 days after immunization, 14 days are observed after virus challenge, pathological changes are observed by dissection, and the comparison results of the safety and the efficacy of the inactivated live mycoplasma gallisepticum vaccine prepared by different culture processes are detailed in a table 3.
TABLE 3
As can be seen from Table 3, the imported control chicken mycoplasma synoviae live vaccine has slight immune side reaction when being subjected to spray immunization, the immune protection rate after challenge is 9/10, the vaccine prepared by the process of the invention has no immune side reaction when being subjected to spray immunization, and the immunized group of chickens are well protected.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the foregoing description only for the purpose of illustrating the principles of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims, specification, and equivalents thereof.
Claims (4)
1. A mycoplasma avium culture medium is characterized in that: the avian mycoplasma culture medium consists of an avian mycoplasma basic culture medium and auxiliary components, wherein the basic culture medium is a conventional avian mycoplasma liquid culture medium, and the auxiliary components consist of gelatin, tween 80 and calcium chloride; the basic culture medium is any one of a conventional CM2 liquid culture medium, a CM culture medium or a modified Frey's culture medium; the dosage of the auxiliary components is the following components in the mass volume ratio (m: V) of the finished product culture medium:
0.2 to 0.4 percent of gelatin,
tween 800.5-1%,
0.2 to 0.4 percent of calcium chloride.
2. A preparation method of mycoplasma avium bacterial liquid is characterized by comprising the following steps: the strain for production is mycoplasma gallisepticum or mycoplasma synoviae, and the bacterial liquid comprises the following steps:
(1) inoculating qualified mycoplasma avium production seeds into the mycoplasma avium culture medium according to the volume ratio (V: V) of 10%, fully and uniformly mixing, standing and culturing at 36-37 ℃, and culturing for 6-8 hours;
(2) raising the culture temperature to 38-39 ℃, stirring and culturing for 8-12 hours at 50-100r/min, stopping stirring when the pH value of the culture medium is reduced to 7.2, reducing the culture temperature to 22 ℃, standing for 2-4 hours, and harvesting the culture solution at the lower layer, wherein the harvesting amount is 1/3-1/2 of the total volume of the culture solution;
(3) supplementing 2-3 times of concentrated mycoplasma avium culture medium of claim 1 to the rest culture solution, repeating the step (2) to continue subculture, and collecting the lower layer culture solution with total volume of 1/3-1/2; continuously feeding and culturing for 4-6 generations according to the method to obtain the mycoplasma avian bacterial liquid.
3. Use of the avian mycoplasma culture medium of claim 1 in the preparation of an avian mycoplasma vaccine.
4. The application of the mycoplasma avium bacterial liquid prepared by the preparation method of the mycoplasma avium bacterial liquid in preparing mycoplasma avium vaccines according to claim 2.
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