CN114134098A - Inactivation method of mycoplasma hyopneumoniae - Google Patents
Inactivation method of mycoplasma hyopneumoniae Download PDFInfo
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Abstract
The invention discloses an inactivation method of mycoplasma hyopneumoniae, which comprises the step of adding diethyleneimine (BEI) into a mycoplasma hyopneumoniae bacterial solution subjected to ultrasonic crushing for inactivation for 24-48 hours. The inactivation method of the mycoplasma hyopneumoniae has the advantages of thorough antigen inactivation and short time consumption, and the vaccine prepared from the inactivated mycoplasma hyopneumoniae has high protein purity, less immune stress and high immune response, and is suitable for market popularization and use.
Description
Technical Field
The invention particularly relates to an inactivation method of mycoplasma hyopneumoniae.
Background
Mycoplasma hyopneumoniae (MPS) is a chronic, contact respiratory infectious disease, also known as swine enzootic pneumonia or swine enzootic pneumonia, the etiology of which was first isolated by Mare, Switzer (1965) and Goodwin et al (1965) from the lung tissue of pneumonia pigs. Mycoplasma hyopneumoniae (Mhp) is a main pathogen of MPS, belongs to mycoplasma, is negative in gram stain, and has the characteristics of wide distribution, high morbidity, low mortality and the like. The mycoplasma hyopneumoniae mainly infects domestic pigs, is mainly transmitted by mosquito bites, needle heads, respiratory bites or mating in swinery, has the infection rate of 80-90 percent, and easily causes the large-area infection and even death of the swinery. Vaccine immunization is one of the most important control measures in mycoplasma hyopneumoniae, wherein the inactivated vaccine can enable pathogens to lose pathogenicity and still keep the immunogenicity of the pathogens, and the immunogenicity of antigens can be effectively improved by selecting a proper purification and inactivation process.
At present, the swine mycoplasma pneumonia live vaccine strains in the market of China comprise 168 strains, RM48 strains and lapinized attenuated strains. Some attenuated live vaccines still retain certain toxicity, are easy to induce certain diseases in some immunodeficiency individuals, and have large stress injury to swinery, troublesome operation and large workload. The inactivated vaccine is fed and injected, the antibody of the inactivated vaccine is delivered to the lung by means of blood circulation, and the mycoplasma hyopneumoniae in the body fluid of the piglet and in the blood circulation system can be neutralized, so that the inactivated vaccine has a good immune effect. In addition, the multiple inactivated vaccines are continuously researched and developed in China, so that multiple prevention and control by one needle are realized, the labor force is reduced, and the prevention and control cost is reduced.
The formaldehyde is one of common mycoplasma hyopneumoniae inactivators, the inactivated virus can achieve a good inactivation effect, but the formaldehyde is a carcinogen, and a series of stress reactions can be generated after the residual free formaldehyde enters the body along with the vaccine. Research shows that the indexes of the BEI inactivated vaccine such as respiratory tract morbidity, lung lesion score, feed conversion rate, daily gain, pig serum antibody level and the like are all better than those of the formaldehyde inactivated vaccine, and the immunity effect and the economic benefit of the mycoplasma hyopneumoniae inactivated vaccine prepared by the BEI inactivation method are better than those of the formaldehyde inactivation method, but the overall immunogenicity is still not ideal.
Disclosure of Invention
In order to solve the problems, the invention provides a method for inactivating mycoplasma hyopneumoniae, which comprises the following steps:
1) taking mycoplasma hyopneumoniae bacterial liquid, concentrating and purifying;
2) and (2) taking the concentrated and purified bacterial liquid obtained in the step 1), carrying out ultrasonic crushing, diluting with a diluent, adding diethylene imine (BEI) for inactivation for 24-48 h, and finally adding sodium thiosulfate to terminate inactivation.
Further, the concentration and purification in the step 1) are performed by concentrating with 100-500 kDa hollow fiber, centrifuging, washing the precipitate with TN buffer for 2-5 times, and freeze-thawing at-80 ℃ for 1 time.
Further, the concentration and purification are performed by concentrating with 300kDa hollow fiber, centrifuging, washing the precipitate with TN buffer 3 times, and freeze-thawing at-80 ℃ 1 time.
Further, the diluent in the step 2) is TN buffer; and diluting to 1/10-1/100 of the volume of the original mycoplasma hyopneumoniae bacterial liquid.
Further, the power of the ultrasonic crushing in the step 2) is 200-500W, the frequency is 2-3 seconds per ultrasonic wave at intervals of 3-4 seconds, the times are 3-5 times, and each time lasts for 8 minutes.
Further, the final concentration of the diethylene imine (BEI) in the step 2) is 1-5 mmol/L, and 3mmol/L is preferable.
Further, the inactivation temperature in the step 2) is 37 ℃, and the time is 24-48 h.
Furthermore, the final concentration of the sodium thiosulfate is 1-5 mmol/L, and preferably 3 mmol/L.
Further, cooling to 2-8 ℃ after inactivation is stopped.
The inactivation method of the mycoplasma hyopneumoniae has the advantages of thorough antigen inactivation and short time consumption, and the vaccine prepared from the inactivated mycoplasma hyopneumoniae has high protein purity, less immune stress and high immune response, and is suitable for market popularization and use.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Detailed Description
Example 1 inactivation of Mycoplasma hyopneumoniae
1) Concentrating and purifying thallus
And (3) taking the mycoplasma hyopneumoniae bacterial liquid, concentrating the mycoplasma hyopneumoniae bacterial liquid by using 300kDa hollow fibers, centrifuging the concentrated mycoplasma hyopneumoniae bacterial liquid to obtain a precipitate, and washing the precipitate for 3 times by using TN buffer. Freezing and thawing once at minus 80 ℃;
2) inactivating
And (3) carrying out ultrasonic crushing on the concentrated and purified bacterial liquid (frequency: ultrasonic treatment is carried out at 200-500W power every time for 2 seconds at intervals of 3 seconds, ultrasonic treatment time is 8 minutes, ultrasonic treatment times are 3 times), diluting the bacterial liquid to 1/10-1/100 of the original volume by using TN buffer, adding BEI until the final concentration is 1-5 mmol/L, inactivating the bacterial liquid at 37 ℃ for 24-48 hours, adding sodium thiosulfate until the final concentration is 1-5 mmol/L, terminating inactivation, and rapidly cooling the bacterial liquid to 2-8 ℃ after termination to obtain the microbial inoculum.
Example 2 inactivation of Mycoplasma hyopneumoniae
1) Concentrating and purifying thallus
And (3) taking the mycoplasma hyopneumoniae bacterial liquid, concentrating the mycoplasma hyopneumoniae bacterial liquid by using 300kDa hollow fibers, centrifuging the concentrated mycoplasma hyopneumoniae bacterial liquid to obtain a precipitate, and washing the precipitate for 3 times by using TN buffer. Freezing and thawing once at minus 80 ℃;
2) inactivating
And (3) taking the concentrated and purified bacterial liquid, carrying out ultrasonic crushing (frequency: ultrasonic treatment is carried out for 2 seconds at 200-500W power every time and is carried out for 3 seconds; ultrasonic treatment time is 8 minutes; ultrasonic treatment times are 4 times), diluting to 1/10-1/100 of the original volume by using TN buffer, adding BEI until the final concentration is 1mmol/L, inactivating for 24-48 hours at 37 ℃, adding sodium thiosulfate until the final concentration is 1mmol/L, terminating inactivation, and rapidly cooling to 2-8 ℃ after termination to obtain the microbial inoculum.
Example 3 inactivation method of Mycoplasma hyopneumoniae of the invention
1) Concentrating and purifying thallus
And (3) taking the mycoplasma hyopneumoniae bacterial liquid, concentrating the mycoplasma hyopneumoniae bacterial liquid by using 300kDa hollow fibers, centrifuging the concentrated mycoplasma hyopneumoniae bacterial liquid to obtain a precipitate, and washing the precipitate for 3 times by using TN buffer. Freezing and thawing once at minus 80 ℃;
2) inactivating
And (3) taking the concentrated and purified bacterial liquid, carrying out ultrasonic crushing (frequency: ultrasonic treatment is carried out for 2 seconds at 200-500W power every time and is carried out for 3 seconds; ultrasonic treatment time is 8 minutes; ultrasonic treatment times are 5 times), diluting to 1/10-1/100 of the original volume by using TN buffer, adding BEI until the final concentration is 5mmol/L, inactivating for 24-48 hours at 37 ℃, adding sodium thiosulfate until the final concentration is 5mmol/L, terminating inactivation, and rapidly cooling to 2-8 ℃ after termination to obtain the microbial inoculum.
The advantageous effects of the present invention are described below by way of test examples.
Test example 1
1 bacterial liquid culture
Inoculating the mycoplasma hyopneumoniae TB strain into a FRIIS culture medium containing 20% of serum and 3mg/ml of glucose according to an inoculation ratio of 10% (v/v), culturing at 37 ℃ for 48-72 hours, and harvesting a bacterial liquid when the color of the culture medium is changed into yellow and the pH value is about 6.8.
2 concentration and purification
Concentrating the prepared mycoplasma hyopneumoniae TB strain bacterial liquid by 300kDa hollow fiber, centrifuging, taking out precipitate, repeatedly washing for 3 times by using TN buffer, and freezing and thawing once at-80 ℃.
3 inactivation mode
3.1 physical inactivation the concentrated and purified bacteria liquid is crushed by an ultrasonic system, ultrasonic treatment is carried out for 3 seconds at intervals of 2 seconds at 270W power every time, ultrasonic treatment is carried out for 8 minutes, and ultrasonic treatment is carried out for 3 times in total.
3.2BEI (diethylene imine) inactivation the concentrated and purified bacterium liquid is inactivated for 24 hours at 37 ℃ by 3mmol/L BEI, the inactivation is stopped by a sodium thiosulfate solution with equal concentration (3mmol/L), and the bacterium liquid is rapidly cooled to 2-8 ℃ for standby after the inactivation is stopped.
3.3 inactivation of Formaldehyde to a final concentration of 0.15% formaldehyde solution at 37 ℃ for 24 hours.
3.4 physical and BEI inactivation the concentrated and purified bacterial liquid is broken by an ultrasonic system, ultrasonic treatment is carried out for 3 seconds at intervals of 2 seconds at 270W power every time, ultrasonic treatment is carried out for 8 minutes, and ultrasonic treatment is carried out for 3 times in total. Diluting the ultrasonically-crushed antigen with TN buffer, adding 3mmol/L BEI, inactivating at 37 ℃ for 24 hours, stopping inactivation with an equal-concentration (3mmol/L) sodium thiosulfate solution, and quickly cooling to 2-8 ℃ for later use after stopping.
4 inactivation test
Respectively taking 1.0ml of inactivated samples of different inactivation modes, adding the inactivated samples into 50ml of mycoplasma hyopneumoniae liquid culture medium, culturing and observing for 5 days at 37 ℃, then taking 0.5ml of the inactivated samples, inoculating the inactivated samples into 4.5ml of mycoplasma hyopneumoniae liquid culture medium, culturing and observing for 10 days at 37 ℃, simultaneously taking 0.2ml of the inoculated mycoplasma hyopneumoniae solid culture medium, and culturing and observing for 10 days at 37 ℃. The experimental result shows that: inactivation was not complete with the sonicated samples. The culture medium color of the two times of liquid culture of other inactivation modes is not changed, and the solid culture sterile colony grows, and the inactivation test is qualified. Specific results are shown in table 1:
TABLE 1 results of inactivation test for different inactivation modes
Note: "+" indicates a color reaction or colony growth; "-" indicates no color reaction or growth of a sterile colony.
5 preparation of inactivated vaccine and detection of efficacy
And (3) respectively taking the inactivated antigen liquid of the BEI inactivated group, the formaldehyde inactivated group and the physical and BEI inactivated groups to prepare vaccines, and performing immune challenge tests on the vaccines. Screening healthy susceptible piglets of 14-21 days old, and having good appearance growth and development and no clinical symptoms such as cough, asthma and the like; the experimental piglets which are negative to the mycoplasma hyopneumoniae antibody are 25, and are randomly divided into 5 groups, and each group has 5 piglets. The 1 st to 3 th groups are vaccine immunization groups, the 4 th group is a challenge control group, the 5 th group is a blank control group, and the groups are separately fed under the same conditions. The specific grouping is shown in table 2 below:
TABLE 2 Experimental grouping
All experimental pigs were injected intratracheally with 5.0ml of virulent strain for mycoplasma hyopneumoniae test (containing 10 minimal pathogenic doses) 21 days after immunization. The observation is continued for 28 days after the challenge. During observation period, dead pigs are roughly dissected to observe lung lesions, all live pigs are sacrificed after the observation is finished, and the lung lesions are roughly dissected to be observed. And (3) judging according to 28-point marking standard of the mycoplasma hyopneumoniae lung lesion index, and calculating the lung lesion reduction rate of each group (shown in a table 3). The results showed that the reduction rate of the lesion index of the pigs in the BEI inactivated group was 57.7%, the reduction rate of the lesion index of the pigs in the formaldehyde inactivated group was 47.4%, and the reduction rate of the lesion index of the pigs in the physical formaldehyde inactivated group was 79.4%. The experimental pigs in the formaldehyde inactivation group have severe stress reactions, and the immunogenicity of the physical inactivation group and the BEI inactivation group is weaker than that of the physical and BEI inactivation groups, so that the vaccine prepared by the BEI inactivated antigen after ultrasonic crushing can generate good immune response to piglets, and the optimal protection effect is achieved.
TABLE 3 inactivated antigen immunogenicity results
6 optimization of physical and BEI inactivation conditions
6.1 screening under inactivation conditions
Adding BEI with different concentrations into the antigen subjected to ultrasonic disruption with different powers, inactivating at 37 ℃, sampling at different times for inactivation termination, and detecting the inactivation thoroughness. The inactivation condition information for each group is shown in table 4.
TABLE 4 inactivation Condition optimization information
6.2 inactivation assay
Respectively taking 1.0ml of inactivated samples of different inactivation tests, adding the inactivated samples into 50ml of mycoplasma hyopneumoniae liquid culture medium, culturing and observing for 5 days at 37 ℃, then taking 0.5ml of inactivated samples, inoculating the inactivated samples into 4.5ml of mycoplasma hyopneumoniae liquid culture medium, culturing and observing for 10 days at 37 ℃, simultaneously taking 0.2ml of inoculated mycoplasma hyopneumoniae solid culture medium, and culturing and observing for 10 days at 37 ℃. The results of the inactivation test for each group are shown in Table 5. Carrying out ultrasonic treatment for 3 seconds at the power of 200W-500W for 2 seconds, carrying out ultrasonic treatment for 8 minutes, and carrying out ultrasonic treatment for 3-5 times. Diluting the ultrasonically-crushed antigen by using TN buffer, adding 1-5 mmol/L BEI, inactivating at 37 ℃ for 24-48 hours, and stopping inactivation by using sodium thiosulfate solution with equal concentration, so that the mycoplasma hyopneumoniae can be effectively inactivated.
TABLE 5 physical and BEI Combined inactivation test results
In conclusion, the inactivation method solves the problems of incomplete physical inactivation and poor immunogenicity of BEI inactivation, does not generate the stress reaction of formaldehyde inactivation, and has thorough antigen inactivation and short time consumption. The vaccine prepared from the inactivated mycoplasma hyopneumoniae has high protein purity, less immune stress and high immune response, and is suitable for market popularization and use.
Claims (9)
1. A method for inactivating mycoplasma hyopneumoniae is characterized by comprising the following steps:
1) taking mycoplasma hyopneumoniae bacterial liquid, concentrating and purifying;
2) and (2) taking the concentrated and purified bacterial liquid obtained in the step 1), carrying out ultrasonic crushing, diluting with a diluent, adding diethylene imine (BEI) for inactivation for 24-48 h, and finally adding sodium thiosulfate to terminate inactivation.
2. The inactivation method of claim 1, wherein: the concentration and purification in the step 1) are carried out by concentrating with 100-500 kDa hollow fiber, centrifuging, washing the precipitate with TN buffer for 2-5 times, and freeze-thawing at-80 ℃ for 1 time.
3. The inactivation method of claim 2, wherein: the concentration and purification are carried out by using 300kDa hollow fiber for concentration, centrifugation, washing the precipitate for 3 times by using TN buffer and freeze thawing for 1 time at minus 80 ℃.
4. The inactivation method of claim 1, wherein: the diluent in the step 2) is TN buffer; the diluted solution is 1/10-1/100 of the original volume.
5. The inactivation method of claim 1, wherein: the power of the ultrasonic crushing in the step 2) is 200-500W, the frequency is 2-3 seconds per ultrasonic and 3-4 seconds apart, the frequency is 3-5 times, and each time is 8 minutes.
6. The method of claim 1, wherein: the final concentration of the diethylene imine (BEI) in the step 2) is 1-5 mmol/L, and 3mmol/L is preferred.
7. The inactivation method of claim 1, wherein: and 2) inactivating at 37 ℃ for 24-48 h.
8. The inactivation method according to claim 1 or 8, wherein: the final concentration of the sodium thiosulfate is 1-5 mmol/L, and 3mmol/L is preferred.
9. The inactivation method of claim 8, wherein: and cooling to 2-8 ℃ after inactivation is stopped.
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| CN110438029A (en) * | 2019-06-25 | 2019-11-12 | 成都天邦生物制品有限公司 | A kind of novel ablation method of mycoplasma hyopneumoniae |
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| CN104271154A (en) * | 2012-04-04 | 2015-01-07 | 硕腾有限责任公司 | Mycoplasma hyopneumoniae vaccine |
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