Swine mycoplasmal pneumonia vaccine strain and application thereof
By optimizing culture conditions and adjuvant use, the CJ03 strain of porcine mycoplasma pneumoniae vaccine was provided, solving the existing problems of isolation and culture of porcine mycoplasma pneumonia vaccines, and achieving efficient and low-cost vaccine production and excellent immune protection.
Patent Information
- Application Number
- CN202510963666.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-14
- Publication Date
- 2025-10-21
AI Technical Summary
The existing porcine mycoplasma pneumonia vaccines are difficult to isolate, have high cultivation requirements and are costly, and the inactivated vaccines on the market are expensive, making it difficult to meet the demand for efficient and low-cost vaccine production.
A vaccine strain of Mycoplasma hyopneumoniae CJ03 is provided. By optimizing culture conditions and adjuvant use, it achieves rapid growth, high protein content and high immunogenicity, and can be administered via a simple intramuscular injection method.
The cultivation time was shortened, the production cost was reduced, the immune protection effect of the vaccine was improved, the pneumonia lesion reduction rate reached 92.00%, and the vaccine had good safety and immunogenicity.
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Figure CN120818461A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a porcine mycoplasma pneumonia vaccine strain and application thereof, belonging to the field of biotechnology. Background Art
[0002] Mycoplasma pneumonia, also known as porcine enzootic pneumonia, is a chronic, contagious respiratory disease caused by Mycoplasma hyopneumoniae (Mhp). It is one of the primary causes of porcine respiratory disease syndrome (PRS). Numerous studies have demonstrated that Mhp interacts with other pathogens and environmental factors to synergize with other pathogens, and that Mhp-infected pathogens can increase the severity and potential persistence of related diseases, exacerbating respiratory illnesses and leading to respiratory disease syndromes, increased mortality, and significant economic losses for the swine industry. Furthermore, Mhp can cause immunosuppression, rendering other vaccines ineffective. Therefore, vaccination remains the preferred treatment for this disease, and the development of relevant vaccines is crucial for its prevention and control.
[0003] Currently, commercially available attenuated vaccines for porcine mycoplasma pneumonia require intrathoracic or nasal administration, requiring high technical expertise. Inactivated vaccines for porcine mycoplasma pneumonia can be administered via simpler methods, such as intramuscular injection. However, these vaccines are currently relatively expensive. Therefore, isolating a safe, immunogenic strain that can be used to prepare an inactivated porcine mycoplasma pneumonia vaccine is crucial in the field. However, the isolation of Mycoplasma hyopneumoniae is susceptible to the influence of other pathogens, such as Mycoplasma hyorhinis, making it difficult to isolate. Furthermore, its in vitro cultivation requires high nutritional requirements and results in a slow growth rate. Therefore, isolating vaccine strains with excellent growth performance, high antigen content, and low production costs is more beneficial for the production, manufacture, and application of porcine mycoplasma pneumonia vaccines.
[0004] In view of the above problems, the present invention is proposed. Summary of the Invention
[0005] To address the above technical problems, the present invention provides a porcine mycoplasma pneumonia vaccine strain and its use in the preparation of a porcine mycoplasma pneumonia vaccine. The porcine mycoplasma pneumonia vaccine strain has a fast growth rate, a high viable count harvested from culture, a high protein content, and good safety and immunogenicity.
[0006] The first object of the present invention is to provide a porcine mycoplasma pneumonia vaccine strain, named Mycoplasma hyopneumoniae CJ03 strain (Mycoplasma hyopneumoniae CJ03), which was deposited in the Wuhan University Collection Center on July 5, 2025, with a deposit number of CCTCC NO. V202528, and the deposit address is the Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province (opposite the First Affiliated Primary School of Wuhan University).
[0007] The second object of the present invention is to provide the use of the porcine mycoplasma pneumonia vaccine strain in preparing vaccines.
[0008] The third object of the present invention is to provide a porcine mycoplasma pneumonia vaccine, which comprises the porcine mycoplasma pneumonia vaccine strain.
[0009] In some embodiments, the vaccine further comprises a pharmaceutically acceptable adjuvant, immunopotentiator or other auxiliary material.
[0010] In some embodiments, the adjuvant is M103 adjuvant.
[0011] In some preferred embodiments, the volume ratio of the antigen to the adjuvant is 3:1.
[0012] The third object of the present invention is to provide a method for preparing a porcine mycoplasma pneumonia vaccine, which comprises the following steps:
[0013] 1) Dissolving the porcine Mycoplasma pneumonia vaccine strain in culture medium, subculture the strain in Friis medium at a ratio of 1:15, and incubate the culture in a constant temperature shaking incubator at 36-38° C. at 50-75 rpm for 30-36 hours. When the bacterial solution changes color and the pH drops to 6.7-6.8, collect the bacterial solution and use it as primary seed;
[0014] 2) Take the first-level seeds and subculture them in Friis medium at a ratio of 1:15. Place them in a constant temperature shaking incubator at 36-38°C and shake at 50-75 rpm for 30-36 hours. When the bacterial solution changes color and the pH drops to 6.7-6.8, collect the bacterial solution and use it as the second-level seeds.
[0015] 3) Take the secondary seeds, subculture them in Friis medium at a ratio of 1:15, culture them at 36-38°C for 36 hours, harvest them, and concentrate them into a 1 / 100 bacterial suspension.
[0016] In some embodiments, the culture is a shake flask culture, a small-scale or large-scale reactor culture;
[0017] In some embodiments, the shake flask culture condition is shaking culture at 50-75 r / min, and the reactor culture condition is stirring culture at 25-50 r / min.
[0018] In some embodiments, the preparation method further includes inactivation and seedling preparation steps.
[0019] In some embodiments, the inactivation step is to add formaldehyde solution to the bacterial suspension prepared in claim 6 at a rate of 0.2% of the total amount of the prepared bacterial suspension, and inactivate the suspension at 36-38° C. for 2 hours.
[0020] In some embodiments, the seedling preparation step is to mix the inactivated bacterial solution with the adjuvant according to claim 5 at a volume ratio of 3:1 and stir evenly for 1 hour.
[0021] Compared with the prior art, the present invention has the following beneficial effects:
[0022] 1) The porcine mycoplasma pneumonia vaccine strain provided by the present invention has a fast growth rate, a short culture time, and a strong reproductive capacity. It can be cultured to reach the highest concentration of bacterial content within 36 hours, which greatly shortens the culture time and saves production costs.
[0023] 2) The porcine mycoplasma pneumonia vaccine strain provided by the present invention has a high concentration of protein. The antigen content measured by ELISA after culture can be as high as 681-743 ng / mL, and the protein content of 1 / 100 bacterial suspension measured by BCA method can reach 7.27-9.65 mg / mL, ensuring that it has good immunogenicity after being prepared into an inactivated vaccine.
[0024] 3) The porcine mycoplasma pneumonia vaccine strain provided by the present invention has complete antigenic fragments after inactivation, which can ensure that the prepared inactivated vaccine has excellent immunogenicity.
[0025] 4) The porcine mycoplasma pneumonia vaccine strain provided by the present invention has excellent immune protection effect, with a protection effect of 5 / 5 on clinical symptoms and a pneumonia lesion reduction rate of 92.00%. It can not only effectively protect animals from clinical symptoms, but also effectively reduce the severity of lesions at the site of disease.
[0026] Deposit of biological materials:
[0027] It was named Mycoplasma hyopneumoniae-CJ03 and was deposited in the Wuhan University Collection Center on July 5, 2025. The deposit number is CCTCC NO. V202528. The deposit address is the Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province (opposite the First Affiliated Primary School of Wuhan University). BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 This is a graph showing the PCR identification results in Example 1 of the present invention, where M is DL 2000 DNA Marker, - is a negative control, + is a positive control (Mycoplasma hyopneumoniae J strain), and 1 to 20 are diseased samples;
[0029] Figure 2This is a graph showing the results of multiplex PCR identification in Example 1 of the present invention, wherein M represents DL 2000 DNA Marker, - represents a negative control, Mhp represents a positive control for Mycoplasma hyopneumoniae (strain J), Mhr represents a positive control for Mycoplasma hyorhinis, Mfl represents a positive control for Mycoplasma suis, and 3 to 18 represent 14 diseased material samples;
[0030] Figure 3 This is a diagram showing the results of SDS-PAGE analysis in Example 1 of the present invention, wherein M is a protein marker, 1 is the Mhp-10 strain, and 2 is the Mhp-18 strain;
[0031] Figure 4 This is a graph showing the PCR amplification results of the P36 gene of Mycoplasma hyopneumoniae Mhp-10 strain in Example 2 of the present invention, wherein M represents DL 2000 DNA Marker, - represents the negative control, 1 represents the amplified product of strain J, and 2 represents the amplified product of strain Mhp-10;
[0032] Figure 5 This is a graph showing the PCR identification results of the recombinant plasmid of Mycoplasma hyopneumoniae Mhp-10 strain in Example 2 of the present invention, wherein M is DL 2000 DNA Marker, - is a negative control, 1 is the amplified product of strain J, and 2 is the amplified product of strain Mhp-10;
[0033] Figure 6 This is a comparison chart of the similarity of the P36 gene between the Mycoplasma hyopneumoniae Mhp-10 strain and other strains in Example 2 of the present invention;
[0034] Figure 7 This is a phylogenetic tree diagram of the P36 gene of Mycoplasma hyopneumoniae Mhp-10 strain and other strains in Example 2 of the present invention. DETAILED DESCRIPTION
[0035] The present invention will be further described below with reference to specific examples so that those skilled in the art can better understand the present invention and implement it, but the examples are not intended to limit the present invention.
[0036] Example 1: Pathogen Isolation and Identification
[0037] 1. Preparation of lung tissue suspension: A total of 20 samples of lung tissue suspected of swine mycoplasma pneumonia were collected from a pig farm in Xinjiang. After being soaked in PBS buffer containing 1000 U / mL penicillin, the surface of the lesions was scalded with a hot plate, and the surface tissue was removed with sterile surgical scissors. 0.5 g of lung tissue was minced and placed in a mortar. 5 mL of Friis medium was added to prepare a lung tissue suspension.
[0038] 2. PCR identification of lung tissue suspension:
[0039] Nucleic acid was extracted from the lung tissue suspension according to the instructions of the nucleic acid extraction kit (Takara, catalog number 9766), and PCR and multiplex PCR identification of the P36 gene were performed.
[0040] The expected length of the amplified fragment is about 427 bp, and the upstream and downstream primers are:
[0041] P1: 5'-TTACAGCGGGAAGACC-3' (SEQ ID NO. 1);
[0042] P2: 5'-CGGCGAGAAACTGGATA-3' (SEQ ID NO. 2);
[0043] The PCR amplification system consisted of 2 μl of template DNA, 0.5 μl of 10 μmol / L primer P1, 0.5 μl of 10 μmol / L primer P2, and 12.5 μl of PCR reaction solution, which was made up to 25 μl with ddH2O. The PCR reaction conditions were: initial denaturation at 95°C for 5 minutes; denaturation at 94°C for 1 minute, annealing at 62°C for 1 minute, and extension at 72°C for 1 minute, repeated for 35 cycles of denaturation / annealing; and extension at 72°C for 10 minutes. The PCR product was stored at 2–8°C and subjected to electrophoresis on a 1.0% agarose gel.
[0044] The DNA extracted from the collected lung tissue suspension was subjected to PCR reaction according to the above amplification system. The results showed that the target fragment of about 427 bp was amplified from the nucleic acid of 14 lung tissue suspensions (amplification results are shown in Figure 1 ).
[0045] 3. Multiplex PCR identification:
[0046] The following method was used to perform multiplex PCR identification on the 14 lung tissue suspensions identified as containing the target fragments. The samples were further screened. The target fragments amplified from Mycoplasma hyopneumoniae were 1000 bp in length, 1129 bp in length for Mycoplasma hyorhinis, and 754 bp in length for Mycoplasma suis. The primers for the multiplex PCR were:
[0047] mhp-f: 5'-TTCAAAGGAGCCTTCAAGCTTC-3' (SEQ ID NO.3);
[0048] mhr-f: 5'-GGGAAGAAAAAAATTAGGTAGGG-3' (SEQ ID NO. 4);
[0049] mfl-f: 5'-CGGGATGTAGCAATACATTCAG-3' (SEQ ID NO.5);
[0050] mr: 5'-AGAGGCATGATGATTTGACGTC-3' (SEQ ID NO.6);
[0051] The multiplex PCR amplification system consisted of 2 μl of template DNA, 8 pmol of each upstream primer, 12 pmol of each downstream primer, and 12.5 μl of PCR reaction solution, which was made up to 25 μl with ddH₂O. The PCR reaction conditions were: initial denaturation at 95°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute; the denaturation / annealing cycle was repeated for 35 cycles, followed by extension at 72°C for 10 minutes. The PCR products were stored at 2–8°C and subjected to 1.0% agarose gel electrophoresis.
[0052] Amplification results such as Figure 2 As shown, 5 samples, namely, samples 4, 6, 10, 13 and 18, were Mycoplasma hyopneumoniae strains without contamination by Mycoplasma hyorhinis and Mycoplasma suis.
[0053] 4. Isolation and cultivation of strains:
[0054] The supernatants of the five identified lung tissue suspensions were inoculated into Friis medium containing 50% hyorhinitis Mycoplasma-specific serum and cultured in a constant temperature shaking incubator at 36-38°C. Blind culture was performed every 5 days, and the pH value of the culture medium was observed daily. 50% hyorhinitis Mycoplasma-specific serum was added to the first three generations. When the culture medium was passed to the 6th to 8th generations, the color change time of the culture medium of two strains (Mhp-10 and Mhp-18) was stable. 0.1 mL of the harvested culture was taken out and inoculated onto a solid culture medium plate. The plate was cultured in a 36-38°C 5% CO2 incubator for 10 days. Pinpoint-sized colonies were visible. Single colonies with neat edges, loose peripheral texture, and dense center were picked under a microscope and inoculated into Friis medium for culture. The strain grew stably on the culture medium for 2-3 days, and the bacterial count could reach 1.0×10 9-10 CCU / mL, and the strain was saved after three replicates.
[0055] 5. Identification of strains:
[0056] The two preserved strains (Mhp-10 and Mhp-18) were identified as follows:
[0057] Cultural and morphological characteristics: The two strains were cultured in Friis medium for 2-3 days, and the cultures were slightly turbid. Liquid culture smears stained with Giemsa stain revealed typical polymorphic forms, including rings, short chains, and bipolar shapes, under a high-power microscope. After culturing on Friis solid medium for 7-10 days, pinpoint-sized colonies were visible to the naked eye. Microscopic observation revealed circular colonies with a loose periphery and a dense center, and a rough, granular surface, consistent with mycoplasma characteristics.
[0058] Growth inhibition test: Two strains were spread on Friis solid medium and kept at 36-38°C to allow the agar surface to dry slightly. A paper disc soaked with Mycoplasma hyopneumoniae antiserum was placed on the agar surface. After 14 days of culture, both strains showed inhibition zones larger than 2.0 mm, consistent with the characteristics of mycoplasma.
[0059] Biochemical test: The biochemical reactions of the two strains are the same as those of the standard strain of Mycoplasma hyopneumoniae (strain J), which is consistent with the characteristics of mycoplasma. The results of biochemical test identification are shown in Table 1:
[0060] Table 1 Biochemical test results of two strains
[0061] project Standard strain (J strain) Strain 1 (Mhp-10 strain) Strain 2 (Mhp-18 strain) Hemolytic - - - Glucose fermentation + + + Hydrolyzed arginine - - - Film and spot formation - - - Hematoadsorption + + +
[0062] Note: “-” indicates negative; “+” indicates positive.
[0063] Virulence test: Two strains were injected into the trachea of five healthy susceptible pigs. Autopsies were performed 28 days after infection. Some piglets became ill and showed lung lesions similar to suis pneumonia. "Meat-like" or "pancreatic-like" parenchymal lesions appeared in the cardiac lobe, apical lobe, anterior 1 / 3 of the diaphragmatic lobe, and middle lobe of the lungs.
[0064] Immunogenicity testing: Fifteen healthy, susceptible pigs aged 3-4 weeks were divided into three groups of five pigs each. Two groups received 2 mL of vaccine prepared from two strains injected intramuscularly into the neck, while one group received no injection as a control. The pigs were housed in isolation under the same conditions. Twenty-eight days after immunization, the pigs, along with the control pigs, were injected with Mycoplasma hyopneumoniae CJ strain histotoxin into their trachea. The pigs were observed for 28 consecutive days, then autopsied. Pneumonia lesions were scored using a 28-point scoring system, and the percentage reduction in pneumonia lesions was calculated. The percentage reduction in pneumonia lesions was 82.61% for the Mhp-10 strain and 68.12% for the Mhp-18 strain, indicating that the Mhp-10 strain exhibited superior immunogenicity.
[0065] After separation by SDS-PAGE, it was found that the Mhp-10 strain had a complete range of antigenic fragments. In comparison, the Mhp-18 strain had a smaller coverage of antigenic fragments (see the results). Figure 3 ).
[0066] Based on the above results, the Mhp-10 strain was identified as a candidate vaccine strain.
[0067] Example 2: Gene Sequencing
[0068] The P36 gene of the Mhp-10 strain obtained by screening was amplified according to the PCR amplification method in Example 1 (see Figure 4), and the amplified products were recovered and sequenced to further confirm their evolutionary relationship. The steps of product recovery and sequencing are as follows: the target fragment was recovered using a gel recovery kit (Tian Gen, product number DP209), cloned into the pMT19-T vector, and the recombinant plasmid was constructed. After the correctness was confirmed by PCR (see Figure 5 ) was sequenced by Sangon Biotech (Shanghai) Co., Ltd., and the sequence of the P36 gene fragment of the isolated Mycoplasma hyopneumoniae Mhp-10 strain is shown in SEQ ID NO.7.
[0069] The results of gene similarity and gene evolution tree were analyzed by NCBI BLAST. Figure 6 and 7 As shown, its homology with the standard strain J was 99.5%, with the 168 strain was 99.3%, with the 232 strain was 98.8%, and with the 7448 strain was 99.8%, further confirming that the Mhp-10 strain was Mycoplasma hyopneumoniae.
[0070] Example 3: Screening of adapted strains of Mycoplasma hyopneumoniae CJ strain
[0071] The Mhp-10 strain was inoculated into Friis medium at a ratio of 1:15 and incubated at 36-38°C in a constant-temperature shaking incubator at 75 rpm for 48 hours. The culture was then serially subcultured according to the above conditions. Starting from the fourth generation, the inoculation ratio (1:10-1:20), harvest time (30-48 hours), and aeration were adjusted, along with viable counts and protein content determinations, and the strain was preserved, to screen for adapted strains of the CJ strain of Mycoplasma hyopneumoniae.
[0072] The method for counting viable bacteria is as follows: take the bacterial strain and serially dilute it 10 times until the dilution reaches 10 -1 , 10 -2 , 10 -3 ...10 -12 Use Friis medium as a control and culture in a constant-temperature shaking incubator at 36-38°C for 14 days. Observe and record changes in the culture medium color and turbidity daily until the pH of the culture remains stable. The dilution at which the color change occurs is the CCU titer of the culture.
[0073] The BCA method for protein content determination is as follows: harvested bacterial suspension is centrifuged at 2000 rpm at 4°C for 10 minutes, the pellet is discarded, and the supernatant is centrifuged at 10,000 rpm at 4°C for 60 minutes. The pellet is resuspended in PBS buffer, washed once, and then resuspended in PBS buffer to prepare a 1 / 100 bacterial suspension. After inactivation and ultrasonic dispersion, the suspension is centrifuged at 10,000 rpm for 10 minutes, and the supernatant is collected for protein concentration determination using the BCA method.
[0074] The specific test results of the six adapted strains are shown in Table 2.
[0075] Table 2 Screening results of subcultured adaptive strains
[0076]
[0077]
[0078] As can be seen from Table 2, among the above 6 strains, the number of viable cells and protein content of the Mhp-10-03 strain at different generations of culture were significantly higher than those of the other strains, and it was speculated that it had better immunogenicity and protection ability. Therefore, the Mhp-10-03 strain was determined to be the vaccine strain of the present invention and was named Mycoplasma hyopneumoniae CJ03 strain.
[0079] Comparative Example 1
[0080] CJ03 strain P6 and standard strain J (ATCC) were inoculated at a ratio of 1:15 into Friis medium and ATCC 1699 medium, respectively. The cultures were incubated at 36-38°C in a constant-temperature shaking incubator at 75 rpm for 48 hours. Three subcultures were performed under these conditions. Viable counts, protein content, and antigen content were measured in the third generation to compare the differences between CJ03 and standard strain J. This experiment was repeated three times. The results are shown in Table 3.
[0081] The method for counting viable bacteria is as follows: take the bacterial strain and serially dilute it 10 times until the dilution reaches 10 -1 , 10 -2 , 10 -3 ...10 -12 Use Friis medium as a control and culture in a constant-temperature shaking incubator at 36-38°C for 14 days. Observe and record changes in the culture medium color and turbidity daily until the pH of the culture remains stable. The dilution at which the color change occurs is the CCU titer of the culture.
[0082] The BCA method for protein content determination is as follows: harvested bacterial suspension is centrifuged at 2000 rpm at 4°C for 10 minutes, the pellet is discarded, and the supernatant is centrifuged at 10,000 rpm at 4°C for 60 minutes. The pellet is resuspended in PBS buffer, washed once, and then resuspended in PBS buffer to prepare a 1 / 100 bacterial suspension. After inactivation and ultrasonic dispersion, the suspension is centrifuged at 10,000 rpm for 10 minutes, and the supernatant is collected for protein concentration determination using the BCA method.
[0083] The ELISA method for determining the antigen content is as follows: the harvested bacterial liquid is tested according to the instructions of the Mycoplasma hyopneumoniae sandwich ELISA antigen detection kit (Jiangsu Academy of Agricultural Sciences).
[0084] Table 3 Detection results of CJ03 strain and J strain
[0085]
[0086] As can be seen from Table 3, the viable cell count, protein content and antigen content of the CJ03 strain isolated in the present invention are much higher than those of the standard strain J, and it has better immunogenicity.
[0087] Comparative Example 2
[0088] CJ03 strain P6 and standard strain J (ATCC) were inoculated at a ratio of 1:15 into Friis medium and ATCC 1699 medium, respectively. The culture was placed in a 36-38°C shaking incubator with shaking at 75 rpm for 48 hours. Two generations were subcultured under the same conditions. Growth curves were performed at the third generation, including viable counts and protein content determination.
[0089] The growth curve determination method is as follows: samples are taken 1 to 3 days after culture, and Friis medium and ATCC 1699 medium are used to count viable bacteria and determine protein content.
[0090] The method for counting viable bacteria is as follows: take the bacterial strain and serially dilute it 10 times until the dilution reaches 10 -1 , 10 -2 , 10 -3 ...10 -12 Use Friis medium as a control and culture in a constant-temperature shaking incubator at 36-38°C for 14 days. Observe and record changes in the culture medium color and turbidity daily until the pH of the culture remains stable. The dilution at which the color change occurs is the CCU titer of the culture.
[0091] The BCA method for protein content determination is as follows: harvested bacterial suspension is centrifuged at 2000 rpm at 4°C for 10 minutes, the pellet is discarded, and the supernatant is centrifuged at 10,000 rpm at 4°C for 60 minutes. The pellet is resuspended in PBS buffer, washed once, and then resuspended in PBS buffer to prepare a 1 / 100 bacterial suspension. After inactivation and ultrasonic dispersion, the suspension is centrifuged at 10,000 rpm for 10 minutes, and the supernatant is collected for protein concentration determination using the BCA method.
[0092] The specific test results are shown in Table 4.
[0093] Table 4 Growth curve determination results
[0094]
[0095] As can be seen from Table 4, the CJ03 strain of the present invention has excellent growth performance, and the viable cell count and protein content can reach the highest level in 36 hours, with a viable cell count of 10 11 CCU / mL, and the protein content was 9.65mg / mL, which was much higher than that of the standard strain J, and was of great significance for improving production efficiency and saving production costs.
[0096] Comparative Example 3
[0097] 1. Preparation of inactivated vaccine against porcine mycoplasma pneumonia (CJ03 strain)
[0098] Take the CJ03 strain P6 bacteria, dissolve it with Friis medium, subculture it in Friis medium at a ratio of 1:15, place it in a constant temperature shaking incubator at 36-38°C, and shake and culture it at 50-75r / min for 30-36 hours. When the color of the bacterial solution changes and the pH value drops to 6.7-6.8, collect the bacterial solution as the first-level seed; perform purity test according to the appendix of the "Chinese Veterinary Pharmacopoeia", and it should be pure; subculture should not exceed 5 generations.
[0099] Secondary seed propagation and identification: Take the first-level seeds, subculture them in Friis medium at a ratio of 1:15, place them in a constant temperature shaking incubator at 36-38℃, and shake and culture at 50-75r / min for 30-36 hours. When the color of the bacterial solution changes and the pH value drops to 6.7-6.8, collect the bacterial solution as the second-level seeds; conduct purity test according to the appendix of the "Chinese Veterinary Pharmacopoeia", and it should be pure.
[0100] Preparation of bacterial suspension for seedling production: Take secondary seeds and subculture them in Friis medium at a ratio of 1:15. Harvest after 36 hours of incubation at 36-38°C (shake flasks at 50-75 rpm, reactors at 25-50 rpm). Concentrate into a 1 / 100 bacterial suspension. Determine the protein content of the 1 / 100 bacterial suspension by the BCA method. Dilute the 1 / 100 bacterial suspension based on the determination results. The protein content should be no less than 50 μg / mL. Perform a purity test according to the appendix of the current "Chinese Veterinary Pharmacopoeia" to ensure that there is no bacterial or fungal contamination.
[0101] Inactivation: Add 0.2% formaldehyde solution to the total volume of the prepared bacterial solution and inactivate at 36-38°C for 2 hours. Store the inactivated bacterial solution at 2-8°C. Add the inactivated bacterial solution to Friis medium and incubate in a constant temperature shaking incubator at 36-38°C for 7 days. If the culture does not change color, it is considered to have passed the inactivation test.
[0102] Vaccine preparation: Mix the inactivated bacterial solution with Isco M103 adjuvant in a volume ratio of 3:1, stir evenly for 1 hour, store at 2-8°C, and aseptically divide into portions, seal with a lid, and affix labels.
[0103] 2. Finished product inspection
[0104] Appearance: milky white suspension;
[0105] Sterility test: Test according to the appendix of the current Chinese Veterinary Pharmacopoeia, and no bacteria growth;
[0106] Safety test: Five healthy susceptible pigs aged 3 to 4 weeks were injected intramuscularly with an inactivated Mycoplasma pneumonia vaccine (CJ03 strain) at 4 mL per pig. The pigs were observed for 14 consecutive days, and no adverse reactions or deaths were reported.
[0107] Efficacy test: 5 healthy susceptible pigs aged 3 to 4 weeks were injected intramuscularly with 2.0 mL / head. 28 days after immunization, 5 control pigs were injected into each trachea with Mycoplasma hyopneumoniae CJ strain lung tissue toxin. After 28 days of challenge, the pigs were autopsied and the pneumonia lesions of each pig were scored according to the 28-point scoring method; the pneumonia lesion reduction rate of the immunized pigs was calculated according to the above formula = (the arithmetic mean score of pneumonia in the control pigs - the arithmetic mean score of pneumonia in the immunized pigs) / the arithmetic mean score of pneumonia in the control pigs × 100%.
[0108] Determination of formaldehyde residue: Determined according to the appendix of the current "Chinese Veterinary Pharmacopoeia" and in compliance with regulations.
[0109] 3. Effectiveness test
[0110] Bacteria used for testing: Mycoplasma hyopneumoniae CJ strain lung tissue toxin, kept and supplied by Tiankang Biopharmaceutical Co., Ltd.
[0111] Experimental animals: 15 healthy susceptible pigs aged 3 to 4 weeks.
[0112] Experimental design: The experiment was divided into four groups, with 5 pigs in each group. The first to third groups of experimental animals were injected intramuscularly with the inactivated porcine mycoplasma pneumonia vaccine (CJ03 strain) of the present invention, imported porcine mycoplasma pneumonia inactivated vaccine, and domestic porcine mycoplasma pneumonia inactivated vaccine, respectively, at 2.0 mL / head. The fourth group was the challenge control group and did not receive any treatment. 28 days after immunization, each pig, along with the challenge control group, was injected with the lung tissue toxin of Mycoplasma hyopneumoniae CJ strain through the trachea.
[0113] Observation of clinical symptoms: After the infection, each group of experimental animals was observed at regular times and locations for 28 consecutive days. The infection control group had clinical symptoms such as coughing and abdominal breathing; none of the five pigs in the Tiankang Pharmaceutical vaccine immunization group showed typical clinical symptoms of porcine mycoplasma pneumonia; one pig in the imported vaccine immunization group showed mild coughing and abdominal breathing symptoms, and the other four did not show typical clinical symptoms of porcine mycoplasma pneumonia; three pigs in the domestic vaccine immunization group showed mild coughing and abdominal breathing symptoms, and the other two did not show typical clinical symptoms of porcine mycoplasma pneumonia.
[0114] Efficacy comparison test: 28 days after the challenge, all the test animals were killed and dissected. The pneumonia lesion reduction rates of the pigs in the immunization groups with the vaccine strain of the present invention, the imported inactivated porcine mycoplasma pneumonia vaccine and the domestic inactivated porcine mycoplasma pneumonia vaccine were 92.00%, 84.00% and 61.33%, respectively. The specific pneumonia lesion scores are shown in Table 5.
[0115] Table 5 Efficacy test results
[0116]
[0117] Note: N / A (not Available) means not applicable.
Claims
1. A porcine mycoplasma pneumonia vaccine strain, characterized in that: The vaccine strain is Mycoplasma hyopneumoniae CJ03, which was deposited in the Wuhan University Collection Center on July 5, 2025, with the deposit number CCTCC NO.V202528.
2. Use of the porcine mycoplasma pneumonia vaccine strain according to claim 1 in the preparation of vaccines.
3. A porcine mycoplasma pneumonia vaccine, characterized in that: The vaccine comprises the porcine mycoplasma pneumonia vaccine strain described in claim 1.
4. The vaccine according to claim 3, characterized in that The vaccine also contains pharmaceutically acceptable adjuvants, immunopotentiators or other auxiliary materials.
5. The vaccine according to claim 4, characterized in that The adjuvant is M103 adjuvant; preferably, the volume ratio of the adjuvant to the antigen is 3:
1.
6. A method for preparing a porcine mycoplasma pneumonia vaccine, characterized in that: The following steps are included: 1) Dissolve the porcine mycoplasma pneumonia vaccine strain described in claim 1 in culture medium, subculture the strain in Friis medium at a ratio of 1:15, and culture the strain in a constant temperature shaking incubator at 36-38° C. at 50-75 rpm for 30-36 hours. When the bacterial solution changes color and the pH drops to 6.7-6.8, collect the bacterial solution and use it as primary seed; 2) Take the first-level seeds and subculture them in Friis medium at a ratio of 1:
15. Place them in a constant temperature shaking incubator at 36-38°C and shake at 50-75 rpm for 30-36 hours. When the bacterial solution changes color and the pH drops to 6.7-6.8, collect the bacterial solution and use it as the second-level seeds. 3) Take the secondary seeds and subculture them in Friis medium at a ratio of 1:
15. After culturing at 36-38°C for 36 hours, harvest and concentrate into a 1 / 100 bacterial suspension; Preferably, the culture is shake flask culture, small-scale or large-scale reactor culture; More preferably, the shake flask culture condition is shaking culture at 50-75 r / min, and the reactor culture condition is stirring culture at 25-50 r / min.
7. The preparation method according to claim 6, characterized in that The preparation method also includes the steps of inactivation and seedling preparation.
8. The preparation method according to claim 7, characterized in that The inactivation step comprises adding formaldehyde solution according to 0.2% of the total amount of the prepared bacterial suspension to the bacterial suspension prepared in claim 6, and inactivating the solution at 36-38° C. for 2 hours.
9. The preparation method according to claim 7, characterized in that The seedling preparation step is to mix the inactivated bacterial solution with the adjuvant according to claim 5 in a volume ratio of 3:1 and stir evenly for 1 hour.