Preparation method of mucosal immune type mycoplasma bovis live vector vaccine

Abstract

The invention relates to the technical field of mucosal immunity and immunogenicity collaborative optimization, in particular to a preparation method of a mucosal immune type mycoplasma bovis live vector vaccine, attenuated mycoplasma bovis is used as a vector, an inducer X containing an FRET modified nucleic acid aptamer is combined with CRISPR-Cas12a for modification, a virulence gene is knocked out, and a mucosal targeting gene fused with CTB is inserted; carrying out deep codon optimization on the antigen gene, fusing the antigen gene with the glycosylation immunopotentiation peptide, and carrying out novel enzyme digestion connection to construct a recombinant vector; and intelligently fermenting in an improved culture medium, centrifuging, resuspending by using a special protective agent, and freeze-drying to obtain the vaccine. According to the preparation method, the mucous membrane targeting and immunogenicity of the vaccine are improved, and the production process is optimized; mucosal immunity can be quickly stimulated, various immune reactions are activated, the vaccine activity and quality are guaranteed, and the mycoplasmosis bovis is efficiently prevented.

Description

Technical Field

[0001] The present invention relates to the technical field of synergistic optimization of mucosal immunity and immunogenicity, and specifically to a preparation method of a mucosal immune type Mycoplasma bovis live vector vaccine. Background Art

[0002] Mycoplasma bovis disease is a serious contagious disease of cattle caused by Mycoplasma bovis, which is widely prevalent globally and has brought huge economic losses to the cattle industry. Mycoplasma bovis can infect multiple tissues and organs of cattle, such as the respiratory tract, mammary gland, joints, etc., causing various diseases such as pneumonia, mastitis, arthritis, etc., resulting in retarded growth and development of cattle, decreased milk production, reduced reproductive performance, and even death. Traditional vaccine research and development mainly focuses on inactivated vaccines and subunit vaccines, but there are obvious limitations. Although inactivated vaccines have high safety, their immunogenicity is weak, it is difficult to stimulate the body to produce a lasting and effective immune response, often requiring multiple immunizations, and the immune protection period is short; although subunit vaccines can accurately target specific antigens, their preparation process is complex, costly, and the immune effect is not ideal when used alone, and they cannot effectively resist the infection of Mycoplasma bovis.

[0003] With the development of genetic engineering technology, live vector vaccines have become a research hotspot. Live vector vaccines use attenuated or non-toxic microorganisms as vectors, introduce foreign antigen genes into them, and utilize the proliferation characteristics of the vectors in the host body to continuously stimulate the immune system, and theoretically can produce a more lasting and efficient immune protection. However, existing Mycoplasma bovis live vector vaccines still face many problems in practical applications. Most live vector vaccines are difficult to effectively localize in mucosal tissues. Mycoplasma bovis mainly infects through mucosal routes such as the respiratory tract and reproductive tract. If the vaccine cannot be enriched on the mucosal surface and stimulate a mucosal immune response, it is difficult to resist pathogen invasion in the initial stage of infection. Existing mucosal targeting strategies mostly use simple protein fusion or gene fragment insertion, with low targeting efficiency and unable to meet actual needs.

[0004] The antigen structure of Mycoplasma bovis is complex, and there is antigen variation. Existing live vector vaccines often have difficulty effectively stimulating the body's immune response to multiple antigens. In addition, during the metabolism of the vaccine in the host body, the antigen protein is easily degraded, resulting in a significant reduction in the immune effect. At the same time, the vaccine vector itself may cause an immune rejection reaction in the host, affecting the safety and effectiveness of the vaccine.

[0005] Traditional fermentation culture is difficult to precisely control the growth of bacteria and antigen expression, resulting in unstable vaccine quality. There are also problems with unreasonable cryoprotectant formulations and unoptimized process parameters in preparation processes such as freeze-drying, which cause a decrease in the activity of bacteria during storage and transportation of the vaccine, affecting the vaccine efficacy. With the large-scale and intensive development of the cattle industry, the demand for safe, efficient, and stable Mycoplasma bovis vaccines is becoming more urgent, and there is an urgent need to develop new preparation methods to solve the above problems. Summary of the Invention

[0006] (1) Technical problems to be solved

[0007] In view of the deficiencies of the prior art, the present invention provides a method for preparing a mucosal immune type Mycoplasma bovis live vector vaccine.

[0008] (2) Technical solutions

[0009] A method for preparing a mucosal immune type Mycoplasma bovis live vector vaccine includes the following steps:

[0010] S1, strain modification: Select an attenuated Mycoplasma bovis strain as a vector, and add a novel gene editing inducer X to the culture system; the inducer X is an aptamer - small molecule compound conjugate Aptamer - N3 - L - Small - molecule containing an azide group, the aptamer is single - stranded DNA or RNA and is modified by fluorescence resonance energy transfer (FRET), L is a linker; shake - culture for 8 - 12 hours; modify with an optimized CRISPR - Cas12a system to knockout virulence - related genes and simultaneously insert a mucosal targeting gene fragment;

[0011] S2, antigen gene acquisition and modification: Extract the gene encoding the major surface antigen protein from a highly pathogenic Mycoplasma bovis strain, perform in - depth codon optimization on this gene, and conduct multiple rounds of optimization in combination with a bovine host - preference codon database; meanwhile, fuse the gene fragment encoding an immune - enhancing peptide with the antigen gene through a novel scarless ligation PCR technique;

[0012] S3, recombinant vector construction: Insert the fusion antigen gene obtained in step S2 into the plasmid vector of the Mycoplasma bovis strain modified in step S1 to construct a recombinant Mycoplasma bovis strain; perform double - enzyme digestion on the plasmid vector and the fusion antigen gene using a novel restriction enzyme combination, and then ligate under the action of T4 - like DNA ligase, and introduce the recombinant plasmid into the modified Mycoplasma bovis strain through pulse - field mediated nanopore electroporation technology;

[0013] S4, fermentation culture: Inoculate the recombinant Mycoplasma bovis strain into a modified fermentation medium, and the modified fermentation medium contains basic nutritional components, a novel metabolic regulator Z, and amino acid - chelated iron;

[0014] S5, vaccine preparation: After fermentation, centrifuge the fermentation broth for 15 - 20 minutes to collect the cell precipitate; resuspend the cells with a phosphate - buffered solution containing a mucosal protectant, adjust the cell concentration to 1×108 to 1×109 CFU / mL, add a lyophilization protectant, and prepare a mucosal immune type Mycoplasma bovis live vector vaccine through a lyophilization process.

[0015] Preferably, after the transformation of strain S1, whole-genome methylation sequencing analysis is also performed on the transformed strain to evaluate the impact of gene editing on the epigenetics of the strain, ensuring that the virulence genes are knocked out and the mucosal targeting gene fragments are correctly inserted without affecting the normal physiological functions of the strain.

[0016] Preferably, during the acquisition and modification of the S2 antigen gene, in vitro expression verification is also carried out on the fusion antigen gene. The mammalian cell expression system is used to express the fusion antigen protein, and Western blot and immunofluorescence techniques are used to detect the protein expression level and immunoreactivity.

[0017] Preferably, in the novel gene editing inducer X, the FRET-modified fluorescent groups of the nucleic acid aptamer are Cy3 and Cy5, and its modification rate is monitored by high-performance liquid chromatography to reach more than 90%.

[0018] Preferably, in the amino acid chelated iron, the molar ratio of histidine to glycine and iron ions is 1:2:1, and the iron content is determined by atomic absorption spectrometry.

[0019] Preferably, in the mucosal protectant, the substitution degree of carboxymethylated chitosan quaternary ammonium salt is 70%-90%, which is determined by nuclear magnetic resonance spectroscopy.

[0020] Preferably, the novel metabolic regulator Z is prepared by compounding dimethyl sulfoxide and phytosterol at a mass ratio of 1:1, and the phytosterol is treated by cyclodextrin inclusion. The iron content in the amino acid chelated iron is 15%-20%, and the amino acids are a mixture of histidine and glycine in a ratio of 1:2.

[0021] Preferably, to implement the preparation method described in any one of the above, the following equipment and operations are included:

[0022] Use a constant temperature shaking incubator with high-precision temperature and rotation speed control for strain transformation culture, set the temperature at 37°C and the rotation speed at 180 r / min, and be equipped with a gene editing reagent adding device and a fluorescence real-time monitoring system for monitoring the binding of the nucleic acid aptamer to the target gene;

[0023] Use a PCR instrument with high-fidelity and rapid amplification capabilities for the acquisition and modification of antigen genes, use a nucleic acid electrophoresis instrument with high resolution to detect the size and purity of gene fragments, and be equipped with a gel imaging system for image acquisition and analysis at the same time;

[0024] Use a clean bench for recombinant vector construction operations, be equipped with a nanopore electroporator with precise voltage and pulse control for recombinant plasmid introduction, and be equipped with a single-cell sequencer for gene analysis of the transformed strain at the single-cell level;

[0025] Fermentation culture is carried out in a fermenter equipped with a multi-sensor fusion monitoring system. The fermenter is equipped with a dissolved oxygen, temperature, pH value, and nutrient concentration monitoring system, as well as an automatic nutrient feeding device. The feeding speed is automatically adjusted through an intelligent algorithm.

[0026] Centrifugation of the fermentation broth is carried out using a centrifuge with low-temperature and high-speed centrifugation function, and freeze-drying process is carried out using a freeze-dryer with in-situ freeze-drying and plugging functions. The pre-freezing temperature is set at -40°C, the vacuum degree is 10 - 20 Pa, and the freeze-drying time is 24 - 36 hours during the freeze-drying process. At the same time, a real-time monitoring system for temperature and vacuum degree is equipped.

[0027] Preferably, the volume of the fermenter is 200 L, the stirring paddle is combined, the stirring speed adjustment range is 100 - 800 r / min, and the ventilation volume adjustment range is 0.5 - 2 vvm.

[0028] Preferably, the cold trap temperature of the freeze-dryer can reach below -80°C, it has an automatic defrosting function, and the temperature control accuracy during the freeze-drying process is ±0.5°C.

[0029] (III) Beneficial technical effects

[0030] Compared with the existing technology, the beneficial effects of the present invention are:

[0031] 1. By inserting a mucosal targeting gene fragment fused with cholera toxin B subunit into the carrier strain and combining with a protein containing RGD-4C sequence, the adhesion and colonization ability of the vaccine in mucosal tissues such as the bovine respiratory tract and reproductive tract is greatly enhanced, and it can rapidly stimulate mucosal immune response at the initial stage of infection, forming the first line of defense against the invasion of Mycoplasma bovis.

[0032] 2. The immune-enhancing peptide with deep codon optimization and glycosylation modification is fused with the antigen gene, significantly improving the expression level and stability of the antigen protein. At the same time, various modification methods enable the vaccine to effectively cope with the antigen variation of Mycoplasma bovis. The vaccine can simultaneously activate humoral immunity and cellular immunity in vivo. Especially the secreted IgA produced through mucosal immunity can neutralize pathogens on the mucosal surface, and the immune protection effect is long-lasting and efficient.

[0033] 3. The intelligent feedback batch feeding fermentation combined with multi-sensor fusion technology can accurately control the growth of bacteria and antigen expression, improving the yield and quality stability of the vaccine. The application of new components such as gene editing inducers and metabolic regulators, as well as innovative formulation and freeze-drying processes, effectively ensure the activity of bacteria in the vaccine during storage and transportation, and extend the shelf life of the vaccine.

[0034] 4. In the process of strain modification and vaccine preparation of the present invention, through multi-dimensional detection and verification means, such as whole-genome methylation sequencing, single-cell gene analysis, etc., the safety and effectiveness of the vaccine are ensured. Compared with traditional vaccines, this mucosal immune type Mycoplasma bovis live vector vaccine can more efficiently prevent Mycoplasma bovis disease, reduce the economic losses caused by diseases in the cattle industry, and promote the healthy and sustainable development of the cattle industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a flow chart of the preparation method of a mucosal immune type Mycoplasma bovis live vector vaccine;

[0036] Figure 2 is a bar chart comparing the mucosal adhesion rate and immune antibody titer of the examples and the comparative examples;

[0037] Figure 3 is a line chart comparing the vaccine stability and immune protection rate of the examples and the comparative examples;

[0038] Figure 4 is a bar-line chart comparing the vaccine cost and yield of the examples and the comparative examples. DETAILED DESCRIPTION OF THE INVENTION

[0039] According to Figures 1 to 4 , the specific implementation manners of the present invention are as follows:

[0040] Example 1

[0041] Strain modification: Select 100 mL of an attenuated Mycoplasma bovis strain culture in the logarithmic growth phase, and add a novel gene editing inducer X with a mass concentration of 0.01%. The nucleic acid aptamer in inducer X has a length of 25 nucleotides, is modified by FRET, and the fluorescent groups are Cy3 and Cy5. The modification rate is monitored by high performance liquid chromatography and reaches more than 90%. Shake and culture at 37 °C and 180 r / min for 8 hours. Use the optimized CRISPR-Cas12a system for gene editing, knock out the virulence-related genes, and insert a mucosal targeting gene fragment encoding a protein containing the RGD-4C sequence and fused with the cholera toxin B subunit (CTB). After the culture is completed, perform whole-genome methylation sequencing analysis on the modified strain to ensure that the gene editing is correct and does not affect the normal physiological functions of the strain.

[0042] Antigen gene acquisition and modification: Extract the gene encoding the major surface antigen protein from a highly pathogenic Mycoplasma bovis strain, and perform three rounds of codon optimization according to the bovine host-preferred codon database. Fuse the gene fragment encoding the glycosylation-modified immune-enhancing peptide (amino acid sequence: YIGSR) with the antigen gene by a novel seamless ligation PCR technique. Use a mammalian cell expression system (HEK293 cells) to verify the in vitro expression of the fused antigen gene, and detect the protein expression level and immunoreactivity by Western blot and immunofluorescence techniques.

[0043] Recombinant vector construction: Double-digest the plasmid vector and the fused antigen gene with a novel restriction enzyme combination (MseI and PstI), and then ligate them under the action of thermostable T4-like DNA ligase. Introduce the recombinant plasmid into the modified Mycoplasma bovis strain by pulse field-mediated nanopore electroporation technology, with the transformation conditions of voltage 1500V and pulse time 5ms. After transformation, use a single cell sequencer to perform gene analysis on the transformed strain at the single cell level.

[0044] Fermentation culture: Inoculate the recombinant Mycoplasma bovis strain into 1L of modified fermentation medium, which contains basic nutrients, 0.2% (mass concentration) of a novel metabolic regulator Z (phytosterol treated by cyclodextrin inclusion), and 0.1% of amino acid chelated iron (iron content 15%, amino acids are histidine and glycine mixed in a ratio of 1:2). Under the conditions of 36°C and pH 7.0, adopt an intelligent feedback fed-batch fermentation method. When the cell concentration reaches OD 600 = 0.6, start to supplement the nutrient feed containing glucose, yeast extract and vitamin B complex, with a feeding rate of 5 mL / h and a fermentation time of 24 hours. During the fermentation process, continuously monitor the concentrations of key nutrients such as dissolved oxygen, glucose and amino acids in the fermentation broth. When the dissolved oxygen is lower than 30%, maintain the dissolved oxygen at 30%-50% by adjusting the ventilation volume and stirring speed.

[0045] Vaccine preparation: After fermentation, centrifuge the fermentation broth at 4°C and 8000 r / min for 15 minutes to collect the cell precipitate. Resuspend the cells with phosphate buffer containing 0.05% mucosal protectant (quaternary ammonium salt of chitosan modified by carboxymethylation, substitution degree 70%), adjust the cell concentration to 1×10 8 CFU / mL, add a freeze-drying protectant with a final concentration of 0.5% (composed of trehalose, mannitol and polyethylene glycol 6000 in a mass ratio of 1:1:0.5), and prepare a mucosal immunogenic Mycoplasma bovis live vector vaccine by freeze-drying technology. The freeze-drying process is set with a pre-freezing temperature of -40°C, a vacuum degree of 10 Pa, and a freeze-drying time of 24 hours.

[0046] Example 2

[0047] Strain modification: Select 150 mL of an attenuated Mycoplasma bovis strain culture in the logarithmic growth phase, and add a new gene editing inducer X with a mass concentration of 0.03%. The nucleic acid aptamer has a length of 30 nucleotides, and the FRET modification is the same as in Example 1. Incubate with shaking at 37 °C and 180 r / min for 10 hours. Perform gene editing operations to knockout virulence genes and insert mucosal targeting gene fragments, and then perform whole-genome methylation sequencing analysis.

[0048] Antigen gene acquisition and modification: After extracting the antigen gene, perform four rounds of codon optimization, fuse the glycosylation immune-enhancing peptide gene fragment with the antigen gene, and the in vitro expression verification process is the same as in Example 1.

[0049] Recombinant vector construction: The double digestion and ligation operations are the same as in Example 1. The nanopore electroporation conditions are a voltage of 1600 V and a pulse time of 6 ms. After transformation, perform single-cell gene analysis.

[0050] Fermentation culture: Inoculate the recombinant strain into 1.5 L of a modified fermentation medium. The mass concentration of the new metabolic regulator Z in the medium is 0.3%, and the content of amino acid chelated iron is 0.2% (iron content 18%). Ferment at 37 °C and a pH value of 7.2. When the cell concentration reaches OD 600 = 0.7, start feeding at a rate of 7 mL / h, and the fermentation time is 30 hours. The control of parameters such as dissolved oxygen during fermentation is the same as in Example 1.

[0051] Vaccine preparation: The centrifugation conditions are centrifugation at 4 °C and 9000 r / min for 18 minutes. The concentration of the mucosal protectant is 0.07% (the degree of substitution of chitosan quaternary ammonium salt is 80%), and the final concentration of the lyophilization protectant is 0.7%. The lyophilization process parameters are the same as in Example 1.

[0052] Example 3

[0053] Strain modification: Select 200 mL of an attenuated Mycoplasma bovis strain culture in the logarithmic growth phase, and add a new gene editing inducer X with a mass concentration of 0.05%. The nucleic acid aptamer has a length of 35 nucleotides, and the FRET modification is the same as in Example 1. Incubate with shaking at 37 °C and 180 r / min for 12 hours. Perform gene editing and analyze the whole-genome methylation.

[0054] Antigen gene acquisition and modification: Perform five rounds of codon optimization to complete the fusion of the immune-enhancing peptide gene and the antigen gene and in vitro expression verification.

[0055] Recombinant vector construction: The double digestion and ligation operations are the same as before. The nanopore electroporation conditions are a voltage of 1700 V and a pulse time of 7 ms. The single-cell gene analysis is the same as in Example 1.

[0056] Fermentation culture: Inoculate into 2 L of improved fermentation medium, with the mass concentration of the novel metabolic regulator Z being 0.5% and the content of amino acid chelated iron being 0.3% (iron content 20%). Ferment at 38 °C and pH 7.4. When the cell concentration reaches OD 600 = 0.8, start feeding, with the feeding rate being 10 mL / h and the fermentation time being 36 hours. Control parameters such as dissolved oxygen content in the same manner as in Example 1.

[0057] Vaccine preparation: Centrifuge at 4 °C and 10,000 r / min for 20 minutes. The concentration of the mucosal protectant is 0.1% (degree of substitution of chitosan quaternary ammonium salt is 90%), and the final concentration of the lyophilization protectant is 1%. The lyophilization process parameters are the same as in Example 1.

[0058] Comparative example

[0059] Adopt the traditional preparation method of live Mycoplasma bovis vector vaccine. Select an attenuated Mycoplasma bovis strain, without treatment with gene editing inducer, directly use the conventional CRISPR-Cas9 system to knock out virulence genes and insert a simple mucosal targeting gene fragment. Do not perform in-depth codon optimization and glycosylation modification on the antigen gene, and directly ligate it with the vector. The fermentation medium is a common medium, without adding the novel metabolic regulator and amino acid chelated iron. Adopt the traditional batch fermentation method without intelligent feeding. For the vaccine preparation, do not use special mucosal protectant and lyophilization protectant, and directly perform lyophilization.

[0060] Performance test

[0061] The present invention demonstrates the significant advantages of the novel live Mycoplasma bovis vector vaccine through three examples and one comparative example. In terms of mucosal adhesion rate, Examples 1 to 3 reach 85%, 90% and 95% respectively, which is much higher than 30% of the comparative example, proving that the modified RGD-4C-CTB fusion protein can efficiently target mucosal tissues. In the immune antibody titer test, the example groups show a stepwise increase, with the logarithmic titer value increasing from 10 to 14, while the comparative example is only 6, indicating that the antigen expression induced by gene editing significantly enhances the humoral immune response. In terms of vaccine stability, the viable bacteria rate of the examples remains at 80% to 90% after storage at 4 °C for 6 months, which is more than doubled compared to 40% of the comparative example, verifying the effectiveness of the lyophilization process and the protectant formulation. Comprehensive data show that this vaccine has broken through the traditional technical bottlenecks in mucosal targeting, immunogenicity and storage stability.

[0062] Comprehensive comparison of the vaccine performance of the examples and the comparative example is as follows in the table:

[0063] Table 1

[0064] Item Example 1 Example 2 Example 3 Comparative Example Mucosal adhesion rate (%) 85 90 95 30 <![CDATA[Immune antibody titer (log2)]]> 10 12 14 6 Vaccine stability (viable bacteria rate after 6 months at 4°C) 80％ 85％ 90％ 40％ Immune protection rate (after challenge) 80 85 90 50

[0065] Conclusion: This table comprehensively shows the differences between the examples and the comparative examples in multiple vaccine performance indicators. Examples 1-3 are significantly superior to the comparative examples in terms of mucosal adhesion rate, immune antibody titer, vaccine stability, and immune protection rate. Moreover, with the optimization of process parameters, Example 3 shows the best performance in all indicators, fully demonstrating the advantages of the preparation method of the present invention.

[0066] The comparison of the vaccine costs and yields between the examples and the comparative examples is as follows in the table:

[0067] Table 2

[0068] Item Example 1 Example 2 Example 3 Comparative Example Cost per dose of vaccine (yuan) 10 9 8 12 Yield per batch of vaccine (doses) 5000 6000 7000 4000

[0069] Conclusion: This table compares the situations of the examples and the comparative examples in terms of vaccine cost and yield. Examples 1-3 not only have lower costs than the comparative examples, but also have higher vaccine yields per batch, indicating that the preparation method of the present invention has significant effects in reducing costs and increasing yields, and is more competitive in the market.

[0070] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A preparation method of a mucosal immune type Mycoplasma bovis live vector vaccine, characterized in that, It includes the following steps: S1, Strain modification: Select an attenuated Mycoplasma bovis strain as a vector, and add a novel gene editing inducer X to the culture system; the inducer X is an aptamer-small molecule compound conjugate Aptamer-N3-L-Small-molecule containing an azide group, the aptamer is single-stranded DNA or RNA, and is modified by fluorescence resonance energy transfer (FRET), L is a linker; shake culture for 8-12 hours; modify with an optimized CRISPR-Cas12a system to knockout virulence-related genes and insert a mucosal targeting gene fragment at the same time; S2, Antigen gene acquisition and modification: Extract the gene encoding the major surface antigen protein from a highly pathogenic Mycoplasma bovis strain, perform deep codon optimization on this gene, and conduct multiple rounds of optimization in combination with a bovine host preference codon database; at the same time, fuse the gene fragment encoding the immune enhancing peptide with the antigen gene through a novel seamless ligation PCR technique; S3, Recombinant vector construction: Insert the fusion antigen gene obtained in step S2 into the plasmid vector of the Mycoplasma bovis strain modified in step S1 to construct a recombinant Mycoplasma bovis strain; use a novel restriction enzyme combination to perform double digestion on the plasmid vector and the fusion antigen gene, and then ligate under the action of T4-like DNA ligase, and introduce the recombinant plasmid into the modified Mycoplasma bovis strain through pulse field-mediated nanopore electroporation technology; S4, Fermentation culture: Inoculate the recombinant Mycoplasma bovis strain into a modified fermentation medium, and the modified fermentation medium contains basic nutrient components, a novel metabolic regulator Z, and amino acid chelated iron; S5, Vaccine preparation: After fermentation, centrifuge the fermentation broth for 15-20 minutes to collect the cell precipitate; resuspend the cells with a phosphate buffer containing a mucosal protectant, adjust the cell concentration to 1×108 to 1×109 CFU / mL, add a freeze-drying protectant, and prepare a mucosal immune type Mycoplasma bovis live vector vaccine through a freeze-drying process.

2. The preparation method of the mucosal immune type Mycoplasma bovis live vector vaccine according to claim 1, characterized in that, It also includes, after the strain modification in S1, performing whole-genome methylation sequencing analysis on the modified strain to evaluate the impact of gene editing on the epigenetics of the strain, ensuring that the virulence gene knockout and the mucosal targeting gene fragment are correctly inserted and do not affect the normal physiological functions of the strain.

3. The preparation method of the mucosal immune type Mycoplasma bovis live vector vaccine according to claim 1, characterized in that, It also includes, during the antigen gene acquisition and modification in S2, performing in vitro expression verification on the fusion antigen gene, expressing the fusion antigen protein using a mammalian cell expression system, and detecting the protein expression level and immunological activity through Western blot and immunofluorescence techniques.

4. The preparation method of the mucosal immune type Mycoplasma bovis live vector vaccine according to claim 1, characterized in that, The FRET-modified fluorescent groups of the aptamer in the novel gene editing inducer X are Cy3 and Cy5, and its modification rate is monitored by high performance liquid chromatography to reach more than 90%.

5. The preparation method of the mucosal immune type Mycoplasma bovis live vector vaccine according to claim 1, characterized in that, In the amino acid chelated iron, the molar ratio of histidine to glycine and iron ions is 1:2:1, and the iron content is determined by atomic absorption spectrometry.

6. The preparation method of the mucosal immune type Mycoplasma bovis live vector vaccine according to claim 1, characterized in that, The degree of substitution of carboxymethylated chitosan quaternary ammonium salt in the mucosal protectant is 70%-90%, which is determined by nuclear magnetic resonance spectroscopy.

7. The preparation method of the mucosal immune type Mycoplasma bovis live vector vaccine according to claim 1, characterized in that, The novel metabolic regulator Z is prepared by compounding dimethyl sulfoxide and phytosterol in a mass ratio of 1:1, and the phytosterol is treated by cyclodextrin inclusion. The iron content in the amino acid chelated iron is 15%-20%, and the amino acids are a mixture of histidine and glycine in a ratio of 1:

2.

8. The production process of a mucosal immune type Mycoplasma bovis live vector vaccine, characterized in that, The preparation method according to any one of claims 1-6 is implemented by the following equipment and operations: A constant temperature shaking incubator with high-precision temperature and rotation speed control is used for strain transformation culture. The temperature is set at 37°C and the rotation speed is 180 r / min. It is equipped with a gene editing reagent addition device and a fluorescence real-time monitoring system for monitoring the binding of the nucleic acid aptamer to the target gene; A PCR instrument with high-fidelity and rapid amplification capabilities is used to obtain and modify the antigen gene. A nucleic acid electrophoresis instrument with high resolution is used to detect the size and purity of the gene fragment, and a gel imaging system is simultaneously equipped for image acquisition and analysis; The operation of constructing a recombinant vector is carried out using a laminar flow hood. A nanopore electroporator with precise voltage and pulse control is equipped for introducing the recombinant plasmid, and a single-cell sequencer is simultaneously equipped for gene analysis of the transformed strain at the single-cell level; Fermentation culture is carried out in a fermenter equipped with a multi-sensor fusion monitoring system. The fermenter is equipped with a dissolved oxygen, temperature, pH value, and nutrient concentration monitoring system, as well as an automatic nutrient feeding device, and the feeding speed is automatically adjusted through an intelligent algorithm; A centrifuge with a low-temperature and high-speed centrifugation function is used to centrifuge the fermentation broth. A freeze dryer with in-situ freezing and plugging functions is used for the freeze-drying process. The pre-freezing temperature is set at -40°C, the vacuum degree is 10-20 Pa, and the freeze-drying time is 24-36 hours. A temperature and vacuum degree real-time monitoring system is simultaneously equipped.

9. The production process according to claim 7, characterized in that, The volume of the fermenter is 200 L, the stirring paddle is a combined type, the stirring speed adjustment range is 100-800 r / min, and the ventilation volume adjustment range is 0.5-2 vvm.

10. The production process according to claim 7, characterized in that, The cold trap temperature of the freeze dryer can reach below -80°C, it has an automatic defrosting function, and the temperature control accuracy during the freeze-drying process is ±0.5°C.

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