Gene directional editing system and method applied to mycoplasma bovis

Abstract

The invention relates to the technical field of biological genes, in particular to a gene directed editing system applied to mycoplasma bovis, which is an editing system for expressing a combination of a single plasmid of GP35 single-chain annealing protein and a targeting fragment ssDNA (single-stranded deoxyribonucleic acid), and comprises an expression plasmid of a GP35 gene and the ssDNA of the targeting fragment. The invention relates to a gene directed editing method applied to mycoplasma bovis. The gene directed editing method comprises the following steps: constructing a mycoplasma bovis strain for expressing GP35 single-chain annealed protein, constructing an ssDNA single chain of a targeting fragment, and performing gene editing on a target gene to obtain a pdhC gene deleted strain. According to the invention, a KanR gene is successfully inserted at the 158bp position of a pdhC gene coding region of a mycoplasma bovis PG45 strain, so that a mycoplasma bovis pdhC gene oriented deletion strain is obtained. According to the invention, the metabolism-related gene directed deletion strain M. bovis PG45 [delta] pdhC is successfully constructed, and the pdhC protein is characterized to have the ability of regulating the growth rate of mycoplasma bovis and maintaining colonial morphology.

Description

Technical Field

[0001] This invention relates to the field of biogenetics, specifically to a gene-directed editing system and method for bovine mycoplasma. Background Technology

[0002] Mycoplasma bovis is a major bacterial pathogen causing multi-systemic inflammation in cattle, including pneumonia, arthritis, mastitis, and otitis media. Through global animal trade, it has spread to all continents and is a significant cause of respiratory diseases in cattle and mastitis in dairy cows in Europe and America. Since an outbreak of bovine mycoplasma in Hubei Province in 2008, it has become prevalent in most parts of my country, with some areas showing a long-term epidemic trend. Chronic, debilitating, and recurrent infections are characteristic of bovine mycoplasma infection. Once infected, it is very difficult to eradicate, causing significant economic impact and production losses to the local cattle industry, particularly the beef and dairy sectors. Antibiotic treatment for bovine mycoplasma is not ideal. This is partly because bovine mycoplasma lacks a cell wall, making it naturally resistant to all antibiotics that target cell walls (such as β-lactams), and partly because increasing reports indicate a growing problem of antibiotic resistance in bovine mycoplasma. Vaccines are the most effective way to control pathogen infection; however, an effective vaccine for bovine mycoplasma remains lacking. Therefore, the development of bovine mycoplasma vaccines and novel drugs is an ongoing and challenging task, mainly because the pathogenic mechanism of bovine mycoplasma and the molecular mechanisms involved in pathogen-host interactions remain unclear.

[0003] The lack of effective gene manipulation tools for *Mycoplasma bovis* has hindered research on its pathogenicity and immune mechanisms compared to other pathogens. While modern genetic engineering techniques are widely used for genome manipulation of various pathogens, strains difficult to manipulate in vitro are considered challenging for gene manipulation, and *Mycoplasma bovis* is a prime example. Currently, common bacterial gene editing tools (such as homologous recombination and the CRISPR / Cas system) have failed to successfully edit fragments of the *Mycoplasma bovis* gene. Single-base mutation technology has been used for targeted gene editing in *Mycoplasma bovis*, but it suffers from non-specific mutations involving random deamination. Therefore, the main gene manipulation tools and methods currently used for *Mycoplasma bovis* gene function research remain transposon random mutation technology and the application of mutant libraries. However, the randomness of transposon mutation technology hinders its application in studying the pathogenic mechanisms of *Mycoplasma bovis*. Therefore, the development and establishment of targeted gene manipulation tools for *Mycoplasma bovis* is urgently needed, as this will provide crucial technical support for fundamental research on the pathogenic mechanisms of *Mycoplasma bovis*. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a gene-directed editing system and method for bovine mycoplasma, aiming to achieve gene editing of bovine mycoplasma based on overexpression of GP35 single-stranded annealing protein and the use of the targeting fragment ssDNA single strand.

[0005] To achieve the above objectives, the following technical solution is provided: A gene-directed editing system for bovine mycoplasma is an editing system consisting of a single plasmid expressing GP35 single-stranded annealing protein and a targeting fragment ssDNA, including an expression plasmid of the GP35 gene and a single-stranded ssDNA of the targeting fragment.

[0006] The expression plasmid contains the single-stranded annealing protein expression element GP35 gene, and the targeting fragment of the ssDNA single strand includes the kanamycin resistance gene (KanR) and 500 nt upstream and downstream homologous arms of the targeting gene site.

[0007] The expression plasmid is pIRR45-GP35 plasmid, and the GP35 gene in the plasmid is used to drive the gene editing of bovine mycoplasma by targeting the ssDNA fragment.

[0008] The GP35 gene is derived from Bacillus subtilis phage, and its amino acid sequence is shown in SEQ ID NO:1: MATKKQEELKNALAQQNGAVPQTPVKPQDKVKGYLERMMPAIKDVLPKHLDADRLSRIAMNVIRTNPKLLECDTASLMGAVLESAKLGVEPGLLGQAYILPYTNYKKKTVEAQFILGYKGLLDLVRRSGHVSTISAQTVYKNDT FEYEYGLDDKLVHRPAPFGTDRGEPVGYYAVAKMKDGGYNFLVMSKQDVEKHRDAFSKSKNREGVVYGPWADHFDAMAKKTVLRQLINYLPISVEQLSGVAADERTGSELHNQFADDDNIINVDINTGEIIDHQEKLGGETNE.

[0009] The expression plasmid for the GP35 gene also contains the bovine mycoplasma PG45 origin of replication. oriC .

[0010] The construction of the ssDNA targeting fragment includes pdhC The 500 bp sequences upstream and downstream of the 158 bp position of the gene are used as homologous arms of the targeting fragment, and the KanR gene is inserted in the middle of the homologous arms.

[0011] The upstream homologous arm sequence of the target gene is shown in SEQ ID NO:2: TATAAAACAAAAAGTGTAAATATGCTCTTTGTTTTATAATAATTTACGTAGATTTATTTACTAAATCTACGTTTTTAAATTTAAAAACTTAATAAATATAACCGTACGGAGGTAAATATGTTATTTAAAGACACTGGCGAAGAAAAGTCAAGCTGCAAAAAAGCTGATGCGCCTGCTGTTGTAGGTCAAATTCAAGTTTCTGATGAATTACTTGATTTTTCTGCATTCGCTAAGCCAAAGAAGAAAGA AACATCATGCAAGGAAAGTGGTGCTTCTCAAAGGATGAAGAAAACCTTACACCTCGTGAAAGAGCTATTAGAGCAAGAAAAGAAGGTAAATAATGGCTGTTGAGCTCGCAAAAGTTAAACCACTAGAAGAAAAAGAAGCTCCTATTTCAGGAATTCGTAAAGCTATTGCTAAAAACTTAAAAGAAGTTTTAGAAACATCTGCATATTGTTCATTAGTTTTAAAAGCTGATGTTACAAATCTTTGAAACT.

[0012] The downstream homologous arm sequence of the targeting gene is shown in SEQ ID NO:3: TACGTGCAAAAGTTAAAGACAAAGTTTTTGAAGAACACAATGTTAAATTAACATTCTTATCATGAATAGTTAAAGCCTCGGCTATTGCTTTAAGCGAATACCCATCTTTTGCTGCTAGATGAGATGGTGTTGAAGGTAAAGTATACTACCCTGGAACACTTAACATTGCTATAGCAGTTGACACACCTTTTGGTCTATTTGTACCTGTAATTAGAGGTGTTGAAAACCTAAGTATAATTGAAATTCAAAAAGAAATTGTTAGATTATCAACACTTGCAAGAGAAAAGAAGCTAAAAATGTCAGATATGACAGGCGGCTGCTTTGCTATTACAAACGTTGGTAGTGCCGGTGTATTATTTGGTTCTCCAATTATGAACAAAGGCAATACAGCAATTTCAGCTACTGGTGCAATCATTGATGAGTTAAAATTAAACAAAGAAGGTGCTGTTGAAAACAGAAAAGTAATGTACTTATCAATAGCAGCTGACCACCAATGAGTT。

[0013] The KanR gene sequence is shown in SEQ ID NO: 4:

[0014] A method for targeted gene editing of bovine mycoplasma, characterized by comprising the following steps: Step 1: Construct a bovine mycoplasma strain expressing GP35 single-chain annealing protein; Step 2: Construct the ssDNA single strand of the target fragment and perform gene editing on the target gene to obtain... pdhC Gene deletion strains; Specifically, the steps include the following: Step 1: Construct the GP35 gene to contain PG45. oriC From the pIRR45 plasmid, the pIRR45-GP35 recombinant plasmid was obtained; Step 2: The pIRR45-GP35 recombinant plasmid was transformed into bovine mycoplasma competent cells treated with CaCl2 using polyethylene glycol 8000 (PEG8000). After tetracycline resistance (TetR) screening, the bovine mycoplasma strain PG45-GP35 containing the pIRR45-GP35 plasmid was successfully obtained. Step 3: Prepare ssDNA single strands of the target fragment. Use the 500bp upstream and downstream sequences of the inserted fragment as the upper and lower homologous arms of the target site. Insert the KanR gene between the upper and lower homologous arms to assemble dsDNA. Prepare ssDNA by nucleic acid amplification or enzyme digestion. Step 4: The prepared ssDNA single strands were transformed into the bovine mycoplasma PG45-GP35 strain containing the pIRR45-GP35 plasmid using PEG 8000. Step 5: Screening for deletion strains using KanR. Specifically, bovine mycoplasma strains with inserted and disrupted genes were successfully screened from mycoplasma culture medium containing kanamycin. pdhC Gene deletion strain.

[0015] Bovine mycoplasma obtained by the above gene-directed editing method pdhC The gene deletion strain is named: Mycoplasma bovis PG45Δ pdhC It is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number M20252202 and deposit date of October 11, 2025.

[0016] In summary, this invention utilizes the combination of GP35 single-stranded annealing protein and the ssDNA single-stranded system of the target fragment to achieve gene editing of bovine mycoplasma, providing an effective technical means for studying the etiology and pathogenic mechanism of bovine mycoplasma.

[0017] This invention successfully isolated bovine mycoplasma. pdhCThe KanR gene was inserted at position 158 bp in the coding region of the gene, thereby obtaining bovine mycoplasma. pdhC Gene-directed deletion strain. Growth curve and colony morphology analysis showed that, compared with the wild-type strain, pdhC The deletion strain showed a significantly reduced growth rate and smaller colony size.

[0018] The beneficial effects of this invention are as follows: Addressing the current lack of an effective targeted gene editing system for Bovine Mycoplasma, this invention successfully establishes a targeted gene deletion technology for Bovine Mycoplasma based on GP35 single-chain annealing protein, enabling the construction of targeted gene deletion strains of Bovine Mycoplasma. Using this technology, targeted deletion strains of metabolism-related genes have been successfully constructed. M. bovis PG45Δ pdhC The study also demonstrated that the pdhC protein has the ability to regulate the growth rate of bovine mycoplasma and maintain colony morphology. Attached Figure Description

[0019] Figure 1 pIRR45-GP35 plasmid and pdhC Schematic diagram of gene deletion construction.

[0020] Figure 2 Nucleic acid electrophoresis identification of pIRR45-GP35 plasmid; where lane M: DL 2000 Marker; lanes 1-4: positive clones transformed by pIRR45-GP35 plasmid; lane 5: ddH2O.

[0021] Figure 3 PCR identification of Mycoplasma bovis PG45-GP35 strain; lane M: DL 2000 Marker; lane 1: Mycoplasma bovis PG45 strain; lanes 2-4: positive clones; lane 5: ddH2O.

[0022] Figure 4 for pdhC Electrophoresis image of targeted ssDNA nucleic acid; where lane M: DL 5000 Marker; lane 1: pdhC Targeting dsDNA fragments; Lane 2: pdhC Targeting the ssDNA chain.

[0023] Figure 5 for M. bovis PG45Δ pdhC Nucleic acid electrophoresis identification of gene-directed insertion / deletion strains; where lane M: DL 5000 Marker; lanes 1-8: M. bovis PG45Δ pdhC Deletion strain genome; Lane 9: Mycoplasma bovis parent strain PG45 genome; Lane 10: ddH2O.

[0024] Figure 6 Western blot identification of wild-type strain PG45 and M. bovis PG45Δ pdhC In strains pdhC The gene expression product is pdhC protein; lane M: protein marker; lane 1: M. bovis PG45Δ pdhC Strains; Lane 2: PG45 strain.

[0025] Figure 7 wild plants and M. bovis PG45Δ pdhC Growth curve determination of the strain.

[0026] Figure 8 wild plants and M. bovis PG45Δ pdhC Colony morphology of the strain under an optical microscope, scale bar 100 μm. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0028] The following detailed description of the construction process of the gene editing system of the present invention, with reference to the accompanying drawings, and further illustrates the usage method and experimental results of the gene editing system of the present invention through the examples.

[0029] Example 1: Construction and identification of pIRR45-GP35 overexpression plasmid This invention employs a seamless cloning method to construct the complete coding sequence of the GP35 gene into the bovine mycoplasma overexpression plasmid pIRR45. Figure 1 (Left), where the pIRR45 plasmid, kindly provided by Glenn Browning of the University of Melbourne, contains a tetracycline resistance marker gene. After obtaining the pIRR45-GP35 plasmid, it was transformed into DH5α competent cells for in vitro plasmid amplification and PCR identification. Figure 2 As shown, using primers pIRR45GP35JD-F / R (TCCAAATAGTCGGATAGA / GAATTGTGAGCGGATAAC), the target band of 1373 bp could be amplified in all four randomly selected positive clones.

[0030] Example 2 Identification of Mycoplasma bovis strain PG45-GP35 To obtain a bovine mycoplasma strain expressing the GP35 single-chain annealing protein, the constructed pIRR45-GP35 plasmid was transformed into the bovine mycoplasma PG45 strain. Since the pIRR45-GP35 plasmid carries a tetracycline resistance gene, the transformed strains were plated on a tetracycline-resistant selection medium for antibiotic stress selection, thus obtaining tetracycline-resistant strains. These resistant strains were then identified by PCR using GP35 gene identification primers. Figure 3 As shown, primers pIRR45GP35JD-F / R amplified the target band of 1373 bp in all eight randomly selected resistant clones. The positions of these bands were consistent with expectations, indicating that the present invention successfully constructed and obtained the bovine mycoplasma PG45-GP35 strain.

[0031] Example 3 pdhC Construction of shooting segments This invention is planned to target strain PG45. pdhC The KanR gene is inserted at position 158 of the gene fragment, thereby disrupting... pdhC The function of genes. First, design. pdhC Homologous arms of 500 bp each upstream and downstream of position 158 of the gene were constructed, and the resistance gene was constructed between the upstream and downstream homologous arms. Figure 1 right).

[0032] Using the constructed targeting fragment as a template, PCR amplification was performed using primers pdhCSYT-F / pdhCXYT-R (TATAAAACAAAAAGTGTAAATATGCT / AACTCATTGGTGGTCAGC) to complete the preparation of the targeted double-stranded dsDNA. Subsequently, ssDNA was prepared by nucleic acid amplification or enzyme digestion. Figure 4 As shown, the electrophoretic bands of the targeted double-stranded fragments match the expected bands. pdhC No obvious impurities were observed in the single-stranded electrophoresis bands of the target fragment, indicating that the prepared... pdhC The targeted ssDNA contains fewer double strands, making it suitable for constructing deletion strains.

[0033] Example 4 pdhC Construction and identification of directional insertion / deletion strains The built pdhCTargeted ssDNA was transformed into *Mycoplasma bovis* strain PG45-GP35 using PEG 8000 and plated on PPLO solid medium containing kanamycin for culture. Subsequently, colonies were selected and subcultured, and the genome was extracted for PCR amplification and sequencing using specific primers. Deletion strains were identified. Results showed that amplification of the PG45 strain genome using pdhCSYT-F / pdhCXYT-R specific primers yielded a nucleic acid fragment of approximately 1000 bp. However, for… pdhC The amplified fragment of the gene deletion strain is approximately 2500 bp. Figure 5 Using NP40 lysis buffer, wild-type PG45 and... pdhC Whole-cell protein was obtained by lysing the gene-deleted bacterial strain, followed by immunoblotting. Primary antibody incubation was performed using a prepared pdhC mouse polyclonal antibody at a ratio of 1:1000, followed by secondary antibody incubation using goat anti-mouse IgG (H+L) at a ratio of 1:5000. Finally, ECL exposure was performed. Results showed... pdhC pdhC protein cannot be expressed in gene-deleted strains. Figure 6 This indicates that we have successfully obtained... pdhC Gene-deleted strains, bovine mycoplasma obtained by the above gene-directed editing method pdhC The gene deletion strain is named: Mycoplasma bovis PG45Δ pdhC ( M. bovis PG45Δ pdhC It is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number M 20252202 and deposit date of October 11, 2025.

[0034] Example 5 M. bovis PG45Δ pdhC Plant growth curve determination Preparation of cryopreserved bacterial culture: Bovine mycoplasma PG45 strain and M. bovis PG45Δ pdhC The strain was revived and cultured twice. The strain from the last subculture was cultured to mid-logarithmic growth, followed by centrifugation, washing, CFU counting, and cryopreservation.

[0035] Preparation of bacterial culture before experiment: Dilute according to the CFU count results before freezing, and unify the initial bacterial culture concentration to 10. 3 CFU / mL, total volume 25 mL, samples were taken every 8 or 12 h; 100 μL of bacterial suspension was diluted and plated, and CFU results were counted after 5-7 days of incubation to plot growth curves. The results showed a significant difference in growth rate between the two strains. The wild-type strain exhibited a logarithmic growth phase from 0-24 h, a plateau phase from 24-64 h, and subsequently entered a decline phase, while... M. bovis PG45Δ pdhC The gene-deleted strain experienced a logarithmic growth phase from 0 h to 64 h, a plateau phase from 64 h to 104 h, and subsequently entered a decline phase. Figure 7 The above results suggest that: pdhC The absence of [the substance] significantly affected the growth rate and growth titer of bovine mycoplasma.

[0036] Example 6 M. bovis PG45Δ pdhC Observation of colony morphology Logarithmic-phase bacterial cells were spread on PPLO solid medium and incubated at 37 °C for 9 days. Colonies were observed and photographed using an optical microscope on days 3, 6, and 9. Both strains formed pinpoint-shaped "fried egg" colonies with neat edges. M. bovis PG45Δ pdhC The colonies of the strain were significantly smaller than those of the wild strain. Figure 8 ),hint pdhC The absence of this substance significantly affects the proliferation and colony morphology of Mycoplasma bovis.

[0037] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A gene-directed editing system for bovine mycoplasma, characterized in that, It includes an expression plasmid and an ssDNA targeting fragment. The expression plasmid contains the single-stranded annealing protein expression element GP35 gene, and the ssDNA targeting fragment includes a kanamycin resistance gene (KanR) and 500 nt upstream and downstream homologous arms of the targeting gene site.

2. The gene-directed editing system for bovine mycoplasma according to claim 1, characterized in that, The expression plasmid is pIRR45-GP35, and the GP35 single-stranded annealing protein in the plasmid is used to drive the targeting ssDNA for gene editing of bovine mycoplasma.

3. The gene-directed editing system for bovine mycoplasma according to claim 2, characterized in that, The GP35 gene is derived from Bacillus subtilis phage, and its amino acid sequence is shown in SEQ ID NO:

1.

4. The gene-directed editing system for bovine mycoplasma according to claim 3, characterized in that, The expression plasmid for the GP35 gene also contains the bovine mycoplasma PG45 origin of replication. oriC .

5. A gene-directed editing system for bovine mycoplasma according to claim 4, characterized in that, The construction of the ssDNA targeting fragment includes pdhC The 500 bp sequences upstream and downstream of the 158 bp position of the gene are used as homologous arms of the targeting fragment, and the KanR gene is inserted in the middle of the homologous arms; the upstream homologous arm sequence of the targeting gene is shown in SEQ ID NO:2, the downstream homologous arm sequence of the targeting gene is shown in SEQ ID NO:3, and the KanR gene sequence is shown in SEQ ID NO:

4.

6. A method for targeted gene editing applied to bovine mycoplasma, characterized in that, This includes constructing a bovine mycoplasma strain expressing GP35 single-stranded annealing protein and constructing a single-stranded ssDNA of the targeting fragment, followed by gene editing of the target gene to obtain... pdhC Gene deletion strain.

7. A method for targeted gene editing of bovine mycoplasma according to claim 6, characterized in that, Specifically, the steps include the following: Step 1: Construct the GP35 gene to contain PG45. oriC From the pIRR45 plasmid, the pIRR45-GP35 recombinant plasmid was obtained; Step 2: The pIRR45-GP35 recombinant plasmid was transformed into bovine mycoplasma competent cells treated with CaCl2 using polyethylene glycol 8000. After tetracycline resistance screening, the bovine mycoplasma strain PG45-GP35 containing the pIRR45-GP35 plasmid was successfully obtained. Step 3: Prepare ssDNA single strands of the target fragment. Use the 500bp upstream and downstream sequences of the inserted fragment as the upper and lower homologous arms of the target site. Insert the KanR gene between the upper and lower homologous arms to assemble dsDNA. Prepare ssDNA by nucleic acid amplification or enzyme digestion. Step 4: The prepared ssDNA single strands are transformed into the bovine mycoplasma basal bacteria PG45-GP35 containing the pIRR45-GP35 plasmid using PEG8000; Step 5: Select and obtain bovine mycoplasma with inserted and disrupted genes using mycoplasma culture medium containing kanamycin. pdhC Gene deletion strain.

8. Bovine mycoplasma obtained by the gene-directed editing method according to claim 7 pdhC The gene deletion strain is named: Mycoplasma bovis PG45Δ pdhC It is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number M20252202 and deposit date of October 11, 2025.

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