Signal peptides, methods, and applications for guiding the secretion and expression of exogenous proteins in mycoplasma

Abstract

This invention discloses a signal peptide, a method, and its application for guiding the secretory expression of exogenous proteins in mycoplasma. The method for guiding the secretory expression of exogenous proteins in mycoplasma includes the step of introducing a signal peptide at one end of the exogenous protein. This invention screens signal peptides with high efficiency in guiding the secretory expression of exogenous proteins and demonstrates that the exogenous proteins are normally expressed and active in mycoplasma. The signal peptide of this invention can be used to guide the secretory expression of mycoplasma and its protective antigens against co-infecting pathogens, and thus for vaccine preparation.

Description

Technical Field

[0001] This invention belongs to the field of animal infectious disease prevention and control, and in particular relates to signal peptides, methods and applications for guiding the secretion and expression of exogenous proteins in mycoplasma. Background Technology

[0002] Previous studies have found that Gram-positive and Gram-negative bacteria primarily rely on the Sec and Tat secretion systems to secrete proteins. However, related research has identified two main secretion pathways in mycoplasma. The first is the secretion of extracellular membrane vesicles composed of lipopolysaccharides, lipids, membrane-associated proteins, genetic material, and other virulence-related factors (Kulpand Kuehn 2010). For example, 37 proteins were identified as virulence factors in extracellular vesicles released by *Acholepsis reesei* PG8. Extracellular vesicles are associated with mycoplasma resistance to fluoroquinolones (Chernov et al 2014). The ability of *Mycoplasma hyopneumoniae* to secrete extracellular vesicles is enhanced under oxidative stress, indicating that the production of extracellular vesicles contributes to microbial survival (deSouza et al 2023). Another approach involves the precursor proteins of mycoplasma secretory proteins anchoring to the cell membrane via structures similar to type I signal peptides. After specific cleavage by peptidases, these precursors generate mature, immunologically functional secretory proteins or signal peptides (Hu et al., 2017). For example, fermented mycoplasma lipoprotein MALP-404, after lipid modification, anchors to the outer surface of the plasma membrane. Site-specific hydrolysis of this protein leads to the production of the immunostimulatory lipopeptide MALP-2, and it was shown that the residual RF peptide is soluble and released into the supernatant (Davis and Wise 2002). However, no secretory signal peptide has yet been identified that can guide protein secretion without cleavage.

[0003] The mycoplasma genome differs significantly from other bacteria and host genomes. The mycoplasma genome has a GC content of approximately 30%, and it uses UGA to encode tryptophan, while UGA is a stop codon in other bacteria and hosts. This makes it unlikely that mycoplasma, as a vector for expressing exogenous proteins, will have its genes acquired and utilized by other microorganisms. However, in recent years, the frequency of co-infections of mycoplasma with other pathogens has been increasing. Therefore, there is an urgent need to develop drugs to treat co-infections of mycoplasma with other pathogens. Summary of the Invention

[0004] To address at least some of the technical problems in the prior art, this invention screened four signal peptides—SMbovP280, SMbovP374, SMbovP475, and SMbovP739—from the bovine mycoplasma genome. Experiments demonstrated that SMbovP280, SMbovP374, and SMbovP475 can guide the secretion of exogenous proteins into the bovine mycoplasma culture supernatant, with SMbovP475 exhibiting the highest efficiency in guiding exogenous protein secretion. Specifically, this invention includes the following:

[0005] A first aspect of the present invention provides a method for guiding the secretory expression of a foreign protein in mycoplasma, comprising the step of introducing a signal peptide at one end of the foreign protein, the signal peptide having any one of the amino acid sequences shown in (I)-(III):

[0006] (I) The amino acid sequence shown in SEQ ID NO. 2, 4 or 6;

[0007] (II) has at least 90% homology with the amino acid sequence shown in (I) and has the same function;

[0008] Amino acid sequences with the same function obtained by modifying, substituting, deleting, or adding one or more amino acids of the amino acid sequences shown in (III), (I), or (II).

[0009] In some embodiments, according to the method of the present invention, the signal peptide is a non-cleavage signal peptide.

[0010] In some embodiments, according to the method of the present invention, the signal peptide is prepared by artificial synthesis or genetic engineering.

[0011] In some embodiments, the method according to the present invention includes the following steps:

[0012] (1) Construct a recombinant expression vector containing the nucleotide sequence of the signal peptide and the exogenous protein;

[0013] (2) Transfect the recombinant expression vector into mycoplasma;

[0014] (3) The mycoplasma is cultured under conditions suitable for the expression of the exogenous protein, so that the exogenous protein is secreted and expressed in the mycoplasma.

[0015] In some embodiments, according to the method of the present invention, the nucleotide sequence comprises a codon-optimized sequence.

[0016] A second aspect of the invention provides the use of a signal peptide in the preparation of a medicament for the prevention or treatment of pathogenic bacterial infections, wherein the signal peptide has any one of the amino acid sequences shown in (I)-(III):

[0017] (I) The amino acid sequence shown in SEQ ID NO. 2, 4 or 6;

[0018] (II) has at least 90% homology with the amino acid sequence shown in (I) and has the same function;

[0019] Amino acid sequences with the same function obtained by modifying, substituting, deleting, or adding one or more amino acids of the amino acid sequences shown in (III), (I), or (II).

[0020] In some embodiments, according to the application described in the present invention, the drug comprises a vaccine.

[0021] In some embodiments, according to the application described in the invention, the prevention or treatment is achieved by administering an immunologically effective dose of vaccine to the subject.

[0022] In some embodiments, according to the application described in the present invention, the vaccine comprises a vector vaccine.

[0023] In some embodiments, according to the application described in the present invention, the vector vaccine uses mycoplasma as a vector.

[0024] This invention screened signal peptides that highly efficient at guiding the secretion and expression of exogenous proteins in mycoplasma, and demonstrated the normal expression and activity of the exogenous proteins in mycoplasma using Western blotting and a fluorescence microplate reader. The signal peptides of this invention can be used to guide the secretion and expression of protective antigens from mycoplasma and co-infecting pathogens, and subsequently for use in vaccine preparation. Attached Figure Description

[0025] Figure 1 This is an expression detection map of SMbovP475 fused with the exogenous protein mCherry in this invention. WT represents the wild-type strain HB0801; mCherry is the exogenous protein mCherry without a signal peptide; SMbovP280-mCherry is the exogenous protein mCherry fused with the SMbovP280 signal peptide; SMbovP374-mCherry is the exogenous protein mCherry fused with the SMbovP374 signal peptide; SMbovP475-mCherry is the exogenous protein mCherry fused with the SMbovP475 signal peptide; and SMbovP739-mCherry is the exogenous protein mCherry fused with the SMbovP739 signal peptide.

[0026] Figure 2 To detect the secretion of the exogenous protein mCherrry in bovine mycoplasma culture supernatant using the Western blotting method, PPLO was used as a blank culture medium control.

[0027] Figure 3 To detect the secretion of different groups of exogenous protein mCherrry in bovine mycoplasma culture supernatant using a fluorescent microplate reader. Detailed Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] method

[0032] One aspect of the present invention provides a method for guiding the secretory expression of a foreign protein in mycoplasma, comprising the step of introducing a signal peptide at one end of the foreign protein, wherein the signal peptide may be located at the N-terminus or C-terminus of the foreign protein. Preferably, a sequence encoding a signal peptide is introduced (operably linked) immediately adjacent to a sequence encoding the foreign protein, the signal peptide having any one of the amino acid sequences shown in (I)-(III):

[0033] (I) The amino acid sequence shown in SEQ ID NO. 2, 4 or 6;

[0034] (II) has at least 90% homology with the amino acid sequence shown in (I) and has the same function;

[0035] Amino acid sequences with the same function obtained by modifying, substituting, deleting, or adding one or more amino acids of the amino acid sequences shown in (III), (I), or (II).

[0036] In this invention, "exogenous protein" includes therapeutic proteins (e.g., antibodies or protein drugs) or immunogenic substances (e.g., antigens).

[0037] Guided secretion of exogenous proteins refers to the ability of the signal peptide of this invention to direct the passage of newly synthesized target proteins across the cell membrane, typically including the inner membrane or both the inner and outer membranes of mycoplasma. This allows exogenous proteins, such as antigens, to be secreted extracellularly into the mycoplasma cell or into the culture medium.

[0038] To obtain signal peptides that guide the secretion and expression of exogenous proteins in mycoplasma, this invention first uses bioinformatics tools to predict secretory lipoproteins in the genome of bovine mycoplasma HB0801. Through screening, four secretion signal peptides of secretory lipoproteins, namely SMbovP280, SMbovP374, SMbovP475, and SMbovP739, were selected. By constructing a shuttle plasmid to express the fusion protein of the signal peptides and the exogenous protein mCherry in bovine mycoplasma, it was demonstrated that SMbovP280, SMbovP374, and SMbovP475 can guide the secretion of the exogenous protein mCherry into the culture supernatant of bovine mycoplasma, and SMbovP475 has the highest efficiency in guiding the secretion of exogenous proteins.

[0039] In this document, the terms "homology" and "identity" are used interchangeably. Homologous sequences include amino acid sequences that are at least 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequences of this invention. To determine sequence identity, sequence alignment can be performed, which can be done in various ways known to those skilled in the art, such as using BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms required to achieve optimal alignment across the full-length sequences being compared.

[0040] In this document, the modified amino acid sequences also fall within the scope of protection of this invention. The term "modification" refers to any chemical modification of an amino acid sequence, such as substitution, deletion, insertion, and / or addition of amino acids. The term "substitution" refers to replacing one or more amino acids with a different amino acid. "Deletion" refers to the reduction of one or more amino acids in an amino acid sequence. "Insertion" or "addition" refers to a change in the amino acid sequence resulting in an increase of one or more amino acids compared to a naturally occurring molecule.

[0041] In a preferred embodiment, the signal peptide is a non-cleaving signal peptide. The term "non-cleaving signal peptide" refers to a signal peptide of the present invention that is not cleaved by a signal peptidase after initiating the expression of a foreign protein.

[0042] In this invention, the signal peptide can be prepared artificially or through genetic engineering, and there is no particular limitation thereto. In some embodiments, the signal peptide of this invention is obtained by artificial synthesis. Methods for artificially synthesizing signal peptides are known in the art, for example, the signal peptide of this invention is obtained by direct amino acid synthesis. In some embodiments, the signal peptide of this invention is prepared through genetic engineering. Methods for preparing signal peptides through genetic engineering are known in the art, for example, preparing the gene sequence of the signal peptide, constructing a vector expressing the gene sequence, and expressing it after transformation into host cells.

[0043] In a preferred embodiment, the method for guiding the secretory expression of exogenous proteins in mycoplasma according to the present invention includes the following steps:

[0044] (1) Construct a recombinant expression vector containing nucleotide sequences encoding the signal peptide and the exogenous protein;

[0045] (2) Transfect the recombinant expression vector into mycoplasma;

[0046] (3) The mycoplasma is cultured under conditions suitable for the expression of the exogenous protein, so that the exogenous protein is secreted and expressed in the mycoplasma.

[0047] In a preferred embodiment, the nucleotide sequence comprises a codon-optimized sequence.

[0048] In this invention, "codon optimization" refers to changing the codons of polynucleotides encoding proteins to codons that are preferentially used in a specific organism, thereby enabling the encoded protein to be effectively expressed in the organism of interest. In a preferred embodiment, the purpose of codon optimization is to enable the effective expression of a fusion protein containing a signal peptide and a foreign protein in mycoplasma.

[0049] The vectors described herein are well known to those skilled in the art, and in a preferred embodiment, the vector is a plasmid. It should be understood that any vector capable of guiding the expression of a foreign gene in a host cell can be used to introduce the nucleotide sequences encoding the signal peptide and the foreign protein provided by this invention into mycoplasma, thereby obtaining the foreign protein, particularly a fusion protein of the signal peptide and the foreign protein. The expression vector carrying the nucleotide sequences encoding the signal peptide and the foreign protein can be used to transform mycoplasma using conventional biological methods such as plasmids, calcium chloride transformation, microinjection, and electrocoagulation, and the mycoplasma can be cultured under conditions suitable for the expression of the foreign protein, allowing the foreign protein to be secreted and expressed in the mycoplasma. The isolated foreign protein can then be obtained through conventional collection, purification, and concentration methods.

[0050] application

[0051] One aspect of the present invention provides the use of a signal peptide in the preparation of a medicament for the prevention or treatment of pathogenic bacterial infections, wherein the signal peptide has any one of the amino acid sequences shown in (I)-(III):

[0052] (I) The amino acid sequence shown in SEQ ID NO. 2, 4 or 6;

[0053] (II) has at least 90% homology with the amino acid sequence shown in (I) and has the same function;

[0054] Amino acid sequences with the same function obtained by modifying, substituting, deleting, or adding one or more amino acids of the amino acid sequences shown in (III), (I), or (II).

[0055] The term "prevention or treatment" as used in this invention refers to improving a condition before or after the onset of a disease or dysfunction. This degree of relief or prevention, measured by any standard technique, is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% compared to an untreated control group under equivalent conditions. In this invention, the term "treatment" refers to therapeutic treatment and preventative or therapeutic measures aimed at preventing or mitigating (reducing) undesirable physiological changes or disorders. Beneficial or desired clinical outcomes include, but are not limited to, the following, whether detectable or undetectable: symptom relief, reduction in disease severity, stabilization of the disease state, improvement or mitigation of the disease state, and reduction (whether partial or complete). Those requiring treatment include those who already have the condition or disorder, those who are susceptible to the condition or disorder, or those who need to prevent the condition or disorder.

[0056] In this invention, the pathogen infection includes mycoplasma infection, as well as co-infection with mycoplasma and other pathogens.

[0057] In a preferred embodiment, the medicament of the present invention includes a vaccine.

[0058] In a preferred embodiment, the vaccine of the present invention comprises a gene sequence expressing a fusion protein containing a signal peptide and a foreign protein.

[0059] In another preferred embodiment, the vaccine of the present invention comprises a vector containing a gene sequence of a fusion protein.

[0060] In yet another preferred embodiment, the vaccine of the present invention comprises a host cell containing a vector encoding a sequence of a fusion protein.

[0061] In this invention, the prevention or treatment is achieved by administering an immune-effective dose of vaccine to the subject.

[0062] In this invention, an "immunely effective dose" is the amount of medicine that produces the desired therapeutic effect in a subject (e.g., prevention or treatment of a target condition or relief of symptoms associated with that condition). A precise immunoeffective dose is the amount of composition that produces the most effective result in terms of therapeutic effect in a given subject. This amount varies depending on a variety of factors, including but not limited to, the characteristics of the therapeutic medicine (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, type and stage of disease, overall physical condition, response to a given dose, and type of medicine), the pharmaceutically acceptable carrier in the formulation or the nature of the carrier, and the route of administration. Those skilled in the art in the clinical and pharmacological fields will be able to determine the immunoeffective dose through routine experiments, i.e., by monitoring the subject's response to administration of the compound and adjusting the dosage accordingly.

[0063] In this invention, the subjects include mammals, such as mice, rabbits, cattle, sheep, pigs, and humans.

[0064] In a preferred embodiment, the vaccine comprises a vector vaccine. Methods of administration of vector vaccines are known in the art, such as subcutaneous or intramuscular injection.

[0065] In a preferred embodiment, the vector vaccine uses mycoplasma as a vector.

[0066] Example 1

[0067] The following illustrates the construction of a recombinant plasmid encoding a bovine mycoplasma signal peptide fused with an exogenous protein and its transformation into bovine mycoplasma.

[0068] The mycoplasma used in this embodiment is Mycoplasma bovis HB0801, which was deposited at the China Center for Type Culture Collection, Wuhan University, China on February 1, 2010, with accession number CCTCC NO: M2010040.

[0069] Due to the codon preference of Bovine Mycoplasma, the nucleotide sequence encoding mCherry fluorescent protein in this invention is optimized according to the codon preference of Bovine Mycoplasma. The nucleotide sequences of SMbovP475, SMbovP374, and SMbovP280, along with the optimized nucleotide sequence encoding mCherry fluorescent protein, were sent to a company for synthesis. The amino acid sequence of SMbovP475 is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.1; the amino acid sequence of SMbovP374 is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.3; the amino acid sequence of SMbovP280 is shown in SEQ ID NO.6, and the nucleotide sequence is shown in SEQ ID NO.5.

[0070] Take 1 μg of the above DNA product and digest it with NotⅠ enzyme. At the same time, digest the pOH plasmid with Xho I and BamHI. Ligate the digested DNA product and pOH plasmid with DNA ligase to obtain the recombinant plasmid.

[0071] Bovine mycoplasma competent cells were prepared using the CaCl2 method, and the operation steps are as follows:

[0072] (1) Take strain HB0801 stored at -80℃, inoculate it into 1mL PPLO at a ratio of 1:100, and incubate it in a 37℃, 5% CO2 incubator for 24h.

[0073] (2) Transfer the above bacterial culture to 1 mL PPLO at a ratio of 1:100 and continue to culture in a 37℃, 5% CO2 incubator for 48 h;

[0074] (3) Centrifuge the cultured HB0801 at 4℃, 10,000×g for 20min, discard the supernatant, and wash the cell pellet with pre-cooled DPBS.

[0075] (4) Centrifuge at 4℃, 10,000×g for 10 min, and repeat the washing steps twice;

[0076] (5) Resuspend the bovine mycoplasma precipitate in 375 μL of CaCl2 (0.1 mol / L) in an ice bath and incubate on ice for 30 min.

[0077] The constructed recombinant plasmid was transformed into bovine mycoplasma HB0801 competent cells to construct a bovine mycoplasma strain expressing the exogenous gene. The specific steps are as follows:

[0078] (1) Take 100 μL of the prepared bovine mycoplasma competent cells and add them to a 1.5 mL EP tube. Add 1 μL of yeast tRNA and 3 μg of recombinant plasmid, and gently pipette to mix.

[0079] (2) Transfer the mixed sample to a 50 mL conical centrifuge tube containing 1 mL of 50% PEG8000 and incubate at room temperature for 1 min;

[0080] (3) Add 5 mL of PPLO medium, mix gently, and incubate at 37°C in a 5% CO2 incubator for 3 h;

[0081] (4) Centrifuge the culture at 25℃, 10,000×g for 8 min, discard the supernatant, and resuspend the precipitate in 1 mL PPLO;

[0082] (5) Take 315 μL of resuspended bacterial solution and spread it on a PPLO solid plate containing 10 μg / mL puromycin. Incubate at 37℃ and 5% CO2 for 3-8 days.

[0083] (6) Pick a single colony and incubate it in 1 mL of PPLO liquid medium containing 10 μg / mL puromycin for 48 h. Identify the colony after the medium turns yellow.

[0084] Example 2

[0085] The following shows the detection of the expression of the exogenous protein mCherry in bovine mycoplasma.

[0086] Wild-type Mycoplasma bovis strain HB0801 and strain expressing exogenous genes were inoculated into 2 mL PPLO and cultured to the logarithmic phase. After centrifugation at 12,000 × g for 5 min at 4 °C, the supernatant was discarded, and the samples were resuspended in 30 μL PBS. SDS-PAGE loading buffer was added, the samples were mixed, and the mixture was boiled in water for 10 min before Western blotting. After SDS-PAGE (10 μL / well) electrophoresis and membrane transfer, the membrane was washed three times with 1×TBST solution on a horizontal shaker at room temperature for 5 min each time. The membrane was then placed in 5% skim milk diluted with 1×TBST and blocked at room temperature or in a 37°C incubator for 2 h. After blocking, the PVDF membrane was washed 3-5 times with 1×TBST solution on a horizontal shaker at room temperature for 5 min each time. After washing, the PVDF membrane was incubated overnight at 4°C with anti-mCherry or NOX primary antibody (1×TBST diluted 1:1,000). After primary antibody incubation, the PVDF membrane was washed 4-6 times with 1×TBST solution on a horizontal shaker at room temperature for 5 min each time. After washing, the PVDF membrane was incubated for 1 h at room temperature with horseradish peroxidase-labeled mouse or rabbit secondary antibody (1×TBST diluted 1:5,000). After secondary antibody incubation, the PVDF membrane was removed and washed three times with 1×TBST solution on a horizontal shaker at room temperature for 5 min each time. Finally, use filter paper to absorb the moisture on the PVDF membrane, then evenly drop the mixed ECL luminescent liquid onto the membrane, and use a chemiluminescence detector to expose, image, and preserve the image.

[0087] The results were obtained using the Western blotting method. Figure 1 As shown, mCherry, SMbovP280-mcherry, SMbovP374-mcherry, SMbovP475-mcherry, and SMbovP739-mcherry, which do not contain the signal peptide, can all be expressed in Mycoplasma bovis, while no mCherry signal was detected in the wild-type strain HB0801(WT) without transformation of the shuttle plasmid.

[0088] Example 3

[0089] The following shows the detection of the secretion amount of the exogenous protein mCherry in the culture supernatant of bovine mycoplasma.

[0090] 1. Western blotting method for detecting mCherry secretion in culture supernatant

[0091] Wild-type Mycoplasma bovis strain HB0801 and strain expressing the exogenous gene were inoculated into 10 mL of PPLO and cultured to the logarithmic phase. The cultures were then centrifuged at 12,000 × g for 5 min at 4 °C. The supernatant was collected and concentrated using an ultrafiltration tube with a molecular cutoff of 3 KD at 4,000 × g and 4 °C. When the remaining supernatant volume was 1 mL, 80 μL of the concentrated supernatant was added, mixed with SDS-PAGE loading buffer, and boiled in water for 10 min before Western blotting. The detection method was the same as in Example 2.

[0092] The results are as follows Figure 2 As shown, mCherry without the signal peptide could not be detected in the culture supernatant; only a small amount of the SMbovP374-mcherry fusion protein was detected in the supernatant; the MbovP280-mcherry and SMbovP475-mcherry fusion proteins were detected in large quantities in the supernatant, with the SMbovP475-mcherry fusion protein having the highest content; while no mCherry signal was detected in the blank PPLO medium and the culture supernatant of the wild-type strain HB0801(WT) without shuttle plasmid transformation.

[0093] 2. Detection of mCherry secretion in culture supernatant using a fluorescent microplate reader.

[0094] Wild-type Mycoplasma bovis strain HB0801 and strain expressing the exogenous gene were inoculated into 10 mL PPLO and cultured to the logarithmic phase. The cultures were then centrifuged at 12,000 × g for 5 min at 4 °C. The culture supernatant was collected, with 100 μL of supernatant from each experimental group placed in a 96-well plate, and three replicates were set for each experimental group. The mCherry fluorescence intensity was detected using a fluorescence microplate reader.

[0095] The results are as follows Figure 3 As shown, the fluorescence signals in the culture supernatants of *Mycoplasma bovis* strains expressing mCherry (without signal peptide), SMbovP280-mcherry, SMbovP374-mcherry, SMbovP475-mcherry, and SMbovP739-mcherry were compared. No difference in fluorescence values ​​was found between the culture supernatants of *Mycoplasma bovis* strains expressing mCherry (without signal peptide) and SMbovP739-mcherry and the wild-type strain HB0801. The fluorescence values ​​of the culture supernatants of *Mycoplasma bovis* strains expressing SMbovP280-mcherry, SMbovP374-mcherry, and SMbovP475-mcherry were significantly higher than those of the wild-type strain HB0801 (**p<0.01), with the highest fluorescence value observed in the culture supernatant expressing SMbovP475-mcherry.

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for guiding the secretory expression of exogenous proteins in bovine mycoplasma, characterized in that, The method includes the step of introducing a signal peptide at one end of the exogenous protein, wherein the signal peptide is a non-cleavage signal peptide, and the amino acid sequence of the signal peptide is shown in SEQ ID NO.

2. The method comprises: (1) Construct a recombinant expression vector containing nucleotide sequences encoding the signal peptide and the exogenous protein; (2) Transfect the recombinant expression vector into bovine mycoplasma; (3) The bovine mycoplasma is cultured under conditions suitable for the expression of the exogenous protein, so that the exogenous protein is expressed in the bovine mycoplasma.

2. The method according to claim 1, characterized in that, The signal peptide is prepared through artificial synthesis or genetic engineering.

3. The method according to claim 1, characterized in that, The nucleotide sequence includes a codon-optimized sequence.

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