Composition for preventing and treating mycoplasma hyorhinis infection, and preparation method therefor

Abstract

The present invention provides a composition for preventing and treating a mycoplasma hyorhinis infection. The composition uses the N-terminal-XylF fusion protein of C-terminal-P72 of DnaK as an active ingredient. The present invention further provides an expression vector and a method required for producing the fusion protein.

Description

Composition for preventing and treating Mycoplasma hyorhinis infection and preparation method thereof Technical Field

[0001] The present application relates to a composition for preventing and treating Mycoplasma infection, in particular to a composition for preventing and treating Mycoplasma hyorhinis infection and a method for preparing the same. Background Art

[0002] Mycoplasma spp. are the smallest known prokaryotic microorganisms capable of extracellular self-replication. They range in size from spherical forms (0.3 to 0.9 μm in diameter) to filaments up to 1 μm in length. Mycoplasma spp. are fastidious bacteria, difficult to culture, and grow very slowly. Several species of Mycoplasma spp. are known to be associated with various human and animal diseases.

[0003] According to literature reports, several species of Mycoplasma are associated with various human and animal diseases. Among them, Mycoplasma hyorhinis is a respiratory pathogen in pigs, with an infection rate ranging from 25% to 93%. It can cause swine enzootic pneumonia (SEP), a highly contagious chronic respiratory disease with a low mortality rate. M. hyorhinis is also associated with polyserositis, arthritis, and meningitis.

[0004] Pigs infected solely with Mycoplasma hyorhinis usually do not die, but they will suffer from reduced feed conversion rate, slowed growth and development, inflammatory reactions, immunosuppression, and secondary infections with other bacterial or viral pathogens, causing huge economic losses to the pig farming industry.

[0005] Currently, an inactivated Mycoplasma hyorhinis vaccine is available on the market for use in the pig farming industry. However, Mycoplasma hyorhinis is difficult to culture, the culture medium used is expensive, Mycoplasma hyorhinis may have the risk of inducing human cancer, and high-standard protective measures are required during the vaccine preparation process. Therefore, the development of highly safe and low-cost subunit vaccines is of great significance and demand in this field.

[0006] In previous research, the inventors of this case screened 17 antigens and identified three suitable as active ingredients in subunit vaccines: XylF, DnaK, and P72. However, from a vaccine manufacturing perspective, the production process for these multiple antigens presents bottlenecks that consume both manpower and time. Furthermore, the optimal production conditions for each antigen, such as bacterial fermentation and protein purification, require tailored processes tailored to the specific antigen's characteristics.

[0007] Summary of the Invention

[0008] To address the needs in the field, this application provides a composition for preventing and treating Mycoplasma hyorhinis infection. By developing a subunit vaccine containing a fusion protein, the vaccine production process and costs are significantly reduced. Pig challenge trials demonstrated that the vaccine prepared with the fusion protein is highly safe and effectively mitigates the pathological changes caused by Mycoplasma hyorhinis infection.

[0009] To achieve the above objectives, the present invention provides a composition for preventing and treating Mycoplasma hyorhinis infection, which may include: (a) a fusion protein of the C-terminus of DnaK of Mycoplasma hyorhinis - the N-terminus of P72 - XylF; and (b) a pharmaceutically acceptable adjuvant.

[0010] Preferably, the concentration of the fusion protein in the composition may be 50 to 500 μg / mL, 60 to 400 μg / mL, 70 to 300 μg / mL, 80 to 200 μg / mL, or 90 to 100 μg / mL. In one embodiment, the concentration of the fusion protein may be 100 μg / mL.

[0011] Preferably, in the composition, the C-terminus of DnaK may include the sequence shown in SEQ ID NO: 9; the N-terminus of P72 may include the sequence shown in SEQ ID NO: 11; or the XylF may include the sequence shown in SEQ ID NO: 13.

[0012] Preferably, the composition, wherein the fusion protein may include the sequence shown in SEQ ID NO: 7.

[0013] Preferably, the pharmaceutically acceptable adjuvant of the composition can be: Freund's complete or incomplete adjuvant, aluminum gel, surfactant, anionic polymer, peptide, oil emulsion or a combination thereof.

[0014] Preferably, the composition may further include pharmaceutically acceptable additives. In one embodiment, the pharmaceutically acceptable additives may include solvents, stabilizers, diluents, preservatives, antibacterial agents, antifungal agents, isotonic agents, absorption delaying agents, or combinations thereof.

[0015] Another object of the present application is to provide an expression vector for expressing the aforementioned fusion protein, which may include: (a) a nucleotide sequence encoding a DnaK C-terminus-P72 N-terminus-XylF fusion protein; (b) a replicon gene; and (c) an expression element, which may include a promoter, a terminator, and a ribosome binding site.

[0016] Preferably, in the expression vector, the nucleotide sequence encoding the C-terminus of DnaK can encode the amino acid sequence shown in SEQ ID NO: 9; the nucleotide sequence encoding the N-terminus of P72 can encode the amino acid sequence shown in SEQ ID NO: 11; and the nucleotide sequence encoding XylF can encode the amino acid sequence shown in SEQ ID NO: 13.

[0017] Preferably, in the expression vector, the nucleotide sequence encoding the C-terminus of DnaK may include the nucleotide sequence shown in SEQ ID NO: 8 or the reverse complementary sequence of the sequence; the nucleotide sequence encoding the N-terminus of P72 may include the nucleotide sequence shown in SEQ ID NO: 10 or the reverse complementary sequence of the sequence; the nucleotide sequence encoding XylF may include the nucleotide sequence shown in SEQ ID NO: 12 or the reverse complementary sequence of the sequence.

[0018] Preferably, the nucleotide sequence encoding the fusion protein in the expression vector can encode the amino acid sequence shown in SEQ ID NO: 7.

[0019] Preferably, the nucleotide sequence encoding the fusion protein in the expression vector may include the nucleotide sequence shown in SEQ ID NO: 6 or the reverse complementary sequence of the sequence.

[0020] Preferably, the expression vector may include the nucleotide sequence shown in SEQ ID NO: 5.

[0021] Preferably, the fusion protein in the expression vector may further include a tag. In one embodiment, the tag may include a histidine tag (His-tag), a glutathione-S-transferase tag (GST-tag), a FLAG tag, a maltose binding protein tag (MBP-tag), a hemagglutinin tag (HA-tag), or a combination thereof.

[0022] Preferably, the expression vector may further include a screening gene. In one embodiment, the screening gene may include a drug resistance screening gene, a non-drug resistance screening gene, or a combination thereof.

[0023] Preferably, the expression vector, wherein the fusion protein can be expressed in Escherichia coli.

[0024] Another object of the present application is to provide a method for producing a DnaK C-terminus-P72 N-terminus-XylF fusion protein, which may include transcribing the aforementioned nucleotide sequence in an expression system and translating it to obtain the fusion protein.

[0025] Another object of the present application is to provide a use of a DnaK C-terminus-P72 N-terminus-XylF fusion protein for preparing a composition for preventing and treating symptoms caused by Mycoplasma hyorhinis infection.

[0026] Preferably, the symptoms caused by the infection with Mycoplasma hyorhinis for use may include pleurisy, peritonitis, pericarditis, arthritis, or a combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1. Construction process of pET29-MHY30.

[0028] Figure 2. Analysis of the expression of DnaK C-terminus-P72 N-terminus-XylF fusion protein in E. coli by protein electrophoresis (A) and immunoblotting (B). TM Prestained Protein Ladder (Thermo Fisher Scientific, USA); C: control group, total cell lysate of E. coli BL21 (DE3) (pET29a); T: total cell lysate of E. coli BL21 (DE3) (pET29-MHY30); S: soluble protein of E. coli BL21 (DE3) (pET29-MHY30).

[0029] Figure 3. Purification of DnaK C-terminus-P72 N-terminus-XylF fusion protein. DETAILED DESCRIPTION

[0030] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those skilled in the art. In case of conflict, the definitions contained in this specification will control.

[0031] When referring to concentration in this application, "percentage", "%", etc., as used herein, may refer to mass concentration or volume percentage concentration; mass concentration is defined as the mass of the solute divided by the percentage of the volume of the corresponding mixture (including the volume of the solute), and may be expressed herein by "percentage", "%", etc. followed by "(w / v)"; volume percentage concentration is defined as the percentage of the volume of the solute to the total volume of the solution, and may be expressed herein by "percentage", "%", etc. followed by "(v / v)".

[0032] As used in this application, the term "produced from" is synonymous with "comprising." As used herein, the terms "includes," "including," "comprises," "comprising," "has," "having," "contains," "containing," or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that contains a plurality of listed elements is not necessarily limited to only those listed elements but may include other elements not expressly listed but inherent to the composition, process, method, article, or apparatus. The term "comprising" is generally used in an inclusive sense, that is, allowing for the presence of one or more additional features or components.

[0033] The indefinite articles "a" and "an" preceding an element or component in the present application are intended to indicate the number of instances (i.e., the number of occurrences) of the element or component without limitation. Therefore, "a" or "an" should be understood to include one or at least one, and the singular form of the element or component also includes the plural unless the number is obviously intended to be singular.

[0034] The present application provides many sequences, including primer sequences, nucleotide sequences and amino acid sequences. When the functions and effects of these sequences in organisms are not changed, they should be allowed to have some mutations. That is to say, even if the sequences are different, but the functions and effects produced are the same as those provided in the present application, they should be considered to be the same sequence. For example: in the primer sequence, even if the primer sequences are slightly different, the primers that can amplify the same product should be considered to be the same; in the nucleotide sequence, if a gene undergoes a silent mutation, the amino acid sequence transcribed from it is the same as before the mutation, it should be considered to be the same sequence; for example, in the amino acid sequence, amino acid bases with similar physical properties (such as the same electrical properties, the same hydrophilicity or hydrophobicity, benzene rings, etc.) mutate with each other without affecting their original structure and function, and should be considered to be the same sequence. The above situations are only examples and should not be interpreted as limitations.

[0035] The present application aims to provide a composition for preventing and treating Mycoplasma hyorhinis infection, comprising: (a) a fusion protein comprising the C-terminus of DnaK and the N-terminus of P72 and XylF from Mycoplasma hyorhinis; and (b) a pharmaceutically acceptable adjuvant. By expressing this fusion protein containing multiple antigens in an expression system (e.g., Escherichia coli) as the active ingredient of a vaccine, the vaccine production process and costs are significantly reduced, thereby optimizing the process previously proposed by the inventors and providing further advantages in the field.

[0036] As used herein, the term "fusion protein" refers to an amino acid sequence composed of multiple different proteins. The order of the proteins may vary without affecting the intended efficacy of the fusion protein in this application. For example, from N-terminus to C-terminus, it is "N-terminus of P72 - C-terminus of DnaK - XylF"; or "XylF - N-terminus of P72 - C-terminus of DnaK," etc. In one embodiment, the fusion protein provided herein is a DnaK C-terminus - P72 N-terminus - XylF fusion protein, and its amino acid sequence, from N-terminus to C-terminus, is "C-terminus of DnaK," "N-terminus of P72," and "XylF." The above is merely an example and should not be construed as limiting.

[0037] As used herein, the term "DnaK C-terminus-P72 N-terminus-XylF fusion protein" may be referred to as "MHY-30." If the nucleotide sequence encoding the fusion protein is referred to, it may be referred to as "the nucleotide sequence of MHY-30." Similarly, if the amino acid sequence of the fusion protein is referred to, it may be referred to as "the amino acid sequence of MHY-30."

[0038] In one embodiment, the fusion protein concentration in the composition can be 50 to 500 μg / mL; in a preferred embodiment, the fusion protein concentration in the composition can be 100 μg / mL, based on the total volume of the composition. However, those skilled in the art will appreciate that these concentrations may vary depending on the intended use. For example, for ease of shipping and storage, one skilled in the art may prepare the composition with a high concentration of the active ingredient and then dilute it prior to use.

[0039] As used herein, the terms "C-terminus of DnaK" and "N-terminus of P72" refer to portions of an amino acid sequence. Those skilled in the art define the amino acid sequence with a free α-amino group as the "N-terminus" and the amino acid sequence with a free α-carboxyl group as the "C-terminus." Amino acid sequences are commonly written from N-terminus to C-terminus, so "N-terminus" can represent the beginning of a sequence, while "C-terminus" refers to the end of a sequence.

[0040] For example, if a protein is 266 amino acids long, its "N-terminus" could be amino acids 1-50, 37-100, or 23-115; and its "C-terminus" could be amino acids 150-235, 122-198, or 178-234. Indicating the position of an amino acid sequence using the N-terminus or C-terminus only roughly indicates whether it is at the "beginning" or "end" of the protein; it does not precisely define the position. Therefore, the detailed position information should be based on the definitions in this specification.

[0041] For example, as used herein, "the C-terminus of DnaK" may include amino acids 291 to 597 of the DnaK protein (e.g., NCBI reference sequence WP_013302427.1); "the N-terminus of P72" may include amino acids 26 to 265 of the P72 protein (e.g., NCBI reference sequence WP_014335516.1); and "XylF" may include amino acids 25 to 447 of the XylF protein (e.g., NCBI reference sequence WP_027332477.1). For protein sequences, see Patent Publication No. TWI606839, which is incorporated herein by reference in its entirety.

[0042] It is worth noting that the amino acid sequence used herein may not be the full length of the protein. Without changing the efficacy of the fusion protein to be achieved in this application, this application should include amino acid sequences of different lengths of the above-mentioned protein.

[0043] In one embodiment, the C-terminus of DnaK of the fusion protein of the composition may include the sequence shown in SEQ ID NO: 9; the N-terminus of P72 may include the sequence shown in SEQ ID NO: 11; and XylF may include the sequence shown in SEQ ID NO: 13.

[0044] The amino acid sequences provided in this application may include amino acid sequences that do not belong to the protein. For example, amino acids 1 to 10 (MRGSHHHHHH) of the C-terminus of DnaK (SEQ ID NO: 9) may include a tag sequence used for protein purification, while amino acids 318 to 332 (EAAAKEAAAKEAAAK) may be a linker peptide, which has been found to increase the yield of the fusion protein. Similar situations exist for amino acids 241 to 255 (EAAAKEAAAKEAAAK) of the N-terminus of P72 (SEQ ID NO: 11) and amino acids 424 to 429 (HHHHHH) of XylF (SEQ ID NO: 13). These amino acid sequences do not affect the effects of the fusion protein.

[0045] In one embodiment, the fusion protein of the composition may include the sequence set forth in SEQ ID NO: 7. Within the fusion protein, a few amino acids (e.g., one, two, or three) may separate the proteins. These are simply restriction enzyme cleavage sites artificially added to the amino acid-encoding nucleotides to facilitate genetic engineering. For example, the nucleotide cleavage site GGTACC for the restriction enzyme KpnI will encode amino acids such as glycine (G) and threonine (T), which does not affect the effectiveness of the fusion protein.

[0046] In one embodiment, the composition of the present invention may further include a pharmaceutically acceptable adjuvant and / or pharmaceutically acceptable additive. Pharmaceutically acceptable adjuvants are used to enhance the immunogenicity of the active ingredient, maintain the stability of the active ingredient, and improve the safety of the composition for vaccine use. Pharmaceutically acceptable adjuvants may include, but are not limited to, Freund's complete or incomplete adjuvant, aluminum gel, surfactants, anionic polymers, peptides, oil emulsions, or combinations thereof. Pharmaceutically acceptable additives may include, but are not limited to, solvents, stabilizers, diluents, preservatives, antibacterial agents, antifungal agents, isotonic agents, absorption delaying agents, or combinations thereof.

[0047] To express the aforementioned fusion protein, the present application provides an expression vector, which may include: (a) a nucleotide sequence encoding a DnaK C-terminus-P72 N-terminus-XylF fusion protein; (b) a replicon gene; and (c) an expression element, which may include a promoter, a terminator, and a ribosome binding site.

[0048] As used herein, the term "expression vector" refers to a vector into which the nucleotide sequence provided herein can be inserted and which can be replicated in large quantities in a host or translated into a fusion protein. Useful expression vectors may include, but are not limited to, pET series vectors for Escherichia coli expression systems, pPIC series vectors for methanol-loving yeast expression systems, baculovirus pOET series transfer vectors (transfer plasmids) for insect cell expression systems, pCMV vectors for mammalian cell expression systems, and shuttle vectors that can replicate and express between different hosts. Any expression vector can be used as long as the nucleotide sequence can be inserted. Those skilled in the art will appreciate that when using an expression vector to express a desired protein, various expression vectors can be selected based on considerations such as the characteristics of the protein, the desired yield, and cost. Therefore, the embodiments provided in this application are merely examples and should not be construed as limiting this application.

[0049] As used herein, the term "replicon gene" refers to a region where DNA replication originates, which at least includes the origin of replication.

[0050] As used herein, the term "expression element" refers to the elements required to initiate transcription and translation processes in an expression system. The aforementioned expression element should at least include a promoter, a terminator, and a ribosome binding site. Preferably, the expression element may additionally include: an operator, transcription / translation enhancer sequences, or a combination thereof. In a preferred embodiment, the aforementioned expression vector is used in an Escherichia coli expression system, and the aforementioned expression element can be recognized by Escherichia coli.

[0051] In one embodiment, the nucleotide sequence encoding the C-terminus of DnaK in the expression vector may encode the amino acid sequence shown in SEQ ID NO: 9; the nucleotide sequence encoding the N-terminus of P72 may encode the amino acid sequence shown in SEQ ID NO: 11; and the nucleotide sequence encoding XylF may encode the amino acid sequence shown in SEQ ID NO: 13.

[0052] In one embodiment, the nucleotide sequence encoding the C-terminus of DnaK of the expression vector may include the nucleotide sequence shown in SEQ ID NO: 8 or the reverse complementary sequence of the sequence; the nucleotide sequence encoding the N-terminus of P72 may include the nucleotide sequence shown in SEQ ID NO: 10 or the reverse complementary sequence of the sequence; the nucleotide sequence encoding XylF may include the nucleotide sequence shown in SEQ ID NO: 12 or the reverse complementary sequence of the sequence.

[0053] In one embodiment, the nucleotide sequence encoding the fusion protein of the expression vector may encode the amino acid sequence set forth in SEQ ID NO: 7. In one embodiment, the nucleotide sequence encoding the fusion protein of the expression vector may include the nucleotide sequence set forth in SEQ ID NO: 6 or its reverse complement. In one embodiment, the expression vector may include the nucleotide sequence set forth in SEQ ID NO: 5.

[0054] The term "reverse complementary sequence of a sequence" as used herein means that, in general, nucleotide sequences are expressed in the art from the 5' end to the 3' end, because when nucleotides are transcribed or translated, most polymerases transcribe or translate them in the order from the 5' end to the 3' end. Therefore, the nucleotide sequences encoding fusion proteins or proteins provided in this application, such as SEQ ID NO: 6, 8, 10, 12 and other sequences, are all expressed in accordance with the conventions in the art. However, since the expression vector is a double-stranded nucleotide sequence, in some cases it is necessary to insert the sequence in a reverse complementary manner to match the transcription direction of the promoter. For example, in the examples provided in this application, in order to match the transcription direction of the T7 promoter, the nucleotide sequences encoding the fusion proteins are all inserted in the reverse complementary form. A sequence and its reverse complementary sequence are complementary nucleotide chains to each other, so the two can be regarded as the same sequence in the art.

[0055] In one embodiment, the fusion protein of the expression vector may further include a tag; which may include: a histidine tag (His-tag), a glutathione-S-transferase tag (GST-tag), a FLAG tag, a maltose binding protein tag (MBP-tag), a hemagglutinin protein tag (HA-tag), or a combination thereof.

[0056] As used herein, the term "tag" refers to a nucleotide sequence inserted into an expression vector along with the nucleotide sequence encoding a fusion protein. After transcription and translation of the tagged fusion protein, the tag binds to a specific resin through affinity, facilitating purification. For example, the histidine tag used in this application contains multiple repeating histidines, which have a high affinity for nickel-ion affinity columns, allowing for the separation and purification of the fusion protein.

[0057] As used herein, the term "affinity column" refers to a column containing a resin that has an affinity for a tag and is used to separate proteins. In addition to the nickel ion column mentioned above, glutathione affinity columns, Flag antibody affinity columns, maltose-binding protein affinity columns, and hemagglutinin protein affinity columns are all commonly used in the art. Fusion proteins can be purified using affinity columns.

[0058] In one embodiment, the expression vector may further include a selection gene, such as a kanamycin resistance gene, but is not limited thereto.

[0059] In one embodiment, the fusion protein of the expression vector can be expressed in E. coli.

[0060] In one embodiment, a method for producing a DnaK C-terminus-P72 N-terminus-XylF fusion protein is provided, which may include transcribing the aforementioned nucleotide sequence in an expression system and translating to obtain the fusion protein.

[0061] As used herein, the term "expression system" refers to an expression vector and its associated host that enables protein expression. An example of this is the E. coli expression system provided in this application. The fusion proteins provided in this application can also be produced using common expression systems in the art, including methanolic yeast expression systems, insect cell expression systems, and mammalian cell expression systems.

[0062] In one embodiment, a DnaK C-terminus-P72 N-terminus-XylF fusion protein is provided for use in preparing a composition for preventing and treating symptoms caused by Mycoplasma hyorhinis infection. Preferably, the symptoms caused by Mycoplasma hyorhinis infection include pleuritis, peritonitis, pericarditis, arthritis, or a combination thereof.

[0063] The following disclosures are examples of the claimed invention, which serve as specific examples of the present application and should not limit the present application by the following examples.

[0064] Example 1: A composition for preventing and treating Mycoplasma hyorhinis infection, comprising:

[0065] (a) a fusion protein of DnaK C-terminus and P72 N-terminus and XylF of Mycoplasma hyorhinis; and

[0066] (b) a pharmaceutically acceptable adjuvant.

[0067] Example 2: The composition of the preceding example, wherein the concentration of the fusion protein is 50 to 500 μg / mL.

[0068] Example 3: The composition as described in the above example, wherein the C-terminus of DnaK comprises the sequence shown in SEQ ID NO: 9; the N-terminus of P72 comprises the sequence shown in SEQ ID NO: 11; and the XylF comprises the sequence shown in SEQ ID NO: 13.

[0069] Example 4: The composition as described in the preceding example, wherein the fusion protein comprises the sequence shown in SEQ ID NO: 7.

[0070] Example 5: The composition as described in the above examples, wherein the pharmaceutically acceptable adjuvant is: Freund's complete or incomplete adjuvant, aluminum gel, a surfactant, an anionic polymer, a peptide, an oil emulsion or a combination thereof.

[0071] Example 6: The composition as described in the above example further comprises a pharmaceutically acceptable additive.

[0072] Example 7: The composition as described in the above examples, wherein the pharmaceutically acceptable additives include solvents, stabilizers, diluents, preservatives, antibacterial agents, antifungal agents, isotonic agents, absorption delaying agents or combinations thereof.

[0073] Example 8: An expression vector for expressing the fusion protein as described in any one of the above examples, comprising:

[0074] (a) Nucleotide sequence encoding the DnaK C-terminus-P72 N-terminus-XylF fusion protein;

[0075] (b) replicon genes, and;

[0076] (c) Expression elements, including promoter, terminator and ribosome binding site.

[0077] Example 9: The expression vector as described in the previous example, wherein the nucleotide sequence encoding the C-terminus of DnaK encodes the amino acid sequence shown in SEQ ID NO: 9; the nucleotide sequence encoding the N-terminus of P72 encodes the amino acid sequence shown in SEQ ID NO: 11; and the nucleotide sequence encoding XylF encodes the amino acid sequence shown in SEQ ID NO: 13.

[0078] Example 10: The expression vector as described in the preceding example, wherein the nucleotide sequence encoding the C-terminus of DnaK comprises the nucleotide sequence shown in SEQ ID NO: 8 or the reverse complementary sequence thereof; the nucleotide sequence encoding the N-terminus of P72 comprises the nucleotide sequence shown in SEQ ID NO: 10 or the reverse complementary sequence thereof; and the nucleotide sequence encoding XylF comprises the nucleotide sequence shown in SEQ ID NO: 12 or the reverse complementary sequence thereof.

[0079] Example 11: The expression vector as described in the above example, wherein the nucleotide sequence encoding the fusion protein encodes the amino acid sequence shown in SEQ ID NO: 7.

[0080] Example 12: The expression vector as described in the above example, wherein the nucleotide sequence encoding the fusion protein comprises the nucleotide sequence shown in SEQ ID NO: 6 or the reverse complementary sequence of the sequence.

[0081] Example 13: The expression vector as described in the above example, wherein the expression vector comprises the nucleotide sequence shown in SEQ ID NO: 5.

[0082] Example 14: The expression vector as described in the above example, wherein the fusion protein further includes a tag; the tag includes: a histidine tag (His-tag), a glutathione-S-transferase tag (GST-tag), a FLAG tag, a maltose binding protein tag (MBP-tag), a hemagglutinin protein tag (HA-tag), or a combination thereof.

[0083] Example 15: The expression vector as described in the above example further comprises a screening gene; the screening gene comprises a drug-resistance screening gene, a non-drug-resistance screening gene, or a combination thereof.

[0084] Example 16: The expression vector as described in the preceding example, wherein the fusion protein is expressed in Escherichia coli.

[0085] Example 17: A method for producing a DnaK C-terminus-P72 N-terminus-XylF fusion protein, comprising transcribing the nucleotide sequence described in the above example in an expression system and translating to obtain the fusion protein.

[0086] Example 18: Use of a DnaK C-terminus-P72 N-terminus-XylF fusion protein for preparing a composition for preventing and treating symptoms caused by Mycoplasma hyorhinis infection.

[0087] Example 19: The use according to the preceding example, wherein the symptoms caused by the Mycoplasma hyorhinis infection include pleurisy, peritonitis, pericarditis, arthritis, or a combination thereof.

[0088] The materials, methods and examples in this application are illustrative only and, unless otherwise specified, are not intended to limit the present invention. Although methods or materials similar or equivalent to those described herein can be used to implement or test the present invention, those described herein are more appropriate methods and materials.

[0089] Example 1: Expression vector construction, fusion protein expression and solubility analysis

[0090] 1.1. Expression vector construction

[0091] GeneDireX, Inc., USA, was commissioned to synthesize the DNA sequence of the DnaKC-P72N-XylF fusion protein, namely the MHY-30 DNA sequence, based on the optimized codons of E. coli. Using the plasmid containing the MHY-30 DNA sequence as a template, the DNAKCHISNDEIF (SEQ ID NO: 1) / XYLHISSALR (SEQ ID NO: 2) primer combination was used. PCR reaction was performed using High-Fidelity DNA Polymerase Reagent (NEB, USA). A 100 μL PCR reaction mixture contained 1x Q5 Reaction buffer, 200 μM dNTPs, 0.5 μM amplification primers, 40 ng pUC57-MHY30, and 2 U High-Fidelity DNA polymerase. PCR reaction conditions are as described in Table 1. After the reaction, the presence of DNA fragments of the expected size was confirmed by 0.7% (w / v) agarose gel electrophoresis, and the PCR product was recovered using the Plus Gel Eluted Kit (GMbiolab, Taiwan).

[0092] Table 1: PCR reaction conditions

[0093] The PCR product was cut with NdeI and SalI, and then recovered using a PCR Clean Up Kit (GMbiolab, Taiwan). The DNA fragment was then ligated into pET29a(+) (abbreviated as pET29a) (Merck Millipore, Germany) cut with the same restriction enzymes using T4 DNA ligase. The ligated product was transformed into ECOS TM 9-5 (Yeastern, Taiwan). After colonies were established, colony polymerase chain reaction (PCR) was performed using a T7 promoter primer (SEQ ID NO: 3) / T7 terminator primer (SEQ ID NO: 4) combination with DreamTaq DNA polymerase reagent (Thermo Fisher Scientific, USA) to select transformants that may have inserted DNA. After confirming the presence of the inserted DNA in the recombinant plasmids in the transformants using 0.7% (w / v) agarose gel electrophoresis, the plasmids were extracted from the transformants using the Plasmid Miniprep Purification Kit II (GMbiolab, Taiwan) for DNA sequencing. The plasmid with the correct DNA sequence was named pET29-MHY30.

[0094] The construction process of pET29-MHY30 is shown in Figure 1; the sequence of the expression vector pET29-MHY30 (SEQ ID NO: 5), the MHY-30 DNA sequence in the expression vector (SEQ ID NO: 6), and the amino acid sequence of MHY-30 (SEQ ID NO: 7) are shown below.

[0095] Sequence of expression vector pET29-MHY30 (SEQ ID NO: 5)

[0096] T7 promoter: 3317-3335 (boxed); MHY-30 gene: 179-3250 (bold); T7 terminator: 26-73 (boxed in italics); lactose repressor gene: 3724-4808 (boxed in bold); pBR322 replication region: 6238-6826 (gray background); kanamycin resistance gene: 6948-7763 (bold italics).

[0097] MHY-30 DNA sequence in the expression vector (SEQ ID NO: 6)

[0098] 5' end ~ KpnI (GGTACC): C-terminal DNA sequence of DnaK; KpnI (GGTACC) ~ BamHI (GGATCC): N-terminal DNA sequence of P72; BamHI (GGATCC) ~ 3' end: XylF DNA sequence, the termination code TAA does not produce amino acids; bold: His-tag DNA sequence; bold box: restriction enzyme cutting site.

[0099] Amino acid sequence of MHY-30 (SEQ ID NO: 7)

[0100] Bold: C-terminus of DnaK; italics: N-terminus of P72; bold italics: XylF; gray background: histidine tag; box: amino acid sequence generated by restriction enzyme cutting site.

[0101] 1.2. Fusion protein expression

[0102] Transform the expression vector into E. coli BL21 (DE3). Select a single colony and inoculate it into 5 mL of LB medium containing 30 μg / mL kanamycin. Culture at 37°C and 180 rpm. After overnight culture, take 50 μL of the pre-culture solution and add it to 5 mL of LB medium containing 30 μg / mL kanamycin. Culture with shaking (37°C, 180 rpm) until the OD reaches 0. 600The OD value is about 0.4 to 0.6. Then add 0.1mM isopropyl-β-D-thiogalactoside (IPTG) and induce the expression of fusion protein at 25℃. After 4 hours of induction, take 1mL OD 600 = 2 was centrifuged (20,630 × g, 5 min, 4°C), and the bacterial fraction was collected.

[0103] Solubility analysis

[0104] The collected cells were suspended in 500 μL of SET buffer (50 mM glucose, 10 mM EDTA, 25 mM Tris, pH 8.0) and disrupted using an ultrasonic disruptor (Sonics VCX-750, USA) with an amplitude of 20% and intermittent treatment (9 seconds of treatment followed by 9 seconds of rest) for a total treatment time of 2 minutes.

[0105] Mix 100 μL of cell lysis solution with 25 μL of 5× Sample Buffer to obtain the total cell lysate. Centrifuge 100 μL of cell lysate (20,630 × g, 10 minutes, 4°C) and mix the supernatant with 25 μL of 5× Sample Buffer to obtain the soluble protein. Heat 25 μL of the total cell lysate and 25 μL of the soluble protein sample at 100°C for 10 minutes. Analyze the protein solubility by protein electrophoresis and immunoblotting.

[0106] The results showed ( FIG. 2 ) that the fusion protein could be expressed in E. coli and was mainly a soluble protein.

[0107] Example 2: Expression and purification of fusion protein

[0108] 2.1. Expression of fusion proteins

[0109] A single colony was selected and inoculated into 12 mL of LB medium containing 30 μg / mL kanamycin and cultured at 37°C and 180 rpm. After overnight culture, 10 mL of the pre-culture solution was added to 1 L of LB medium containing 30 μg / mL kanamycin and cultured with shaking (37°C, 180 rpm) until the OD 600 The expression of the fusion protein was induced by adding 0.1 mM isopropyl-β-D-thiogalactopyranoside at 25°C. After 4 hours of induction, the bacterial cells were collected by centrifugation (10,000 × g, 10 minutes, 4°C).

[0110] Mix 2L shake flask cultured cells with 96mL of Lysis buffer (20mM sodium phosphate, 500mM NaCl, 1% (v / v) Triton X-100, pH 7.4). Disrupt the cells using a high-pressure cell disruptor at 2,000 bar. Centrifuge the cell disruptor (20,630×g, 4°C, 30 minutes) and collect the supernatant. Remove cell debris from the supernatant using a 0.22μm polyethersulfone (PES) filter coupled with a peristaltic pump.

[0111] Purification of fusion protein

[0112] Taking advantage of the fact that the fusion protein has a histidine tag that can form a coordinated covalent bond with nickel or cobalt ions, the fusion protein is purified using immobilized-metal ion affinity chromatography. The purification method of the fusion protein is to use a protein liquid chromatography system. Prime plus (GE Healthcare, Sweden) was used with a 5 mL HiTrap Excel column (GE Healthcare, Sweden). The column was first equilibrated with 25 mL of equilibration buffer (20 mM sodium phosphate, 500 mM NaCl, pH 7.4), and the filtered supernatant was injected into the column. After sample injection, nonspecifically bound proteins were washed with 100 mL of wash buffer A (20 mM sodium phosphate, 500 mM NaCl, 30 mM imidazole, Triton-114, pH 7.4), followed by further washing with 100 mL of wash buffer B (20 mM sodium phosphate, 500 mM NaCl, 30 mM imidazole, pH 7.4). The fusion protein on the resin was then eluted with 100 mL of a mixture of wash buffer B and elution buffer (20 mM sodium phosphate, 500 mM NaCl, 250 mM imidazole, pH 7.4) at 30 to 250 mM imidazole. Finally, the column was eluted with 50 mL of elution buffer. Protein purification was assessed by electrophoresis. The concentration of the purified protein was determined using the Protein Assay Kit II (Bio-Rad, USA). The purity of the purified protein was analyzed using ImageQuant software.

[0113] Taking advantage of the fact that the fusion protein carries a histidine tag that can form a coordinated covalent bond with nickel or cobalt ions, immobilized metal ion affinity chromatography was used to purify the soluble fusion protein in E. coli. TMThe fusion protein was purified using a Ni Excel column (Figure 3).

[0114] Example 3: Preparation of subunit vaccine and pig immune challenge test

[0115] 3.1. Preparation of subunit vaccines

[0116] A subunit vaccine was prepared using the fusion protein described in Example 2 as the active ingredient and tested in vivo in pigs to determine its protective immune response. The fusion protein was uniformly mixed with an adjuvant to prepare the subunit vaccine. Each dose of the vaccine contained 200 μg of the fusion protein, and the dose was 2 mL.

[0117] 3.2. Pig immune challenge test

[0118] Twelve pigs from a commercial piggery were randomly assigned to groups of six before the trial. The immunized group received a single dose (2 mL) of vaccine intramuscularly at 1 and 3 weeks of age, while the control group received an equal volume of saline intramuscularly at the same time. At 8 weeks of age, all pigs were challenged with a culture medium containing a field isolate of Mycoplasma hyorhinis. Anatomical pathology was performed 3 weeks after challenge (11 weeks of age), and the percentage of pigs with lesions including peritonitis, pleuritis, pericarditis, and arthritis was calculated. Gross lesion scoring was performed according to the method described by Magnusson et al. (Vet. Immunol. Immunopathol., 61:83-96, 1998). During the trial, pigs were observed daily for clinical signs and weighed regularly to assess vaccine safety. Serum was collected regularly for antibody testing to assess changes in serum antibodies following vaccination.

[0119] Results from the pig challenge trial showed that the vaccinated pigs showed good spirits and activity after vaccination, with no adverse reactions such as redness, swelling, or inflammation at the immunization site, demonstrating the vaccine's high safety. Analysis of lesions showed that the vaccine reduced the severity of peritonitis, pericarditis, and arthritis in the pigs (Table 2), indicating that the vaccine induces a protective immune response and is effective in preventing and treating Mycoplasma hyorhinis infection.

[0120] Table 2. Macroscopic lesion scoring results of the pig immune challenge test

Claims

1. A composition for preventing and treating Mycoplasma hyorhinis infection, comprising: The C-terminus of DnaK of Mycoplasma hyorhinis-the N-terminus of P72-XylF fusion protein; as well as A pharmaceutically acceptable adjuvant. 2 . The composition of claim 1 , wherein the concentration of the fusion protein is 50 to 500 μg / mL.

3. The composition of claim 1, wherein the C-terminus of DnaK comprises the sequence shown in SEQ ID NO: 9; the N-terminus of P72 comprises the sequence shown in SEQ ID NO: 11; or the XylF comprises the sequence shown in SEQ ID NO:

13. The composition of claim 3 , wherein the fusion protein comprises the sequence shown in SEQ ID NO:

7.

5. The composition of claim 1, wherein the pharmaceutically acceptable adjuvant is: Freund's complete or incomplete adjuvant, aluminum gel, surfactant, anionic polymer, peptide, oil emulsion or a combination thereof. The composition of claim 1 , further comprising a pharmaceutically acceptable additive.

7. The composition of claim 6, wherein the pharmaceutically acceptable additive comprises a solvent, a stabilizer, a diluent, a preservative, an antibacterial agent, an antifungal agent, an isotonic agent, an absorption delaying agent, or a combination thereof.

8. An expression vector for expressing the fusion protein as claimed in any one of claims 1 to 7, comprising: A nucleotide sequence encoding a DnaK C-terminus-P72 N-terminus-XylF fusion protein; Replicon genes; as well as Expression elements include promoter, terminator and ribosome binding site.

9. The expression vector of claim 8, wherein the nucleotide sequence encoding the C-terminus of DnaK encodes the amino acid sequence shown in SEQ ID NO: 9; the nucleotide sequence encoding the N-terminus of P72 encodes the amino acid sequence shown in SEQ ID NO: 11; and the nucleotide sequence encoding XylF encodes the amino acid sequence shown in SEQ ID NO:

13.

10. The expression vector according to claim 8, wherein the nucleotide sequence encoding the C-terminus of DnaK comprises the nucleotide sequence shown in SEQ ID NO: 8 or the reverse complementary sequence of the sequence; the nucleotide sequence encoding the N-terminus of P72 comprises the nucleotide sequence shown in SEQ ID NO: 10 or the reverse complementary sequence of the sequence; the nucleotide sequence encoding XylF comprises the nucleotide sequence shown in SEQ ID NO: 12 or the reverse complementary sequence of the sequence.

11. The expression vector according to claim 8, wherein the nucleotide sequence encoding the fusion protein encodes the amino acid sequence shown in SEQ ID NO:

7.

12. The expression vector according to claim 11, wherein the nucleotide sequence encoding the fusion protein comprises the nucleotide sequence shown in SEQ ID NO: 6 or the reverse complementary sequence of the sequence.

13. The expression vector of claim 8, wherein the expression vector comprises the nucleotide sequence shown in SEQ ID NO:

5.

14. The expression vector of claim 8, wherein the fusion protein further comprises a tag; the tag comprises: Histidine tag (His-tag), glutathione-S-transferase tag (GST-tag), FLAG tag, maltose binding protein tag (MBP-tag), hemagglutinin protein tag (HA-tag), or a combination thereof.

15. The expression vector of claim 8, further comprising a screening gene; the screening gene comprises a drug resistance screening gene, a non-drug resistance screening gene, or a combination thereof. The expression vector of claim 8 , wherein the fusion protein is expressed in Escherichia coli.

17. A method for producing a DnaK C-terminus-P72 N-terminus-XylF fusion protein, comprising transcribing the nucleotide sequence as claimed in any one of claims 8 to 16 in an expression system, and translating to obtain the fusion protein.

18. Use of a DnaK C-terminus-P72 N-terminus-XylF fusion protein for preparing a composition for preventing and treating symptoms caused by Mycoplasma hyorhinis infection.

19. The use according to claim 18, wherein the symptoms caused by the infection with Mycoplasma hyorhinis include pleurisy, peritonitis, pericarditis, arthritis, or a combination thereof.

Images