Fusion protein for diagnosing mycoplasma bovis infection as well as preparation method and application of fusion protein

Abstract

The invention discloses a fusion protein for diagnosing mycoplasma bovis infection as well as a preparation method and application of the fusion protein. According to the fusion protein disclosed by the invention, dominant B cell epitope peptides of two strong immunogenic lipoproteins of mycoplasma bovis are connected, mycoplasma bovis infection can be effectively distinguished, and the fusion protein is further used for preparing a detection product for diagnosing mycoplasma bovis infection. A detection product prepared from the fusion protein disclosed by the invention has excellent sensitivity and specificity, and is of great significance for improving the economic benefit of the cattle breeding industry.

Description

Technical Field

[0001] This invention belongs to the field of animal infectious disease detection technology, and in particular relates to a fusion protein for diagnosing bovine mycoplasma infection, its preparation method, and its application. Background Technology

[0002] Bovine mycoplasma ( Mycoplasma bovis , M. bovis Mycoplasma bovis is a minimal pathogenic microorganism capable of self-replication outside the body. It can infect beef cattle, dairy cattle, yaks, and other livestock, causing various diseases such as pneumonia, arthritis, mastitis, and conjunctivitis. Mycoplasma bovis often mixes with other respiratory pathogens, significantly increasing morbidity and mortality, making it a key pathogen threatening the global cattle industry and causing enormous economic losses. Currently, pneumonia and mastitis caused by Mycoplasma bovis remain widespread worldwide. Because Mycoplasma bovis lacks a cell wall structure, it is insensitive to β-lactam antibiotics and all antimicrobial agents that target the cell wall. Furthermore, it has developed resistance to tetracyclines, macrolides, aminoglycosides, and fluoroquinolones.

[0003] Bovine mycoplasma infection is an infectious disease caused by Mycoplasma bovis, and it is one of the important infectious diseases affecting the economic benefits of cattle farming. Its transmission routes are diverse, generally through the respiratory and digestive tracts. After infecting healthy cattle, Mycoplasma bovis mainly parasitizes the nasal cavity and mammary gland tissue, persisting in the host's body for a long time and forming a chronic infection. Currently, the methods for diagnosing bovine mycoplasma both domestically and internationally mainly focus on etiological diagnosis, immunological diagnosis (immunohistochemistry and Western blotting), and serological diagnosis (enzyme-linked immunosorbent assay and colloidal gold immunochromatography). However, these methods still face challenges such as slow diagnostic speed, low sensitivity, inability to differentiate infection status, and complex procedures.

[0004] Therefore, developing new diagnostic technologies and control measures for bovine mycoplasma disease is of great significance to the long-term stable development of the cattle breeding industry. Summary of the Invention

[0005] To address at least some of the technical problems in the prior art, the present invention provides a fusion protein for diagnosing bovine mycoplasma infection, a method for its preparation, and its applications. Specifically, the present invention includes the following:

[0006] In a first aspect, the present invention provides a fusion protein comprising a first B-cell epitope peptide and a second B-cell epitope peptide derived from an immunogenic lipoprotein, the first B-cell epitope peptide having the amino acid sequence shown in SEQ ID NO.1 and the second B-cell epitope peptide having the amino acid sequence shown in SEQ ID NO.2.

[0007] In some embodiments, the fusion protein according to the present invention further comprises a linker peptide.

[0008] In some embodiments, according to the fusion protein of the present invention, the fusion protein is selected from one of the following: (I) The amino acid sequence shown in SEQ ID NO.3; (II) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in (I) and has the same function; (III) An amino acid sequence that has the same function as the amino acid sequence shown in (I) or (II) obtained by modifying, substituting, deleting or adding one or more amino acids.

[0009] A second aspect of the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein described in the first aspect of the invention.

[0010] A third aspect of the invention provides a carrier molecule comprising the nucleic acid molecule described in the second aspect of the invention.

[0011] In a fourth aspect, a host cell is provided that comprises a nucleic acid molecule as described in the second aspect of the invention or a carrier molecule as described in the third aspect of the invention.

[0012] A fifth aspect of the present invention provides a method for preparing a fusion protein according to a first aspect of the present invention, wherein the fusion protein is prepared by artificial synthesis or genetic engineering.

[0013] A sixth aspect of the invention provides a detection product for diagnosing bovine mycoplasma infection, comprising the fusion protein described in the first aspect of the invention.

[0014] In some embodiments, the detection product according to the present invention includes a reagent kit, test strip, or protein chip.

[0015] A seventh aspect of the invention provides the use of the fusion protein described in the first aspect of the invention in the preparation of products for diagnosing bovine mycoplasma infection.

[0016] This invention connects the dominant B-cell epitope peptides of two highly immunogenic lipoproteins from bovine mycoplasma to obtain a fusion protein for detecting bovine mycoplasma. This fusion protein can effectively distinguish bovine mycoplasma infection and can then be used as a diagnostic antigen in the preparation of diagnostic products for bovine mycoplasma infection. The fusion protein detection product of this invention exhibits excellent sensitivity and specificity, which is of great significance for improving the economic benefits of cattle farming. Attached Figure Description

[0017] Figure 1The images show the reactions of immunogenic lipoproteins with bovine mycoplasma-positive and bovine mycoplasma-negative sera, where A represents the ELISA results and B represents the Western blot results.

[0018] Figure 2 The results of B-cell epitope prediction and surface accessibility analysis for the highly immunogenic lipoproteins MbovP537 (left) and MbovP468 (right) are shown. The red boxes represent B-cell epitopes predicted to be located on the protein surface, and the blue boxes represent B-cell epitopes predicted to be located inside the protein.

[0019] Figure 3 The image shows the recombinant plasmid pTrxA-MPfusion2M prepared in this invention.

[0020] Figure 4 Figures A and B are gel images and Western Blot verification images of the purified recombinant MPfusion protein of this invention, respectively. In Figure A, MK is the protein marker and 1 is the purified MPfusion fusion protein. In Figure B, MK is the protein marker, 1 is 1 μl of purified MPfusion fusion protein, 2 is 0.5 μl of purified MPfusion fusion protein, and 3 is the positive control.

[0021] Figure 5 To demonstrate the effectiveness of the MPfusion fusion protein of this invention in identifying bovine mycoplasma infection, A represents the ROC curve analysis results; B represents the ELISA detection results of negative and positive serum samples. Detailed Implementation

[0022] 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.

[0023] 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.

[0024] 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.

[0025] Fusion protein In one aspect, the present invention provides a fusion protein for detecting bovine mycoplasma, the fusion protein comprising a first B-cell epitope peptide and a second B-cell epitope peptide derived from immunogenic lipoproteins, the first B-cell epitope peptide and the second B-cell epitope peptide being derived from different highly immunogenic lipoproteins, wherein the first B-cell epitope peptide has the amino acid sequence shown in SEQ ID NO. 1, and the second B-cell epitope peptide has the amino acid sequence shown in SEQ ID NO. 2.

[0026] In a preferred embodiment, the fusion protein of the present invention has any one of the amino acid sequences shown in (I)-(III): (I) The amino acid sequence shown in SEQ ID NO.3; (II) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in (I) and has the same function; (III) An amino acid sequence that has the same function as the amino acid sequence shown in (I) or (II) obtained by modifying, substituting, deleting or adding one or more amino acids.

[0027] 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.

[0028] 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 different amino acids. "Deletion" refers to the reduction of one or more amino acids in the 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 the naturally occurring molecule. It should be noted that in the modified fusion proteins provided by this invention, the modification preferably occurs in regions other than the dominant antigenic epitope, and the modified protein still retains the desired antigenic functional properties of the fusion protein of this invention, or has improved antibody-binding properties.

[0029] In this invention, conserved amino acid substitutions are preferred, and these conserved polypeptide variants are preferably generated by amino acid substitutions according to Table 1: Table 1 In a preferred embodiment, the amino acid sequence shown in SEQ ID NO.1 and the amino acid sequence shown in SEQ ID NO.2 are linked by a linker peptide. The sequence of the linker peptide is not particularly limited and can be any flexible linker peptide. Preferably, the linker peptide can be (GS)n, (GGGS)n, or (GGGGS)n, where n is an integer from 1 to 5. In a specific embodiment, the linker peptide has the sequence shown in SEQ ID NO.5. Those skilled in the art will understand that the specific linking method of the first B-cell epitope peptide and the second B-cell epitope peptide derived from immunogenic lipoprotein is not particularly limited; it can be in the form of first B-cell epitope peptide-linker peptide-second B-cell epitope peptide, or it can be in the form of second B-cell epitope peptide-linker peptide-first B-cell epitope peptide.

[0030] It is understood that fusion proteins with added tags for purposes such as purification, increased expression levels, simplified identification, and enhanced solubility are also within the scope of protection of this invention. Examples of such tag proteins include, but are not limited to, MBP, GST, NusA, Poly-His Tag, DsbA / B / C, etc.

[0031] Nucleic acid molecules In one aspect, a nucleic acid molecule is provided that comprises a nucleotide sequence encoding the fusion protein described herein.

[0032] As used in this invention, the term "nucleic acid" is intended to include polymeric forms of nucleotides of any length containing deoxyribonucleotides, ribonucleotides, and / or their analogues, including DNA, RNA, and DNA / RNA hybrids, and also including DNA or RNA analogues, such as those containing a modified backbone (e.g., peptide nucleic acid (PNA) or phosphate thioester) or modified bases. Therefore, the nucleic acids of this invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc.

[0033] In this invention, the full-length nucleotide sequence or fragment thereof can typically be obtained using PCR amplification, recombination, or artificial synthesis. Currently, the DNA sequence encoding the fusion protein of this invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0034] carrier molecules In one aspect, the present invention provides a carrier molecule comprising the nucleic acid molecule described herein.

[0035] The vector of this invention refers to an artificial construct capable of delivering and preferably expressing one or more target genes or sequences in a host cell. The vector of this invention is not limited and can be an expression vector, viral vector, etc. In some embodiments, the vector contains a target gene encoding the fusion protein of this invention, a promoter, a terminator, or optionally a marker gene. The vector can be a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenovirus vectors, AAV viral vectors, etc.

[0036] host cells In one aspect, the present invention provides a host cell comprising the nucleic acid molecule or the carrier molecule described in the present invention.

[0037] The host cell of this invention refers to any cell type suitable for transformation, transfection, transduction, etc., using a nucleic acid construct or expression vector containing the nucleic acid molecules of this invention. The host cell includes any offspring of the parent cell that differs from the parent cell due to mutations occurring during replication.

[0038] Preparation method In one aspect, the present invention provides a method for preparing the fusion protein described herein. The preparation method is not particularly limited, and includes preparation via artificial synthesis or genetic engineering.

[0039] In some embodiments, the fusion protein of the present invention is obtained by artificial synthesis. Methods for artificially synthesizing fusion proteins are known in the art. Specifically, these include both liquid-phase and solid-phase methods. Liquid-phase synthesis methods include those using BOC protection and Z protection. Compared to solid-phase synthesis, it has many advantages such as a wider selection of protecting groups, lower cost, and easier scale-up.

[0040] In some embodiments, the fusion protein of the present invention is synthesized via a solid-phase method, including both FMOC protection and BOC protection. This method offers advantages such as convenient and rapid synthesis, and ease of automation.

[0041] In some embodiments, the fusion protein of the present invention is obtained through recombinant technology, i.e., through genetic engineering expression. Genetic engineering expression systems include prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Examples of prokaryotic cell expression systems include the *E. coli* expression system. Eukaryotic cell expression systems include enzyme expression systems, insect cell expression systems, and mammalian cell expression systems.

[0042] In this invention, the recombinant DNA technology generally includes the following steps: (1) Transform or transduce suitable host cells with the polynucleotide (or variant) encoding the fusion protein of the present invention, or with a recombinant expression vector containing the polynucleotide; (2) Culture the host cells in a suitable culture medium; (3) Isolate and purify proteins from culture media or cells.

[0043] Fusion proteins can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, recombinant proteins can be isolated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeabilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0044] Product testing In one aspect, the present invention provides a detection product comprising the fusion protein described herein for detecting (or quantifying) bovine mycoplasma. The detection product includes a kit, test strip, or protein chip.

[0045] In a preferred embodiment, the test product further includes a specification for implementing the test method of the present invention.

[0046] In this document, the term "kit" refers to a combination of reagents and other materials. A kit is intended to contain reagents such as buffers, protein stabilizing agents, signal generation systems (e.g., fluorescence signal generation systems), antibodies or antigen-binding fragments thereof, control proteins, and test containers (e.g., microtiter plates, etc.). The term "kit" is not limited to a specific combination of reagents and / or other materials; for example, a kit may also include instructions for using the reagents. Kits can be packaged in any suitable manner, typically having components in a single container or (if necessary) in multiple containers, along with instructions for performing the assay. Kits can be prepared using a variety of methods known in the art.

[0047] In some embodiments, the kit may further include a washing solution, a substrate solution, a diluent, a calibration solution, an enzyme-labeled secondary antibody, and a stop solution. The washing solution is not particularly limited in composition, but examples include, but are not limited to, buffer solutions, surfactants, and preservatives. The substrate solution may use known substrates, including, but not limited to, chromogenic substrates, fluorescent substrates, and luminescent substrates. The diluent is not particularly limited in composition, but examples include, but are not limited to, buffer solutions and surfactants. The calibration solution is not particularly limited in composition, but examples include, but are not limited to, BSA solution, trehalose solution, and animal serum. The enzyme-labeled secondary antibody is not particularly limited in composition, but examples include, but are not limited to, goat anti-bovine secondary antibody, horse anti-bovine secondary antibody, rabbit anti-bovine secondary antibody, and mouse anti-bovine secondary antibody. The stop solution is not particularly limited in composition, but examples include, but are not limited to, sulfuric acid solution, hydrochloric acid solution, sodium hydroxide solution, and EDTA solution.

[0048] application One aspect of the present invention provides the use of the fusion protein according to the invention in the preparation of products for diagnosing bovine mycoplasma infection. Examples of such products include, but are not limited to, reagents, kits, test strips, protein chips, etc. Examples of such kits include, but are not limited to, ELISA kits, plate-based chemiluminescence immunoassay kits, fully automated chemiluminescence immunoassay kits, radioimmunoassay kits, fluorescence immunoassay kits, chemiluminescence immunoassay kits, immunoblotting kits, immunoprecipitation kits, flow cytometry kits, colloidal gold assay kits, bioluminescent immunoassay kits, immunochromatographic assay kits, electrophoresis kits, latex-based kits, and kits based on direct or indirect competitive methods, etc.

[0049] Example 1 This example demonstrates the detection of immunogenicity of bovine mycoplasma protein.

[0050] 1. ELISA detection method 250 ng of purified protein was coated into each well of a 96-well ELISA plate and incubated overnight at 4°C. After washing with PBST, 5% skim milk powder was added, and the plate was blocked at 37°C for 1 h. After washing three times with PBST, 100 μl of bovine mycoplasma-positive serum (1:100 dilution) was added to each well, and bovine mycoplasma-negative serum (1:100 dilution) was added to the corresponding negative control wells. The plates were incubated at 37°C for 1 h. After washing three times with PBST, 100 μl of HRP-labeled goat anti-bovine IgG (1:5000 dilution) was added, and the plates were incubated at 37°C for 1 h. After washing five times with PBST, 100 μl of TMB chromogenic solution was added to each well, and the plates were incubated at room temperature for 10 min. Finally, 50 μl of stop solution was added to terminate the reaction, and the OD was measured using an ELISA reader. 630 .

[0051] The results are as follows Figure 1 As shown in A, MbovP537 and MbovP468 proteins exhibit strong immunogenicity.

[0052] 2. Western blotting (protein immunoblotting) The purified protein was measured at 5 μg / well by SDS-PAGE. MbovP739 protein was used as a positive control (5 μg). The protein was then transferred to a PVDF membrane, blocked with 5% skim milk at room temperature for 2 h, and bovine mycoplasma-positive serum was added as the primary antibody (1:500 dilution). The membrane was incubated at room temperature for 2 h, washed three times with PBST, and HRP-labeled goat anti-bovine IgG was added as the secondary antibody (1:5000 dilution). The membrane was incubated at room temperature for 1 h, washed with PBST, and then chemiluminescently detected using ECL chromogenic solution to observe the reaction between the protein and bovine mycoplasma-positive serum.

[0053] The results are as follows Figure 1 As shown in B, MbovP537 and MbovP468 proteins exhibit the strongest immunogenicity.

[0054] Example 2 This example demonstrates the prediction of B-cell linear epitopes of the bovine mycoplasma MbovP537 and MbovP468 proteins.

[0055] 1. Prediction of B-cell antigenic epitopes in Mycoplasma bovis MbovP537 and MbovP468 proteins The online software BepiPred (BepiPred 2.0-DTU Health Tech-Bioinformatic Services) was used to predict the B-cell antigenic epitopes of Mycoplasma bovis MbovP537 and MbovP468 proteins. The prediction results are shown in Table 2. Table 2. Prediction results of B-cell antigenic epitopes In the Protein analysis module of DNAstar software, the Kyte-Doolittle method, Karplus-Schulz method, Emini method, and Jameson-Wolf method were used to predict the hydrophilic region, backbone flexibility, surface accessibility, and antigenicity of MbovP537 and MbovP468 proteins, respectively. The results are as follows: Figure 2 As shown.

[0056] Based on the above prediction results, the B-cell epitopes in the 193-204 peptide of MbovP537 protein and the 103-114, 304-310, and 440-570 regions of MbovP468 protein are all located inside the three-dimensional structure of the protein and have a low probability of being displayed on the surface; while the remaining B-cell epitopes have a high probability of being located on the protein surface.

[0057] 2. Constructing the MPfusion fusion protein by tandemly using B-cell dominant antigenic epitopes of Bovine Mycoplasma MbovP537 and MbovP468 proteins. Based on the above B cell epitope and accessibility prediction results, and considering the requirements of the detection experiment on protein conformation, this invention connects the dominant antigenic epitope in the 46-296 region of the MbovP537 protein peptide chain with the dominant antigenic epitope in the 35-440 region of the MbovP468 protein peptide chain through a linker to obtain the MPfusion fusion amino acid sequence, and the linker sequence is GGGGSGGGGSGGGGS (SEQ ID NO.5).

[0058] Example 3 This embodiment illustrates the construction of a prokaryotic expression system for recombinant MPfusion fusion protein.

[0059] 1. Construction of pTrxA-MPfusion2M plasmid To increase the expression level of the fusion gene MPfusion2M of this invention in Escherichia coli and promote its soluble expression, the applicant designed a recombinant plasmid pTrxA-MPfusion2M containing a thioredoxin (TrxA) tag. Figure 3 The nucleic acid sequence was optimized based on E. coli codon preference for expression and purification of MPfusion fusion protein.

[0060] 2. pTrxA-MPfusion2M plasmid was transformed into E. coli (1) Thawing competent cells: Take out BL21(DE3) competent cells from -80℃ and thaw them on ice for 2 min; (2) Plasmid addition: Add 0.5 μl of pTrxA-MPfusion2M plasmid to 100 μl of thawed competent cells; (3) Ice incubation: Place on ice for 30 min; (4) Heat shock: Place in a 42℃ metal bath for 45 s; (5) Cooling recovery: Quickly transfer to ice and incubate for 3 min; (6) Resuscitation of bacterial culture: Add 450 μl of LB medium to the mixture and incubate at 37°C with shaking for 45 min; (7) Spreading on plates: After centrifuging the bacterial culture at 3500 rpm / min for 5 min, discard the supernatant, resuspend the precipitate and spread it evenly on LB solid medium containing ampicillin resistance, and incubate the plates in a constant temperature incubator at 37℃ for 12-16 h.

[0061] 3. Induction of recombinant MPfusion fusion protein expression and Western blot validation 3.1 Induced expression of recombinant MPfusion fusion protein (1) Pick a single colony of recombinant positive bacteria BL21 and place it in LB liquid medium containing ampicillin resistance. Incubate at 37°C and 200 rpm / min on a shaker until OD. 600 The value reached 0.6; (2) Add IPTG to the bacterial culture to a final concentration of 0.8 mM for induction, and continue to incubate in a shaker at 37℃ and 200 rpm / min for 4 h; (3) Uninduced pTrxA-MPfusion2M BL21(DE3) and induced Frd-TrxA empty vector BL21(DE3) were used as controls; (4) The bacterial culture was centrifuged at 12000 rpm / min for 1 min, the bacterial precipitate was collected, resuspended in PBS, and then an equal volume of 2×SDS-PAGE loading buffer was added and mixed. The sample was boiled in a metal bath at 100℃ for 10 min and then sampled for SDS-PAGE analysis.

[0062] 3.2 Soluble expression of recombinant MPfusion fusion protein (1) After expression under the above induction conditions, collect the induced bacterial culture; (2) Ultrasonic disruption of Escherichia coli, disruption conditions: power 390 W, working for 2 s, stopping for 4 s, time for 15 min; (3) The bacterial culture was centrifuged at 9000 rpm / min and 4℃ for 10 min, and the supernatant and precipitate were collected separately (dissolved in denaturing buffer); (4) Take the bacterial culture before and after induction and the supernatant precipitate after disruption, add an equal volume of 2×SDS-PAGE loading buffer and mix well. Boil the sample in a metal bath at 100℃ for 10 min. Take the sample for SDS-PAGE analysis to determine that most of the recombinant MPfusion fusion protein is expressed in the supernatant.

[0063] 3.3 Western Blot Identification of Recombinant MPfusion Fusion Protein (1) Samples: 12% SDS-PAGE was performed on samples before induction, after induction, supernatant, precipitate and positive control containing His protein; (2) Electrophoresis: Stacking gel at a constant voltage of 90 V for about 20 min, then use bromophenol blue to separate the gel interface; then use a constant voltage of 120 V to separate the gel until the bromophenol blue reaches about 1 cm from the bottom of the gel. (3) Transfer: Wet transfer method was adopted, with U=220 V and I=250 mA (constant current) set, and the transfer was carried out at 4℃ for 60 min; (4) Sealing: Use TBST to prepare 3% skim milk powder and shake slowly on a shaker at room temperature for 60 minutes; (5) Washing the membrane: Wash 3 times with TBST, 10 min each time; (6) Antibody incubation: HRP-Mouse anti-His mAb was diluted 1:2000 with BSA-TBST, and streptavidin HRP was diluted 1:5000 with 1% BSA-TBST. Incubation was carried out at room temperature for 1.5 h. (7) Elution of antibodies: Wash 3 times with TBST, 10 min each time; (8) Color development and exposure: Imaging with a color luminescence analyzer for 1 min.

[0064] 4. Purification and Western Blot Validation of Recombinant MPfusion Fusion Protein 4.1 Purification of recombinant MPfusion fusion protein After activating the normally expressing bacterial culture, expand the culture to 600 ml. When OD... 600 When the value reaches 0.6, the supernatant is collected according to the induction expression conditions mentioned in Section 3.1 and the disruption conditions mentioned in Section 3.2. The supernatant is filtered through a 0.22 μm filter, and the filtrate is used as the sample for nickel column loading before purification.

[0065] The specific purification steps for the recombinant MPfusion fusion protein are as follows: (1) Equilibrate the packed nickel column purification column with 5 column volumes of equilibration buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0), repeating 2-3 times. (2) Add the sample to the balanced gravity column and incubate at room temperature for 1 h to ensure that the sample and the medium are in full contact. Collect the effluent. (3) Wash with 15 column volumes of washing solution (50 mM NaH2PO4, 300 mM NaCl, pH 8.0) to remove non-specifically adsorbed proteins and collect the washing solution. (4) Perform gradient elution sequentially using 10 column volumes of elution buffer 1 (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) / elution buffer 2 (50 mM NaH2PO4, 300 mM NaCl, 50 mM imidazole, pH 8.0) / elution buffer 3 (50 mM NaH2PO4, 300 mM NaCl, 100 mM imidazole, pH 8.0) / elution buffer 4 (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0), and collect the eluent in segments. (5) After elution, rinse with equilibration buffer for 10 column volumes, then preserve the purified column with 20% ethanol and store at 2-8℃. (6) The eluent containing the target protein was dialyzed with PBS and concentrated to a suitable volume. The final protein purity and concentration were detected by SDS-PAGE.

[0066] Purification results are as follows Figure 4 As shown in Figure A, the molecular weight of the fusion protein is 93.8 KD, and the main protein band is obvious.

[0067] 4.2 Western Blot Validation of Recombinant MPfusion Fusion Protein Purification The method is the same as in Section 3.3.

[0068] The verification results are as follows Figure 4 As shown in Figure B, the positive signal is located near the 100KD Marker and is a single band; based on the combined results of the SDS-PAGE gel image, it can be determined that the complete fusion protein was purified with a molecular weight of 93.8KD.

[0069] Example 4 This embodiment demonstrates the application of the bovine mycoplasma MPfusion fusion protein in the identification of bovine mycoplasma infection. Recombinant MPfusion fusion protein (100 ng / well) was used as the antigen to coat 96-well ELISA plates overnight at 4°C. After washing with PBST, 5% skim milk was added, and the plates were blocked at 37°C for 1 h. After washing with PBST, serum samples (1:100 dilution) were added and incubated at 37°C for 1 h. After washing with PBST, goat anti-bovine secondary antibody (1:5000 dilution) was added and incubated at 37°C for 1 h. After washing with PBST, substrate chromogenic solution was added, and the plates were developed in the dark for 10 min. The reaction was terminated by adding stop solution. OD was measured at 630 nm using an ELISA reader. 630 The obtained OD values ​​were used to determine the threshold of the ROC curve using an online tool. The results are as follows: Figure 5 As shown, based on the ROC curve, when the threshold is 0.582, the specificity of this diagnostic method is 94.7% and the sensitivity is 96.2%, which can effectively distinguish between bovine mycoplasma infection and non-infection. Among them, the number of positive samples is 26 and the number of negative samples is 19.

[0070] In summary, this invention detected the immunogenicity of bovine mycoplasma lipoproteins MbovP280, MbovP290, MbovP458, MbovP468, MbovP475, MbovP537, MbovP682, MbovP838, and MbovP739. Among these, MbovP468 and MbovP537, which exhibited stronger immunogenicity, were selected. Their immunogenicity was verified by Western blotting. Based on the prediction of B-cell epitopes of bovine mycoplasma MbovP468 and MbovP537 proteins using multiple methods, two dominant epitope peptides were selected and tandemly linked using a flexible linker. The fusion gene sequence was optimized according to the codon preference of *E. coli*, and the plasmid vector pTrxA-MPfusion2M was constructed. The optimized nucleotide sequence is shown in SEQ ID NO.1. After plasmid extraction, the protein was transformed into *E. coli* BL21(DE3) and expressed under IPTG induction. The amino acid sequence of the recombinant MPfusion fusion protein is shown in SEQ ID NO.2. Therefore, the purified *Mycoplasma bovis* MPfusion fusion protein of this invention can be used as a diagnostic antigen and for the preparation of diagnostic products for *Mycoplasma bovis* infection.

[0071] 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 fusion protein, characterized in that, The fusion protein includes a first B-cell epitope peptide and a second B-cell epitope peptide derived from immunogenic lipoproteins, wherein the first B-cell epitope peptide has the amino acid sequence shown in SEQ ID NO.1 and the second B-cell epitope peptide has the amino acid sequence shown in SEQ ID NO.

2.

2. The fusion protein according to claim 1, characterized in that, The fusion protein further includes a linker peptide.

3. The fusion protein according to claim 1, characterized in that, The fusion protein is selected from one of the following: (I) The amino acid sequence shown in SEQ ID NO.3; (II) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in (I) and has the same function; (III) An amino acid sequence that has the same function as the amino acid sequence shown in (I) or (II) obtained by modifying, substituting, deleting or adding one or more amino acids.

4. A nucleic acid molecule, characterized in that, It contains a nucleotide sequence encoding the fusion protein of any one of claims 1-3.

5. A carrier molecule, characterized in that, It comprises the nucleic acid molecule as described in claim 4.

6. A host cell, characterized in that, It comprises the nucleic acid molecule of claim 4 or the carrier molecule of claim 5.

7. The method for preparing the fusion protein according to any one of claims 1-3, characterized in that, Prepared through artificial synthesis or genetic engineering.

8. A detection product for diagnosing bovine mycoplasma infection, characterized in that, It comprises the fusion protein as described in any one of claims 1-3.

9. The testing product according to claim 8, characterized in that, The detection products include reagent kits, test strips, or protein chips.

10. The use of the fusion protein according to any one of claims 1-3 in the preparation of products for diagnosing bovine mycoplasma infection.

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