Ovine disease protein chip, kit and application thereof

Abstract

The present application relates to the technical field of biology. Specifically, the present application discloses a sheep disease protein chip, a kit and application thereof. The sheep disease protein chip comprises a substrate and antigens connected to the substrate. The antigens comprise mycobacterium avium subsp. paratuberculosis antigens, brucella melitensis antigens, pseudotuberculosis corynebacterium antigens and foot-and-mouth disease virus type A antigens. The sheep disease protein chip can realize high-throughput detection of sheep disease serology and rapid screening of sheep diseases.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a protein chip for sheep diseases, a reagent kit, and their applications. Background Technology

[0002] With the transformation of modern animal husbandry towards intensification and large-scale operations, the epidemiological characteristics of infectious diseases in sheep flocks have undergone significant changes. The increasing complexity and diversification of pathogens has become a key bottleneck restricting the healthy development of the sheep farming industry. Accurate and rapid diagnosis is the primary line of defense for blocking the spread of diseases and reducing economic losses. Traditional serological methods (such as ELISA) are mostly based on a one-disease-one-test model, which is not only low-throughput and time-consuming, but also difficult to effectively distinguish between vaccine immune antibodies and wild-type virus infection antibodies. While molecular biological detection (such as PCR) is sensitive, it has high requirements for laboratory environment and operators, making it difficult to promote on a large scale at the grassroots level. At the same time, co-infection with multiple pathogens is extremely common in the clinical treatment of sheep diseases. For example, the clinical symptoms of respiratory diseases (which may involve Mycoplasma pneumoniae, Pasteurella, Mansonia, and parainfluenza virus, etc.) and reproductive disorders (which involve Brucella, Chlamydia, Toxoplasma, etc.) highly overlap and are difficult to distinguish by visual observation alone. In addition, pox-like lesions of the skin and mucous membranes are easily confused with diseases such as peste des petits ruminants, sheep pox, and oral thrush in the early stages. On the other hand, mixed infections have become the norm. The synergistic effect of multiple pathogens not only exacerbates the disease but also interferes with the accuracy of single-pathogen detection, thus delaying treatment or leading to immunization failure. Therefore, there is an urgent need to develop a screening tool that can cover multiple core pathogens and has both high throughput and high sensitivity to meet the pressing need for simultaneous detection and differential diagnosis of multiple pathogens in modern sheep disease prevention and control. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a sheep disease protein chip with the advantages of high throughput and high sensitivity, providing a technical solution for the simultaneous screening and detection of multiple pathogenic sheep diseases.

[0004] To address the aforementioned technical problems, as a first aspect of the present invention, the present invention provides a sheep disease protein chip, which includes a substrate and an antigen connected thereto. The antigens include Mycobacterium avium paratuberculosis subspecies antigen, Brucella mesenteriae antigen, Corynebacterium pseudotuberculosis antigen, and Foot-and-mouth disease virus type A antigen. The antigen of Mycobacterium paratuberculosis subspecies is one or more of the following: Mycobacterium paratuberculosis subspecies plasma membrane vesicles, MAP1693c, MAP4308c, MAP2677c, and MAP1197; MAP1693c, MAP4308c, MAP2677c, and MAP1197 are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 44~47; The *Brucella melitensis* antigen is one or more of *Brucella melitensis* lipopolysaccharide, *B. melitensis-Omp25*, *B. melitensis-Omp31*, and *B. melitensis-Dps*; *B. melitensis-Omp25*, *B. melitensis-Omp31*, and *B. melitensis-Dps* are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 61~63, respectively. The pseudotuberculous corynebacterium antigen is one or more of CLA-PLD, CLA-CP40, and CLA-SlpA; CLA-PLD, CLA-CP40, and CLA-SlpA are respectively encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 71~73. The type A foot-and-mouth disease virus antigen is one or more of FMDV-A-1, FMDV-A-2, and FMDV-A-3; the amino acid sequences of FMDV-A-1, FMDV-A-2, and FMDV-A-3 are shown in SEQ ID NO: 85~87, respectively.

[0005] As an improvement to the above technical solution, the antigens also include goatpox virus antigen, sheep infectious pustular virus antigen, bluetongue virus antigen, small ruminant plague virus antigen, pseudorabies virus antigen, Nipah virus antigen, Rift Valley fever virus antigen, caprine arthritis-encephalitis virus antigen, Japanese encephalitis virus antigen, Medi-Visna virus antigen, Nairobi sheep disease virus antigen, sheep abortive salmonella antigen, Mycoplasma filamentosa caprine subspecies antigen, Mycoplasma hyopneumoniae antigen, Clostridium perfringens antigen, Mycoplasma agalactiae antigen, Chlamydia psittaci antigen, Chlamydia abortus antigen, Echinococcus granulosus antigen, Listeria monocytogenes antigen, Toxoplasma gondii antigen, Pasteurella multocida antigen, Mansonia hemolyticus antigen, Bacillus anthracis antigen, type O foot-and-mouth disease virus antigen, and Mycobacterium tuberculosis bovine variant antigen. The goatpox virus antigen is one or more of GTPV-P32, GTPV-R122, GTPV-A34, and GTPV-A36; GTPV-P32, GTPV-R122, GTPV-A34, and GTPV-A36 are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 1~4, respectively. The sheep infectious pustular virus antigen is one or more of ORFV-B2L, ORFV-F1L, ORFV-020, and ORFV-125; ORFV-B2L, ORFV-F1L, ORFV-020, and ORFV-125 are respectively encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 5~8; The bluetongue virus antigen is one or more of BTV-VP7, BTV-VP2, and BTV-NS3; BTV-VP7, BTV-VP2, and BTV-NS3 are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 9~11. The small ruminant plague virus antigen is one or more of PPRV-N, PPRV-F, and PPRV-H; PPRV-N, PPRV-F, and PPRV-H are respectively encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 12~14; The pseudorabies virus antigen is one or more of PRV-gD, PRV-gB, and PRV-gE, and PRV-gD, PRV-gB, and PRV-gE are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 15~17. The Nipah virus antigen is NiV-N and / or NiV-G; NiV-N and NiV-G are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 18~19, respectively. The Rift Valley fever virus antigen is one or more of RVFV-NP, RVFV-NSs, and RVFV-NSm; RVFV-NP, RVFV-NSs, and RVFV-NSm are respectively encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 20~22; The capillary arthritis-encephalitis virus antigen is one or more of CAEV-CA, CAEV-gp135, and CAEV-MA; CAEV-CA, CAEV-gp135, and CAEV-MA are respectively encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 23~25. The Japanese encephalitis virus antigen is one or more of JEV-C, JEV-E, and JEV-NS1; JEV-C, JEV-E, and JEV-NS1 are respectively encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 26~28; The Medi-Visna virus antigen is one or more of MVV-P15, MVV-P25, MVV-P28, and MVV-P135; MVV-P15, MVV-P25, MVV-P28, and MVV-P135 are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 29~32. The Nairobi sheep disease virus antigen is NSD-N and / or NSD-Gc / Gn; NSD-N and NSD-Gc / Gn are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 33~34, respectively. The Salmonella abortus antigen of the sheep abortion is S.Abortusovis-fljB-H and / or S.Abortusovis-OMP; S.Abortusovis-fljB-H and S.Abortusovis-OMP are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 35~36, respectively; The Mycoplasma filamentosa goat subspecies antigen is M.mycoides-LPPA and / or M.mycoides-SecA; M.mycoides-LPPA and M.mycoides-SecA are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 37~38, respectively. The ovine mycoplasma pneumoniae antigen is one or more of M.ovi-P70, M.ovi-Enolase, M.ovi-P113, M.ovi-P60, and M.ovi-pdhD; M.ovi-P70, M.ovi-Enolase, M.ovi-P113, M.ovi-P60, and M.ovi-pdhD are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 39~43. The Clostridium perfringens antigen is Alpha toxin and / or Beta toxin; Alpha toxin and Beta toxin are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 48~49, respectively. The Clostridium perfringens antigen is NetB and / or Epsilon toxin; NetB and Epsilon toxin are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 50-51, respectively. The agalactiae mycoplasma antigen is one or more of M. agalacttiae-PDHB, M. agalacttiae-P80, and M. agalacttiae-P48; M. agalacttiae-PDHB, M. agalacttiae-P80, and M. agalacttiae-P48 are respectively encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 52~54. The psittaci-chlamydia psittaci antigen is one or more of C. psittaci-MOMP, C. psittaci-TARP, and C. psittaci-CPAF; C. psittaci-MOMP, C. psittaci-TARP, and C. psittaci-CPAF are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 55~57, respectively. The abortive chlamydia antigen is one or more of C. abortus-MIP, C. abortus-MOMP, and C. abortus-POMP90B; C. abortus-MIP, C. abortus-MOMP, and C. abortus-POMP90B are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 58~60; The Echinococcus granulosus antigen is one or more of EgM123, EgSeverin, Eg14-3-3ζ, and Eg-AgB8 / 3; EgM123, EgSeverin, Eg14-3-3ζ, and Eg-AgB8 / 3 are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 64~67; The Listeria monocytogenes antigen is one or more of LLO, P60, and InlB; LLO, P60, and InlB are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 68~70. The Toxoplasma gondii antigen is one or more of SAG1, GRA6, and SAG2A; SAG1, GRA6, and SAG2A are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 74~76, respectively. The Pasteurella multocida antigen is Pm-OmpA and / or Pm-OmpH; Pm-OmpA and Pm-OmpH are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 77~78, respectively. The hemolytic Mansell's antigen is M. haemolytica-Lkt and / or M. haemolytica-NlpI; M. haemolytica-Lkt and M. haemolytica-NlpI are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 79~80, respectively. The anthrax Bacillus antigen is B. anthracis-PA63, which is encoded by a DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 81; The type O foot-and-mouth disease virus antigen is one or more of FMDV-O-1, FMDV-O-2, and FMDV-O-3; the amino acid sequences of FMDV-O-1, FMDV-O-2, and FMDV-O-3 are shown in SEQ ID NO: 82~84, respectively. The Mycobacterium tuberculosis bovine variant antigen is Mycobacterium tuberculosis bovine variant lipoarabinomannan.

[0006] As improvements to the above technical solutions, the following are also mentioned: GTPV-P32, GTPV-R122, GTPV-A34, GTPV-A36, ORFV-B2L, ORFV-F1L, ORFV-020, ORFV-125, BTV-VP7, BTV-VP2, BTV-NS3, PPRV-N, PPRV-F, PPRV-H, PRV-gD, PRV-gB, PRV-gE, NiV-N, NiV-G, RVFV-NP, RVFV-NSs, RVFV-NSm, CAEV-CA, CAEV-gp135, CAEV-MA, JEV-C, JEV-E, JEV-NS1, MVV-P15, MVV-P25, MVV-P28, MVV-P135, NSD-N, NSD-Gc / Gn, S. Abortusovis-fljB-H, S. Abortusovis-OMP, M. mycoides-LPPA, M. mycoides-SecA, M.ovi-P70, M.ovi-Enolase, M.ovi-P113, M.ovi-P60, M.ovi-pdhD, MAP1693c, MAP4308c, MAP2677c, MAP1197, Alpha toxin, Beta toxin, NetB, Epsilon toxin, M. agalactiae-PDHB, M. agalactiae-P80, M. agalactiae-P48, C. psittaci-MOMP, C. psittaci-TARP, C.psittaci-CPAF, C. abortus-MIP, C. abortus-MOMP, C. abortus-POMP90B, B. melitensis-Omp25, B. melitensis-Omp31,B. melitensis-Dps, EgM123, EgSeverin, Eg14-3-3ζ, Eg-AgB8 / 3, LLO, P60, InlB, CLA-PLD, CLA-CP40, CLA-SlpA, SAG1, GRA6, SAG2A, Pm-OmpA, Pm-OmpH, M.haemolytica-Lkt, M.haemolytica-NlpI, and B.anthracis-PA63 are recombinant expressed and purified proteins.

[0007] As an improvement to the above technical solution, the substrate is a nitrocellulose-modified glass slide, an aldehyde-modified glass slide, or an epoxy-modified glass slide; and / or The antigen is diluted with spotting buffer to a spotting concentration of 0.25 mg / L to 1.5 mg / L; and / or The spotting buffer is selected from one or more of glycerol aqueous solution, PBS, and TBS.

[0008] As an improvement to the above technical solution, the substrate is a nitrocellulose-modified glass slide; and / or The antigen is diluted with spotting buffer to a spotting concentration of 0.25 mg / L to 1.0 mg / L; and / or The spotting buffer solution is an 80 wt% aqueous glycerol solution.

[0009] As a second aspect of the present invention, the present invention provides a kit comprising the above-described sheep disease protein chip.

[0010] As an improvement to the above technical solution, it also includes sample diluent, blocking solution, washing solution, secondary antibody and fluorescent triple antibody.

[0011] As an improvement to the above technical solution, the sample diluent is PBST containing 0.5wt%~2wt% BSA or a mixture of fish skin gelatin and water containing 0.5wt%~2wt% fish skin gelatin; and / or The blocking solution is PBS containing 1 wt% to 5 wt% BSA or a mixture of fish skin gelatin and water containing 2 wt% to 5 wt% fish skin gelatin; and / or The washing solution is PBS, PBST, or TBST; and / or The secondary antibody is rabbit anti-goat IgG; and / or The fluorescent triple antibody is a mouse anti-rabbit IgG labeled with a fluorescent group; the fluorescent group is selected from FITC, TRITC, Cy, FAM or R-PE; and / or The dilution of the secondary antibody is 1:2000 to 1:3000; and / or The dilution of the fluorescent triple antibody is 1:2000~1:3000.

[0012] As an improvement to the above technical solution, the sample diluent is PBST containing 1 wt% BSA; and / or The blocking solution is PBS containing 3 wt% BSA; and / or The washing solution is PBST; and / or The secondary antibody is diluted at a ratio of 1:2000; and / or The dilution of the fluorescent triple antibody is 1:2000.

[0013] As a third aspect of the present invention, the present invention discloses the application of the above-mentioned sheep disease protein chip in (1) or (2): (1) Sheep disease detection for non-diagnostic purposes; (2) Screening or evaluating vaccines for sheep diseases.

[0014] As a fourth aspect of the present invention, the present invention discloses the application of the above-described reagent kit in (1) or (2): (1) Sheep disease detection for non-diagnostic purposes; (2) Screening or evaluating vaccines for sheep diseases.

[0015] Implementing this invention has the following beneficial effects: The sheep disease protein chip constructed in this invention employs a multi-target joint detection strategy, designing one or more specific antigens for each pathogen, which greatly reduces the risk of missed detection and improves the sensitivity and specificity of detection. This chip enables simultaneous screening of dozens of sheep disease pathogens in a single experiment, making it suitable not only for large-scale epidemiological surveys but also for providing precise data support for vaccine efficacy evaluation, early disease warning, and complex etiological analysis of mixed infections, demonstrating significant clinical application value. Attached Figure Description

[0016] Figure 1 This is a diagram showing the antigen screening results in Example 1 of the present invention; Figure 2 This is a diagram showing the substrate screening results in Embodiment 2 of the present invention; Figure 3 This is a graph showing the antigen concentration screening results in Example 4 of the present invention; Figure 4 This is a graph showing the screening results of the spotting buffer solution in Example 5 of the present invention; Figure 5 This is a graph showing the optimized reaction conditions in Example 6 of the present invention; Figure 6 This is a comparison chart of the detection results of the sheep disease protein chip based on the present invention and the traditional ELISA detection in Embodiment 8 of the present invention. Detailed Implementation

[0017] To facilitate understanding of the present invention, it will be described in more detail below. However, it should be understood that the present invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. Rather, these embodiments or examples are provided to make the disclosure of the present invention more thorough and complete.

[0018] Unless otherwise defined, 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. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments or examples only and is not intended to limit the invention. The optional range of the term "and / or" as used herein includes any one of two or more of the related listed items, as well as any and all combinations of the related listed items, including any two related listed items, any more related listed items, or a combination of all related listed items.

[0019] The following embodiments are provided for the purpose of illustrating various embodiments of the present invention and are not intended to limit the invention in any way. Those skilled in the art will understand that variations and other uses as defined in the claims are included within the spirit and scope of the invention. Unless otherwise specified, the materials, reagents, etc., used in the following embodiments are commercially available. The promoter and terminator sequences mentioned in the embodiments can also be downloaded from NCBI, and the specific sequence start positions can be determined from the primers in the primer table. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.

[0020] Unless otherwise specified, the percentage content involved in this invention refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures.

[0021] Unless otherwise specified, all percentage concentrations mentioned in this invention refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of that component.

[0022] Unless otherwise specified, the temperature parameters in this invention can be either constant temperature processing or processing within a certain temperature range. The constant temperature processing allows temperature fluctuations within the precision range controlled by the instrument.

[0023] Example 1 Preparation of Antigen This embodiment aims to prepare highly specific antigens for sheep disease detection. The selected pathogenic microorganisms are all derived from common or serious diseases affecting sheep flocks, covering three major categories: viruses, bacteria (including mycoplasma and chlamydia), and parasites, specifically classified as follows: Viral pathogens include: Goat pox virus (GTPV), Orfvirus (ORFV), Blue tongue virus (BTV), Peste despetitsruminants virus (PPRV), Pseudorabies virus (PRV), Nipah virus (NiV), Rift Valley fever virus (RVFV), Caprine arthritis-encephalitis virus (CAEV), Japanese encephalitis virus (JEV), Medi-Visner virus (MVV), Nairobi sheep disease virus (NSD), and Foot-and-mouth disease virus (FMDV, including types O and A).

[0024] Bacterial, mycoplasmal, and chlamydial pathogens: mainly including Mycoplasma ovineis, which causes respiratory and systemic infections. Mycoplasma ovipneumoniae Mycoplasma filamentosa goat subspecies ( M. mycoides subsp. capri ), Mycoplasma agalactiae ( Mycoplasma agalactiae Pasteurella multocida ( ) Pasteurella multocida Pm) and hemolytic Mansonephritis ( Mannheimia haemolytica Brucella ovis, which is involved in zoonotic and reproductive system diseases ( ); Brucella melitensis Chlamydia abortus ( ) Chlamydia abortus ), Chlamydia psittaci ( Chlamydia psittaci ) and Salmonella abortus in sheep ( Salmonella Abortusovis ); and other important pathogens such as Mycobacterium avium paratuberculosis subsp. (MAP), Mycobacterium tuberculosis bovine variant ( M. bovis Clostridium perfringens ( ) Clostridium perfringens Corynebacterium pseudotuberculosis ( Corynebacterium pseudotuberculosis Bacillus anthracis ( Bacillus anthracis ) and Listeria monocytogenes ( Listeria monocytogenes ).

[0025] Parasitic pathogens: Echinococcus granulosus was selected ( Echinococcus granulosus ) and Toxoplasma gondii ( Toxoplasma gondii More specifically, the various antigens involved in this application are shown in Tables 1 to 3.

[0026] Table 1 Information on various antigens involved in this application.

[0027] Table 2 Information on various antigens involved in this application.

[0028] Table 3 Information on various antigens involved in this application.

[0029] (I) Preparation of prokaryotic expression antigens 1. Construction of expression vectors and screening of strains: Protein sequences of target pathogens (bacteria, mycoplasma, chlamydia, parasites, and some viral nucleocapsids) were retrieved from the Uniprot database. Signal peptides and transmembrane domains were predicted using bioinformatics analysis. For target proteins, highly hydrophobic transmembrane regions and signal peptide sequences were removed to improve solubility. For *Escherichia coli* (…),… E. coli Codon optimization was performed based on preference. The synthesized gene fragment was inserted into the multiple cloning site of the pET32a vector using seamless cloning technology to construct a recombinant plasmid fused with a His tag and a Trx tag (containing a PP restriction site) at the N-terminus. The plasmid was transformed into Rosetta (DE3) competent cells, screened by LB plates (containing 100 μg / mL ampicillin) and identified by PCR. Positive single clones were inoculated into 96-well plates and cultured overnight. Glycerol bacteria (final concentration 20%) were then prepared and stored at -80℃.

[0030] 2. High-throughput expression and solubility analysis: Culture and induction: After resuscitating the bacterial strain in a 96-well plate, the culture was transferred to a 24-well plate (3 mL LB medium per well containing antibiotics) for expansion. When the OD600 of the bacterial culture reached 0.5-0.6, IPTG was added to a final concentration of 0.5 mM, and the culture was induced for expression for 16 h in a 16°C shaker (120 rpm).

[0031] 3. Expression Identification: Bacterial cells were collected, washed with TBS and PBS, treated with bacterial lysis buffer, and the supernatant and precipitate were separated by centrifugation. Protein expression was detected by SDS-PAGE and Western Blot (primary antibody: anti-His tag antibody; secondary antibody: HRP-labeled goat anti-mouse IgG). Results showed that the target proteins were expressed solublely or efficiently in inclusion body form under optimized conditions (inclusion bodies were denatured and renatured), and the molecular weight was consistent with the theoretical value.

[0032] 4. Purification of recombinant protein: Positive engineered bacteria were inoculated at a ratio of 1% into 1L LB medium for amplification and induction. After sonication and centrifugation, the supernatant (or renatured protein solution) was loaded onto a Ni-NTA affinity chromatography column. The eluted target protein was desalted and replaced with PBS buffer (pH 7.2-7.4), and PreScission Protease (PP enzyme) was added and digested overnight at 4°C to remove the fusion tag. The digested product was then reverse-filtered through a nickel column for purification (removing the cleaved tag and uncleaved protein), and the flow-through was collected. Finally, the protein was concentrated through a 3kDa ultrafiltration tube and the buffer was replaced with PBS. After quantification by BCA method and purity verification by SDS-PAGE, the concentration was adjusted to 1 mg / mL, aliquoted, and stored at -80°C.

[0033] (II) Preparation of eukaryotic expression antigens 1. Vector Construction: For viral antigens requiring glycosylation modification or maintenance of native conformation (such as envelope proteins and glycoproteins), a eukaryotic expression strategy was employed. After sequence optimization and removal of the signal peptide, the target gene was cloned into the pCDNA3.1(+) vector. Kpn I and Xho Between the I sites, a 6×His tag is fused at the C-terminus. The recombinant plasmid is transformed into TOP10 competent cells, positive clones are screened, and endotoxin-free plasmids are prepared.

[0034] 2. Transient transfection and expression: A 293F cell suspension culture system was used. Cells were resuscitated to the logarithmic growth phase, and the recombinant plasmid was introduced into the cells using PEI transfection reagent (cell density approximately 3.5~4×10⁶). 6 (cells / mL). Fresh medium was added 4 h after transfection, and the cells were cultured at 37 °C and 5% CO2 for 96 h with shaking.

[0035] 3. Purification and Identification: Cell culture supernatant was collected, centrifuged at 10000g, and filtered through a 0.45μm filter. Secretory His-tagged proteins were captured using Ni-NTA affinity chromatography. The eluent was desalted by PBS and concentrated by ultrafiltration. Specific bands were verified by SDS-PAGE and Western Blot, ultimately yielding high-purity eukaryotic recombinant protein antigen.

[0036] (III) Preparation of polypeptide antigens For foot-and-mouth disease virus (FMDV) of types O and A, a total of 6 polypeptide sequences (FMDV-O-1~3, FMDV-A-1~3) with a purity greater than 90% were synthesized. They were dissolved in DMSO at a final concentration of 10 mg / mL and stored at -80°C. Before use, they were diluted 10 times with TBS. The sequence information is shown in Table 3.

[0037] (iv) Preparation of extracted antigens 1. Extraction of lipoarabinomannan (LAM) from Mycobacterium tuberculosis bovine variant: BCG (Bacillus Calmette-Guérin) was statically cultured in 7H10 liquid medium until a bacterial film formed. The bacterial cells were collected and subjected to multiple defatting and lysis processes using chloroform / methanol mixtures (ratios of 2:1, 1:1, and 1:2). After drying the precipitate, the cells were disrupted, extracted by hot ethanol reflux, and then by hot phenol-water extraction. The aqueous phase was dialyzed, then digested sequentially with DNase / RNase and proteinase K, dialyzed again, and purified using a Superdex-75 molecular sieve. The target peak was collected and freeze-dried for later use.

[0038] 2. Extraction of plasma membrane vesicles (pTB) from *Mycobacterium paratuberculosis* subspecies: *Mycobacterium paratuberculosis* strain K10 (MAP K-10, ATCC BAA-968) was inoculated into a specific culture medium (containing mycosin) and incubated statically for approximately 2 months until a bacterial film formed. The bacterial cells and culture supernatant were collected and sterilized by 0.22 μm filtration. The filtrate was concentrated using a 100 kDa ultrafiltration membrane, and PBS was added for multiple buffer replacements and desalting. The retentate was collected as the purified plasma membrane vesicle antigen, quantified using the BCA method, and then used for further processing.

[0039] 3. Extraction of Brucella lipopolysaccharide (LPS): A virulent strain of Brucella A19-△virB6 (CGMCC No: 46097, deposited at the China General Microbiological Culture Collection Center on August 8, 2024) was used. After the strain was expanded and inactivated, extraction was performed using the hot phenol-water method. The bacterial culture was mixed with an equal volume of 90% hot phenol (66℃), stirred, and centrifuged to separate the layers. The upper aqueous phase was collected, and crude LPS was precipitated by adding cold methanol containing sodium acetate. The precipitate was resuspended in water, dialyzed to remove phenol, and then thoroughly digested with proteinase K to remove impurities. Finally, it was dialyzed thoroughly in deionized water and freeze-dried for storage.

[0040] 4. Preparation of PPA antigen from Mycobacterium avium paratuberculosis subspecies: After washing and resuspending the cultured Mycobacterium avium paratuberculosis subspecies in pre-cooled PBS, the cultured Mycobacterium avium paratuberculosis subspecies was homogenized using a high-pressure homogenizer under low temperature protection to release soluble proteins. The homogenized mixture was centrifuged at 20,000 rcf / min at 4°C to remove undisturbed bacterial cells and cell wall fragments. The collected supernatant was the PPA antigen. Finally, it was filtered through a 0.22 μm filter membrane for sterilization, the protein concentration was measured, and the antigen was aliquoted and stored at -80°C for later use.

[0041] (v) Screening of antigens Protein chips and ELISA differ in the properties of the solid-phase carrier and the antigen coating method. This embodiment uses both indirect ELISA and the protein chip method of this invention in parallel to compare their reaction performance on different platforms and screen for antigens suitable for the protein chip system. In the ELISA assay, candidate antigens are coated onto 96-well microplates, blocked, and reacted with standard positive serum to measure OD. 450nm Numerical values; Protein chip detection: Candidate antigens from the same batch were spotted onto a PATH substrate, blocked, and reacted with the same positive serum. The fluorescence signal intensity was then scanned and read. Using foot-and-mouth disease, paratuberculosis, Brucella, and Corynebacterium pseudotuberculosis antigens as examples, the detection results are as follows: Figure 1 As shown, antigens that showed good protein chip reaction were selected for subsequent validation.

[0042] Example 2: Substrate Selection 1. Establishment of Antigen Activity Validation and Detection Procedure: Given the diversity of antigen types involved in this chip (covering recombinant proteins, synthetic peptides, lipopolysaccharide (LPS), lipoarabinomannan (LAM), and plasma membrane vesicles, etc.), different substrates may exhibit varying adsorption properties. Therefore, this embodiment selected small ruminant virus N protein (PPRV-N), goatpox virus P32 protein (GTPV-P32), Mycobacterium tuberculosis bovine variant lipoarabinomannan (LAM), Mycobacterium avium paratuberculosis subspecies plasma membrane vesicles (pTB), Brucella lipopolysaccharide (LPS), and foot-and-mouth disease virus peptides (FMDV-O-1 / FMDV-A-1) as representative antigens to screen and validate substrate types and coating systems. The specific operating procedure is as follows: (1) Microarray preparation: The concentration of the seven representative antigens was adjusted to 1 mg / mL using PBS buffer. In a 384-well source plate, each antigen was mixed with 80% glycerol at a 1:1 volume ratio (6 μL each), and centrifuged at 1600 rpm to remove air bubbles. The samples were printed onto the surface of the substrate to be screened using a non-contact microarray spotting instrument. Each chip was planned with 12 subarrays, and each sample had 3 replicates. PBS blank control, BSA negative control, and goat IgG positive spot were also set up. After spotting, the chips were incubated overnight at 4°C to complete antigen fixation, and then sealed and stored at -80°C.

[0043] (2) Blocking: Take out the frozen chip, warm it up, and then immerse it in blocking buffer (prepared with PBS containing 3% BSA). Place it on a side-shaking table at room temperature for 3 hours (20~30 rpm) to block non-specific binding sites.

[0044] (3) Cleaning: After sealing, the chip was cleaned in PBS, 0.2 times PBS and ultrapure water for 5 minutes each. Then it was placed in a 50 mL centrifuge tube, centrifuged at 3000 rpm for 4 minutes and dried, and stored at 4℃ for later use.

[0045] (4) Serum incubation: The chip was separated using a sample grid. Positive sera for peste des petits ruminants (PPR), sheep pox, brucellosis, paratuberculosis, and foot-and-mouth disease (type O / A) were pretreated by centrifugation at 12000 g and then diluted 1:200 with sample diluent (PBST containing 1% BSA). An equal volume of the above positive serum mixture was added to the positive control wells, and healthy sheep serum was added to the negative control wells. The sample was added to the corresponding well (0.2 mL / well) and incubated with shaking at room temperature for 1 hour.

[0046] (5) Cleaning: Remove the liquid from the reaction wells and wash the chip repeatedly with PBST by vigorous blowing. After removing the grid, immerse the chip in PBST and shake it rapidly for 15 seconds, then wash it slowly on a shaker 3 times (10 minutes each time) to completely remove unbound serum proteins.

[0047] (6) Secondary antibody incubation: Add rabbit anti-sheep IgG (concentration adjusted to 1 μg / mL) prepared with dilution solution, place on a side-shaking incubator, and incubate at room temperature in the dark for 1 hour.

[0048] (7) Washing: Discard the secondary antibody solution, transfer the chip to the washing box, and add 30 mL of PBST. First, shake the chip rapidly for 15 seconds (repeat twice), then wash it three times at 100-110 rpm in the dark (10 minutes each time).

[0049] (8) Triple antibody incubation and signal amplification: Add Cy3-labeled mouse anti-rabbit IgG (Mouse anti-Rabbit IgG-Cy3) diluted to 1 μg / mL, place on a side-shaking incubator, and incubate at room temperature in the dark for 1 hour.

[0050] (9) Washing and drying: Discard the triple antibody solution, wash 3 times with PBST according to the standard procedure above (including fast and slow steps); then add 30 mL of ultrapure water and wash twice (5 minutes each time) to remove salt ions. Finally, put the chip into a centrifuge tube, centrifuge at 3000 rpm for 4 minutes to remove water, and store in the dark.

[0051] (10) Data acquisition: The chip was scanned using a laser confocal scanner (excitation wavelength 532 nm, Cy3 channel). A pre-scan was performed to determine the signal area and background intensity. After adjusting the PMT gain value, a fine scan was performed, and the image data was saved and a signal-to-noise ratio analysis was performed.

[0052] 2. Substrate Screening Results Analysis: Based on the detection process constructed above, the performance differences of three solid-phase supports—nitrocellulose membrane-modified slides (PATH), epoxy-modified slides, and aldehyde-modified slides (Aldehyde)—were compared and analyzed (see results for details). Figure 2 ).

[0053] Data analysis showed that in the detection of the positive control (sheep IgG), all three substrates exhibited good protein adsorption capacity, with no statistically significant difference in detection signals against mixed positive serum. Simultaneously, the fluorescence signal values ​​of the negative control (BSA) and blank control (PBS) remained at extremely low levels (<10), confirming that the surface treatment processes of the three substrates all possessed good anti-nonspecific adsorption properties. Secondly, regarding the response to different types of antigens, the detection sensitivity of the three substrates for recombinant protein antigens (PPRV-N, GTPV-P32) was essentially equivalent. For non-protein or small molecule antigens (including FMDV peptides, extracted LAM, pTB, and LPS), the PATH substrate showed a significant advantage, with its fluorescence signal intensity being significantly higher than that of the Epoxy and Aldehyde substrates. In conclusion, the nitrocellulose membrane-modified slide (PATH) has broader antigen compatibility and can simultaneously achieve effective immobilization of various biomolecules such as recombinant proteins, synthetic peptides, polysaccharides, and lipid complexes, thus establishing it as the preferred substrate of this invention.

[0054] Example 3: Establishment of the detection system and optimization of working concentration 1. Determination of the testing system Based on the present invention's requirement for high specificity and high sensitivity, and to ensure the stability and standardization of reagent supply, this embodiment directly selects commercially available antibodies to construct the detection system.

[0055] (1) Secondary antibody: Rabbit anti-sheep IgG (purchased from Sigma, catalog number: AP147P) was selected to specifically capture sheep-derived antibodies bound to the substrate.

[0056] (2) Triple antibody: Mouse anti-rabbit IgG (purchased from Sigma, catalog number: R1008) was selected and labeled with Cy3 fluorescence for signal amplification and fluorescence coloring.

[0057] 2. Optimization of working concentration To determine the optimal concentration of the aforementioned commercially available antibodies in the chip system, a checkerboard titration method was used for testing.

[0058] (1) Chip preparation: PATH substrates with representative antigens such as PPRV-N, GTPV-P32 and Brucella LPS were prepared according to the method in Example 2.

[0059] (2) Group design: Secondary antibody dilution: Set three gradients: 1:1000, 1:2000, and 1:4000.

[0060] Three-antibody dilution: set three gradients: 1:1000, 1:2000, and 1:4000.

[0061] Samples: Control tests were conducted using mixed positive sheep serum (P) and negative serum from healthy sheep (N).

[0062] (3) Detection and judgment: Incubation and washing are carried out according to the process described in Example 2.

[0063] The results showed that: with the high concentration combination (secondary antibody 1:1000, triple antibody 1:1000), the positive signal was extremely strong (F532>60000), but the negative control wells showed a slight nonspecific background (Background>300); with the low concentration combination (secondary antibody 1:4000, triple antibody 1:4000), the background was extremely low, but the signal intensity of weak positive samples was attenuated, which may affect the sensitivity; with the medium concentration combination (secondary antibody 1:2000, triple antibody 1:2000), the positive signal intensity remained at a high level (F532≈55000), and the negative background was controlled within the ideal range (Background<50), with the signal-to-noise ratio (SNR) reaching its maximum value.

[0064] 3. Clinical sample validation The optimal working concentrations (secondary antibody 1:2000, tri antibody 1:2000) were used to validate three clinical sheep serum samples (S1, S2, and S3). Results showed that the system accurately identified antibodies against peste des petits ruminants, sheep pox, and Brucella in S3 serum (signal values ​​significantly higher than the threshold), while showing no nonspecific reaction to S1 and S2 negative sera. Conclusion: The antibody detection system based on Sigma AP147P and Sigma R1008 constructed in this invention exhibits good specificity and signal-to-noise ratio, meeting the requirements for high-throughput clinical screening.

[0065] Example 4 Optimization of antigen coating concentration 1. Experimental Design and Chip Fabrication To determine the optimal immobilization concentration of the antigen on the PATH substrate and to balance detection sensitivity with reagent cost, this embodiment sets up three antigen concentration gradients.

[0066] (1) Gradient preparation: The antigen to be tested was diluted to three final concentration levels using PBS buffer: 1.0 mg / mL, 0.5 mg / mL and 0.25 mg / mL.

[0067] (2) Spotting system: In a 384-well source plate, 80% glycerol and the above-mentioned antigen solutions of different concentrations (6 μL each) were added at a 1:1 volume ratio. At the same time, sheep IgG was set as a positive spot, and BSA and PBS were set as negative and blank control spots, respectively.

[0068] (3) Solidification: The spotting operation was completed using a microarray spotter. After the chip was incubated overnight at 4°C, it was stored at -80°C for later use.

[0069] 2. Samples and Testing (1) Quality control sample: Multivalent positive control (P): composed of equal proportions of positive sera from various sheep diseases, including peste des petits ruminants, ovine paratuberculosis, and sheep brucellosis. Negative control (N): Serum from healthy sheep that has passed testing; Test samples: Two clinically collected sheep serum samples were randomly selected.

[0070] (2) Detection process: The mouse anti-sheep IgG monoclonal antibody determined in Example 3 was used as the secondary antibody, and hybridization incubation and signal acquisition were carried out strictly in accordance with the standard procedure described in Example 2.

[0071] 3. Results Analysis Specific results are as follows Figure 3 As shown in the figure. Scanning results indicate that within a concentration range of 0.25 mg / mL to 1.0 mg / mL, the chip background remained at a low level (without non-specific tailing), and the qualitative judgment results for positive samples were completely consistent across groups. This demonstrates that the antigen microarray system of this invention has strong stability, and slight fluctuations in antigen concentration do not significantly interfere with the accuracy of the detection results. However, comparing the fluorescence signal intensity revealed that the 1.0 mg / mL concentration group exhibited the most saturated specific fluorescence signal value (F532) and the best signal-to-noise ratio. To ensure signal stability during long-term storage and testing of complex clinical samples, this invention ultimately selects 1.0 mg / mL as the standard antigen spotting concentration.

[0072] Example 5: Screening and Evaluation of Spotting Buffer Given the complexity of the antigen types covered by this chip (including hydrophilic recombinant proteins, short-chain peptides, and more hydrophobic plasma membrane vesicles such as pTB, lipopolysaccharide (LPS), and lipoarabinomannan (LAM), selecting a universal spotting buffer is crucial to ensuring the uniform immobilization of antigens on the substrate.

[0073] 1. Solution stability test: Representative antigens were selected and mixed with four candidate buffers at a 1:1 volume ratio: Group A: PBS buffer (pH 7.2-7.4); Group B: 80wt% glycerol aqueous solution; Group C: TBS buffer (pH 8.0); Group D: PBS solution containing 10vol% DMSO. The mixtures were incubated at 2-8℃ for 24 hours, followed by high-speed centrifugation (12000g, 30min). Visual observation revealed that all four solutions remained clear and transparent, with no visible precipitate or flocculent material at the bottom of the tubes, indicating good solubility and stability of each antigen in the above systems.

[0074] 2. Chip Performance Evaluation: Chips were prepared using the four buffer solutions described above, and serological testing was performed according to standard procedures. Scanning results are shown below. Figure 4 show: 80wt% glycerol: The antigen spots are the most regular and full (perfect solid circles), with the highest positive signal intensity (F532) and extremely low background noise (optimal signal-to-noise ratio).

[0075] PBS and TBS: The spot morphology was acceptable, but the signal intensity was slightly lower than that of the glycerol group, and some hydrophobic antigen spots showed a slight "hollow" phenomenon.

[0076] PBS solution containing 10 vol% DMSO (DMSO): The background fluorescence value was significantly increased, interfering with the interpretation of weak positive samples.

[0077] Conclusion: Considering spot morphology, signal intensity and background control, this invention preferably uses 80wt% glycerol aqueous solution as the standard spotting buffer.

[0078] Example 6 Optimization and Determination of Reaction Conditions To improve detection efficiency, this embodiment compares the differences between two reaction modes: long-term incubation at room temperature and short-term incubation at 37°C.

[0079] 1. Experimental Design: The chip was prepared according to Example 2, and an orthogonal experimental design was used (see Table 4) to investigate the reaction effects of serum, secondary antibody and tertiary antibody under different temperature and time combinations.

[0080] Condition I: Incubate at room temperature (approximately 25°C) for 60 min; Condition II: Incubate at 37℃ for 30 min.

[0081] 2. Results Analysis: Analysis of the detection data of positive serum showed (trends as follows) Figure 5 As shown in the figure): Under four different combinations of conditions, the detection rate of the target pathogen was 100% and the fluctuation range of the fluorescence signal intensity was within the statistically permissible error.

[0082] 3. Conclusion: Although both conditions can meet the detection requirements, considering the timeliness requirements of large-scale clinical screening, the 37°C incubation for 30 minutes condition can significantly shorten the total detection cycle (the entire process can be controlled within 3.5 hours). Therefore, this invention ultimately standardizes the incubation procedures for serum, secondary antibody, and tertiary antibody to a constant temperature reaction of 37°C for 30 minutes.

[0083] Table 4 Reaction Conditions

[0084] Example 7: Establishment of a standardized protein chip detection procedure Based on the optimization results of the foregoing embodiments, the standard operating procedure for the protein chip of the present invention is established as follows: 1. Microarray chip preparation: The cryopreserved recombinant antigen was slowly thawed in an ice bath and centrifuged at 12000 g for 15 min at 4°C to remove aggregates. The supernatant was collected, and the antigen concentration was adjusted to 1.0 mg / mL using PBS buffer. In a 384-well source plate, each antigen solution was mixed with 80 wt% glycerol at a 1:1 volume ratio (final volume 12 μL), and centrifuged to remove bubbles before use. Using a non-contact microarray printing system, the above samples were spotted onto the surface of nitrocellulose membrane-modified slides (PATH). The array layout was designed as follows: each slide contained 12 independent detection subarrays, and each antigen and control (sheep IgG positive control, BSA negative control, PBS blank control) had 3 replicates. After spotting, the chip was incubated overnight at 4°C and then sealed and stored at -80°C.

[0085] 2. Blocking treatment: Before use, remove the chip and warm it to room temperature. Immerse it in blocking buffer (PBS solution containing 3% BSA) and place it on a side-shaking shaker. Shake for 3 hours at room temperature (25±2℃) (25 rpm) to fully block non-specific binding sites on the membrane surface.

[0086] 3. After pre-washing and discarding the blocking solution, transfer the chip to the cleaning tank. Wash with PBS, 0.2×PBS, and ultrapure water for 5 min each in sequence. Then centrifuge at 3000 rpm for 4 min to remove residual moisture on the surface, and store at 4℃ for later use.

[0087] 4. Primary Antibody Hybridization Reaction (Serium Incubation): Install the chip in a 16-well reaction grid. After centrifuging the sheep serum to be tested at 12000g, take the supernatant and dilute it 1:200 with sample diluent (PBST containing 1% BSA). Add 200μL of the diluted serum sample to each well (mixed positive serum to positive control wells, and healthy sheep serum to negative control wells). Incubate at 37℃ with shaking at 25 rpm for 30 min.

[0088] 5. Wash and remove the reaction solution. Add 300 μL of PBST to each well and wash by pipetting (repeat 3 times). Remove the grid and immerse the chip in a large volume of PBST washing solution. First, shake rapidly and vigorously for 15 seconds, then wash slowly on a shaker 3 times (10 min each time) to completely remove unbound serum proteins.

[0089] 6. Secondary antibody specific binding: Add rabbit anti-goat IgG antibody (Sigma, catalog number: AP147P) diluted to the working concentration (diluent is PBST containing 1% BSA) to the reaction chamber. Place on a side-shaking incubator and incubate at 37°C in the dark for 30 min.

[0090] 7. During the intermediate wash, discard the secondary antibody incubation solution and wash three times (10 min each time) with PBST buffer according to the standard procedure described above to remove free secondary antibody.

[0091] 8. Triple Antibody Signal Amplification: Add Cy3-labeled mouse anti-rabbit IgG antibody (Sigma, catalog number: R1008) diluted to the working concentration. Protect from light throughout the process and incubate at 37°C with shaking for 30 min. This step utilizes the recognition of "rabbit anti-sheep" antibodies bound to the sheep antibody by the "mouse anti-rabbit" antibody, achieving cascade amplification of the fluorescence signal.

[0092] 9. Final washing and drying: Discard the triple antibody and wash three times with PBST (10 min each time). Then add 30 mL of ultrapure water for two rapid washes (5 min each time) to remove salt ion interference. Finally, place the chip in a 50 mL centrifuge tube, centrifuge at 3000 rpm for 4 min, and then dry.

[0093] 10. Signal Acquisition and Analysis: The chip was scanned using a laser confocal scanner (excitation wavelength 532 nm, Cy3 channel). The photomultiplication factor (PMT) gain was adjusted to obtain the optimal signal-to-noise ratio. Fluorescence images were acquired, and the fluorescence intensity values ​​(F532) of each antigen point were extracted using software.

[0094] Example 8: Consistency between protein chip and ELISA detection methods Following Example 7, protein chips were used to detect 10 clinical sheep serum samples. Simultaneously, after coating with the corresponding antigens, ELISA was used for concurrent detection. The F532 and OD450 values ​​of each sample were compared. The detection results are as follows: Figure 6 The results showed a positive correlation between the protein chip detection values ​​and the ELISA detection values. r =0.7689), and the test results are consistent.

[0095] Example 9: Clinical Sample Screening and Validation To further verify the reliability and practicality of the protein chip of this invention in actual clinical applications, 50 clinical serum samples were collected from a large-scale sheep farm suspected of having respiratory diseases and reproductive disorders. Chip testing was performed strictly following the standard operating procedure (SOP) established in the aforementioned embodiments, and a nationally approved commercial ELISA kit was used simultaneously as a control method for parallel verification.

[0096] 1. Analysis of Test Results The detection data from the two methods were compared and analyzed, and the results are as follows: (1) Among the 50 samples, 24 serum samples showed fluorescence signal values ​​(F532) for more than 30 diseases covered by the chip that were all lower than the set cut-off value and were judged as negative. This result is completely consistent with the results of parallel ELISA tests (100% negative concordance rate), which strongly confirms that the chip system has a clean background and is not prone to false positive results caused by non-specific adsorption.

[0097] (2) Three samples showed multiple positive signals, accurately detecting antibodies against Brucella mesenteroides, paratuberculosis, and Mycoplasma ovis, successfully revealing complex mixed infections in clinical practice. One sample was identified as positive for tuberculosis antibodies. Five samples were identified as positive for Brucella antibodies. All the above positive results were confirmed by commercial ELISA kits, and the results were highly consistent, proving that this chip has excellent detection sensitivity.

[0098] 2. Detection throughput and timeliness analysis In this embodiment, a single chip can simultaneously accommodate 10 serum samples through a microarray layout, and perform synchronous scanning of more than 30 pathogens on each sample.

[0099] The method of this invention completes the entire process from sample addition to data analysis for 50 serum samples in just 3.5 to 4.5 hours. In contrast, if a traditional ELISA kit is used to screen the same scale (50 samples × 30 pathogens), the cumulative time is typically as high as 40 to 50 hours due to the varying dilution ratios, incubation temperatures, and reaction times for different pathogens, making simultaneous operation impossible. Furthermore, reagent and labor costs are exorbitant. Therefore, the protein chip of this invention significantly improves detection efficiency while ensuring accuracy, greatly reducing time and economic costs, and has significant clinical application value.

[0100] The above description is a preferred embodiment of the invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the invention, and these improvements and modifications are also considered to be within the scope of protection of the invention.

Claims

1. A protein chip for sheep diseases, characterized in that, Including the substrate and the antigen attached to it; The antigens include Mycobacterium avium paratuberculosis subspecies antigen, Brucella mesenteriae antigen, Corynebacterium pseudotuberculosis antigen, and Foot-and-mouth disease virus type A antigen. The antigen of Mycobacterium paratuberculosis subspecies is one or more of the following: Mycobacterium paratuberculosis subspecies plasma membrane vesicles, MAP1693c, MAP4308c, MAP2677c, and MAP1197; MAP1693c, MAP4308c, MAP2677c, and MAP1197 are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 44~47; The *Brucella melitensis* antigen is one or more of *Brucella melitensis* lipopolysaccharide, *B. melitensis-Omp25*, *B. melitensis-Omp31*, and *B. melitensis-Dps*; *B. melitensis-Omp25*, *B. melitensis-Omp31*, and *B. melitensis-Dps* are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 61~63, respectively. The pseudotuberculous corynebacterium antigen is one or more of CLA-PLD, CLA-CP40, and CLA-SlpA; CLA-PLD, CLA-CP40, and CLA-SlpA are respectively encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 71~73. The type A foot-and-mouth disease virus antigen is one or more of FMDV-A-1, FMDV-A-2, and FMDV-A-3; the amino acid sequences of FMDV-A-1, FMDV-A-2, and FMDV-A-3 are shown in SEQ ID NO: 85~87, respectively.

2. The sheep disease protein chip as described in claim 1, characterized in that, The antigens also include goatpox virus antigen, sheep infectious pustular virus antigen, bluetongue virus antigen, small ruminant plague virus antigen, pseudorabies virus antigen, Nipah virus antigen, Rift Valley fever virus antigen, caprine arthritis-encephalitis virus antigen, Japanese encephalitis virus antigen, Medi-Visna virus antigen, Nairobi sheep disease virus antigen, sheep abortive salmonella antigen, Mycoplasma filamentosa caprine subspecies antigen, Mycoplasma hyopneumoniae antigen, Clostridium perfringens antigen, Mycoplasma agalactiae antigen, Chlamydia psittaci antigen, Chlamydia abortus antigen, Echinococcus granulosus antigen, Listeria monocytogenes antigen, Toxoplasma gondii antigen, Pasteurella multocida antigen, Mansonia hemolyticus antigen, Bacillus anthracis antigen, foot-and-mouth disease virus type O antigen, and Mycobacterium tuberculosis bovine variant antigen. The goatpox virus antigen is one or more of GTPV-P32, GTPV-R122, GTPV-A34, and GTPV-A36; GTPV-P32, GTPV-R122, GTPV-A34, and GTPV-A36 are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 1~4, respectively. The sheep infectious pustular virus antigen is one or more of ORFV-B2L, ORFV-F1L, ORFV-020, and ORFV-125; ORFV-B2L, ORFV-F1L, ORFV-020, and ORFV-125 are respectively encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 5~8; The bluetongue virus antigen is one or more of BTV-VP7, BTV-VP2, and BTV-NS3; BTV-VP7, BTV-VP2, and BTV-NS3 are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 9~11. The small ruminant plague virus antigen is one or more of PPRV-N, PPRV-F, and PPRV-H; PPRV-N, PPRV-F, and PPRV-H are respectively encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 12~14; The pseudorabies virus antigen is one or more of PRV-gD, PRV-gB, and PRV-gE, and PRV-gD, PRV-gB, and PRV-gE are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 15~17. The Nipah virus antigen is NiV-N and / or NiV-G; NiV-N and NiV-G are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 18~19, respectively. The Rift Valley fever virus antigen is one or more of RVFV-NP, RVFV-NSs, and RVFV-NSm; RVFV-NP, RVFV-NSs, and RVFV-NSm are respectively encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 20~22; The capillary arthritis-encephalitis virus antigen is one or more of CAEV-CA, CAEV-gp135, and CAEV-MA; CAEV-CA, CAEV-gp135, and CAEV-MA are respectively encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 23~25. The Japanese encephalitis virus antigen is one or more of JEV-C, JEV-E, and JEV-NS1; JEV-C, JEV-E, and JEV-NS1 are respectively encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 26~28; The Medi-Visna virus antigen is one or more of MVV-P15, MVV-P25, MVV-P28, and MVV-P135; MVV-P15, MVV-P25, MVV-P28, and MVV-P135 are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 29~32. The Nairobi sheep disease virus antigen is NSD-N and / or NSD-Gc / Gn; NSD-N and NSD-Gc / Gn are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 33~34, respectively. The Salmonella antigen in sheep abortion is S. Abortusovis-fljB-H and / or S. Abortusovis-OMP; S.Abortusovis-fljB-H and Abortusovis-OMP are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 35~36, respectively; The Mycoplasma filamentosa goat subspecies antigen is M.mycoides-LPPA and / or M.mycoides-SecA; M.mycoides-LPPA and M.mycoides-SecA are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 37~38, respectively. The ovine mycoplasma pneumoniae antigen is one or more of M.ovi-P70, M.ovi-Enolase, M.ovi-P113, M.ovi-P60, and M.ovi-pdhD; M.ovi-P70, M.ovi-Enolase, M.ovi-P113, M.ovi-P60, and M.ovi-pdhD are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 39~43. The Clostridium perfringens antigen is Alpha toxin and / or Beta toxin; Alpha toxin and Beta toxin are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 48~49, respectively. The Clostridium perfringens antigen is NetB and / or Epsilon toxin; NetB and Epsilon toxin are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 50-51, respectively. The agalactiae mycoplasma antigen is one or more of M. agalacttiae-PDHB, M. agalacttiae-P80, and M. agalacttiae-P48; M. agalacttiae-PDHB, M. agalacttiae-P80, and M. agalacttiae-P48 are respectively encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 52~54. The psittaci-chlamydia psittaci antigen is one or more of C. psittaci-MOMP, C. psittaci-TARP, and C. psittaci-CPAF; C. psittaci-MOMP, C. psittaci-TARP, and C. psittaci-CPAF are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 55~57, respectively. The abortive chlamydia antigen is one or more of C. abortus-MIP, C. abortus-MOMP, and C. abortus-POMP90B; C. abortus-MIP, C. abortus-MOMP, and C. abortus-POMP90B are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 58~60; The Echinococcus granulosus antigen is one or more of EgM123, EgSeverin, Eg14-3-3ζ, and Eg-AgB8 / 3; EgM123, EgSeverin, Eg14-3-3ζ, and Eg-AgB8 / 3 are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 64~67; The Listeria monocytogenes antigen is one or more of LLO, P60, and InlB; LLO, P60, and InlB are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 68~70. The Toxoplasma gondii antigen is one or more of SAG1, GRA6, and SAG2A; SAG1, GRA6, and SAG2A are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 74~76, respectively. The Pasteurella multocida antigen is Pm-OmpA and / or Pm-OmpH; Pm-OmpA and Pm-OmpH are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 77~78, respectively. The hemolytic Mansell's antigen is M. haemolytica-Lkt and / or M. haemolytica-NlpI; M. haemolytica-Lkt and M. haemolytica-NlpI are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 79~80, respectively. The anthrax Bacillus antigen is B. anthracis-PA63, which is encoded by a DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 81; The type O foot-and-mouth disease virus antigen is one or more of FMDV-O-1, FMDV-O-2, and FMDV-O-3; the amino acid sequences of FMDV-O-1, FMDV-O-2, and FMDV-O-3 are shown in SEQ ID NO: 82~84, respectively. The Mycobacterium tuberculosis bovine variant antigen is Mycobacterium tuberculosis bovine variant lipoarabinomannan.

3. The sheep disease protein chip as described in claim 1, characterized in that, GTPV-P32, GTPV-R122, GTPV-A34, GTPV-A36, ORFV-B2L, ORFV-F1L, ORFV-020, ORFV-125, BTV-VP7, BTV-VP2, BTV-NS3, PPRV-N, PPRV-F, PPRV-H, PRV-gD, PRV-gB, PRV-g E, NiV-N, NiV-G, RVFV-NP, RVFV-NSs, RVFV-NSm, CAEV-CA, CAEV-gp135, CAEV-MA, JEV-C, JEV-E, JEV-NS1, MVV-P15, MVV-P25, MVV-P28, MVV-P135, NSD-N, NSD-Gc / Gn, S. Abortusovis-fljB-H,S. Abortusovis-OMP, M. mycoides-LPPA, M. mycoides-SecA, M.ovi-P70, M.ovi-Enolase, M.ovi-P113, M.ovi-P60, M.ovi-pdhD, MAP1693c, MAP4308c, MAP2677c, MAP1197, Alpha toxin, Beta toxin, NetB, Epsilontoxin, M. agalactiae-PDHB, M. agalactiae-P80, M. agalactiae-P48, C. psittaci-MOMP, C. psittaci-TARP, C. psittaci-CPAF, C. abortus-MIP, C. abortus-MOMP, C.abortus-POMP90B, B. melitensis-Omp25, B. melitensis-Omp31, B. melitensis-Dps, EgM123, EgSeverin, Eg14-3-3ζ, Eg-AgB8 / 3, LLO, P60, InlB, CLA-PLD, CLA-CP40, CLA-SlpA, SAG1, GRA6, SAG2A, Pm-OmpA, Pm-OmpH, M.haemolytica-Lkt, M.haemolytica-NlpI, and B.anthracis-PA63 are recombinant expressed and purified proteins.

4. The sheep disease protein chip as described in claim 1, characterized in that, The substrate is a nitrocellulose-modified glass slide, an aldehyde-modified glass slide, or an epoxy-modified glass slide; and / or The antigen is diluted with spotting buffer to a spotting concentration of 0.25 mg / L to 1.5 mg / L; and / or The spotting buffer is selected from one or more of glycerol aqueous solution, PBS, and TBS.

5. The sheep disease protein chip as described in claim 1, characterized in that, The substrate is a nitrocellulose-modified glass slide; and / or The antigen is diluted with spotting buffer to a spotting concentration of 0.25 mg / L to 1.0 mg / L; and / or The spotting buffer solution is an 80 wt% aqueous glycerol solution.

6. A reagent kit, characterized in that, Including the sheep disease protein chip as described in any one of claims 1 to 5.

7. The kit according to claim 6, characterized in that, It also includes sample diluent, blocking solution, washing solution, secondary antibody, and fluorescent triple antibody.

8. The kit according to claim 7, characterized in that, The sample diluent is PBST containing 0.5wt%~2wt% BSA or a mixture of fish skin gelatin and water containing 0.5wt%~2wt% fish skin gelatin; and / or The blocking solution is PBS containing 1 wt% to 5 wt% BSA or a mixture of fish skin gelatin and water containing 2 wt% to 5 wt% fish skin gelatin; and / or The washing solution is PBS, PBST, or TBST; and / or The secondary antibody is rabbit anti-goat IgG; and / or The fluorescent triple antibody is a mouse anti-rabbit IgG labeled with a fluorescent group; the fluorescent group is selected from FITC, TRITC, Cy, FAM or R-PE; and / or The dilution of the secondary antibody is 1:2000 to 1:3000; and / or The dilution of the fluorescent triple antibody is 1:2000~1:3000.

9. The kit according to claim 7, characterized in that, The sample diluent is PBST containing 1 wt% BSA; and / or The blocking solution is PBS containing 3 wt% BSA; and / or The washing solution is PBST; and / or The secondary antibody is diluted at a ratio of 1:2000; and / or The dilution of the fluorescent triple antibody is 1:2000.

10. The use of the sheep disease protein chip as described in any one of claims 1 to 5 or the kit as described in any one of claims 6 to 9 in (1) or (2): (1) Sheep disease detection for non-diagnostic purposes; (2) Screening or evaluating vaccines for sheep diseases.

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