Protein chip, kit and application thereof

Abstract

The invention relates to the technical field of biology. Specifically, the invention discloses a protein chip, a kit and application thereof. The protein chip comprises a substrate, 44 first antigens and 11 second antigens, wherein the 44 first antigens and the 11 second antigens are connected with the substrate; the first antigen is antigen protein, and the second antigen is polypeptide. The protein chip provided by the invention can realize bovine disease serological high-throughput detection and rapid bovine disease screening.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a protein chip, a reagent kit, and their applications. Background Technology

[0002] Bovine diseases pose a significant challenge to the global livestock industry. Their diversity and complexity greatly complicate diagnosis and control, making accurate diagnosis of infectious diseases in cattle a core component of the livestock disease prevention and control system. Currently, widely used clinical serological testing techniques (such as ELISA and agglutination tests) and etiological detection methods (PCR and bacterial culture) have significant limitations: traditional serological tests are typically designed for single pathogens, making simultaneous detection of multiple pathogens difficult, and their ability to differentiate between vaccine immunization and wild-type virus infection is limited; etiological detection is highly dependent on laboratory conditions and time-consuming. It is noteworthy that many bovine diseases present with highly similar clinical symptoms but are caused by different pathogens. For example, respiratory symptoms may be caused by bovine viral diarrhea virus, bovine infectious rhinotracheitis virus, or bovine respiratory syncytial virus infection, while diarrhea may be caused by bovine rotavirus, bovine coronavirus, or Cryptosporidium infection. Traditional methods require separate testing, resulting in low diagnostic efficiency and high costs. Cross-infection often occurs among these pathogens, leading to the co-occurrence of multiple diseases. This not only increases the complexity of clinical diagnosis but also hinders the selection of appropriate treatment plans, thereby affecting the health and productivity of cattle herds. Traditional single-pathogen detection methods are inefficient in the face of such complex infection situations and cannot meet the needs of modern animal husbandry for rapid detection of multiple pathogens. Summary of the Invention

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

[0004] To address the aforementioned technical problems, as a first aspect of the present invention, the present invention provides a protein chip comprising a substrate and 44 first antigens and 11 second antigens connected thereto; The first antigen is an antigen protein, which is: BVDV-E0, BVDV-E1, BVDV-E2, BVDV-p80 (200-550), LSDV-F13, LSDV-A33, LSDV-117, LSDV-B5, LSDV-A12L, BPIV3-NP (1-279), BPIV3-NP (280-516), BPIV3-M (1-192), BPIV3-M (193-351), BPIV3-HN, RPV-H, RPV-N, RPV-M, BoHV-1-gE, BoHV-1-gB, BoHV-1-gD, BoHV-1-gI, BRV-VP6, BRV-VP7, BRSV-G, BRSV-N, BLV-Pr44, BLV-gp60 SU, BEFV-G (470-623), BEFV-N, BCoV-N, AKAV-N, SBV-N, SBV-G, Mycoplasma bovis-P48, Mycoplasma bovis-P30, Pm-OmpA, Pm-OmpH, Pm-plpB, M. haemolytica -lipoproteinE M. haemolytica -NlpI, M. Bovis -MPB70, M. Bovis -MPB83, M. Bovis -CFP10-ESAT6, B. anthracis -PA63; which are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 1~44; The second antigen is a polypeptide, namely: LSDV-H3-1, LSDV-H3-2, LSDV-H3-3, BEFV-G1, BCoV-P, FMDV-O-1, FMDV-O-2, FMDV-O-3, FMDV-A-1, FMDV-A-2, FMDV-A-3; its amino acid sequence is shown in SEQ ID NO: 45~55.

[0005] As an improvement to the above technical solution, three third antigens are also included: Mycobacterium tuberculosis lipoarabinomannan, Mycobacterium avium paratuberculosis subspecies plasma membrane vesicles, and Brucella lipopolysaccharide.

[0006] As an improvement to the above technical solution, the first antigen is a recombinant expressed and purified protein.

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

[0008] As an improvement to the above technical solution, the substrate is a nitrocellulose-coated glass slide; and / or The first antigen, the second antigen, and the third antigen were all diluted with an antigen solvent to a concentration of 0.5 mg / L to 1 mg / L; The antigen solvent is an 80wt% aqueous glycerol solution.

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

[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 PBST or TBST; and / or The secondary antibody is rat anti-bovine IgG or mouse anti-bovine IgG; and / or The fluorescent triple antibody is a fluorescently labeled goat anti-mouse IgG, donkey anti-mouse IgG, or rabbit anti-mouse IgG; the fluorescent group is selected from FITC, TRITC, Cy, FAM, or R-PE.

[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 rat anti-bovine IgG; and / or The fluorescent triple antibody is Cy3-labeled goat anti-mouse IgG.

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

[0015] Implementing this invention has the following beneficial effects: The protein chip of this invention selects one or more antigenic targets for each pathogen, and the combined determination reduces the false negative rate and improves sensitivity and specificity. This protein chip can simultaneously detect multiple pathogens and is suitable for large-scale screening. Furthermore, the protein chip of this invention can also be used for vaccine efficacy evaluation, early diagnosis, and mixed infection analysis. Attached Figure Description

[0016] Figure 1 This is a diagram showing the substrate screening results in Embodiment 2 of the present invention; Figure 2 This is a graph showing the screening results of secondary antibodies in Example 3 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 antigen solvent screening results 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 based on the protein chip of this invention and the traditional ELISA detection in Embodiment 7 of this 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 The viruses, mycoplasma, and bacteria involved in this embodiment are as follows: Bovine viral diarrhea virus (BVDV), Bovine lumpy skin disease virus (LSDV), Bovine parainfluenza virus 3 (BPIV-3), Rinderpest virus (RPV), Bovine herpesvirus 1 (BoHV-1), Bovine rotavirus (BRV), Bovine respiratory syncytial virus (BRSV), Bovine leukemia virus (BLV), Bovine ephemeral fever virus (BEFV), Bovine coronavirus (BCoV), Foot-and-mouth disease virus (FMDV) types O and A, Akabane Disease Virus (AKVA), and Schmallenberg virus. Mycoplasma bovis, Pasteurella multocida (Pm), and Mannheimia haemolytica (BMP) are all bacteria that cause bovine swine fever. M . haemolytica Mycobacterium tuberculosis bovine variant ( Mycobacterium bovis M Bacillus bovis, Bacillus anthracis, B . anthracis ), Brucella abortus B . abortus ), Mycobacterium avium paratuberculosis subsp. Mycobacterium avium subsp Paratuberculosis MAP).

[0024] (I) Preparation of prokaryotic expression antigens 1. Construction of antigen expression vectors: The full protein sequences of the above-mentioned viruses were downloaded from the Uniprot database. Highly conserved viral nucleocapsid proteins, envelope proteins, bacterial secretory proteins, and outer membrane proteins were screened to obtain 76 target proteins. The signal peptide sequence, transmembrane region, and highly hydrophobic region of each protein were analyzed. The signal peptide and transmembrane region of the target proteins were removed, and codon optimization was used to convert them into nucleic acid sequences suitable for expression in *E. coli*. After the sequences were synthesized, they were ligated into the pET32a vector using a seamless cloning method. The N-terminus carried a Trx tag-PP restriction site, and the C-terminus carried a His tag. The vectors were transformed into Rosseta (DE3) competent cells and plated on LB agar (containing 100 μg / mL ampicillin). After incubation at 37°C overnight, single clones that were correctly identified by PCR were picked and transferred to 96-well deep-well plates. After shaking and incubation overnight, glycerol was added to a final concentration of 20%, and the plates were frozen at -80°C.

[0025] 2. High-throughput expression of recombinant proteins (1) Strain revival: Add 0.6 mL of LB liquid medium (containing 100 μg / mL ampicillin) to each well of a 96-well deep plate, and then add 3 μL of the strain preserved at -80℃ per well using a pipette. Revive the strain by shaking at 37℃ and 160 r / min overnight.

[0026] (2) Induction: Add LB liquid medium (containing 100 μg / mL ampicillin) to a 24-well shake plate (3 mL / well), and then add 30 μL of activated bacterial solution to each well. Perform two replicates for each strain. Shake and incubate at 37℃ and 160 r / min for 5 h. At this time, the OD600 of the bacterial solution is between 0.5 and 0.6. Then add 3 μL of 0.5 M IPTG to each well and shake and incubate at 16℃ and 120 r / min for 16 h.

[0027] (3) Protein expression detection: Centrifuge the culture plate at 4000 r / min for 15 min, discard the supernatant, resuspend the bacterial cells with 0.3 mL TBS, combine the bacterial cultures of the same strain, transfer to a 96-well deep-well plate, centrifuge at 4000 r / min for 15 min, discard the supernatant, wash once with 0.5 mL TBS, and add 0.18 mL TBS to each well. After resuspending the bacterial cells in PBS, 0.02 mL of bacterial lysis buffer was added, and the cells were lysed at room temperature for 10 min. The lysis buffer was then transferred to a 0.45 μm 96-well microfiltration plate and centrifuged at 4000 r / min for 10 min. The filtrate was collected and transferred to a 96-well plate. 50 μL of 5× protein loading buffer was added to each well, and the plate was sealed and heated in a 100℃ water bath for 5 min. 20 μL of protein was taken from each well for protein electrophoresis. The protein was transferred to a PVDF membrane, washed three times with TBST, and then blocked overnight at 4℃ with 5% skim milk. After washing three times with TBST, 1 μg / mL of anti-His tag antibody was added and incubated at 37℃ for 1 h. After washing, HRP-goat anti-mouse secondary antibody was added and incubated at 37℃ for 1 h. After washing with TBST, ECL chemiluminescence staining solution was used for color development. The protein bands and expected molecular weights were compared. The results showed that 21 proteins in the 76 constructed clones were soluble and expressed in the E. coli expression system, and their size and molecular weight were consistent. These 21 proteins were preserved (Table 1).

[0028] (4) Purification of recombinant protein: Resuscitate the correctly identified strains and transfer them to 1L LB liquid medium (containing 50μg / mL ampicillin) at a volume ratio of 1%, and culture at 37℃ with shaking at 160r / min until the OD600 of the bacterial solution is between 0.6 and 0.8. Then add 1mL 0.5M IPTG and culture at 16℃ with shaking at 160r / min for 16h. Centrifuge at 4000 rpm for 15 min, discard the supernatant, wash the precipitate three times with 45 mL of TBS, resuspend in 36 mL of TBS, add 8 mL of bacterial lysis buffer, vortex to mix, incubate at room temperature for 10 min, centrifuge at 6000 g for 15 min, filter the supernatant through a 0.45 μm filter to remove impurities, and perform nickel affinity chromatography to purify the target protein. After enriching the target protein eluent, replace the protein with PBS (pH=7.2-7.4) using a desalting column to detect the protein concentration. Add PP enzyme (20 μg PP enzyme for 1 mg target protein, PP enzyme carries a 6×His tag) and digest at 4℃ for 16 h. Perform nickel affinity chromatography on the digested sample, collect the target protein and add it to a 3 kDa ultrafiltration tube, concentrate the protein and replace it with PBS, detect the protein concentration with a BCA protein quantification kit, and detect the protein purity with SDS-PAGE. Adjust the protein concentration to 1 mg / mL with PBS buffer, and store in aliquots at -80℃.

[0029] (II) Preparation of eukaryotic expression antigens 1. Construction of antigen expression vector: The remaining 55 proteins were expressed using a eukaryotic expression system. The signal peptide and transmembrane region of the target protein were removed, and codon optimization was used to convert them into nucleic acid sequences suitable for eukaryotic expression. After sequence synthesis, the sequence was ligated seamlessly between the KpnI and XhoI restriction sites of the pCDNA3.1(+) vector, and a 6×HIS tag was added to the C-terminus of the antigen. The recombinant plasmid was transformed into TOP10 competent cells, plated on LB agar (containing 100 μg / mL ampicillin), and cultured overnight at 37°C. After PCR-identified single clones were picked and transferred to 96-well deep-well plates, cultured overnight with shaking, and then glycerol was added to a final concentration of 20% and stored at -80°C.

[0030] 2. Expression of recombinant proteins: (1) Resuscitate the strains and transfer them to 100 mL LB liquid medium (containing 100 μg / mL ampicillin) at a volume ratio of 1%. Incubate overnight at 37°C with shaking at 160 rpm. Centrifuge at 4000 rpm for 15 min, discard the supernatant, and wash the precipitate three times with 45 mL PBS. Extract recombinant plasmids using an endotoxin-free plasmid extraction kit, determine the concentration, and then freeze in aliquots at -80°C. Dilute 100 μg of plasmid with 1 mL of medium, and simultaneously dilute 600 μL of PEI (1 mg / mL stock solution) with 600 μL of medium. Mix and incubate at room temperature for 5 min. Inoculate with 1×10⁻⁶ 6 293F cells (100 mL) were cultured at a density of 3.5–4 × 10⁶ cells / mL until the cell density reached 3.5–4 × 10⁶ cells / mL. 6 After reaching cell / mL concentration, centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cells in 100 mL of Freestyle 293 medium. Slowly add the plasmid and PEI mixture to the cell suspension, mix thoroughly, and incubate at 37℃, 5% CO2, and 100 rpm for 4 h. After adding another 100 mL of medium, adjust the centrifugation speed to 120 rpm and continue incubation for 96 h. Centrifuge the culture supernatant at 10000g, 4℃ for 15 min to remove cell debris, and then filter through a 0.45 μm filter. The filtrate is used to purify the protein.

[0031] (2) Purification and identification of recombinant protein: The filtered culture medium was subjected to nickel column affinity chromatography to purify the target protein. After enriching the target protein eluent, the protein was replaced with PBS using a desalting column. The protein was then concentrated using a 3kDa ultrafiltration tube. The protein concentration was detected by BCA protein quantification kit and the protein purity was detected by SDS-PAGE. The protein concentration was adjusted to 1 mg / mL with PBS and then stored in separate flasks at -80℃. Add 10 μL of 5× protein loading buffer to each 40 μL protein solution, seal and heat in a 100℃ water bath for 5 min. Take 20 μL from each well for protein electrophoresis, transfer to PVDF membrane, wash 3 times with PBST, add 5% skim milk and block overnight at 4℃, wash 3 times with PBST, add 1 μg / mL of anti-His tag antibody and incubate at 37℃ for 1 h, wash, add HRP-goat anti-mouse secondary antibody and incubate at 37℃ for 1 h, wash with PBST, develop color with ECL chemiluminescence solution, compare protein bands with expected molecular weight, and record the correct clone numbers (Table 1, 23).

[0032] (III) Preparation of polypeptide antigens Since some proteins failed to be expressed successfully, we analyzed the antigenic epitopes of the amino acid sequence and synthesized a total of 11 polypeptide sequences with a purity greater than 90%. These sequences were dissolved in DMSO at a final concentration of 10 mg / mL and stored at -80°C. Before use, they were diluted 10-fold with TBS. The sequence information is shown in Table 1.

[0033] (iv) Preparation of extracted antigens 1. Extraction of Mycobacterium tuberculosis (LAM): BCG was streaked onto 7H10-OADC solid medium. Colonies were picked and transferred to liquid medium (4g asparagine, 2g citric acid, 0.24g magnesium sulfate, 0.5g dipotassium hydrogen phosphate, 0.05g ferric ammonium citrate, 4.825g D-glucose hydrate, 4.825g sodium pyruvate, 60mL glycerol, 0.1mL 0.05% zinc sulfate, deionized water to 1000mL, pH adjusted to 6.8 with ammonia, autoclaved at 115℃ for 30min). The medium was incubated statically at 37-39℃ for 2 months until a milky white bacterial membrane was observed. A silicone tube with a filter was inserted under the bacterial membrane, and the liquid was slowly aspirated using a peristaltic pump to trap the bacteria. The bacteria were washed with PBS, weighed, and temporarily stored at -70℃. For every 1L of culture (approximately 18g of bacterial cells), add 45mL of lysis buffer 1 (methanol:chloroform = 2:1) to resuspend the bacterial cells, and incubate at room temperature in the dark for 24 hours to inactivate the cells. Centrifuge at 27000g for 10 minutes, collect the supernatant, add 50mL of lysis buffer 2 (methanol:chloroform = 1:1) to the precipitate, vortex to mix, and incubate at room temperature in the dark for 24 hours. Centrifuge at 27000g for 10 minutes, collect the supernatant, add 50mL of lysis buffer 3 (methanol:chloroform = 1:2) to the precipitate, vortex to mix, and incubate at room temperature in the dark for 24 hours. Centrifuge at 27000g for 10 minutes, collect the supernatant, dry the precipitate at 55℃ for 24 hours, weigh it (approximately 1.5g), and freeze at -80℃ for 24 hours. If the bacterial cells are not completely dried, they can be freeze-dried once. Transfer the bacterial cells to a glass beaker, add 30 mL of double-distilled water (bacterial wet weight (g): water (mL) = 18:30), and centrifuge at 500 W for 6 s × 7 s × 300 times until the solution is homogenized. Add 300 mL of 50% ethanol, reflux at 65 °C for 4 h, centrifuge at 3000 g for 15 min, collect the supernatant, and repeat the above steps until the supernatant becomes colorless (approximately 1 L of supernatant is collected). The extract was evaporated and dried using a rotary evaporator (55-60℃). 35 mL of PBS was added to dissolve the precipitate, followed by an equal volume of saturated phenol. The mixture was heated and stirred at 80℃ for 2 hours. After the sample cooled to room temperature, it was centrifuged at 3000g for 15 minutes. The aqueous layer was collected and dialyzed against deionized water (6-8 kDa) four times, 6 hours each time. After drying by rotary evaporation at 55-60℃, the precipitate was resuspended in 10 mL of Tris-HCl buffer at pH 8.0. DNase and RNase were added to a final concentration of 50 μg / mL and digested at 37℃ for 8 hours. Proteinase K was then added and digested at 55℃ for 8 hours. The mixture was transferred to a 6-8 kDa dialysis bag and dialyzed against deionized water (6-8 kDa) four times, 6 hours each time.Remove the dialysis bag and concentrate the solution to approximately 2 mL in PEG20000. Centrifuge at 12000g for 15 min. Purify the supernatant using Superdex 75 molecular sieve and collect peak 206. Detect LAM purity by silver staining and quantify LAM using the anthrone sulfate method. Adjust the LAM concentration to 1 mg / mL with sterile, enzyme-free water, lyophilize in aliquots, and store at -15°C for later use.

[0034] 2. Extraction of plasma membrane vesicles from *Mycobacterium avium* subspecies *Parabolica*: Prepare culture medium (4g asparagine, 2g citric acid, 0.24g magnesium sulfate, 0.5g dipotassium hydrogen phosphate, 0.05g ferric ammonium citrate, 4.825g D-glucose hydrate, 4.825g sodium pyruvate, 60mL glycerol, 0.1mL 0.05% zinc sulfate, deionized water to 1000mL, pH adjusted to 6.8 with ammonia), autoclave at 115℃ for 30min, then cool to room temperature, add purified mycobacterium avium to a final concentration of 2μg / mL, inoculate with MAP, and incubate statically at 37℃ for 2 months. A milky white bacterial membrane will be visible. Insert a silicone tube with a filter screen under the bacterial membrane and use a peristaltic pump to slowly aspirate the liquid to retain the bacterial cells. The filtrate was filtered through a 0.22 μM sterile filter bottle and then ultrafiltered using a 100 kDa membrane. The liquid volume was concentrated 20 times, and then 3 times the volume of 0.1×PBS was added for desalting. The purified plasma membrane vesicles can be used as antigens for the diagnosis of paratuberculosis. The protein concentration was determined using a BCA protein quantification kit. When the protein concentration was 1 mg / mL, it was aliquoted and stored at -15°C for later use.

[0035] 3. Extraction of Brucella LPS: Brucella bovis attenuated strain A19-△virB6 (deposited on August 8, 2024, at the China General Microbiological Culture Collection Center, accession number CGMCC No. 46097, biologically classified as Brucella attenuated strain) was identified and preserved by the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences. The strain was streaked onto TSA medium and incubated at 36–37°C for 48–72 hours. 15 mL of physiological saline was added to each plate to wash off the culture. After filtration through four layers of sterile gauze, the culture was collected in sterile glass bottles and inactivated in an 80°C water bath for 2 hours. The inactivated bacterial solution was centrifuged at 10,000 g for 10 min, and the supernatant was discarded. Add 170 mL of distilled water to every 50 g of wet bacteria, mix well, and heat to 66 °C. Then add 190 mL of 90% (v / v) phenol solution at 66 °C, stir for 15 min at 66 °C, centrifuge at 10000 g for 15 min at 4 °C, and discard the lower phenol phase using a long needle. Filter the upper liquid through commercial filter paper and measure the volume of the harvested liquid. Add 3 volumes of cold methanol containing 1% saturated sodium acetate to precipitate LPS. After precipitation at 4 °C for 2 h, centrifuge at 10000 g for 15 min, discard the supernatant, resuspend the precipitate in 1 / 2 volume of distilled water from the original aqueous phase, stir at 4 °C for 18 h, centrifuge at 10000 g for 10 min, collect the supernatant, resuspend the precipitate in an equal volume of sterile distilled water, stir at 4 °C for 2 h, centrifuge at 10000 g for 15 min, collect the supernatant, and combine it with the previous supernatant. Add 5% trichloroacetic acid to the supernatant, stir for 10 min, centrifuge at 10000 g, and collect the supernatant. Dialyze the LPS with 4000 mL of distilled water for 3 hours each time, for a total of 4 dialysis cycles. Add proteinase K to the dialyzed LPS to a final concentration of 15 μg / mL, digest at 55°C for 3 hours, then digest at room temperature for 24 hours. Finally, dialyze the LPS five times with 4000 mL of deionized water for 3 hours each time. This is the purified LPS. Aliquot the LPS antigen into 1 mg / mL portions, lyophilize, and store at -15°C or below for later use.

[0036] The detailed antigen information obtained in this embodiment is shown in Table 1.

[0037] Table 1 Protein chip antigen information

[0038] Example 2: Substrate Selection 1. Antigen Testing and Chip Detection Process: Antigen detection involves proteins, peptides, LPS, LAM, and plasma membrane vesicles. The optimal coating substrate and coating solution may differ for each. We used Mycobacterium tuberculosis bovine variant MPB70 and LAM (MPB70, LAM), Mycobacterium paratuberculosis plasma membrane vesicle antigen (pTB), Brucella LPS (LPS), Bovine viral diarrhea virus whole virus (BVDV), Bovine viral diarrhea virus E0 (E0), Foot-and-mouth disease virus type O peptide (FMDV-O-1), and Foot-and-mouth disease virus type A peptide (FMDV-A-1) as examples to screen substrates and coating solutions, as detailed below: (1) Chip application: Dilute the above 8 antigens to 1 mg / mL with PBS for later use. Add 6 μL of 80% glycerol to each well of a 384-well plate, followed by 6 μL of the diluted antigen. Add blank control (PBS), irrelevant protein control (BSA), and positive control (bovine IgG) at the same time. Centrifuge at 1600 r / min for 1 min. Use a microarray chip application system to apply the above samples to the chip. Set 12 arrays on each chip. Each antigen and control has 3 replicates in each array. Place the completed chip in a chip box, store at 4°C overnight, and then freeze at -80°C.

[0039] (2) Blocking: After removing the chip from -80℃, immerse it in blocking solution (3% BSA-PBS) and block it for 3 hours at room temperature on a side-shaking shaker at a speed of 20~30r / min.

[0040] (3) Washing: Transfer the chip to the washing box and wash it with 30mL PBS, 0.2×PBS and ultrapure water respectively for 5min each time. Then transfer the chip to a 50mL centrifuge tube and centrifuge at 3000r / min at room temperature for 4min to remove excess water. Store at 4℃ for later use.

[0041] (4) Incubation of serum: Fix the chip on the 16-well grid, centrifuge the positive serum of bovine tuberculosis, bovine paratuberculosis, bovine brucellosis, bovine viral diarrhea virus, bovine foot-and-mouth disease type O, and bovine foot-and-mouth disease type A at 12000g for 15min, dilute the supernatant 200 times with PBST containing 1% BSA and add 0.2mL to each sample well, mix the above positive serum in equal volumes as positive control, and fetal bovine serum as negative control, dilute 200 times with PBST containing 1% BSA and add 0.2mL to each positive control well and negative control well, at room temperature at a speed of 20~30r / min on a side-shaking incubator for 1h.

[0042] (5) Washing: Use a pipette to aspirate the reaction solution and discard it. Add 300 μL of PBST to each well and wash 15 times by pipetting and discarding the washing solution. Repeat this washing process 3 times. Remove the fence and immerse the chip in a washing box containing 30 mL of PBST. Shake rapidly for 15 seconds and discard the washing solution. Repeat this washing process 3 times. Add 30 mL of PBST and wash on a side-swinging shaker at 20-30 r / min for 10 min. Repeat this process 3 times to thoroughly remove non-specifically adsorbed proteins from the chip.

[0043] (6) Incubation of secondary antibody: Dilute rat anti-bovine IgG monoclonal antibody to 1 μg / mL with PBST containing 1% BSA, add to chip reaction box, and react on a side-shaking shaker at 20~30 r / min at room temperature in the dark for 1 h.

[0044] (7) Washing: Discard the incubation solution, transfer the chip to the washing box, add 30 mL PBST and wash quickly with shaking for 15 s, repeat the washing twice; add 30 mL PBST and wash on a side-swinging shaker at 100~110 r / min in the dark for 10 min, repeat 3 times.

[0045] (8) Incubation of triple antibodies: Dilute Cy3-goat anti-mouse IgG antibody to 1 μg / mL with PBST containing 1% BSA, add it to the chip reaction box, and react on a side-swinging shaker at 20~30 r / min at room temperature in the dark for 1 h.

[0046] (9) Washing: Discard the incubation solution, transfer the chip to the washing box, add 30 mL PBST and wash quickly with shaking for 15 s, repeat the washing twice; add 30 mL PBST and wash on a side-swinging shaker at 100~110 r / min in the dark for 10 min, repeat 3 times; then add 30 mL ultrapure water and wash on a side-swinging shaker at 100~110 r / min in the dark for 5 min, repeat 2 times; then transfer the chip to a 50 mL centrifuge tube, centrifuge at 3000 r / min at room temperature for 4 min to remove excess water, and store at room temperature in the dark.

[0047] (10) Scanning: Use PMT=532 (Cy3 channel) to pre-scan the chip, determine the fine scan area and scan parameters, then perform fine scanning on the chip, save and analyze the data.

[0048] 2. Substrate Screening: Following the above procedures, spot the test antigen onto nitrocellulose-coated slides (PATH), epoxy-modified slides, and aldehyde-modified slides (Aldehyde), respectively. Compare the specificity and sensitivity of the antigen's reaction with serum antibodies on different substrates. The detection results are as follows: Figure 1The results showed that when detecting positive control sera (six positive sera mixed in equal volumes), there was no significant difference in the detection values ​​among the three substrates, indicating that all three substrates could coat the protein (bovine IgG) under the above detection procedure and could be used to detect antibodies against bovine IgG in serum. The negative control (BSA protein) and blank control (PBS) of the substrates showed readings below 10 when detecting positive control sera, indicating low background and minimal non-specific reactions among the three substrates. When detecting antibody levels against recombinantly expressed MPB70 and E0 in positive serum, there was no significant difference in the detection results among the three substrates; however, when detecting antibodies against polypeptide antigens, whole virus, extracted LAM, pTB, and LPS in positive serum, the reading of the nitrocellulose-coated slide (PATH) was significantly higher than that of the epoxy-modified slide and the aldehyde-modified slide, indicating that the nitrocellulose-coated slide has a wider range of applications and can be used for coating recombinant proteins, polypeptides, polysaccharides, lipopolysaccharides, and plasma membrane vesicles. Therefore, this patent prefers PATH as the substrate.

[0049] In this embodiment, the purification process of whole BVDV virus is as follows: MDBK cells were seeded in a cell factory. After cell adhesion, BVDV virus was added and cultured continuously for 5 days. 2L of virus solution was freeze-thawed three times to completely release virus particles. Cell debris was removed by filtration through a 0.45μm sterile filter flask, followed by filtration through a 100kDa ultrafiltration membrane to concentrate the culture medium to 50-80mL. The medium was then desalted using 500mL of PBS buffer and transferred to the PBS buffer system. After filtration through a 0.45μm filter, β-propiolactone (virus solution:β-propiolactone = 2000:1) was added, and the virus was inactivated at 4℃ for 24h, followed by β-propiolactone decomposition at 37℃ for 2h. MDBK cells were inoculated with the inactivated virus solution, and no cytopathic effect was observed after 5 days, indicating complete virus inactivation. Centrifuge the inactivated virus at 25000 r / min for 2 h, discard the supernatant, add 4 mL of PBS to dissolve overnight, detect the whole viral protein concentration with BCA protein quantification kit, adjust the concentration to 1 mg / mL with PBS, aliquot and freeze at -80℃.

[0050] Example 3 Screening of secondary antibodies (1) Following the procedure in Example 2, mouse anti-bovine IgG antibodies (Bio-Rad, IL-A2 strain) were compared. Figure 2 (Middle anti-A), mouse anti-bovine IgG antibody (Sigma, BG-18 strain) Figure 2 Secondary antibody B), rat anti-bovine IgG monoclonal antibody (Mabtech, MT391 strain), Figure 2 The specificity of the secondary antibody C in protein chip detection system.

[0051] (2) Spot the antigen onto the PATH using a spotting instrument. At the same time, set up a blank control (PBS), an irrelevant protein control (BSA), and a positive control (bovine IgG). Set up 12 arrays on each chip. Each antigen and control has 3 replicates in each array. Place the finished chip into a chip box, store it at 4°C overnight, and then freeze it at -80°C.

[0052] (3) Serum: The volumes of positive serum / immune serum for 21 pathogens were mixed as a positive control (P), fetal bovine serum as a negative control (N), and three serum samples from newborn calves were used as test serum (C1, C2, and C3). The three clinical serum samples were tested by ELISA. C1 and C2 were negative for antibodies against all 21 pathogens, while C3 serum was positive for antibodies against bovine viral diarrhea virus, bovine parainfluenza virus type 3, bovine mycoplasma, bovine variant of Mycobacterium tuberculosis, and subsp. paratuberculosis of avian mycobacterium, and negative for antibodies against other pathogens.

[0053] (4) Following the operating steps of Example 2, the results showed that all three secondary antibodies could recognize bovine IgG protein well. However, when secondary antibody A (IL-A2 strain) or secondary antibody B (BG-18 strain) detected two newborn bovine serum samples C1 and C2 that were negative for pathogen antibodies, the readings of some antigens were high, indicating that these two monoclonal antibodies would have non-specific reactions in the protein chip reaction system. Secondary antibody C (MT391 strain) had a low background and the readings of C1 and C2 serum were lower than those without non-specific reactions, making it more suitable for protein chip detection systems. Figure 2 ).

[0054] Example 4 Screening of antigen concentration (1) Following the procedure in Example 2, the antigens were diluted with PBS to concentrations of 1 mg / mL, 0.5 mg / mL, and 0.25 mg / mL, respectively. In a 384-well plate, 6 μL of 80% glycerol was added to each well, followed by 6 μL of the diluted antigen. Blank control (PBS), irrelevant protein control (BSA), and positive control (bovine IgG) were also included. Using a microarray chip spotting system, the samples were spotted onto the chip. Each chip had 12 arrays. The prepared chips were placed in a chip cassette, stored overnight at 4°C, and then frozen at -80°C.

[0055] (2) Serum: The volumes of positive serum / immune serum of 21 pathogens were mixed as positive control (P), fetal bovine serum was used as negative control (N), and 2 clinical serum samples were used as serum to be tested.

[0056] (3) The selected rat anti-bovine IgG monoclonal antibody (MT391 strain) was used as the secondary antibody. The detection was carried out according to the operation steps of Example 2. The results showed that when the chip was coated with three protein concentrations, the background was clean and the detection results were consistent, indicating that the chip has a wide range of applicability to antigen amount and is not easy to cause result deviation due to changes in antigen amount. However, in order to ensure sufficient antigen, we preferred to use a protein concentration of 1 mg / mL to spot the chip.

[0057] Example 5 Screening of antigen solvents This invention relates to antigens such as proteins, peptides, plasma membrane vesicles, and LPS. To screen for the optimal solvent, antigens were mixed with equal volumes of PBS (pH 7.2-7.4), 80wt% glycerol, TBS (pH 8.0), and 10vol% DMSO. After storage at 2-8℃ for 24 hours, the mixtures were centrifuged at 12000g for 30 minutes to observe for sedimentation. The results showed that the antigens were all clear and without sedimentation. Chips were then prepared following the procedures in Example 2, and positive and negative control sera were tested. The results showed that 80wt% glycerol provided the best detection performance, followed by PBS and TBS, while DMSO was not effective. Therefore, 80wt% glycerol was preferred for preparing the protein chip.

[0058] Example 6 Optimization of Reaction Conditions The chip was prepared according to Example 2. The incubation conditions for serum, secondary antibody, and third antibody were set to room temperature for 1 hour and 37°C for 30 minutes, respectively. An orthogonal experiment was designed to compare the positive serum detection values ​​under different reaction times. The results showed that the detection results were consistent across the four reaction conditions, and there was no significant difference in the positive serum values ​​(e.g., ...). Figure 5 The results indicate that the serum / antibody incubation conditions are relatively lenient and have good applicability. In order to improve the detection efficiency, the incubation conditions for serum, secondary antibody, and tertiary antibody were set to 37℃ for 30 min.

[0059] Table 2 Reaction Conditions

[0060] Example 7: Optimized protein chip detection process (1) Chip application: After thawing the antigen in an ice bath, centrifuge at 12000g for 15 min at 4℃, and dilute the supernatant to 1 mg / mL with PBS. Add 6 μL of 80wt% glycerol to each well of a 384-well plate, followed by 6 μL of diluted antigen. Set up blank control (PBS), irrelevant protein control (BSA), and positive control (bovine IgG). Centrifuge at 1600 r / min for 1 min. Using a microarray chip application system, apply the above samples to the PATH chip. Set up 12 arrays on each chip, with 3 replicates for each antigen and control in each array. Place the completed chips in a chip cassette, store at 4℃ overnight, and then freeze at -80℃.

[0061] (2) Blocking: After removing the chip from -80℃, immerse it in blocking solution (3% BSA-PBS) and block it for 3 hours at room temperature on a side-shaking shaker at a speed of 20~30r / min.

[0062] (3) Washing: Transfer the chip to the washing box and wash it with 30mL PBS, 0.2×PBS and ultrapure water respectively for 5min each time. Then transfer the chip to a 50mL centrifuge tube and centrifuge at 3000r / min at room temperature for 4min to remove excess water. Store at 4℃ for later use.

[0063] (4) Incubation of serum: Fix the chip on the 16-well grid, centrifuge 12000g of serum for 15min, dilute the supernatant 200 times with PBST containing 1% BSA, add 0.2mL to each sample well, mix the above positive serum in equal volumes as positive control, and fetal bovine serum as negative control, dilute 200 times with PBST containing 1% BSA, add 0.2mL to each positive control well and negative control well, and react at 37℃ on a side-shaking incubator at a speed of 20~30r / min for 0.5h.

[0064] (5) Washing: Use a pipette to aspirate the reaction solution and discard it. Add 300 μL of PBST to each well and wash 15 times by pipetting and discarding the washing solution. Repeat this washing process 3 times. Remove the fence and immerse the chip in a washing box containing 30 mL of PBST. Wash the chip on a side-swinging shaker at 20-30 r / min for 10 min. Repeat this process 3 times to thoroughly remove non-specifically adsorbed proteins from the chip.

[0065] (6) Incubation of secondary antibody: Dilute rat anti-bovine IgG monoclonal antibody to 1 μg / mL with PBST containing 1% BSA, add to chip reaction box, and react on a side-shaking shaker at 20~30 r / min at 37℃ in the dark for 0.5 h.

[0066] (7) Washing: Discard the incubation solution, transfer the chip to the washing box, add 30 mL PBST, and wash on a side-shaking shaker at 100~110 r / min in the dark for 10 min. Repeat 3 times.

[0067] (8) Incubation of triple antibodies: Dilute Cy3-goat anti-mouse antibody to 1 μg / mL with PBST containing 1% BSA, add it to the chip reaction box, and react on a side-swinging shaker at 20~30 r / min at 37℃ in the dark for 0.5 h.

[0068] (9) Washing: Discard the incubation solution, transfer the chip to the washing box, add 30 mL PBST, and wash on a side-swinging shaker at 100~110 r / min in the dark for 10 min, repeat 3 times; then add 30 mL ultrapure water, and wash on a side-swinging shaker at 100~110 r / min in the dark for 5 min, repeat 2 times; then transfer the chip to a 50 mL centrifuge tube, centrifuge at 3000 r / min at room temperature for 4 min to remove excess water, and store at room temperature in the dark.

[0069] (10) Scanning: Use PMT=532 (Cy3 channel) to pre-scan the chip, determine the fine scan area and scan parameters, then perform fine scanning on the chip, save and analyze the data.

[0070] Example 7: Consistency between protein chip and ELISA detection methods Following Example 6, protein chips were used to detect 10 clinical bovine 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 results showed a positive correlation between the protein chip detection values ​​and the ELISA detection values. r =0.6741), and the test results are consistent.

[0071] Example 8: Application of protein chips in clinical sample testing Following Example 6, protein chips were used to detect 50 clinical bovine serum samples, and the results were validated using commercial kits or ELISA methods. The results showed that the diagnostic results of the protein chip for clinical samples were consistent with those of ELISA, as detailed below: (1) The protein chip detection results showed that the F532 values ​​of 58 antigens against 21 pathogens in 21 serum samples were all less than 30, which was determined to be antibody negative. This was consistent with the detection results of the ELISA method, confirming that the chip had good detection specificity.

[0072] (2) Twelve serum samples were positive for antibodies against Brucella bovis, Bovine viral diarrhea virus and Mycobacterium avium paratuberculosis subsp. avium, consistent with the results of commercially available ELISA kits, confirming that the chip has good detection sensitivity.

[0073] (3) Thirteen serum samples were positive for bovine viral diarrhea virus antibodies, consistent with the results of commercial ELISA test kits.

[0074] (4) One serum sample was positive for Mycobacterium tuberculosis bovine variant antibody, which was consistent with the results of the commercial kit.

[0075] In this embodiment, one chip can detect antibodies against 21 bovine diseases in 10 clinical serum samples, offering high throughput and accuracy. It can complete the testing of 50 serum samples in just 3.5–4.5 hours. In contrast, when using commercially available kits or established ELISA methods, the serum dilution, reagents, and judgment criteria vary between kits, requiring 40–50 hours to test 50 serum samples. Therefore, protein chips are clearly superior in high-throughput detection.

[0076] 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, characterized in that, It includes a substrate and 44 primary antigens and 11 secondary antigens linked to it; The first antigen is an antigen protein, which is: BVDV-E0, BVDV-E1, BVDV-E2, BVDV-p80 (200-550), LSDV-F13, LSDV-A33, LSDV-117, LSDV-B5, LSDV-A12L, BPIV3-NP (1-279), BPIV3-NP (280-516), BPIV3-M (1-192), BPIV3-M (193-351), BPIV3-HN, RPV-H, RPV-N, RPV-M, BoHV-1-gE, BoHV-1-gB, BoHV-1-gD, BoHV-1-gI, BRV-VP6, BRV-VP7, BRSV-G, BRSV-N, BLV-Pr44, BLV-gp60 SU, BEFV-G (470-623), BEFV-N, BCoV-N, AKAV-N, SBV-N, SBV-G, Mycoplasma bovis-P48, Mycoplasma bovis-P30, Pm-OmpA, Pm-OmpH, Pm-plpB, M. haemolytica -lipoproteinE, M. haemolytica -NlpI, M. Bovis -MPB70, M. Bovis -MPB83, M. Bovis -CFP10-ESAT6, B. anthracis -PA63; which are encoded by DNA molecules with nucleotide sequences as shown in SEQ ID NO: 1~44; The second antigen is a polypeptide, namely: LSDV-H3-1, LSDV-H3-2, LSDV-H3-3, BEFV-G1, BCoV-P, FMDV-O-1, FMDV-O-2, FMDV-O-3, FMDV-A-1, FMDV-A-2, FMDV-A-3; its amino acid sequence is shown in SEQ ID NO: 45~55.

2. The protein chip as described in claim 1, characterized in that, It also includes three third antigens: Mycobacterium tuberculosis lipoarabinomannan, Mycobacterium avium paratuberculosis subspecies plasma membrane vesicles, and Brucella lipopolysaccharide.

3. The protein chip as described in claim 1, characterized in that, The first antigen is a recombinant protein that has been purified through expression.

4. The protein chip as described in claim 2, characterized in that, The substrate is a nitrocellulose-coated glass slide, an aldehyde-modified glass slide, or an epoxy-modified glass slide; and / or The first antigen, the second antigen, and the third antigen were all diluted with an antigen solvent to a concentration of 0.25 mg / L to 1.5 mg / L; The antigen solvent is selected from one or more of glycerol, aqueous glycerol solution, PBS, and TBS.

5. The protein chip as described in claim 1, characterized in that, The substrate is a nitrocellulose-coated glass slide; and / or The first antigen, the second antigen, and the third antigen were all diluted with an antigen solvent to a concentration of 0.5 mg / L to 1 mg / L; The antigen solvent is an 80wt% aqueous glycerol solution.

6. A reagent kit, characterized in that, Including the 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 PBST or TBST; and / or The secondary antibody is rat anti-bovine IgG or mouse anti-bovine IgG; and / or The fluorescent triple antibody is a fluorescently labeled goat anti-mouse IgG, donkey anti-mouse IgG, or rabbit anti-mouse IgG; the fluorescent group is selected from FITC, TRITC, Cy, FAM, or R-PE.

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 rat anti-bovine IgG; and / or The fluorescent triple antibody is Cy3-labeled goat anti-mouse IgG.

10. The use of the 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) Cattle disease testing for non-diagnostic purposes; (2) Screening or evaluating vaccines for bovine diseases.

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