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CN-122017233-A - Protein chip, kit and application thereof

CN122017233ACN 122017233 ACN122017233 ACN 122017233ACN-122017233-A

Abstract

The present invention relates to the field of biotechnology. In particular, 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 are connected with the substrate, the first antigens are antigen proteins, and the second antigens are polypeptides. The protein chip can realize high-throughput detection of bovine disease serology and rapid screening of bovine disease.

Inventors

  • DING JIABO
  • XIN TING
  • JIN JIAXIN
  • ZHANG ZE
  • JIANG HUI
  • FENG YU
  • CHEN SUMENG

Assignees

  • 中国农业科学院北京畜牧兽医研究所

Dates

Publication Date
20260512
Application Date
20251011

Claims (10)

  1. 1. A protein chip comprising a substrate and 44 first antigens and 11 second antigens attached thereto; The first antigens are antigen proteins which are ：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-lipoprotein E,M. haemolytica-NlpI,M. Bovis-MPB70,M. Bovis-MPB83,M. Bovis-CFP10-ESAT6,B. anthracis-PA63; respectively and are encoded by DNA molecules with nucleotide sequences shown as SEQ ID NO. 1-44; the second antigens are polypeptides, the amino acid sequences of which are ：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; respectively as shown in SEQ ID NO. 45-55.
  2. 2. The protein chip of claim 1, further comprising 3 third antigens, which are mycobacterium tuberculosis lipoarabinomannan, mycobacterium avium subspecies paratuberculosis vesicles, and brucella lipopolysaccharide, respectively.
  3. 3. The protein chip of claim 1, wherein said first antigen is a recombinant expressed purified protein.
  4. 4. A protein chip as claimed in claim 2, wherein the substrate is a nitrocellulose cover glass, an aldehyde-modified glass or an epoxy-modified glass, and/or The first antigen, the second antigen and the third antigen are diluted to the concentration of 0.25 mg/L-1.5 mg/L through an antigen solvent; the antigen solvent is one or more of glycerol, glycerol water solution, PBS and TBS.
  5. 5. The protein chip as claimed in claim 1, wherein the substrate is a nitrocellulose-coated glass slide, and/or The first antigen, the second antigen and the third antigen are diluted to a concentration of 0.5 mg/L-1 mg/L through an antigen solvent; The antigen solvent is 80wt% glycerin water solution.
  6. 6. A kit comprising the protein chip according to any one of claims 1 to 5.
  7. 7. The kit of claim 6, further comprising a sample diluent, a blocking solution, a wash solution, a secondary antibody, and a fluorescent tertiary antibody.
  8. 8. The kit according to claim 7, wherein the sample diluent is PBST containing 0.5wt% to 2wt% BSA or a mixture of fish skin gelatin and water containing 0.5wt% to 2wt% fish skin gelatin, and/or The blocking solution is PBS containing 1-5wt% BSA or a mixture of fish skin gelatin and water containing 2-5wt% fish skin gelatin, and/or The washing liquid is PBST or TBST, and/or The secondary antibody is rat anti-bovine IgG or mouse anti-bovine IgG, and/or The fluorescent tri-antibody is goat anti-mouse IgG, donkey anti-mouse IgG or rabbit anti-mouse IgG marked by a fluorescent group, and the fluorescent group is FITC, TRITC, cy, FAM or R-PE.
  9. 9. The kit according to claim 7, wherein the sample diluent is PBST containing 1wt% BSA, and/or The blocking solution is PBS containing 3wt% BSA, and/or The washing liquid is PBST, and/or The secondary antibody is rat anti-bovine IgG, and/or The fluorescent tri-antibody is Cy 3-labeled goat anti-mouse IgG.
  10. 10. Use of a protein chip according to any one of claims 1 to 5 or a kit according to any one of claims 6 to 9 in (1) or (2): (1) Bovine disease detection for non-diagnostic purposes; (2) Vaccines for bovine disease are screened or evaluated.

Description

Protein chip, kit and application thereof Technical Field The invention relates to the technical field of biology, in particular to a protein chip, a kit and application thereof. Background Bovine disease is a major challenge facing the global animal husbandry, the diversity and complexity of the bovine disease bring great difficulty to diagnosis, prevention and control of diseases, and accurate diagnosis of infectious diseases of cattle groups is a core link of an animal husbandry epidemic disease prevention and control system. The serological detection technology (such as ELISA and agglutination test) and the etiology detection method (PCR and bacterial culture) which are widely applied in clinic at present have obvious limitations that the traditional serological detection is usually designed aiming at single pathogen, the synchronous detection of multiple pathogens is difficult to realize, the discrimination capability of vaccine immunity and wild virus infection is limited, and the etiology detection has high dependence on laboratory conditions and takes longer time. It is noted that many bovine clinical symptoms are highly similar, but are caused by different pathogens, for example respiratory symptoms may be caused by infection with bovine viral diarrhea virus, bovine infectious rhinotracheitis virus or bovine respiratory syncytial virus, while diarrhea symptoms may be caused by infection with bovine rotavirus, bovine coronavirus or cryptosporidium, and the conventional methods require separate detection, resulting in low diagnostic efficiency and high cost. Cross infection often occurs between these pathogens, resulting in the co-occurrence of multiple diseases, which not only increases the complexity of clinical diagnosis, but also prevents the selection of the correct therapeutic regimen, thereby affecting the health level and productivity of the herd. Traditional single pathogen detection methods appear inefficient in the face of such complex infection conditions, and it is difficult to meet the requirements of modern animal husbandry for rapid detection of multiple pathogens. Disclosure of Invention The invention aims to solve the technical problem of providing a protein chip which has the advantages of high flux and high sensitivity and provides a technical scheme for synchronously screening and detecting multi-pathogen bovine diseases. In order to solve the above 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 antigens are antigen proteins which are ：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-lipoprotein E,M. haemolytica-NlpI,M. Bovis-MPB70,M. Bovis-MPB83,M. Bovis-CFP10-ESAT6,B. anthracis-PA63; respectively and are encoded by DNA molecules with nucleotide sequences shown as SEQ ID NO. 1-44; the second antigens are polypeptides, the amino acid sequences of which are ：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; respectively as shown in SEQ ID NO. 45-55. As an improvement of the above technical scheme, 3 third antigens are included, which are mycobacterium tuberculosis lipoarabinomannan, mycobacterium avium subspecies paratuberculosis membrane vesicles and brucella lipopolysaccharide, respectively. As an improvement of the above technical scheme, the first antigen is recombinant expression purified protein. As an improvement of the technical proposal, the substrate is a nitrocellulose cover glass, an aldehyde group modified glass or an epoxy group modified glass, and/or The first antigen, the second antigen and the third antigen are diluted to the concentration of 0.25 mg/L-1.5 mg/L through an antigen solvent; the antigen solvent is one or more of glycerol, glycerol water solution, PBS and TBS. As an improvement of the technical proposal, the substrate is a nitrocellulose coated glass slide, and/or The first antigen, the second antigen and the third antigen are diluted to a concentration of 0.5 mg/L-1 mg/L through an antigen solvent; The antigen solvent is 80wt% glycerin water solution. As a second aspect of the present invention, there is provided a kit comprising the protein chip described above. As improvement of the technical scheme, the fluorescent three-antibody fluorescent three-dimensional fluorescent probe further comprises sample diluent, sealing liquid, washing liquid, secondary antibody and fluorescent three-antibody. As an improvement of the technical scheme, the sample diluent is PBST containing 0.5-2wt% of BSA or a mixt