CN116754766A - Kit for detecting feline calicivirus - Google Patents

Kit for detecting feline calicivirus Download PDF

Info

Publication number
CN116754766A
CN116754766A CN202310806217.0A CN202310806217A CN116754766A CN 116754766 A CN116754766 A CN 116754766A CN 202310806217 A CN202310806217 A CN 202310806217A CN 116754766 A CN116754766 A CN 116754766A
Authority
CN
China
Prior art keywords
seq
amino acid
variable region
chain variable
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202310806217.0A
Other languages
Chinese (zh)
Other versions
CN116754766B (en
Inventor
吴茂柏
李婷婷
查丽莎
周宇杭
黄茜
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Hertz Life Science Technology Co ltd
Original Assignee
Shenzhen Hertz Life Science Technology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Hertz Life Science Technology Co ltd filed Critical Shenzhen Hertz Life Science Technology Co ltd
Priority to CN202310806217.0A priority Critical patent/CN116754766B/en
Priority claimed from CN202310806217.0A external-priority patent/CN116754766B/en
Publication of CN116754766A publication Critical patent/CN116754766A/en
Application granted granted Critical
Publication of CN116754766B publication Critical patent/CN116754766B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2470/00—Immunochemical assays or immunoassays characterised by the reaction format or reaction type
    • G01N2470/04—Sandwich assay format
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • Microbiology (AREA)
  • Organic Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention relates to the field of biotechnology detection, in particular to a kit based on detection of feline calicivirus. The invention provides a reagent for detecting feline calicivirus by a double-antibody sandwich enzyme-linked immunosorbent assay. According to the invention, the recombinant antibody based on VP1 protein as an important immunogenic protein of the feline calicivirus is obtained through screening by a phage display technology, and is used for an ELISA detection kit of FCV, and the recombinant antibody has the advantages of high specificity, high sensitivity, good stability and the like.

Description

Kit for detecting feline calicivirus
Technical Field
The invention relates to the field of biotechnology detection, in particular to a kit based on detection of feline calicivirus.
Background
Feline calicivirus infection is a feline viral respiratory infectious disease, and is mainly manifested by upper respiratory symptoms, namely mental depression, serous and mucous rhinorrhea, conjunctivitis, stomatitis, tracheitis, bronchitis, with bipolar fever. Under natural conditions, only felines are susceptible to the virus and frequently occur in cats of 6 to 8 days of age. The main infectious agents are sick cats and cats with poison. The infected cat can discharge a large amount of viruses along with secretions and excretions in the acute stage, and the infected cat is directly infected with the susceptible cat. The cat with poison is treated, symptoms can disappear, but long-term detoxification is the most dangerous infectious source. Furthermore, the intensity variant FCV-VSD of FCV causes systemic infection and increased mortality of animals,
currently, the diagnosis of feline calicivirus infection is based on medical history, clinical symptoms and epidemic characteristics. Laboratory diagnosis can be performed when clinical diagnosis is difficult; scraping the pathological tissue object, staining by fluorescent antibody, and monitoring the existence of antigen. In order to improve the detection effect of the feline calicivirus and reduce the great economic loss brought to the wild animals and pet industry, further research and development of a kit for detecting the feline calicivirus is still a research hotspot in the field.
Disclosure of Invention
In view of the above, the technical problem to be solved by the invention is to provide a double-antibody sandwich ELISA detection kit for feline calicivirus, which has good sensitivity, specificity and stability.
The invention provides a reagent for detecting feline calicivirus, which comprises a capture antibody coated on a solid phase medium and a detection antibody marked with a biomarker or a chemical marker.
Wherein the capture antibody or the detection antibody may be the same or different.
Among the capture antibodies or detection antibodies:
the amino acid sequence of CDR1 of the heavy chain variable region is GX H1 X H2 LSX H3 YX H4 Wherein X is H1 Selected from I or F; x is X H2 Selected from D or S, X H3 Selected from T or S, X H4 Selected from A or V or Y or T;
the amino acid sequence of CDR2 of the heavy chain variable region is IX H5 X H6 X H7 X H8 X H9 X H10 Wherein X is H5 Selected from N or Y; x is X H6 Selected from T or G; x is X H7 Selected from D or S or T or G; x is X H8 Selected from G or A; x is X H9 Selected from S or N or Y or I; x is X H10 Selected from A or T or R;
the amino acid sequence of CDR3 of the heavy chain variable region is X H11 X H12 X H13 X H14 X H15 X H16 X H17 X H18 X H19 GX H20 X H21 X H2 2 X H23 X H24 X H25 Wherein X is H11 Selected from A or none; x is X H12 Selected from R or none; x is X H13 Selected from A or none; x is X H14 Selected from G or none; x is X H15 Selected from A or P or none; x is X H16 Selected from G or R or none; x is X H17 Selected from R or S or E or G or none; x is X H18 Selected from G or Y; x is X H19 Selected from R or V or I or none; x is X H20 Selected from F or L or V or Y or I; x is X H21 Selected from P or T or none; x is X H22 Selected from D or A or N; x is X H23 Selected from L or Y or F; x is X H24 Selected from D or none; x is X H25 Selected from P or S or none;
the amino acid sequence of CDR1 of the light chain variable region is X L1 SX L2 X L3 X L4 X L5 X L6 X L7 Wherein X is L1 Selected from Q or E; x is X L2 Selected from V or I; x is X L3 Selected from Y or none; x is X L4 Selected from S or D or G or N; x is X L5 Selected from D or S or N; x is X L6 Selected from W or N; x is X L7 Selected from Y or E or none;
the amino acid sequence of CDR2 of the light chain variable region is X L8 X L9 S, wherein X L8 Selected from D or Y or A; x is X L9 Selected from V or A;
the amino acid sequence of CDR3 of the light chain variable region is X L10 X L11 X L12 X L13 X L14 YX L15 X L16 X L17 X L18 X L19 X L20 X L21 Wherein X is L10 Selected from C or none; x is X L11 Selected from C or none; x is X L12 Selected from A or S or L; x is X L13 Selected from G or N; x is X L14 Selected from A or S or G; x is X L15 Selected from S or D; x is X L16 Selected from G or D or none; x is X L17 Selected from N or D; x is X L18 Selected from I or V or A; x is X L19 Selected from Y or D; x is X L20 Selected from T or G or A or S or N; x is X L16 Selected from L or A or none.
In some embodiments of the present invention,
in the capture antibody:
the amino acid sequence of CDR1 of the heavy chain variable region is shown as at least one of SEQ ID NO. 29, SEQ ID NO. 35, SEQ ID NO. 41, SEQ ID NO. 47 and/or SEQ ID NO. 53;
the amino acid sequence of CDR2 of the heavy chain variable region is shown as at least one of SEQ ID NO. 30, SEQ ID NO. 36, SEQ ID NO. 42, SEQ ID NO. 48 and/or SEQ ID NO. 54;
the amino acid sequence of CDR3 of the heavy chain variable region is shown as at least one of SEQ ID NO. 31, SEQ ID NO. 37, SEQ ID NO. 43, SEQ ID NO. 49 and/or SEQ ID NO. 55;
the amino acid sequence of CDR1 of the light chain variable region is shown as at least one of SEQ ID NO. 32, SEQ ID NO. 38, SEQ ID NO. 44, SEQ ID NO. 50 and/or SEQ ID NO. 56;
the amino acid sequence of CDR2 of the light chain variable region is shown as at least one of SEQ ID NO. 33, SEQ ID NO. 39, SEQ ID NO. 45, SEQ ID NO. 51 and/or SEQ ID NO. 57;
the amino acid sequence of CDR3 of the light chain variable region is shown as at least one of SEQ ID NO 34, SEQ ID NO 40, SEQ ID NO 46, SEQ ID NO 52 and/or SEQ ID NO 58;
in the detection antibody:
the amino acid sequence of CDR1 of the heavy chain variable region is shown as at least one of SEQ ID NO. 29, SEQ ID NO. 35, SEQ ID NO. 41, SEQ ID NO. 47 and/or SEQ ID NO. 53;
the amino acid sequence of CDR2 of the heavy chain variable region is shown as at least one of SEQ ID NO. 30, SEQ ID NO. 36, SEQ ID NO. 42, SEQ ID NO. 48 and/or SEQ ID NO. 54;
the amino acid sequence of CDR3 of the heavy chain variable region is shown as at least one of SEQ ID NO. 31, SEQ ID NO. 37, SEQ ID NO. 43, SEQ ID NO. 49 and/or SEQ ID NO. 55;
the amino acid sequence of CDR1 of the light chain variable region is shown as at least one of SEQ ID NO. 32, SEQ ID NO. 38, SEQ ID NO. 44, SEQ ID NO. 50 and/or SEQ ID NO. 56;
the amino acid sequence of CDR2 of the light chain variable region is shown as at least one of SEQ ID NO. 33, SEQ ID NO. 39, SEQ ID NO. 45, SEQ ID NO. 51 and/or SEQ ID NO. 57;
the amino acid sequence of CDR3 of the light chain variable region is shown as at least one of SEQ ID NO 34, SEQ ID NO 40, SEQ ID NO 46, SEQ ID NO 52 and/or SEQ ID NO 58;
still further, the method comprises the steps of,
in the detection antibody:
the amino acid sequences of the three CDR regions of the light chain are respectively shown as SEQ ID NO. 32-34; the amino acid sequences of the three CDR regions of the heavy chain are respectively shown in any one of SEQ ID NO. 29-31;
in the capture antibody:
the amino acid sequences of the three CDR regions of the light chain are respectively shown as SEQ ID NO 50-52; the amino acid sequences of the three CDR regions of the heavy chain are shown in any one of SEQ ID NOs 47 to 49 respectively.
More specifically, the method comprises the steps of,
the heavy chain variable region of the detection antibody is shown as SEQ ID NO. 1; the light chain variable region is shown as SEQ ID NO. 2; the heavy chain and the light chain are connected by a linker, and the linker sequence is shown as SEQ ID NO. 11.
The heavy chain variable region of the capture antibody is shown as SEQ ID NO. 7; the light chain variable region is shown as SEQ ID NO. 8; the heavy chain and the light chain are connected by a linker, and the linker sequence is shown as SEQ ID NO. 11.
In the present invention, the chemical label is an isotope, an immunotoxin and/or a chemical drug; the biomarker is biotin, avidin or an enzyme label. The enzyme label is preferably horseradish peroxidase or alkaline phosphatase. The immunotoxin is preferably aflatoxin, diphtheria toxin, pseudomonas aeruginosa exotoxin, ricin, abrin, mistletoe lectin, podophyllotoxin, PAP, sequoyins, gelonin or luffa toxin.
In the present invention, the solid or semi-solid medium refers to any support to which the recombinant antibodies, labeled recombinant antibodies, of the present invention can be attached, including but not limited to nitrocellulose membranes, polyvinylidene difluoride (PVDF) membranes, iPDMS chips, microwell plates, polystyrene plates, microparticles, microcarriers, gels, and the like.
In some embodiments, the capture antibody is coated on an elisa plate and the detection antibody is labeled with HRP.
In some embodiments, the mass ratio of capture antibody to detection antibody is 10:1.
The detection reagent also comprises a coating buffer solution, a washing solution, a sealing solution, TMB color development solution and a termination solution; wherein:
the coating buffer solution is PBS buffer solution with pH of 7.2-7.4,
the washing liquid is PBST solution containing 0.05% Tween-20 by mass fraction.
The sealing liquid is a skimmed milk powder solution with the mass fraction of 2%;
the termination liquid is 2mol/L H 2 SO 4 A solution.
Further, the invention also provides a detection method of the feline calicivirus, which comprises detecting a sample by the detection reagent.
The detection method of the present invention may be for diagnostic purposes or non-diagnostic purposes, and the present invention is not limited thereto. Among them, in the detection method for diagnostic purposes, the sample is derived from an animal body, and in the detection method for non-diagnostic purposes, the sample is derived from an environment such as food, water, laboratory culture, or a swab on the surface of an implement.
In the invention, the detection method specifically comprises the following steps: after capturing the sample by the capture antibody, adding the detection antibody for incubation; the capturing condition is 37 ℃ for 1h; the incubation condition is 37 ℃ for 1h;
in the invention, after the incubation, the steps of TMB color development, termination of color development and reading of absorbance at 450nm are also included.
The detection method of the invention can be a qualitative detection method.
And the qualitative detection is based on whether fluorescence judgment results are generated, and the sample generating fluorescence is a positive sample.
The invention provides a reagent for detecting feline calicivirus by a double-antibody sandwich enzyme-linked immunosorbent assay. According to the invention, the recombinant antibody based on VP1 protein as an important immunogenic protein of the feline calicivirus is obtained through screening by a phage display technology, and is used for an ELISA detection kit of FCV, and the recombinant antibody has the advantages of high specificity, high sensitivity, good stability and the like.
Drawings
FIG. 1 shows construction of FCV VP1-scFv phage library and diversity analysis of immune library provided by the embodiment of the invention, wherein A is antibody titer of 4 times of immunization of New Zealand rabbits; b is amplified bands of VL gene library and VH gene library, M: a Marker;1: VH,2: VL,3: VL-linker-VH destination bands;
FIG. 2 shows the screening of high affinity FCV VP1-scFv provided by the examples of the present invention, wherein A is the result of PCR identification after electrotransformation; b is the diversity of the antibody library after the first round of phage display screening; c is the diversity of the antibody library after the second round of phage display screening; d is a second round of phage ELISA to determine scFv antibodies with high specific binding of screening antigen FCV VP 1;
FIG. 3 shows the expression and affinity verification of Ab-FCV VP1 provided by the examples of the present invention, wherein A is the SDS-PAGE result, M, of Ab-FCV VP 1: marker,1: ab-FCV VP1-1,2: ab-FCV VP1-2,3: ab-FCV VP1-3,4: ab-FCV VP1-4,5: ab-FCV VP1-5; b is EC of Ab-FCV VP1-1 antibody bound with antigen FCV VP1 50 The method comprises the steps of carrying out a first treatment on the surface of the C is EC of Ab-FCV VP1-2 antibody bound with antigen FCV VP1 50 The method comprises the steps of carrying out a first treatment on the surface of the D is EC of Ab-FCV VP1-3 antibody bound with antigen FCV VP1 50 The method comprises the steps of carrying out a first treatment on the surface of the E is EC of Ab-FCV VP1-4 antibody bound with antigen FCV VP1 50 The method comprises the steps of carrying out a first treatment on the surface of the F is EC of Ab-FCV VP1-5 antibody bound with antigen FCV VP1 50 ；
FIG. 4 shows the results of an indirect immunofluorescence experiment between the Ab-FCV VP1-4 recombinant antibody and FCV virus provided by the embodiment of the invention.
FIG. 5 shows the specificity verification of Ab-FCV VP1-4 recombinant antibodies binding to FCV, FPV, FHV virus provided by the examples of the present invention.
FIG. 6 shows plasmid maps of vectors pTT5-V5 and pTT5-V6 containing rabbit antibody skeletons according to the examples of the present invention.
Detailed Description
The invention provides an antibody for detecting feline calicivirus based on VP1 protein and application thereof, and a person skilled in the art can properly improve the technological parameters by referring to the content of the text. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
Heavy chain variable region amino acid sequences of single chain antibody FCV VP1-scFv-1 and recombinant antibody Ab-FCV VP 1-1: QSVKESGGRLVTPGTPLTITCTVSGIDLSTYAMSWVRQDPGKGLEYIGFINTDGSAYYAS WAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCGRGFDLWGQGTLVTVSS (SEQ ID NO: 1);
H1-CDR1:GIDLSTYA(SEQ ID NO:29)；
H1-CDR2:INTDGSA(SEQ ID NO:30)；
H1-CDR3:GRGFDL(SEQ ID NO:31)；
light chain variable region amino acid sequences of single chain antibody FCV VP1-scFv-1 and recombinant antibody Ab-FCV VP 1-1: DGMMTQTPSPVSAAVGGTVTIKCQSSQSVYSDWLSWFQQKPGQPPKRLIYDASTLASG VPSRFKGSGSGTQFTLTISDLECDDAATYYCAGAYSGNIYTFGGGTEVVVK (SEQ ID NO: 2);
L1-CDR1:QSVYSDW(SEQ ID NO:32)；
L1-CDR2:DAS(SEQ ID NO:33)；
L1-CDR3:AGAYSGNIYT(SEQ ID NO:34)；
heavy chain variable region amino acid sequences of single chain antibody FCV VP1-scFv-2 and recombinant antibody Ab-FCV VP 1-2: QSLEESGGRMVTPGTPLTLTCTVSGFSLSSYVVSWVRQAPGKGLEWIGIIYTSGNTYYAN WAKGRFTISKTSTTVDLKITSPTTEDTATYFCGRGGLPALWGQGTLVTVSS (SEQ ID NO: 3);
H2-CDR1:GFSLSSYV(SEQ ID NO:35)；
H2-CDR2:IYTSGNT(SEQ ID NO:36)；
H2-CDR3:GRGGLPAL(SEQ ID NO:37)；
light chain variable region amino acid sequences of single chain antibody FCV VP1-scFv-2 and recombinant antibody Ab-FCV VP 1-2: DGVLTQTPSSTSAAVGGTVTISCQSSESVYDNNYLSWYQQKPGQPPKVLIYYVSTLASGV PSRFKGSGSGTQFTLTISDLECDDAATYYCAGAYSGNIYGFGGGTEVVVK (SEQ ID NO: 4);
L2-CDR1:ESVYDNNY(SEQ ID NO:38)；
L2-CDR2:YVS(SEQ ID NO:39)；
L2-CDR3:AGAYSGNIYG(SEQ ID NO:40)；
heavy chain variable region amino acid sequences of single chain antibody FCV VP1-scFv-3 and recombinant antibody Ab-FCV VP 1-3: QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYAMIWVRQAPGKGLEWIGIIYGTAYRYYASW AKGRFTISKTSTTVDLKITSPTTEDTATYFCARSYVGVTAYDPVGQGTLVTVSS (SEQ ID NO: 5);
H3-CDR1:GFSLSTYA(SEQ ID NO:41)；
H3-CDR2:IYGTAYR(SEQ ID NO:42)；
H3-CDR3:ARSYVGVTAYDP(SEQ ID NO:43)；
light chain variable region amino acid sequences of single chain antibody FCV VP1-scFv-3 and recombinant antibody Ab-FCV VP 1-3: DGVPTQTPSSVSAAVGGTVTISCQSSQSVYSNWLSWFQQKPGQPPKRLIYDASTLTSGVS SRFKGSGSGTQFILTISDLECDDAATYYCAGAYSGNIYAFGGGTEVVVK (SEQ ID NO: 6); L3-CDR1 QSVYSNW (SEQ ID NO: 44);
L3-CDR2:DAS(SEQ ID NO:45)；
L3-CDR3:AGAYSGNIYA(SEQ ID NO:46)；
heavy chain variable region amino acid sequences of single chain antibody FCV VP1-scFv-4 and recombinant antibody Ab-FCV VP 1-4: QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYTMSWVRQAPGKGLEWIGIIYTGGITYYASW AKGRFTISRTSTTVDLKITSPTTEDTATYFCAREGVGYAFDSWGPGTLVTVSS (SEQ ID NO: 7);
H4-CDR1:GFSLSSYT(SEQ ID NO:47)；
H4-CDR2:IYTGGIT(SEQ ID NO:48)；
H4-CDR3:AREGVGYAFDS(SEQ ID NO:49)；
light chain variable region amino acid sequences of single chain antibody FCV VP1-scFv-4 and recombinant antibody Ab-FCV VP 1-4: DGMMTQTPSPVSATVGGTVTIKCQASESIGSWLAWYQQKPGQRPKLLIYAASTLASGVP SRFSGSGSGTEFTLAISGVECADAATYYCQQSNSYSDVDSLFGGGTEVVVK (SEQ ID NO: 8);
L4-CDR1:ESIGSW(SEQ ID NO:50)；
L4-CDR2:AAS(SEQ ID NO:51)；
L4-CDR3:QQSNSYSDVDSL(SEQ ID NO:52)；
heavy chain variable region amino acid sequences of single chain antibody FCV VP1-scFv-5 and recombinant antibody Ab-FCV VP 1-5: QSLEESGGRLVTPGTPLTLTCTASGFSLSSYYMTWVRQAPGKGLEWIGIIYGTGYTYYAS WAKGRFTISKTSTTVDLKMTSLTTEDTATYFCARAGPGGGIGINLWGQGTLVTVSS (SEQ ID NO: 9);
H5-CDR1:GFSLSSYY(SEQ ID NO:53)；
H5-CDR2:IYGTGYT(SEQ ID NO:54)；
H5-CDR3:ARAGPGGGIGINL(SEQ ID NO:55)；
light chain variable region amino acid sequences of single chain antibody FCV VP1-scFv-5 and recombinant antibody Ab-FCV VP 1-5: DGVMTQTESPVSAAVGSTVTINCQSSQSVYNNNELSWYQQKPGQPPKLLIYYASTLASG VSSRFKGSGSGTQFTLTISGVQCDDAATYYCLGGYDDDADNAFGGGTEVVVK (SEQ ID NO: 10);
L5-CDR1:QSVYNNNE(SEQ ID NO:56)；
L5-CDR2:YAS(SEQ ID NO:57)；
L5-CDR3:LGGYDDDADNA(SEQ ID NO:58)；
amino acid sequence of linker peptide linker: GGGGSGGGGGGSSRSS (SEQ ID NO: 11);
VH-scFv-F1：5’-ggtgggggtggttcctctagatcttcccagtcgktggaggagtcc-3’(SEQ ID NO:12)；
VH-scFv-F2：5’-ggtgggggtggttcctctagatcttcccagwcagtgaaggagtcc-3’(SEQ ID NO:13)；
VH-scFv-F3：5’-ggtgggggtggttcctctagatcttcccagtcgctggrggagtcc-3’(SEQ ID NO:14)；
VH-scFv-F4：5’-ggtgggggtggttcctctagatcttcccagtcggtggaggagtcc-3’(SEQ ID NO:15)；
VH-scFv-R1：5’-aggaggctattggccggcctggcctgargagayggtgaccagggtgcc-3’(SEQ ID NO:16)；
VH-scFv-R2：5’-aggaggctattggccggcctggccgcagcagggggccagtgggaagactgac-3’(SEQ ID NO:17)；
VL-scFv-F1：5’-aaaagaggcccaggcggccgagctcgtgmtgacccagactcca-3’(SEQ ID NO:18；
VL-scFv-F2：5’-aaaagaggcccaggcggccgagctcgatmtgacccagactcca-3’(SEQ ID NO:19)；
VL-scFv-F3：5’-aaaagaggcccaggcggccgcycaagtgctgacccag-3’(SEQ ID NO:20)；
VL-scFv-F4：5’-aaaagaggcccaggcggccgcccwagtgatgacccag-3’(SEQ ID NO:21)；
VL-scFv-R1:5’-ggaagatctagaggaaccacccccaccaccgcccgagccaccgccacctttgatttccacattggtgcc-3’(SEQ ID NO:22)；
VL-scFv-R2:5’-ggaagatctagaggaaccacccccaccaccgcccgagccaccgccacctaggatctccagctcggtccc-3’(SEQ ID NO:23)；
overlapping PCR-FP:5'-gaggaggaaaaagaggcccaggcggcc-3' (SEQ ID NO: 24);
overlapping PCR-RP 5'-agaggaggctattggccggcctggcc-3' (SEQ ID NO: 25);
pComb3XSS-FP：5’-gctggtttcgctaccgtggcccaggcggcc-3’(SEQ ID NO:26)；
pComb3XSS-RP：5’-gtgatggtgatggtgctggccggcctggcc-3’(SEQ ID NO:27)；
amino acid sequence of FCV VP1 protein:
MCSTCANVLKYYDWDPHFRLVVNPNKFLSVGFCDNPLLCCYPELLPEFGTVWDCNQSPLQIYLESILGDDDWSSTHEAIDPVVPPMHWDEAGKIFQPHPGVLMHYIVGEVAKAWDPNLPLFRLEADDGSITTPEQGTVVGGVIAEPSAQMAVAADTATGKSVDSEWEAFFSFHTSVNWGTSETQGKILFKQSLGPLLNPYLEHLAKLYVAWSGSIEVRFSISGSGVFGGKLAAIVVPPGVDPVQSTSMLQYPHVLFDARQVEPVIFTIPDLRSTLYHLMSDTDTTSLVIMVYNDLINPYARDSNSSGCIVTVETKPGPDFRFHLLKPPGSMLTHGSVPSDLIPKNSSLWIGNRHWTDITDFIIRPFVFQANRHFDFNQETAGWSTPRFRPITVTISQKDGAKLGIGIATDCIVPGIPDGWPDTTIPSRLTPAGDYAITSGDNSDIATKQQYETADEIKNNTNFKSMYICGALQRAWGDKKVSNTGFITTATISDNQLVPSNVIDQTKIAVFQDNRVSKEVQTSDVTLAILGYTGIGEEAVGADRDKVVRISVLPETGARGGNHPIFYKNSMKLGYVVGSIDVFNSQILHTSRQLSLNNYLLAPDSLAVYRIIDANGSWFDVGIDSDGFSFVGVSHIGKLEFPLSASYMGIQLAKIRLASNIRSNMVKL(SEQ ID NO:28)。
strains, plasmids, test animals and reagents: adult New Zealand rabbits were purchased from Yongkangqing source farms in the county of Anhui province; the FCV VP1 protein is preserved in the laboratory (the amino acid sequence is SEQ ID NO. 28); FCV virus is preserved by the laboratory (GenBank: KT 000003.1); vectors pTT5-V5 and pTT5-V6 containing rabbit antibody frameworks were stored for this laboratory (FIG. 5); HRP-labeled recombinant protein a was purchased from engineering bioengineering (Shanghai) stock, inc; HRP-labeled murine anti-M13K 07 phage were purchased from adzuki biotechnology limited; HRP-labeled goat anti-rabbit IgG was purchased from beginner Mo De biotechnology limited; pComb3XSS plasmid, e.g. dh5α competent cells were purchased from shandong hertz biotechnology limited; M13K07 helper phage, E.coli ER2738 competent cells were purchased from Nanjing He Ding Biotechnology Co., ltd; SDS-PAGE protein gel kit was purchased from Yase Biotechnology Co., ltd; penicillin-streptomycin solutions were purchased from GE company (for cell culture); freund's adjuvant was purchased from Sigma company; clonExpress II One Step Cloning Kit from Nanjinouzan Biotechnology Co., ltd; premix taq, plasmid mini kit, gel Extraction and transfection reagents were all purchased from TaKaRa.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
EXAMPLE 1 animal immunization
Healthy adult New Zealand rabbits are selected for the experiment, vein blood collection is carried out before immunization, and serum is separated to serve as a negative control. Taking 1×10 5 TCID 50 The FCV virus/mL was injected in the back neck of New Zealand rabbits, immunized once a week for 4 times, and preserved by intravenous blood sampling of 0.5mL of the isolated serum before each immunization on days 0, 7, 14, 21. 7 days after the 4 th immunization (i.e. day 28), 0.5mL of the isolated serum was collected intravenously and stored. ELISA results show that the antibody titer of the first immune serum is obviously increased, and the titer of the secondary immune antibody is more than 5 multiplied by 10 4 Three-free rear-reaching 10 5 The titer of the four-immune serum reaches 1.5 multiplied by 10 5 There was a significant increase in serum antibody titers compared to that before immunization (a in fig. 1). New Zealand rabbits were sacrificed on day 28, 0.5mL of blood was collected and isolated serum was saved, the spleen was dissected and removed for lymphocyte separation.
EXAMPLE 2 extraction of Total lymphocyte RNA and amplification of antibody light-heavy chain Gene
Total RNA of lymphocytes is extracted by Trizol reagent, and cDNA is synthesized by reverse transcription using Oligo (dT) as a primer with the total RNA as a template. The coding regions of antibody VH and VL were PCR amplified by designing a primer sequence combination (primer sequences SEQ ID NO. 12-SEQ ID NO. 23) based on the light and heavy chain framework regions of the rabbit antibody sequence. The PCR reaction conditions were: the pre-denatured product was 5min at 95℃for 30s, annealed at 55℃for 30s, extended at 72℃for 60s, and after 35 cycles, extended at 72℃for another 5min. The PCR product was confirmed by 1% agarose gel electrophoresis to have a band of interest of about 350bp, and was recovered by purification (FIG. 1B).
EXAMPLE 3 construction of FCV VPV 1-scFv antibody library
The purified VH and VL are used as templates, a linker (with the sequence of SEQ ID NO. 11) is inserted between the VH and the VL after overlapping PCR, and the VL-linker-VH of the scFv fragment is synthesized, and the two ends of the sequence contain sfiI restriction enzyme sites.
Overlapping extension first round PCR: without primer, 1min was denatured at 98 ℃, annealed at 45 ℃ for 30s, extended at 72 ℃ for 1min, repeated 10 cycles, and finally acted at 72 ℃ for 5min. Adding primer overlapping PCR-FP and overlapping PCR-RP (SEQ ID NO. 24-SEQ ID NO. 25), denaturing at 98 ℃ for 30s, then renaturating at 58 ℃ for 30s, extending at 72 ℃ for 1min, and extending at 72 ℃ for 5min after 30 cycles. The amplified product was recovered by gel recovery of the PCR product by 1% agarose gel electrophoresis, and the band was about 800bp (B in FIG. 1) and stored at-20 ℃. The sequence of the overlapped PCR primer is SEQ ID NO. 24-SEQ ID NO.25.
And respectively carrying out enzyme digestion on the PCR product and the pComb3XSS vector after gum recovery by using restriction enzyme sfiat 50 ℃ for 30min, and carrying out column recovery and purification after 1% agarose gel electrophoresis verification on the enzyme digestion product, and connecting the enzyme digestion product with T4 DNA ligase to obtain the FCV VP1-scFv-pComb3XSS library. The ligation products were transformed into ER2738 competent cells, and positive clones were screened with tetracycline and ampicillin resistant 2YT (2X Yeast extract and Tryptone) solid medium. 20 monoclonal antibodies in the library were randomly selected for PCR identification (primer sequences SEQ ID NO. 26-SEQ ID NO. 27), and the recombination rate of the antibody library was calculated to be 60% (A in FIG. 2). All clones on solid medium were washed off with 2YT medium and stored at 4 ℃.
Example 4 amplification of phages
2mL of the library bacterial solution of all clones washed by the 2YT medium is inoculated into 50mL of the 2YT liquid medium, and activated to OD at 37 ℃ at 200rpm 600 Reaching 0.8-1.0, adding into the mixture with the final concentration of 1 multiplied by 10 12 The pfu/mL M13K07 helper phage was infected, allowed to stand for 30min, kanamycin (final concentration 50. Mu.g/mL) was added, and incubated overnight at 30℃at 200 rpm. Centrifugation was performed for 20min at 10000rpm4℃the next day, the supernatant was retained and 1/2 volume of PEG8000 was added thereto, and the mixture was allowed to stand on ice for 2 hours to precipitate phage. Centrifuging at 10000rpm at 4deg.C for 20min, and discarding supernatant. The pellet was resuspended in 5mL PBS, 3mL PEG8000 was added for further precipitation, centrifugation was repeated, and after resuspension of the pellet with 4mL PBS and sterilization by filtration, the pellet was stored at 4℃for further use.
EXAMPLE 5FCV VP1-scFv screening
FCV VP1 protein was biotinylated in advance. After the amplified FCV VP1-scFv library phage, dynabeads M-280 magnetic beads marked with FCV VP1 protein and 2% nonfat milk powder blocking solution are mixed, the mixture is incubated for 1h at 25+/-5 ℃, and the magnetic beads are washed for 5-10 times by PBST. The bead surface phage was eluted with 0.1M Glycine-HCl (ph=2.0) and after elution neutralized with 1M tris to pH 7.0. ER2738 competent cells in the logarithmic growth phase are infected with the phage neutralized after elution, enriched by culturing on a 2YT solid medium, and colonies are harvested for the next round of screening.
The above experiment was repeated twice. From the two rounds of enrichment affinity screening libraries, 20 monoclonal antibodies were randomly selected for PCR validation, respectively, and the results showed that the established FCV VP1-scFv library had a positive rate of 70% in the first round of panning and 95% in the second round of panning (B and C in FIG. 2).
EXAMPLE 6Phage ELISA
FCV VP1 antigen was coated on the ELISA plate at a concentration of 50. Mu.g/mL per well at 4℃overnight. The cells were washed 5 times with PBST solution containing 0.05% Tween-20, PBST was discarded, and 2% nonfat dry milk blocking solution was added and blocked at 37℃for 1 hour. Washing with PBST solution for 5 times, beating the residual liquid in the hole, and mixing the supernatant of the positive clone antibody to be screened and 2% skim milk powder sealing liquid according to the following steps of 3: mixing at a ratio of 2, standing at 25+ -5deg.C for 10min to remove interference, addingIn the ELISA plate, incubation was performed at 37℃for 1h. Wash 5 times with PBST solution, dry the wells with residual liquid, add HRP-labeled murine anti-M13K 07 antibody (diluted 1:3000 with blocking solution), incubate for 1h at 37 ℃. Washing with PBST solution for 5 times, drying the residual liquid in the wells, adding TMB color development liquid, standing in dark for 15min, and adding 2mol/L H 2 SO 4 The color development was stopped and the absorbance at 450nm was read (Table 1). As shown in FIG. 2D, select OD 450 And sequencing 12 antibodies with high values, and finally obtaining 5 antibodies with different sequences for subsequent experiments.
TABLE 1.96 affinity detection of Ab-FCV VP1-1 antibody-containing phages with antigen FCV VP1
0.7499 0.9347 1.384 1.5467 1.4948 0.4876 1.3691 0.8919 1.3492 0.7921 1.8346 0.1093
1.2354 0.8379 1.0397 1.9346 1.2378 1.6392 1.7381 0.6732 1.8279 0.329 0.9218 0.5369
1.8796 1.2938 1.1289 1.0729 1.5386 1.5936 1.3918 2.6924 1.3926 0.6827 0.612 0.7361
0.5389 0.5379 0.6948 0.5379 1.5097 1.0527 1.3094 1.261 0.7912 1.0724 0.3719 1.7362
1.9346 1.3897 0.7124 0.839 0.973 0.7943 0.9682 1.0739 0.9368 1.2681 0.2768 0.9824
2.3469 0.8967 1.5432 1.549 1.3032 0.5392 1.3768 1.5927 1.0329 0.2917 1.0258 0.6381
1.3672 1.8631 1.7362 1.4739 0.5495 1.7935 2.0361 0.6719 0.5928 0.1368 1.2773 0.0431
0.9256 1.2395 0.3718 1.7935 1.0748 1.6927 1.8531 0.8529 0.3191 0.7183 0.6263 0.0482
EXAMPLE 7 recombination, expression and purification of the recombinant antibody Ab-FCV VP1 of FCV VP1
Amplifying and purifying VH and VL sequences of 5 single-chain antibody FCV VP1-scFv, double-enzyme cutting pTT5-V5 and pTT5-V6 vector containing rabbit antibody skeleton by EcoRI and HindIII restriction endonuclease, and homologous recombination to obtain antibody expression plasmids VH-pTT 5-V5-1-VH-pTT 5-V5-5 and VL-pTT 5-V6-1-VL-pTT 5-V6-5. The recombinant plasmids VH-pTT 5-V5-1-VH-pTT 5-V5-5 and VL-pTT 5-V6-1-VL-pTT 5-V6-5 are in one-to-one correspondence and are transfected into HEK293f cells through PEI for eukaryotic expression. After 48h, the culture supernatant was collected by centrifugation at 10000rpm for 20min at 4℃and the antibody was purified by Protein A affinity chromatography, and finally the fusion proteins were stored in PBS solution, designated Ab-FCV VP1-1, ab-FCV VP1-2, ab-FCV VP1-3, ab-FCV VP1-4, ab-FCV VP1-5, respectively.
The purified sample was added to a reduced protein loading buffer, denatured, and subjected to SDS-PAGE electrophoresis, and the result was shown in FIG. 3A, in which disulfide bonds between the heavy chain and the light chain of the antibody were reduced, and target bands were detected at about 50kDa and 25kDa, consistent with the expected results.
Example 8 detection of binding of recombinant antibody Ab-FCV VP1 to FCV VP1 antigen by indirect ELISA method
The affinity of Ab-FCV VP1 to antigen FCV VP1 was determined by indirect ELISA. Antigen was added to the elisa plate for incubation overnight at 4 ℃ and blocked with 2% nonfat dry milk blocking solution at 37 ℃ for 2h. Antibody Ab-FCV VP1 was diluted with 2% nonfat milk powder and added to the wells in 12 gradients and incubated for 1h at 37 ℃. HRP-labeled goat anti-rabbit IgG secondary antibody was added, color development reaction was performed with TMB, and absorbance at a wavelength of 450nm was detected in a microplate reader (tables 2 to 6).
TABLE 2 EC of Ab-FCV VP1-1 binding to antigen FCV VP1 50
TABLE 3 EC of Ab-FCV VP1-2 binding to antigen FCV VP1 50
TABLE 4 Ab-FCV VP1-3 EC binding to antigen FCV VP1 50
TABLE 5 Ab-FCV VP1-4 EC binding to antigen FCV VP1 50
TABLE 6 Ab-FCV VP1-5 EC binding to antigen FCV VP1 50
The detection of the binding effect of the antibody Ab-FCV VP1 is shown in figures 3B-3F, the dose-response curve is successfully fitted with a regression model, all 5 antibodies of Ab-FCV VP1 can effectively bind to FCV VP1 protein, the affinity of the antibody Ab-FCV VP1-4 is highest, and the antibody Ab-FCV VP1-4 binds to recombinant expressed FCV VP1 protein 50 1.243×10 -1 Mu g/mL, the recombinant antibody has activity and good affinity, and Ab-FCV VP1-4 is used for subsequent experiments.
Example 9 Indirect immunofluorescence detection of recombinant antibodies
Resuscitating CRFK adherent cells with EMEM containing 10% FBS, inoculating FCV venom with MOI of 0.1 when cell fusion degree reaches 80-90%, and inoculating 5% CO at 37deg.C 2 The incubator was co-infected with the virus negative group as a control (i.e., no virus added). An equal volume of 2% FBS EMEM was added and cultured in a cell incubator, after 24h the cells were washed 3 times with PBS. Cells were fixed by adding 500. Mu.L of 4% paraformaldehyde, and after 30min, the cells were washed 3 times with PBS. mu.L of 0.1% Triton X100 was added, and after 15min, the cells were washed 3 times with PBS. Blocking with 3% sodium caseinate in PBS at 37℃for 1h and washing with PBS 3 times. The recombinant antibody Ab-FCV VP1 is used as a primary antibody of indirect immunofluorescence, the concentration is 0.5 mug/mL, the secondary antibody is goat anti-rabbit FITC, the secondary antibody is incubated for 1h at 37 ℃, the plates are washed 3 times, 500ul DAPI is added for dyeing 20min, and the PBS is used for washing the plates 3 times for observation by using an inverted fluorescence microscope. As shown in FIG. 4, the recombinant antibodies Ab-FCV VP1-4 exhibited a distinct specific green fluorescent signal with FCV virus positive samples, whereas no fluorescent signal was seen in virus negative control. Proved that the recombinant antibody Ab-FCV VP1-4 can be specifically combined with FCV virus, and can be applied to specific detection of cat calicivirus FCV.
Example 10 antibody specificity validation
Respectively 1X 10 4 TCID 50 The concentration of the per ml FPV, FCV, FHV cell venom supernatant coating was coated on the ELISA plate at 4℃overnight. The cells were washed 5 times with PBST solution containing 0.05% Tween-20, PBST was discarded, and 2% nonfat dry milk blocking solution was added and blocked at 37℃for 1 hour. The wells were dried by washing 5 times with PBST solution, and recombinant antibodies Ab-FCV VP1-4 and 2% nonfat dry milk blocking solution were prepared according to 3: mixing in proportion, standing at 25+/-5 ℃ for 10min to remove interference, adding into an ELISA plate, and incubating at 37 ℃ for 1h. Wash 5 times with PBST solution, dry the remaining liquid in the wells, add HRP-labeled goat anti-rabbit IgG secondary antibody, incubate for 1h at 37 ℃. Washing with PBST solution for 5 times, drying the residual liquid in the wells, adding TMB color development liquid, standing in dark for 15min, and adding 2mol/L H 2 SO 4 The color development was stopped and the absorbance at 450nm was read (Table 7). As shown in FIG. 6, the recombinant antibody Ab-FCV VP1-4 shows good affinity with FCV virus, has binding level difference with FPV and FHV virus, shows FCV virus specificity, proves that the recombinant antibody Ab-FCV VP1-4 can be specifically bound with FCV virus, can be applied to specific detection of feline calicivirus FCV, is not confused with specific detection of feline parvovirus and feline herpesvirus, and is an excellent feline calicivirus detection antibody.
TABLE 7 verification of Ab-FCV VP1-4 and FCV, FPV, FHV specific binding
1 2 3
FCV 2.0273 2.1523 2.0741
FPV 0.0613 0.0801 0.0609
FHV 0.0536 0.0512 0.0688
Example 11 method for establishing double-antibody sandwich ELISA based on anti-feline calicivirus rabbit single-chain recombinant antibody
Horseradish peroxidase (HRP) labeling treatment method: the procedure was as described for HRP-labeled kit (6012-1).
The specific steps of the double-antibody sandwich method ELISA method are as follows: (1) Anti-feline calicivirus rabbit single-chain recombinant antibodies Ab-FCV VP1-1, ab-FCV VP1-2, ab-FCV VP1-3, ab-FCV VP1-4, ab-FCV VP1-5 are diluted to 5 μg/mL by PBS buffer (pH=7.2-7.4) for coating, incubated overnight at 4 ℃, and the plates are washed by washing liquid; (2) blocking with 2% skim milk powder blocking solution at 37deg.C for 2 hr; (3) Adding 50ng/mL standard sample (FCV VP1 protein), incubating for 1h at 37 ℃ with 100 mu L of each well, and washing the plate with washing solution; (3) Adding 2000-fold diluted Ab-FCV VP1-1-HRP, ab-FCV VP1-2-HRP, ab-FCV VP1-3-HRP, ab-FCV VP1-4-HRP and Ab-FCV VP1-5-HRP detection antibodies respectively, incubating for 1h at 37 ℃ with 100 mu L of each well, and washing the plate with washing solution; (4) Adding TMB color development liquid, developing at normal temperature in dark for 10min, adding 2mol/L H 2 SO 4 The color development was stopped, and the absorbance at 450nm was read to determine the optimal pairing antibody.
TABLE 8 results of antibody pairing experiments
As can be seen from the data in Table 8, when the capturing antibody was Ab-FCV VP1-4 and the detecting antibody was Ab-FCV VP1-1 after pairing 5 antibodies, the OD was measured for the same concentration sample 450 The method has the highest value, so that the rabbit single-chain recombinant antibody Ab-FCV VP1-4 is selected as a capture antibody, and the rabbit single-chain recombinant antibody Ab-FCV VP1-1 marked by horseradish peroxidase is used as a detection antibody, and the two antibodies are combined to be used for enzyme-linked immunosorbent assay by a double antibody sandwich method, so that the method has the advantages of high specificity, high sensitivity, good stability and the like, and has important significance in clinical diagnosis and scientific research application.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. A reagent for detecting feline calicivirus, comprising a capture antibody coated on a solid medium and a detection antibody labeled with a biomarker or a chemical label;
in the capture antibody:
the amino acid sequence of CDR1 of the heavy chain variable region is shown as at least one of SEQ ID NO. 29, SEQ ID NO. 35, SEQ ID NO. 41, SEQ ID NO. 47 and/or SEQ ID NO. 53;
the amino acid sequence of CDR2 of the heavy chain variable region is shown as at least one of SEQ ID NO. 30, SEQ ID NO. 36, SEQ ID NO. 42, SEQ ID NO. 48 and/or SEQ ID NO. 54;
the amino acid sequence of CDR3 of the heavy chain variable region is shown as at least one of SEQ ID NO. 31, SEQ ID NO. 37, SEQ ID NO. 43, SEQ ID NO. 49 and/or SEQ ID NO. 55;
the amino acid sequence of CDR1 of the light chain variable region is shown as at least one of SEQ ID NO. 32, SEQ ID NO. 38, SEQ ID NO. 44, SEQ ID NO. 50 and/or SEQ ID NO. 56;
the amino acid sequence of CDR2 of the light chain variable region is shown as at least one of SEQ ID NO. 33, SEQ ID NO. 39, SEQ ID NO. 45, SEQ ID NO. 51 and/or SEQ ID NO. 57;
the amino acid sequence of CDR3 of the light chain variable region is shown as at least one of SEQ ID NO 34, SEQ ID NO 40, SEQ ID NO 46, SEQ ID NO 52 and/or SEQ ID NO 58;
in the detection antibody:
the amino acid sequence of CDR1 of the heavy chain variable region is shown as at least one of SEQ ID NO. 29, SEQ ID NO. 35, SEQ ID NO. 41, SEQ ID NO. 47 and/or SEQ ID NO. 53;
the amino acid sequence of CDR2 of the heavy chain variable region is shown as at least one of SEQ ID NO. 30, SEQ ID NO. 36, SEQ ID NO. 42, SEQ ID NO. 48 and/or SEQ ID NO. 54;
the amino acid sequence of CDR3 of the heavy chain variable region is shown as at least one of SEQ ID NO. 31, SEQ ID NO. 37, SEQ ID NO. 43, SEQ ID NO. 49 and/or SEQ ID NO. 55;
the amino acid sequence of CDR1 of the light chain variable region is shown as at least one of SEQ ID NO. 32, SEQ ID NO. 38, SEQ ID NO. 44, SEQ ID NO. 50 and/or SEQ ID NO. 56;
the amino acid sequence of CDR2 of the light chain variable region is shown as at least one of SEQ ID NO. 33, SEQ ID NO. 39, SEQ ID NO. 45, SEQ ID NO. 51 and/or SEQ ID NO. 57;
the amino acid sequence of CDR3 of the light chain variable region is shown as at least one of SEQ ID NO 34, SEQ ID NO 40, SEQ ID NO 46, SEQ ID NO 52 and/or SEQ ID NO 58.
2. The detection reagent according to claim 1, wherein,
in the detection antibody:
the amino acid sequences of the three CDR regions of the light chain are respectively shown as SEQ ID NO. 32-34; the amino acid sequences of the three CDR regions of the heavy chain are respectively shown in any one of SEQ ID NO. 29-31;
in the capture antibody:
the amino acid sequences of the three CDR regions of the light chain are respectively shown as SEQ ID NO 50-52; the amino acid sequences of the three CDR regions of the heavy chain are shown in any one of SEQ ID NOs 47 to 49 respectively.
3. The detection reagent according to claim 2, wherein,
the heavy chain variable region of the detection antibody is shown as SEQ ID NO. 1; the light chain variable region is shown as SEQ ID NO. 2;
the heavy chain variable region of the capture antibody is shown as SEQ ID NO. 7; the light chain variable region is shown in SEQ ID NO. 8.
4. The detection reagent according to claim 3, wherein,
in the detection antibody, a heavy chain and a light chain are connected by a linker, and a linker sequence is shown as SEQ ID NO. 11;
in the capture antibody, a heavy chain and a light chain are connected through a linker, and a linker sequence is shown as SEQ ID NO. 11.
5. The detection reagent according to any one of claims 1 to 4, wherein the solid-phase medium is an ELISA plate; the detection antibody is labeled with HRP.
6. The detection reagent according to any one of claims 1 to 4, wherein the mass ratio of the capture antibody to the detection antibody is 10:1.
7. The detection reagent according to any one of claims 1 to 4, further comprising a coating buffer, a washing solution, a blocking solution, a TMB developing solution and a stop solution; wherein:
the coating buffer solution is PBS buffer solution with pH of 7.2-7.4,
the washing liquid is
The sealing liquid is 2% of defatted milk powder solution;
the termination liquid is 2mol/L H 2 SO 4 A solution.
8. A method for detecting feline calicivirus of non-diagnostic interest comprising detecting a sample with the detection reagent of any one of claims 1-7.
9. The method according to claim 8, wherein the sample is captured with a capture antibody, and then incubated with a detection antibody; the capturing condition is 37 ℃ for 1h; the incubation conditions were 37℃for 1h.
10. The method of claim 9, further comprising the steps of TMB development, termination of development and reading of absorbance at 450nm after said incubation.
CN202310806217.0A 2023-06-30 Kit for detecting feline calicivirus Active CN116754766B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310806217.0A CN116754766B (en) 2023-06-30 Kit for detecting feline calicivirus

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310806217.0A CN116754766B (en) 2023-06-30 Kit for detecting feline calicivirus

Publications (2)

Publication Number Publication Date
CN116754766A true CN116754766A (en) 2023-09-15
CN116754766B CN116754766B (en) 2024-10-25

Family

ID=

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117886900A (en) * 2024-03-15 2024-04-16 北京纳百生物科技有限公司 Cat calicivirus VP1 protein truncate and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040058316A1 (en) * 2000-03-09 2004-03-25 Jensen Wayne A. Use of recombinant antigens to determine the immune status of an animal
CN111848788A (en) * 2020-07-10 2020-10-30 青岛博隆基因工程有限公司 Feline calicivirus antibody sequences, tetrapeptide chain molecules, immunoglobulin molecules
CN116041497A (en) * 2022-12-22 2023-05-02 杭州贤至生物科技有限公司 Cat calicivirus (FCV) recombinant protein monoclonal antibody and preparation method thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040058316A1 (en) * 2000-03-09 2004-03-25 Jensen Wayne A. Use of recombinant antigens to determine the immune status of an animal
CN111848788A (en) * 2020-07-10 2020-10-30 青岛博隆基因工程有限公司 Feline calicivirus antibody sequences, tetrapeptide chain molecules, immunoglobulin molecules
CN116041497A (en) * 2022-12-22 2023-05-02 杭州贤至生物科技有限公司 Cat calicivirus (FCV) recombinant protein monoclonal antibody and preparation method thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MICHÈLE BERGMANN 等: "Antibody Response to Feline Calicivirus Vaccination in Healthy Adult Cats", 《VIRUSES》, vol. 11, no. 8, 31 August 2019 (2019-08-31), pages 702 *
MIKHAIL POPKOV 等: "Rabbit immune repertoires as sources for therapeutic monoclonal antibodies: the impact of kappa allotype-correlated variation in cysteine content on antibody libraries selected by phage display", 《J MOL BIOL》, vol. 325, no. 2, 10 January 2003 (2003-01-10), pages 325 - 335 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117886900A (en) * 2024-03-15 2024-04-16 北京纳百生物科技有限公司 Cat calicivirus VP1 protein truncate and application thereof
CN117886900B (en) * 2024-03-15 2024-05-31 北京纳百生物科技有限公司 Cat calicivirus VP1 protein truncate and application thereof

Similar Documents

Publication Publication Date Title
CN116836267A (en) Antibody for detecting feline panleukopenia virus and application thereof
CN114316035A (en) General type foot-and-mouth disease virus structural protein antibody, preparation method and application thereof
CN113698475A (en) Monoclonal antibody of anti-porcine delta coronavirus N protein and porcine delta coronavirus colloidal gold rapid detection test strip
CN116754766B (en) Kit for detecting feline calicivirus
CN116754766A (en) Kit for detecting feline calicivirus
CN112851803B (en) Monoclonal antibody ZJU10-01 for resisting H10 subtype avian influenza virus hemagglutinin protein and application thereof
CN114163521B (en) Monoclonal antibody for identifying hog cholera virus 2.1 subtype virulent strain and antibody thereof
CN116063466A (en) anti-SARS-CoV-2 virus N protein binding antibody and application thereof
CN117607431B (en) ELISA detection reagent for feline herpesvirus
CN116819078B (en) Cat parvovirus detection kit
CN113687073A (en) African swine fever virus p54 blocking ELISA antibody detection kit and application thereof
CN117607431A (en) ELISA detection reagent for feline herpesvirus
CN116655784A (en) VP1 protein-based antibody for detecting feline calicivirus and application thereof
CN116444653B (en) Preparation and application of blocking African swine fever virus monoclonal antibody hybridoma cell strain
CN117229393B (en) Antibodies that specifically bind adeno-associated virus 5 and uses thereof
CN117129675B (en) Reagent or kit for human bocavirus type specific detection or diagnosis
CN117683122B (en) Antibody against monkey poxvirus, and preparation method and application thereof
CN117363582B (en) Hybridoma cell strain secreting anti-peste des petits ruminants virus F protein monoclonal antibody, monoclonal antibody thereof and application
CN117126270B (en) Type 2 human bocavirus type specific antibody and application thereof
CN116535498B (en) Anti-canine parvovirus VP2 protein antibody, and preparation method and application thereof
CN112724249B (en) Monoclonal antibody ZJU9-01 for resisting H9 subtype avian influenza virus hemagglutinin protein and application thereof
CN114891095B (en) Nanobody pair for detecting PRRSV antigen, kit and application thereof
CN117466996A (en) Novel coronavirus detection kit and application
CN116813755A (en) Antibody for detecting 4-type avian adenovirus based on Fiber1 protein
CN116693665A (en) Antibodies for detecting feline herpesvirus based on gB protein

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant