CN117363582B - Hybridoma cell strain secreting anti-peste des petits ruminants virus F protein monoclonal antibody, monoclonal antibody thereof and application - Google Patents
Hybridoma cell strain secreting anti-peste des petits ruminants virus F protein monoclonal antibody, monoclonal antibody thereof and application Download PDFInfo
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Classifications
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- 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
- C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
-
- 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
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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]
-
- 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
- G01N2333/115—Paramyxoviridae, e.g. parainfluenza virus
-
- 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
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- Health & Medical Sciences (AREA)
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- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Virology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
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- General Physics & Mathematics (AREA)
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- Tropical Medicine & Parasitology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The application discloses a hybridoma cell strain secreting anti-peste des petits ruminants virus F protein monoclonal antibody, and monoclonal antibody and application thereof. The hybridoma cell strain is 2D10-F-PPRV, the preservation number is CGMCC No.45615, the monoclonal antibody can be used for detecting PPRV, has strong specificity and high antibody titer, and can perform specific fluorescent reaction with Vero cells infected with PPRV Nigeria 75/1 strain of peste des petits ruminants virus, and does not react with normal Vero cells, sheep herpesvirus I, bovine viral diarrhea virus II and other common sheep viruses.
Description
Technical Field
The application relates to the field of biological detection, in particular to a hybridoma cell strain secreting anti-peste des petits ruminants virus F protein monoclonal antibody, and monoclonal antibody and application thereof in peste des petits ruminants virus infection detection.
Background
Peste des petits ruminants (Peste des petits ruminants, PPR) is a highly contagious viral infectious disease caused by peste des petits ruminants virus (Peste des petits ruminants virus, PPRV), mainly infects goats, sheep and other pestes, wherein goats are highly susceptible, the morbidity is up to 100%, the mortality rate is 100% in severe outbreaks, and serious economic losses are caused for the breeding industry. PPRV belongs to the genus measles virus of the family paramyxoviridae and can be divided into 4 lineages according to the N and F genes, with only one serotype currently.
At present, it has been proved that the existing strains in China are mainly IV lines, and the PPRV vaccine strains belong to II lines, but the vaccine strains have good immune protection effect, and the disease is expected to be removed from China by making reasonable epidemic prevention measures.
Disclosure of Invention
In the art, a rapid, effective and high-throughput PPRV detection method is established, which can provide technical support for the elimination of peste des petits ruminants. The prior PPRV pathogen detection multipurpose conventional RT-PCR and qRT-PCR detection methods need operations such as virus RNA extraction, have complicated operation, long time consumption and high cost, and cannot detect with high flux. Monoclonal antibodies play an important role in the diagnosis and control of animal viruses. The double antibody sandwich ELISA detection method or the indirect immunofluorescence method for detecting the pathogen established by utilizing the monoclonal antibody plays an important role in clinical monitoring and laboratory detection of the pathogen. At present, no monoclonal antibody and high-flux detection method for PPRV pathogen F protein exist in China, so that an effective detection means which is simple, convenient, quick, high in sensitivity and good in specificity is needed to be provided for detecting a large number of samples.
Aiming at the defects of the prior art, the technical problem to be solved by the application is to provide a hybridoma cell strain secreting anti-peste des petits ruminants virus monoclonal antibody and a preparation method thereof.
The technical problem to be solved by the application is to provide the monoclonal antibody secreted by the hybridoma cell strain and a preparation method thereof.
The technical problem to be solved finally in the application is to provide the application of the monoclonal antibody. In particular, the present application relates to the following:
the application provides a hybridoma cell, and the preservation number of the hybridoma cell strain is CGMCC No.45615.
In a specific embodiment of the application, the antigen bound by the hybridoma is PPRV Nigeria 75/1 strain F protein, preferably the amino acid sequence of the epitope bound by the hybridoma comprises a sequence shown as SEQ ID NO.1, more preferably the amino acid sequence of the epitope bound by the hybridoma is shown as SEQ ID NO.1,
the antigen epitope sequence SEQ ID NO.1 is:
ALHQSLMNSQAIESLKTSLEKSNQAIEEIRLANKETILAVQGVQDYINNELVPSVHRMSCELVGHKLSLKLLRYYTEILSIFGPSLRDPIAAEISIQALSYALGGDINKILDKLGYSGGDFLAILESKGIKARVTYVDTRDYFIILSIAYPTLSEIKGVIVHKIEAISYNIGAQEWYTTIPRYVATQGYLISNFDETSCVFTPEGTVCSQNALYPMSPLLQECFRGSTKSCARTLVSGTTSNRFILSKGNLIANCASVLCKCYTTETVINQDPDKLLTVIASDKCPVVEVDGVTIQVGSREYPDSVYLHEIDLGPAISLEKLDVGTNLGNAVTRLENAKELLDASDQILKTVKGVP.
The application provides a monoclonal antibody which is produced by a hybridoma cell strain with a preservation number of CGMCC No. 45615.
In a specific embodiment of the present application, the monoclonal antibody is capable of binding to PPRV Nigeria 75/1 strain F protein, preferably to an epitope comprising the sequence shown as SEQ ID NO.1, preferably to an epitope shown as SEQ ID NO. 1.
In one embodiment of the present application, the monoclonal antibody comprises an antibody heavy chain complementarity determining region comprising: CDR-H1, CDR-H2, CDR-H3, wherein: CDR-H1 has the sequence SEQ ID NO 4: the amino acid sequence of SYGLS, CDR-H2 has the sequence SEQ ID NO 5: TITWNGGSTYYPDTVKG, CDR-H3 has the amino acid sequence of SEQ ID NO 6: ESLLRLPAWFAY.
In one embodiment of the present application, the monoclonal antibody comprises an antibody light chain complementarity determining region comprising: CDR-L1, CDR-L2, CDR-L3, wherein: CDR-L1 has the sequence SEQ ID NO 7: RASKSVSTSGYSYMH, CDR-L2 has the amino acid sequence of SEQ ID NO 8: the amino acid sequence of LVSNLES, CDR-L3 has the sequence SEQ ID NO 9: the amino acid sequence of QHIRELYT.
In one embodiment of the present application, a monoclonal antibody that binds PPRV Nigeria 75/1 strain F protein comprises:
a) An antibody heavy chain complementarity determining region comprising: CDR-H1, CDR-H2, CDR-H3, wherein:
CDR-H1 has the sequence SEQ ID NO 4: the amino acid sequence of SYGLS,
CDR-H2 has the sequence SEQ ID NO 5: the amino acid sequence of TITWNGGSTYYPDTVKG,
CDR-H3 has the sequence SEQ ID NO 6: the amino acid sequence of ESLLRLPAWFAY,
b) An antibody light chain complementarity determining region comprising: CDR-L1, CDR-L2, CDR-L3, wherein:
CDR-L1 has the sequence SEQ ID NO 7: the amino acid sequence of RASKSVSTSGYSYMH,
CDR-L2 has the sequence SEQ ID NO 8: the amino acid sequence of LVSNLES,
CDR-L3 has the sequence SEQ ID NO 9: the amino acid sequence of QHIRELYT.
In a specific embodiment of the present application, the monoclonal antibody comprises an antibody heavy chain variable region HCVR as set forth in SEQ ID NO 10 or an antibody heavy chain variable region HCVR having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR as set forth in SEQ ID NO 11 or an antibody heavy chain variable region LCVR having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO 11, wherein:
HCVR has the sequence SEQ ID NO 10: EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYGLSWVRQTPDKRLELVATITWNGGSTYYPDTVKGRFTISRDSAKNILYLQMSSLKSEDTAMYYCARESLLRLPAWFAYWGQGTLVTVSA amino acid sequence;
LCVR has the sequence SEQ ID NO 11:
DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELYTFGGGTKLEIK.
In a specific embodiment of the present application, the monoclonal antibody comprises a heavy chain as shown in SEQ ID NO 12 or a heavy chain having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO 12 and a light chain as shown in SEQ ID NO 13 or a light chain having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO 13, wherein:
the heavy chain amino acid sequence is SEQ ID NO 12:
AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKL amino acid sequence;
The light chain amino acid sequence is SEQ ID NO 13: RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.
A kit for detecting and/or diagnosing a peste des petits ruminants virus infection in an animal, comprising: the monoclonal antibody or the antibody produced by hybridoma cell with the preservation number of CGMCC No. 45615.
The beneficial effects are that: compared with the prior art, the application has the following advantages:
(1) The monoclonal antibody obtained by the application can be used for detecting PPRV, has strong specificity and high antibody titer, and can perform specific fluorescent reaction with Vero cells infected with PPRV Nigeria 75/1 strain of peste des petits ruminants virus, and does not react with normal Vero cells and sheep herpesvirus I, bovine viral diarrhea virus II and other common sheep viruses.
(2) The application provides a technical means for establishing a rapid, simple and accurate detection method, and in aspects of immune mechanism research, immune function research, detection method establishment and the like, and has important significance for PPRV prevention and control and laboratory research.
Drawings
The drawings are included to provide a better understanding of the present application and are not to be construed as unduly limiting the present application. Wherein:
FIG. 1 shows the identification of pET28a-PPRV-F recombinant protein.
FIG. 2 shows the results of an indirect immunofluorescence assay containing peste des petits ruminants virus.
FIG. 3 shows the reaction of a monoclonal antibody 2D10-F-PPRV strain and PPRV detected by Western blot.
Detailed Description
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.
It should be noted that certain terms are used throughout the description and claims to refer to particular components. Those of skill in the art will understand that a person may refer to the same component by different names. The specification and claims do not identify differences in terms of components, but rather differences in terms of the functionality of the components. As referred to throughout the specification and claims, the terms "include" or "comprising" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description hereinafter sets forth the preferred embodiment for carrying out the present application, but is not intended to limit the scope of the present application in general, as the description proceeds. The scope of the present application is defined by the appended claims.
Technical and scientific terms used in the present specification have the same meaning as commonly understood by one of ordinary skill in the art, and if so conflict, the present specification will control.
Generally, terms used in the present specification have the following meanings.
In the present specification, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homologous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during production of monoclonal antibody preparations), which are typically present in minor amounts. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being derived from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used according to the invention can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals comprising all or part of a human immunoglobulin locus, such methods and other exemplary methods of preparing monoclonal antibodies are described herein.
In the present specification, the term "hybridoma" means a cell obtained by fusing a myeloma cell and a B lymphocyte in the preparation of a monoclonal antibody, and is generally prepared by culturing the hybridoma.
In this specification, the terms "heavy chain" ("CH"), "light chain" ("CL"), "heavy chain constant Region", "light chain variable Region" ("VL"), "heavy chain variable Region" ("VH"), "Framework Region" ("Framework Region, FR"), "complementarity determining Region" ("CDR") are parts of an antibody, and different kinds of antibodies can be formed by different combinations. For example, conventional IgG antibodies are tetramers consisting of two light chains and two heavy chains. Wherein, VL and VH are composed of different CDR and FR, the CDR contains residues contacting antigen, the antigen specificity of VL and VH is determined, the FR is used for maintaining the variable region structure and determining the position of CDR ring. Typically, the heavy chain variable region and the heavy chain constant region comprise a complete heavy chain, and the light chain variable region and the light chain constant region comprise a complete light chain.
In the present specification, the term "label" refers to a molecule that indicates the position or amount of an antibody by color, chemical reaction, excitation light, mass spectrum, or the like after binding of the antibody. Including, for example: alkaline phosphatase, peroxidase, luciferase, luciferin, fluorescent protein, isotopes, and the like.
In this specification, the terms "polynucleotide" or "nucleic acid", "nucleic acid molecule" are used interchangeably and include, but are not limited to DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (microrna), snoRNA (short nucleolar RNA), miRNA (microrna), genomic DNA, synthetic RNA, and/or tRNA.
In the present specification, "enzyme-linked immunosorbent assay (EILSA)" refers to a detection method in which a free hetero protein is bound to a target protein bound to a solid carrier by utilizing the characteristic that an antibody molecule can specifically bind to an antigen molecule, and qualitative or quantitative analysis is performed using a specific label. The principle is as follows: the antigen or antibody can be physically adsorbed on the solid surface and maintain the immunological activity thereof; the antigen or antibody is capable of forming an enzyme conjugate with the enzyme via a covalent bond while maintaining the respective immunological or enzymatic activity; after binding the enzyme conjugate to the corresponding antigen or antibody, the occurrence of an immune response can be determined by a color reaction of the added substrate, the color reaction being in direct proportion to the amount of the corresponding antigen or antibody in the sample. According to the substance to be detected and the conditions of detection, various detection methods of different types can be designed, and the double antibody sandwich method is the most common method for detecting antigens. The antiserum containing known antibody is adsorbed in a small hole on a micro-titer plate and washed once; adding an antigen to be detected, if the antigen and the antigen are specific, combining, and then washing out redundant antibodies; adding an enzyme-linked antibody which specifically reacts with an antigen to be detected to form a sandwich; the addition of the substrate for the enzyme indicates the presence of the corresponding antigen if colored enzymatic products are observed.
In the present specification, the term "plasmid" means a DNA molecule other than a chromosome (or pseudonucleus) in organisms such as bacteria, yeasts and actinomycetes, which exists in cytoplasm or nucleus, has autonomous replication ability, maintains a constant copy number in a daughter cell, and expresses the carried genetic information.
In this specification, the term "vector" refers to a vector by which a polynucleotide sequence (e.g., a foreign gene) can be introduced into a host cell to transform the host and facilitate expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, phage vectors, viral vectors, and the like. Wherein "viral vector" is a vector engineered from a viral genome that introduces a foreign gene into a host cell by viral infection.
The application provides a hybridoma cell strain 2D10-F-PPRV, and the preservation number of the hybridoma cell strain is CGMCC No.45615.
The application also provides a monoclonal antibody which is produced by a hybridoma cell strain with the preservation number of CGMCC No.45615. The hybridoma cell strain is named as 2D10-F-PPRV, is classified and named as hybridoma cell strain, has a preservation number of CGMCC No.45615, and has a preservation date of: 2023, 05, 17, deposit unit: china general microbiological culture Collection center, address Beijing, kogyo, north Chen West Lu No. 1, 3, postal code 100101.
A monoclonal antibody provided herein, wherein the antibody comprises: a) An antibody heavy chain complementarity determining region comprising: CDR-H1, CDR-H2, CDR-H3, wherein: CDR-H1 has the amino acid sequence of SEQ ID NO 4 SYGLS, CDR-H2 has the amino acid sequence of SEQ ID NO 5 TITWNGGSTYYPDTVKG, CDR-H3 has the amino acid sequence of SEQ ID NO 6 ESLLRLPAWFAY; b) An antibody light chain complementarity determining region comprising: CDR-L1, CDR-L2, CDR-L3, wherein: CDR-L1 has the amino acid sequence of SEQ ID NO 7: RASKSVSTSGYSYMH, CDR-L2 has the amino acid sequence of SEQ ID NO 8:LVSNLES, and CDR-L3 has the amino acid sequence of SEQ ID NO 9:QHIRELYT.
The present application also provides a monoclonal antibody that binds PPRV Nigeria 75/1 strain F protein comprising: a) An antibody heavy chain complementarity determining region comprising: CDR-H1, CDR-H2, CDR-H3, wherein: CDR-H1 has the amino acid sequence of SEQ ID NO 4 SYGLS, CDR-H2 has the amino acid sequence of SEQ ID NO 5 TITWNGGSTYYPDTVKG, CDR-H3 has the amino acid sequence of SEQ ID NO 6 ESLLRLPAWFAY; b) An antibody light chain complementarity determining region comprising: CDR-L1, CDR-L2, CDR-L3, wherein: CDR-L1 has the amino acid sequence of SEQ ID NO 7: RASKSVSTSGYSYMH, CDR-L2 has the amino acid sequence of SEQ ID NO 8:LVSNLES, and CDR-L3 has the amino acid sequence of SEQ ID NO 9:QHIRELYT.
In one embodiment of the present application, the monoclonal antibody that binds to PPRV Nigeria 75/1 strain F protein includes but is not limited to that produced by hybridoma cell line with the preservation number of CGMCC No.45615 or obtained by adopting a chemical synthesis method.
In a specific embodiment of the present application, the above-described antibody comprises an antibody heavy chain variable region HCVR as set forth in SEQ ID NO 10 or an antibody heavy chain variable region HCVR having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR as set forth in SEQ ID NO 11 or an antibody heavy chain variable region LCVR having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO 11, wherein:
HCVR has the sequence SEQ ID NO 10:
EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYGLSWVRQTPDKRLELVATITWNGGSTYYPDTVKGRFTISRDSAKNILYLQMSSLKSEDTAMYYCARESLLRLPAWFAYWGQGTLVTVSA amino acid sequence;
LCVR has the sequence SEQ ID NO 11:
DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELYTFGGGTKLEIK.
In a specific embodiment of the present application, the above-described antibody comprises a heavy chain as shown in SEQ ID NO 12 or a heavy chain having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO 12 and a light chain as shown in SEQ ID NO 13 or a light chain having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO 13, wherein:
The heavy chain amino acid sequence is SEQ ID NO 12:
AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKL amino acid sequence;
the light chain amino acid sequence is SEQ ID NO 13:
RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.
In one embodiment of the present application, the method of producing monoclonal antibodies from hybridoma cell line having a accession number of CGMCC No.45615 is conventional in the art.
In one embodiment of the present application, the method for producing monoclonal antibodies from the hybridoma cell line with the preservation number of CGMCC No.45615 is a mouse abdominal water method.
The application also provides a preparation method of the monoclonal antibody, in a specific embodiment of the application, sterilized liquid paraffin is injected into a Balb/c mouse with the age of 6-8 weeks in an intraperitoneal mode; in one embodiment of the present application, the hybridoma cell line 2D10-F-PPRV is injected into the abdominal cavity of the mouse 7 days later; in one embodiment of the present application, ascites in mice with significantly distended abdomen is collected 7 days later; in one embodiment of the present application, ascites from the mice is centrifuged at 4000rpm for 10min, and the supernatant of ascites is collected, packaged, labeled, and stored for later use.
The application also provides application of the monoclonal antibody in peste des petits ruminants virus infection detection.
In one specific embodiment of the present application, the hybridoma cell strain secreting the peste des petits ruminants virus (PPRV) monoclonal antibody is obtained by fusing a mouse myeloma cell with a mouse spleen cell; in one embodiment of the present application, cell fusion takes the form of a PEG cell fusion method. In one embodiment of the present application, the ratio of mouse myeloma cells (SP 2/0) to immunized Balb/c mouse spleen cells is 1:8; in one embodiment of the present application, after centrifugation, the supernatant is discarded and then resuspended in an appropriate amount of serum-free DMEM medium; in one embodiment of the present application, the cells are washed by spinning at 1000rpm for 6min at 25℃and then the supernatant is discarded and the tube bottom is tapped to loosen and homogenize the cells; in one specific embodiment of the present application, uniformly dispersed cells are preheated in a 37 ℃ water bath for 1min, and then 0.6mL of preheated PEG1460 is added thereto; in one embodiment of the present application, the mixture is added and then left to stand in a water bath for 90 seconds, from slow to fast, with the preheated 10 mL DMEM broth; in one embodiment of the present application Standing in an incubator at 37 ℃ for 5 min after uniformly mixing; in one specific embodiment of the application, the washing is repeated for 2 times after standing and centrifugation for 10min at 500 r/min; in one embodiment of the present application, DMEM medium containing 15% FBS and HAT is added to resuspend cells at 100. Mu.L per well into 96 well cell culture plates containing feeder cells, and placed at 37℃in 5% CO 2 Is cultured in a constant temperature incubator;
in one embodiment of the present application, the antigen of the hybridoma cell line that secretes the PPRV monoclonal antibody is a recombinantly expressed PPRV Nigeria 75/1 strain F protein.
In one embodiment of the present application, it is preferable that the amino acid sequence of the epitope bound by the hybridoma cell is shown in SEQ ID NO. 1.
In one embodiment of the present application, the recombinant expressed PPRV Nigeria 75/1 strain F protein is prepared by expressing the PPRV Nigeria 75/1 strain F protein in an E.coli expression system and purifying.
The application also provides an antigen detection kit for resisting peste des petits ruminants virus, which comprises a monoclonal antibody generated by the hybridoma cell strain with the preservation number of CGMCC No.45615, and can be used for detecting PPRV. In one embodiment of the present application, the kit is an in vitro test kit. In a specific embodiment of the present application, the kit is an immunoassay kit, such as an ELISA kit or an immunohistochemical kit, comprising the neutralizing antibody or antigen binding fragment thereof of the PPRV described previously. In a specific embodiment of the present application, the kit is an in vivo noninvasive diagnostic kit comprising the aforementioned complex. In one embodiment of the present application, the in vivo noninvasive diagnostic kit may be used in an imaging procedure selected from the group consisting of: radioimmunoimaging or targeted ultrasound imaging. When used in radioimmunoimaging, the complex is a complex of a neutralizing antibody or antigen binding fragment thereof of the aforementioned PPRV and a radionuclide. When used for targeted ultrasound imaging, the complex is a complex of a neutralizing antibody or antigen binding fragment thereof of the PPRV described previously with an ultrasound contrast agent.
BL21 (DE 3) competent cells used in the examples of this application are competent cells obtained by treating E.coli BL21 (DE 3) with a special process, and can be used for chemical transformation of plasmids
Western immunoblotting, as used in the examples herein, is a method in which proteins are transferred to a membrane and then detected using antibodies. For known expressed proteins, the corresponding antibodies can be used as primary antibodies, and the expression products of the novel genes can be detected by antibodies to the fusion moiety. Western Blot uses polyacrylamide gel electrophoresis, the detected object is protein, the probe is antibody, and the color development is marked secondary antibody. The protein sample separated by PAGE is transferred to a solid phase carrier, the solid phase carrier adsorbs the protein in a non-covalent bond form, and the type of the polypeptide separated by the PAGE and the biological activity of the polypeptide are kept unchanged. The protein or polypeptide on the solid carrier is used as antigen, reacts with the corresponding antibody, then reacts with the enzyme or the second antibody marked by the isotope, and the protein component expressed by the specific target gene separated by electrophoresis is detected through substrate color development or autoradiography.
As used herein, "recombinant expressed PPRV Nigeria 75/1 strain F protein" and "recombinant PPRV-F protein" refer to proteins after expression, which are the same protein.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
EXAMPLE 1 preparation of recombinant F-His protein
1. PCR amplification of PPRV-F Gene
The F protein was analyzed for hydrophilicity, antigenicity, and functional region based on the PPRV Nigeria 75/1 strain gene sequence (KY 628761.1) and the PPRV Clone9 strain gene sequence stored in the laboratory. As a result, the 132AA-487AA protein sequence has relatively concentrated epitope, and can be used as an alternative fragment of the expressed protein. According to the protein structure analysis, nucleotide fragments (1071 bp) of the protein region are selected for codon optimization and synthesis. And BamHI and XhoI sites are introduced into the 3 'and 5' ends, and F gene fragments are amplified by using a homologous recombination method. The primer sequences were as follows:
F:GtggacagcaaatgggtcgCGGATCCGCACTCCATCAGTCATTGATGAAT（SEQ ID No.2）；
CAGTGGTGGTGGTGGTGGTGCTCGAGCAAGGAGTTTTCTAGGTCTTTATGCG (SEQ ID No. 3); the above primers were synthesized by Huada bioengineering Co., ltd. The plasmid after the synonymous optimization of F protein amino acid is used as a template, and the target gene with the carrier sequence homology arm and enzyme cutting site is amplified by using the upstream primer and the downstream primer. According to the KOD PCR enzyme instructions (TOYOBO Co., ltd.) a 50. Mu.L reaction system was used, and the reaction procedure was as follows: 94 ℃ for 2min; reacting at 98 ℃ for 10s,55 ℃ for 30s and 68 ℃ for 50s for 30 cycles; and at 68℃for 7min. The PCR amplification products were detected by 1% agarose gel electrophoresis. The recovery of the destination strip was performed according to the gel recovery kit instructions. The pET-28a (+) prokaryotic expression vector was double digested with BamHI and XhoI (NEB), homologous recombination was performed according to Seamless Cloning Mix homologous recombination instructions (Beyotidme Co.) and reacted at 50℃for 15min. The homologous recombination products were transformed into TOP10 (full gold Biotechnology Co., ltd.) competent cells, plated on LB plates containing kanamycin, and incubated overnight at 37 ℃. Single colonies were picked, cultured with shaking in LB medium containing kanamycin, plasmids were extracted, and identified by PCR and BamHI and XhoI double restriction enzyme. The positive plasmid was sent to the large bioengineering Co.Ltd for sequencing.
The epitope sequence is as follows:
ALHQSLMNSQAIESLKTSLEKSNQAIEEIRLANKETILAVQGVQDYINNELVPSVHRMSCELVGHKLSLKLLRYYTEILSIFGPSLRDPIAAEISIQALSYALGGDINKILDKLGYSGGDFLAILESKGIKARVTYVDTRDYFIILSIAYPTLSEIKGVIVHKIEAISYNIGAQEWYTTIPRYVATQGYLISNFDETSCVFTPEGTVCSQNALYPMSPLLQECFRGSTKSCARTLVSGTTSNRFILSKGNLIANCASVLCKCYTTETVINQDPDKLLTVIASDKCPVVEVDGVTIQVGSREYPDSVYLHEIDLGPAISLEKLDVGTNLGNAVTRLENAKELLDASDQILKTVKGVP（SEQ ID No.1）。
2. construction of recombinant expression Strain BL21 (DE 3) -F and protein expression
E.coli BL21 (DE 3) competent cells were transformed with the correct recombinant plasmid pET28a-F by sequencing, and the monoclonal was selected and inoculated in LB medium containing kanamycin, 37Culturing overnight at the temperature. Inoculating the cultured fungus solution to new culture medium, and culturing at 37deg.C to OD 600nm About 0.6-0.8, adding IPTG with a final concentration of 1mmol/L, inducing expression at 20deg.C for 12h, centrifuging, ultrasonically lysing bacteria, and performing SDS-PAGE identification on the precipitate and supernatant. The protein purification method was performed by referring to the Ni-NTA reagent protocol of GE company. The expression supernatant was purified by affinity chromatography on a nickel column and subjected to SDS-PAGE, and the expression band of the target protein was apparent at about 39kDa (FIG. 1A).
3. Western blot identification of recombinant proteins
After SDS-PAGE was completed, the recombinant proteins were transferred to PVDF membrane (Millipore) by wet transfer, and blocked with PBST containing 2% skim milk for 2h; adding His-Tag Mouse mAb monoclonal antibody (protein) diluted by 1:10000, and incubating for 2h at room temperature; PBST wash 3 times; goat anti-mouse IgG-HRP (Beijing full gold Biotechnology Co., ltd.) diluted 1:10000 was added, and the mixture was gently shaken at room temperature for 1h; PBST was washed 3 times and developed with TMB development kit (SeraCare). The results showed that a specific band was detectable at 39kDa, demonstrating successful expression and purification of the recombinant protein (FIG. 1B). Protein concentration was measured by BCA method to be 0.81mg/mL and stored at-80℃for further use.
Example 2 screening of monoclonal antibody hybridoma cell lines against peste des petits ruminants virus F protein
1. Balb/c mice were immunized: purified recombinant F-His protein was subcutaneously injected into 6-8 week old female BALB/c mice (Beijing vetch Liwa) 3 times in total, 3 weeks apart, and the first immunization was performed with 50. Mu.g of purified recombinant F-His protein and an equal volume of complete Freund's adjuvant (purchased from Sigma), and the second immunization was performed with 100. Mu.g of purified recombinant F-His protein and an equal volume of incomplete Freund's adjuvant (purchased from Sigma); blood is collected 10-14 days after the third immunization, immune serum titer of the mice is detected, the mice with ELISA antibody titer of >106 are selected, the immunization is enhanced once 3 days before cell fusion, and 100 mug of purified recombinant F-His protein is adopted for intraperitoneal injection.
2. Cell fusion: cell fusion the PEG cell fusion method was used. Mouse myeloma cells (SP 2/0) were taken and the ratio of immunized Balb/c mouse spleen cells was 1:8 (fineCell number ratio), centrifuging at 25deg.C for 6min at 1000rpm, removing supernatant, adding appropriate amount of serum-free DMEM culture medium, centrifuging at 25deg.C for 6min at 1000rpm to clean cells, removing supernatant, tapping the bottom of tube, loosening and homogenizing cells, preheating in 37deg.C water bath for 1min, adding 0.6mL of PEG1460 preheated in 37deg.C water bath in advance, adding fusion agent, standing in water bath for 90s, adding preheated 10 mL DMEM culture solution from slow to fast, standing in 37 deg.C incubator for 5 min after mixing, centrifuging for 10min at 500r/min, and washing repeatedly for 2 times. The DMEM medium containing 15% FBS and HAT was added to resuspend cells at 100. Mu.L per well into 96 well cell culture plates containing feeder cells, and the plates were placed at 37℃with 5% CO 2 Is cultured in a constant temperature incubator. During the period, the condition of cells in the wells is observed, and when the cell colonies grow to 1/2 of the cell wells, cell culture supernatants are taken for antibody detection.
3. Selection of hybridoma cells: the ELISA plate was coated with 0.05mol/L of carbonate buffer at pH 9.6 as coating solution, 2. Mu.g/L of purified F protein diluted in a double manner as coating antigen, 100. Mu.L/well, and overnight at 4 ℃. Washing with PBST for 3 times, and drying; adding the supernatant of the fusion cells, balb/c immunized mice positive serum diluted by 1:1000 (v/v) and the negative serum diluted by 1:1000 (v/v) into corresponding holes, 100 mu L/hole, allowing the mixture to act for 1h at 37 ℃, washing by PBST for 3 times, and drying by beating; horseradish peroxidase (HRP) -labeled goat anti-mouse IgG (purchased from Beijing full gold Biotechnology Co., ltd.) diluted 1:5000 (v/v) was added, 100. Mu.L/well incubated for 1h at 37℃and PBST washed 3 times, and the mixture was patted dry. Adding a substrate TMB (SeraCare), 100 mu L/hole, and developing for 10min at room temperature in a dark place; the reaction was terminated by adding 50. Mu.L of 2mol/L sulfuric acid per well. ELISA plate OD (optical density) measurement by ELISA plate 450nm Value, P is OD of each detection well 450nm Value, N is OD4 of negative serum 50nm Value, as negative serum OD 450nm The value is less than or equal to 0.1 and the OD of positive serum 450nm Value and negative serum OD 450nm On the premise that the ratio of the P/N is more than or equal to 2.1, namely that the negative and positive controls are both established, the negative and positive of the detection hole are judged by the judgment standard that the P/N is more than or equal to 2.1 is judged as the positive hole. Detecting once again after 2d interval, and selecting the hybridoma cell strain with positive detection results for subcloning.
4. Hybridoma cellsCloning: the screened positive well cell strain is dyed with trypan blue, counted, diluted into 100 cells/10 mL culture medium by DMEM culture medium containing 15% FBS, the diluted cell suspension is added into 96-well plates with feeder cells laid in advance, 100 mu L of each well is placed at 37 ℃ and 5% CO is added 2 And culturing in an incubator, observing the cell strain during culturing, and timely performing ELISA detection according to the established indirect ELISA method when the cell strain grows to 1/2 of the bottom area of the 96-well plate. Recording positive holes of monoclonal cells, and performing subcloning for more than 3 times until all cloned cell strain supernatants after cloning are positive and OD detected by each hole 450nm The values are closer. The cloned PPRV F specific monoclonal antibody hybridoma cell strain is subjected to expansion culture, frozen and named as hybridoma cell strain 2D10-F-PPRV.
EXAMPLE 3 preparation and purification of monoclonal antibodies
The ascites preparation method is adopted: sterilized liquid paraffin was intraperitoneally injected into Balb/c mice (purchased from Beijing velocin Lihua Co.) at 6 to 8 weeks old, 0.5 mL/mouse, and after 7 days, hybridoma cell lines 2D10-F-PPRV were injected into the intraperitoneally of the mice, each 0.5mL (10 6 Individual hybridoma cells). Collecting ascites of mice with obviously bulged abdomen after 7 days, centrifuging at 4000rpm for 10min, collecting supernatant of ascites, packaging, labeling and storing at-80deg.C for use.
Treating the crude extract with saturated ammonium sulfate to obtain IgG: adding the ascites into a 100mL beaker with a rotor, adding PBS according to the same volume as the ascites, placing the beaker on ice, and placing the beaker on a magnetic stirrer; slowly adding 2 times of saturated ammonium sulfate solution with the volume of the ascites sample dropwise, stirring while adding, and stirring for more than 4 hours at the temperature of 4 ℃; centrifuging 10000g for 10min, discarding supernatant, re-suspending the precipitate with PBS equal to ascites volume, loading the re-suspended liquid into dialysis bag, and sealing two ends of the dialysis bag; the IgG samples were dialyzed against PBS at 4 ℃ for more than about 12 hours, during which time at least 2 changes of PBS were made to remove residual ammonium sulfate from the samples.
Further purification of crude IgG: 10000G of the dialyzed crude IgG sample was centrifuged for 10min, and the supernatant was collected and purified using Protein G Resin. The Protein G Resin column was washed with PBS until the OD280nm of the effluent was less than 0.01. Adding the sample into the column, controlling the flow rate to be 5 s/drop, preparing an effluent of the empty tube for recovering the sample in advance, repeatedly adding the effluent after primary combination into the ProteinG chromatographic column, repeatedly adding for 3 times, and keeping the effluent at 4 ℃ for the last time; the heteroproteins were eluted using PBS at a flow rate of 5 s/drop until the effluent OD280nm was less than 0.01. And taking a certain amount of glycine (such as 10 ml), and adding the volume of Tris-HCI to ensure that the PH of the mixture after neutralization is 7-8. Eluting the target protein by using glycine solution, collecting the eluted target protein by using a sterilized centrifuge tube, immediately neutralizing the collected sample by using a pre-determined neutralization volume ratio and adding Tris-HCI to ensure that the PH is 7-8, and stopping collecting the sample when the OD280nm of effluent is less than 0.1; the collected sample was dialyzed with PBS at 4℃for more than about 12 hours, during which time the PBS was replaced at least 2 times, and finally the purified antibody of interest was collected and stored at-20 ℃.
Example 4 analysis of antibody Properties
1. Monoclonal antibody potency detection
The hybridoma cell culture supernatant obtained in example 3 and the ascites titer were measured by an indirect ELISA method, and the results are shown in Table 1, wherein the ascites titer induced in mice is 1:32000, and the hybridoma cell culture supernatant is 1:320.
Table 1 monoclonal antibody titer detection
。
2. Indirect immunofluorescence assay (IFA)
After culturing PPRV Nigeria75/1 strain infected Vero cells and normal cells for 4 days respectively, discarding the supernatant, washing 3 times by PBS, fixing the cells with 4% paraformaldehyde (100 mu L/hole) for 6-8min, and washing 3 times by PBS; 100 u L/hole 2% BSA closed cells, room temperature closed for 2 hours, removing the closed liquid, PBS washing 3 times; adding 100 mu L of hybridoma cell supernatant into each hole, and reacting at 37 ℃ for 1h by taking SP2/0 cell supernatant and pre-immune mouse serum as negative and positive controls; washed 3 times with PBS and reacted with FITC-labeled goat anti-mouse IgG (1:200, sigma Co.) at 37℃for 1h; washed 3 times with PBS, and then dried by beating, and observed under a fluorescence microscope.
Indirect immunofluorescence experiments show that the SP2/0 cell supernatant and the pre-immune mouse serum react with the Vero cells infected by the PPRV Nigeria75/1 strain and normal cells, and the recombinant PPRV-F protein is used for generating specific fluorescence after the reaction between the recombinant PPRV-F protein and the Vero cells infected by the PPRV Nigeria75/1 strain, so that the negative control and the positive control are established. The monoclonal antibody 2D10-F-PPRV reacted with Vero cells infected with Nigeria75/1 strain of peste des petits ruminants virus, and a distinct specific fluorescent reaction was observed, but not with normal Vero cells, as shown in FIG. 2, wherein A: vero cells infected with peste des petits ruminants virus; b: normal Vero cells.
3. Specificity experiments
The indirect immunofluorescence experiment is used for respectively carrying out specificity experiments on sheep herpesvirus type I (CHpV-1), bovine viral diarrhea virus type I (BVDV-1) and bovine viral diarrhea virus type II (BVDV-2) stored in the laboratory, and the result shows that the monoclonal antibody 2D10-F-PPRV reacts positively with PPRV but does not react with other viruses, thus indicating that the method has good specificity. The detailed results are shown in Table 2.
TABLE 2 specificity test results
。
4. Immunoblotting detection
After culturing PPRV Nigeria75/1 strain infected Vero cells and normal cells for 4 days respectively, the supernatant was discarded, washed 3 times with PBS, and then the cells were lysed with 100. Mu.L/Kong Liejie solution (Biyun biotechnology Co., ltd.), and then a protein electrophoresis loading buffer was added and boiled for 5min to prepare a protein sample for electrophoresis. After SDS-PAGE was completed, proteins were transferred to PVDF membrane (Millipore) by wet transfer method, and blocked with PBST containing 2% skim milk for 2 hours; respectively adding 2D10-F-PPRV monoclonal antibodies diluted by 1:2000, and incubating for 2 hours at room temperature; PBST wash 3 times; goat anti-mouse IgG-HRP (Beijing full gold Biotechnology Co., ltd.) diluted 1:10000 was added, and the mixture was gently shaken at room temperature for 1h; PBST was washed 3 times and developed with TMB development kit (SeraCare).
The assay showed that after incubation of 2D10-F-PPRV mab, the PPRV-infected Vero cell samples appeared to be a distinct band at 48-63kDa (predicted size 58 kDa), but the normal Vero cell samples were not (FIG. 3). The monoclonal antibody can be used for laboratory Western blot detection.
5. Neutralization activity assay
The working concentration of immobilized virus (PPRV Nigeria75/1 was 100 TCID) 50 0.1 ml) and the method of diluting ascites were subjected to a neutralization test to identify the neutralizing activity of monoclonal antibody. 2D10-F-PPRV ascites was diluted 1:20, 1:40, 1:80, 1:160 and the neutralization titers were calculated by the Reed-Muench method.
The result shows that the ascites titer of the 2D10-F-PPRV strain monoclonal antibody is 1:50.1, which indicates that the 2D10-F-PPRV monoclonal antibody has the activity of neutralizing PPRV. The detailed results are shown in Table 3.
Table 3 neutralization activity test results
。
In conclusion, the results of indirect immunofluorescence detection and neutralization experiments show that the monoclonal antibody can react with the Vero cells infected with PPRV Nigeria75/1 strain specifically, but does not react with normal Vero cells, so that the monoclonal antibody obtained by the application can react with PPRV specifically and has neutralization activity, and the neutralization titer is 1:50.1. It was further found that the monoclonal antibodies obtained in the present application do not react with the sheep herpesvirus, bovine viral diarrhea virus type i, bovine viral diarrhea virus type ii, indicating that the monoclonal antibodies in the present application have good specificity.
The present application provides a hybridoma cell strain secreting anti-peste des petits ruminants virus monoclonal antibody, and ideas and methods for its monoclonal antibody and application, and the method and means for implementing the technical scheme are numerous, and the above description is only a preferred embodiment of the present application, and it should be pointed out that, for those skilled in the art, several improvements and modifications can be made without departing from the principles of the present application, and these improvements and modifications should also be regarded as the protection scope of the present application. The components not explicitly described in this embodiment can be implemented by using the prior art.
Claims (33)
1. A hybridoma cell, wherein the preservation number of the hybridoma cell strain is CGMCC No.45615.
2. The hybridoma cell according to claim 1, wherein the antigen to which the hybridoma cell binds is PPRV Nigeria 75/1 strain F protein.
3. The hybridoma cell according to claim 2, wherein the amino acid sequence of the epitope bound by the hybridoma cell comprises a sequence as shown in SEQ ID No. 1.
4. Use of a hybridoma cell line according to claims 1-3 for the preparation of a reagent for the detection and/or diagnosis of animals infected with peste des petits ruminants virus.
5. A monoclonal antibody, wherein the monoclonal antibody is produced by a hybridoma cell strain with a preservation number of CGMCC No. 45615.
6. The monoclonal antibody of claim 5, wherein the monoclonal antibody is capable of binding PPRV Nigeria 75/1 strain F protein.
7. The monoclonal antibody of claim 6, wherein the monoclonal antibody is capable of binding an epitope comprising the sequence shown as SEQ ID No. 1.
8. The monoclonal antibody of claims 5-7, wherein the antibody comprises:
a) An antibody heavy chain complementarity determining region comprising: CDR-H1, CDR-H2, CDR-H3, wherein:
CDR-H1 has the sequence SEQ ID NO 4: the amino acid sequence of SYGLS,
CDR-H2 has the sequence SEQ ID NO 5: the amino acid sequence of TITWNGGSTYYPDTVKG,
CDR-H3 has the sequence SEQ ID NO 6: the amino acid sequence of ESLLRLPAWFAY,
b) An antibody light chain complementarity determining region comprising: CDR-L1, CDR-L2, CDR-L3, wherein:
CDR-L1 has the sequence SEQ ID NO 7: the amino acid sequence of RASKSVSTSGYSYMH,
CDR-L2 has the sequence SEQ ID NO 8: the amino acid sequence of LVSNLES,
CDR-L3 has the sequence SEQ ID NO 9: the amino acid sequence of QHIRELYT.
9. A monoclonal antibody that binds PPRV Nigeria 75/1 strain F protein, wherein the antibody comprises:
a) An antibody heavy chain complementarity determining region comprising: CDR-H1, CDR-H2, CDR-H3, wherein:
the amino acid sequence of CDR-H1 is shown as SEQ ID NO 4: the expression "SYGLS" is used,
the amino acid sequence of CDR-H2 is shown as SEQ ID NO 5: as shown in figure TITWNGGSTYYPDTVKG,
the amino acid sequence of CDR-H3 is shown as SEQ ID NO 6: as shown in figure ESLLRLPAWFAY,
b) An antibody light chain complementarity determining region comprising: CDR-L1, CDR-L2, CDR-L3, wherein:
the amino acid sequence of CDR-L1 is shown as SEQ ID NO 7: as shown in figure RASKSVSTSGYSYMH,
the amino acid sequence of CDR-L2 is shown as SEQ ID NO 8: as shown in the LVSNLES,
the amino acid sequence of CDR-L3 is shown as SEQ ID NO 9: QHIRELYT.
10. The monoclonal antibody of claim 8 or 9, wherein the antibody comprises an antibody heavy chain variable region HCVR as set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR as set forth in SEQ ID NO 11, wherein:
HCVR has the sequence SEQ ID NO 10: EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYGLSWVRQTPDKRLELVATITWNGGSTYYPDTVKGRFTISRDSAKNILYLQMSSLKSEDTAMYYCARESLLRLPAWFAYWGQGTLVTVSA amino acid sequence;
LCVR has the sequence SEQ ID NO 11:
DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELYTFGGGTKLEIK.
11. The monoclonal antibody of claim 10, wherein the antibody further comprises an antibody heavy chain variable region HCVR having 90% identity to the amino acid sequence set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR having 90% identity to the amino acid sequence set forth in SEQ ID NO 11.
12. The monoclonal antibody of claim 10, wherein the antibody further comprises an antibody heavy chain variable region HCVR having 91% identity to the amino acid sequence set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR having 91% identity to the amino acid sequence set forth in SEQ ID NO 11.
13. The monoclonal antibody of claim 10, wherein the antibody further comprises an antibody heavy chain variable region HCVR having 92% identity to the amino acid sequence set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR having 92% identity to the amino acid sequence set forth in SEQ ID NO 11.
14. The monoclonal antibody of claim 10, wherein the antibody further comprises an antibody heavy chain variable region HCVR having 93% identity to the amino acid sequence set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR having 93% identity to the amino acid sequence set forth in SEQ ID NO 11.
15. The monoclonal antibody of claim 10, wherein the antibody further comprises an antibody heavy chain variable region HCVR having 94% identity to the amino acid sequence set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR having 94% identity to the amino acid sequence set forth in SEQ ID NO 11.
16. The monoclonal antibody of claim 10, wherein the antibody further comprises an antibody heavy chain variable region HCVR having 95% identity to the amino acid sequence set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR having 95% identity to the amino acid sequence set forth in SEQ ID NO 11.
17. The monoclonal antibody of claim 10, wherein the antibody further comprises an antibody heavy chain variable region HCVR having 96% identity to the amino acid sequence set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR having 96% identity to the amino acid sequence set forth in SEQ ID NO 11.
18. The monoclonal antibody of claim 10, wherein the antibody further comprises an antibody heavy chain variable region HCVR having 97% identity to the amino acid sequence set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR having 97% identity to the amino acid sequence set forth in SEQ ID NO 11.
19. The monoclonal antibody of claim 10, wherein the antibody further comprises an antibody heavy chain variable region HCVR having 98% identity to the amino acid sequence set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR having 98% identity to the amino acid sequence set forth in SEQ ID NO 11.
20. The monoclonal antibody of claim 10, wherein the antibody further comprises an antibody heavy chain variable region HCVR having 99% identity to the amino acid sequence set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR having 99% identity to the amino acid sequence set forth in SEQ ID NO 11.
21. The monoclonal antibody of claim 8 or 9, wherein the antibody comprises a heavy chain as set forth in SEQ ID NO 12 and a light chain as set forth in SEQ ID NO 13, wherein:
the heavy chain amino acid sequence is SEQ ID NO 12:
AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKL amino acid sequence;
The light chain amino acid sequence is SEQ ID NO 13: RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.
22. The monoclonal antibody of claim 21, wherein the antibody further comprises a heavy chain having 90% identity to the amino acid sequence set forth in SEQ ID NO 12 and a light chain having 90% identity to the amino acid sequence set forth in SEQ ID NO 13.
23. The monoclonal antibody of claim 21, wherein the antibody further comprises a heavy chain having 91% identity to the amino acid sequence set forth in SEQ ID NO 12 and a light chain having 91% identity to the amino acid sequence set forth in SEQ ID NO 13.
24. The monoclonal antibody of claim 21, wherein the antibody further comprises a heavy chain having 92% identity to the amino acid sequence set forth in SEQ ID NO 12 and a light chain having 92% identity to the amino acid sequence set forth in SEQ ID NO 13.
25. The monoclonal antibody of claim 21, wherein the antibody further comprises a heavy chain having 93% identity to the amino acid sequence set forth in SEQ ID NO 12 and a light chain having 93% identity to the amino acid sequence set forth in SEQ ID NO 13.
26. The monoclonal antibody of claim 21, wherein the antibody further comprises a heavy chain having 94% identity to the amino acid sequence set forth in SEQ ID NO 12 and a light chain having 94% identity to the amino acid sequence set forth in SEQ ID NO 13.
27. The monoclonal antibody of claim 21, wherein the antibody further comprises a heavy chain having 95% identity to the amino acid sequence set forth in SEQ ID NO 12 and a light chain having 95% identity to the amino acid sequence set forth in SEQ ID NO 13.
28. The monoclonal antibody of claim 21, wherein the antibody further comprises a heavy chain having 96% identity to the amino acid sequence set forth in SEQ ID NO 12 and a light chain having 96% identity to the amino acid sequence set forth in SEQ ID NO 13.
29. The monoclonal antibody of claim 21, wherein the antibody further comprises a heavy chain having 97% identity to the amino acid sequence set forth in SEQ ID NO 12 and a light chain having 97% identity to the amino acid sequence set forth in SEQ ID NO 13.
30. The monoclonal antibody of claim 21, wherein the antibody further comprises a heavy chain having 98% identity to the amino acid sequence set forth in SEQ ID NO 12 and a light chain having 98% identity to the amino acid sequence set forth in SEQ ID NO 13.
31. The monoclonal antibody of claim 21, wherein the antibody further comprises a heavy chain having 99% identity to the amino acid sequence set forth in SEQ ID NO 12 and a light chain having 99% identity to the amino acid sequence set forth in SEQ ID NO 13.
32. Use of a monoclonal antibody according to claim 5 or 9 for the preparation of a reagent for detecting and/or diagnosing animals infected with peste des petits ruminants virus.
33. A kit for detecting and/or diagnosing a peste des petits ruminants virus infection in an animal, comprising: the monoclonal antibody of any one of claims 5 to 31 or an antibody produced by the hybridoma of claims 1 to 3.
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