CN107216372A - A kind of PPR virus HN Protein Epitopes peptide H362 and its determination, preparation method and application - Google Patents

A kind of PPR virus HN Protein Epitopes peptide H362 and its determination, preparation method and application Download PDF

Info

Publication number
CN107216372A
CN107216372A CN201710500780.XA CN201710500780A CN107216372A CN 107216372 A CN107216372 A CN 107216372A CN 201710500780 A CN201710500780 A CN 201710500780A CN 107216372 A CN107216372 A CN 107216372A
Authority
CN
China
Prior art keywords
cell
hole
positive
added
protein
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
CN201710500780.XA
Other languages
Chinese (zh)
Other versions
CN107216372B (en
Inventor
窦永喜
梁忠祥
朱学亮
蒙学莲
张志东
才学鹏
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.)
Lanzhou Veterinary Research Institute of CAAS
Original Assignee
Lanzhou Veterinary Research Institute of CAAS
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 Lanzhou Veterinary Research Institute of CAAS filed Critical Lanzhou Veterinary Research Institute of CAAS
Priority to CN201710500780.XA priority Critical patent/CN107216372B/en
Publication of CN107216372A publication Critical patent/CN107216372A/en
Application granted granted Critical
Publication of CN107216372B publication Critical patent/CN107216372B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18311—Metapneumovirus, e.g. avian pneumovirus
    • C12N2760/18322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18311—Metapneumovirus, e.g. avian pneumovirus
    • C12N2760/18334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The present invention relates to a kind of PPR virus HN Protein Epitopes peptide H362 and its determination, preparation method and application, the amino acid sequence of epitope peptide is：H362:362EANWVVPSTDVRDL375；The present invention have detected the reactionogenicity and the specificity of antibody of monoclonal antibody and PPR virus, detection monoclonal antibody has good reactionogenicity with rPPRV HN F proteins, the B cell epitope of HN albumen is predicted with reference to Immunoinformatics, candidate's epi-position and monoclonal antibody 10E3 are have detected using Aminated ELISA Plate, establish the corresponding epitope H362 of 10E3, the determination of epitope peptide of the present invention, is that PPR virus epiposition vaccine antigen and diagnostic reagent antigen prepare and provides theoretical foundation.

Description

A kind of PPR virus HN Protein Epitopes peptide H362 and its determination, preparation Methods and applications
Technical field
The invention belongs to bioinformatics and immunological technique field, and in particular to a kind of PPR virus HN eggs White epitope peptide H362 and its determination, preparation method and application.
Background technology
PPR virus (PPRV) belongs to Morbillivirus (Morbillivirus), is to cause goat and sheep etc. The cause of disease of small ruminant acute infectious disease, the sick feature is high incidence and high mortality, by OIE (OIE) statutory report zoonosis is classified as, China is classified as I class animal epidemics.Initial report PPR virus Only infection goat and sheep, but the case that viral inter-species was propagated in recent years is constantly reported that the current disease is distributed mainly on Africa And Asia, it is positive to threaten Europe.
At present, the main preventive means of the disease is attenuated vaccine immunity, but the vaccine there is heat endurance and virulence is anti- Strong the problems such as, and it is unfavorable for the global elimination plan of PPR.HN is the glycoprotein being embedded on PPRV cyst membranes, is constituted The fibre of virion surface is dashed forward.HN albumen can be invaded with the SLAM acceptor interactions mediate retroviral on lymphocyte, therefore Decision host's preferendum of HN albumen.In addition, factor receptor SLAM is on the immunocytes such as major part and lymphocyte, it may be possible to PPR virus causes the main cause that immunity of organism suppresses.The cell that PPR virus can cause body strong is exempted from Epidemic disease and humoral immune response, and HN albumen is then main protective antigens.HN albumen is that vaccine design and detection method are set up Important target.Therefore, the epitope collection of illustrative plates of HN albumen is drawn and Antibody preparation is significant.
The content of the invention
It is an object of the invention to provide a kind of PPR virus HN Protein Epitopes peptides.
Another object of the present invention is to provide PPR virus HN Protein Epitopes peptide H362 to ruminate small Application in epizootic disease virus epitopes vaccine antigen and the preparation of diagnostic reagent antigen.
Another object of the present invention is to provide determination, the preparation side of PPR virus HN Protein Epitopes Method.
The technical solution adopted by the present invention is：A kind of PPR virus HN Protein Epitopes peptides, the antigen table The amino acid sequence of position peptide is H362:362EANWVVPSTDVRDL375。
A kind of determination of PPR virus HN Protein Epitopes, preparation method, methods described comprise the following steps：
Calculating simulation：
Step one, virtual HN albumen head 3D structures are built with molecular simulation software：Utilize Discovery Studio V4.5 is to the two target proteins --- and street strain PPRV HN and vaccine strain PPRV HN albumen carry out Blast search；Wherein PPRV Hw, GenBank accession number are FJ905304；PPRV Hv, GenBank accession number：X74443；
Step 2, search pattern：PDB databases (www.pdb.org) are searched for, the homologous egg parsed by experiment is found White structure, selection sequence identity is more than 30%, and the longer protein structure of aligned sequences is as formwork structure, carries out Follow-up calculating；
Step 3, adjusts sequence alignment：Choose after template protein, the space coordinate of albumen obtained from PDB databases, Obtained template protein sequence is compared with target protein；
Step 4, modeling：Comparison result is submitted into server, according to the space coordinate of template protein and the similitude of sequence Speculate structure, target protein structure is obtained so as to calculate, i.e., each mould builds the different target protein structure of generation at least five, The model for selecting DOPE Score minimum while asking PDF Total Energy relatively low is subsequently calculated；
Step 5, optimization：Using the CHARMm field of forces, first carry out Steepest Decent and optimize 5000 steps, then carry out Conjugate Gradient optimize 2000 steps；
Step 6, evaluation structure：The reasonability of protein structure is assessed using Laplace conformation figure analysis method, mustn't if being located at Can area non-glycine/proline proportion be no more than 5%, then illustrate simulation protein structure be rational, can carry out Follow-up calculating analysis；
Prediction based on Immunoinformatics：
Step one, PPRV-HN protein B cell antigen epitopes are predicted：By the amino acid sequence IEDB of PPRV HN albumen, Immunomedicine Group and BepiPred Immunoinformatics analysis softwares are analyzed, and comprehensive analysis predicts its B cell Epitope；
Step 2, the synthesis of epitope：GeneScript companies are sent by the epitope amino acid sequence of software prediction Synthesized with Peptide synthesizer, after synthesis with high performance liquid chromatography (high performance liquid chromatography, HPLC) purity of analysis Peptide systhesis and the correctness of amino acid；
The preparation of monoclonal antibody：
Step one, animal is immunized：6-8 week old females Balb/c is immunized with reactionogenicity purpose antigen rPPRV-HN-F Mouse, the subcutaneous 4 points of injections of nape part, immune protein sample size is 50 μ g/, and the μ L/ of volume 200 are immunized only；First immunisation is used Isometric Freund's complete adjuvant emulsification, secondary immunity and three times it is immune with isometric incomplete Freund's adjuvant adjuvant emulsion；Every 14 days it is immune once, three times it is immune 14 days after antibody in mouse docking blood sampling detection serum, treat that antibody titer is high by 1：After 10000 Four can be carried out to exempt from, four exempt from without adjuvant, intraperitoneal injection；
Step 2, cell fusion：Mouse antibodies potency is higher than 1:10 000, the 4th immune mouse prepares small after three days Mouse splenic lymphocytes；After mouse anesthesia, eyeball is gone to take mouse peripheral blood, it is sterile to win mouse spleen simultaneously for separating serum Grinding；SP2/0 cells are mixed about 1 with Mouse spleen cells:5-1:10 ratio is mixed, and 800rpm centrifugations abandon supernatant in 4 minutes, Cell is mixed again, centrifuge tube is put into 40 DEG C of deionized water, 1mL polyethylene glycol cells were uniformly added into 1 minute and are melted Mixture, 1mL1640 cell culture fluids were added in 5 seconds, and 15mL1640 cell culture fluids are added, 90 seconds by slow-to-fast, Terminate fusion reaction；Incubation at room temperature 10 minutes, 800rmp centrifugations abandon supernatant in 4 minutes；With 1640/ containing Peritoneal Cells of Mice The cell of 20%FBS/HAT culture mediums suspension fusion is laid in 96 orifice plates, 37 DEG C, 5%CO2 environment cultures；
Step 3, the screening and cloning of hybridoma positive colony：Fused cell culture is entered after 14 days to positive cell strain Row screening, cell culture fluid is detected by rPPRV-HN-F albumen coated elisa plate with indirect elisa method；Select indirect elisa method The positive hole of detection, then with indirect immunofluorescene assay and the reactionogenicity of PPR virus；Choose Immunofluorescence test sun Property hole is subcloned；Positive monoclonal cell line is obtained with Endpoint Dilution Method；The positive hole of Immunofluorescence test is trained with 1640 Nutrient solution, which suspends, to be moved into 1.5mL centrifuge tubes, and cell counting count board adjusts the concentration of cell to the cell count in positive cell hole Cell is diluted to 1/100 μ L to 0.5-2/μ L, then with 1460/20%FBS/HT cell culture fluids, 96 hole cells are laid on In culture plate, per the μ L1460/20%FBS/HT cell culture fluids of hole 100；Finally, Peritoneal Cells of Mice is contained in addition 1460/20%FBS/HT nutrient solutions 100mL；After 14 days, positive cell strain is screened with same method, such as no positive monoclonal is thin Born of the same parents' strain, continuation is subcloned to positive hole until obtaining positive monoclonal cell line；It is pre- using limiting dilution assay screening secretion Determine the cell of monoclonal antibody specific；The positive of monoclonal antibody needed for being produced using indirect ELISA and immunofluorescence screening Hybridoma；Clonal expansion finally is carried out to required positive hybridoma cell；
Step 4, monoclonal antibody hypotype and titration：
Hypotype is detected according to the specification of monoclonal antibody hypotype detection kit；
Sample-adding：Positive monoclonal cell line cell conditioned medium is added in ELISA Plate sample well, per the μ L of hole 50, without being incubated；
Enzyme-added labeling antibody：1 × IgM+IgG-HRP of sheep anti mouse dilutions are added in ELISA Plate sample well, per the μ L of hole 50, Mix；
It is incubated：Shrouding film is capped, 1 hour is incubated at room temperature；
Washing：Liquid is removed, is washed 3 times, 2 minutes every time, dried with PBST；
Colour developing：The nitrite ion prepared is added into sample well, per the μ L of hole 100；
It is incubated：The ELISA Plate for having added nitrite ion is placed in light protected environment, room temperature 20 minutes；
Terminate：Terminate liquid is added, per the μ L of hole 100；
Read：Read OD450 or observe, color is most deep or OD value highests are corresponding hypotype；
As a result：The hypotype of 10E3 monoclonal antibodies is IgG2b；
Antibody titer measure is carried out to positive monoclonal cell line cell conditioned medium with indirect ELISA method, it is thin with SP2/0 Born of the same parents' supernatant is negative control+2SD as negative control, critical value, and potency is 1:800；
It is prepared by step 5, monoclonal antibody batch
Culture is enlarged to the positive monoclonal cell line of acquisition, cell and culture medium is collected, cell is noted with abdominal cavity The mode penetrated is injected into the culture of adult dams abdominal cavity, and the peritoneal fluid of mouse is collected after about 7 days.
The identification of epitope：
The epitope of prediction passes through biosynthesis, and identification meets requirement of experiment, solid phase carrier is used as using Aminated ELISA Plate Indirect ELISA method carry out；
Dilution：The deionized water synthesized and lyophilized epitope peptide is pressed through with height is diluted to 1 μ g/ μ L；
Epitope peptide is coated with：The epitope peptide diluted is coated on Aminated ELISA Plate, per the μ L coating buffers of hole 50, antigen Content is 5 μ g, and 4 DEG C overnight；
Board-washing：Liquid in enzyme mark hole is got rid of to the greatest extent as far as possible, about 250 μ LPBST are then added per hole, 3 minutes are stood, weight It is multiple three times, finally ELISA Plate is patted dry on gauze；
Closing：The μ L of 1 × confining liquid 150 are added into enzyme mark hole, 37 DEG C of incubations are after 1.5 hours, by enzyme mark hole Confining liquid is got rid of to the greatest extent as far as possible, standby after finally ELISA Plate is patted dry on gauze；
It is incubated primary antibody：Monoclonal antibody is pressed 1 with confining liquid:500 dilutions, add ELISA Plate per the μ L of hole 50, place 37 DEG C It is incubated 1 hour in incubator；
Board-washing：Ibid walk board-washing；
It is incubated secondary antibody：HRP is marked into anti-mouse IgG secondary antibodies by 1 with confining liquid:5 000 dilutions, add ELISA Plate per the μ of hole 50 L, places in 37 DEG C of incubators and is incubated 1 hour；
Board-washing：Ibid walk board-washing；
Colour developing：50 μ L TMB nitrite ions are added per hole, 37 DEG C of lucifuge is incubated 15 minutes；
Terminate：50 μ L terminate liquids, color development stopping are added per hole；
Read：ELISA Plate is detected at wavelength 450nm with end-point method, OD450nm absorbances are read.
Beneficial effects of the present invention：
PPR virus is inoculated with VeroE6 cells, the reaction of detection monoclonal antibody and PPR virus is former The specificity of property and antibody.As a result show, monoclonal antibody 10E3 (Fig. 1 left parts) is ruminated with small in Vero cells Epizootic disease virus reaction, shows stronger green fluorescence, does not have fluorescence (Fig. 1 right parts) with Vero cell effects；SP2/0 is thin Born of the same parents' culture medium does not react (Fig. 1 center sections) with the PPR virus in Vero cells, without fluorescence.Show, monoclonal The specific antibody of antibody PPR virus.The monoclonal antibody 10E3 of acquisition is entered with Western-Blotting methods The specific detection based on Prokaryotic expression, purification rPPRV-HN-F albumen is gone, empty carrier expresses bacterium as negative control, as a result Show that single band occurs in rPPRV-HN-F albumen swimming lane, and empty carrier expression bacterium swimming lane does not have band (Fig. 2), shows Dan Ke Grand antibody has good reactionogenicity with rPPRV-HN-F albumen, and is not reacted with empty carrier expression bacterium.Utilize Discovery Studio v4.5 predict the head construction (figure of the strain HN albumen of PPR virus Nigeria 75/1 3) the B cell epitope of HN albumen, is predicted with reference to Immunoinformatics, candidate's epi-position H362 (Fig. 3 blue portions) is determined, it is artificial synthesized High-purity small peptide.Candidate's epi-position and monoclonal antibody 10E3 (Fig. 4) are have detected using Aminated ELISA Plate, 10E3 correspondences are established Epitope H362.
Brief description of the drawings
Fig. 1 is the anti-of fluoroscopic examination 10E3 monoclonal antibodies and rPPRV-HN-F polyclonal antibodies and PPR virus Answer originality and specificity；
Fig. 2 is 10E3 monoclonal antibodies and the reactionogenicity of rPPRV-HN-F albumen；
Fig. 3 is positions of the candidate's epi-position H362 in PPRV-HN structures；
Fig. 4 is that monoclonal antibody 10E3 indirect ELISAs detect HN Protein Epitopes H362 reactionogenicities.
Embodiment
A kind of PPR virus HN Protein Epitopes peptides of the present invention, the amino acid of the epitope peptide Sequence is H362:362EANWVVPSTDVRDL375。
The determination of epitope of the present invention, preparation method, method comprise the following steps：
Calculating simulation：
Step one, virtual HN albumen head 3D structures are built with molecular simulation software：Utilize Discovery Studio V4.5 is to the two target proteins --- and street strain PPRV HN and vaccine strain PPRV HN albumen carry out Blast search；Wherein PPRV Hw, GenBank accession number are FJ905304；PPRV Hv, GenBank accession number：X74443；
Step 2, search pattern：PDB databases (www.pdb.org) are searched for, the homologous egg parsed by experiment is found White structure, selection sequence identity is more than 30%, and the longer protein structure of aligned sequences is as formwork structure, carries out Follow-up calculating；
Step 3, adjusts sequence alignment：Choose after template protein, the space coordinate of albumen obtained from PDB databases, Obtained template protein sequence is compared with target protein；
Step 4, modeling：Comparison result is submitted into server, according to the space coordinate of template protein and the similitude of sequence Speculate structure, target protein structure is obtained so as to calculate, i.e., each mould builds the different target protein structure of generation at least five, The model for selecting DOPE Score minimum while asking PDF Total Energy relatively low is subsequently calculated；
Step 5, optimization：Using the CHARMm field of forces, first carry out Steepest Decent and optimize 5000 steps, then carry out Conjugate Gradient optimize 2000 steps；
Step 6, evaluation structure：The reasonability of protein structure is assessed using Laplace conformation figure analysis method, mustn't if being located at Can area non-glycine/proline proportion be no more than 5%, then illustrate simulation protein structure be rational, can carry out Follow-up calculating analysis；
Prediction based on Immunoinformatics：
Step one, PPRV-HN protein B cell antigen epitopes are predicted：By the amino acid sequence IEDB of PPRV HN albumen, Immunomedicine Group and BepiPred Immunoinformatics analysis softwares are analyzed, and comprehensive analysis predicts its B cell Epitope；
Step 2, the synthesis of epitope：GeneScript companies are sent by the epitope amino acid sequence of software prediction Synthesized with Peptide synthesizer, after synthesis with high performance liquid chromatography (high performance liquid chromatography, HPLC) purity of analysis Peptide systhesis and the correctness of amino acid；
The preparation of monoclonal antibody：
Step one, animal is immunized：6-8 week old females Balb/c is immunized with reactionogenicity purpose antigen rPPRV-HN-F Mouse, the subcutaneous 4 points of injections of nape part, immune protein sample size is 50 μ g/, and the μ L/ of volume 200 are immunized only；First immunisation is used Isometric Freund's complete adjuvant emulsification, secondary immunity and three times it is immune with isometric incomplete Freund's adjuvant adjuvant emulsion；Every 14 days it is immune once, three times it is immune 14 days after antibody in mouse docking blood sampling detection serum, treat that antibody titer is high by 1：After 10000 Four can be carried out to exempt from, four exempt from without adjuvant, intraperitoneal injection；
Step 2, cell fusion：Mouse antibodies potency is higher than 1:10 000, the 4th immune mouse prepares small after three days Mouse splenic lymphocytes；After mouse anesthesia, eyeball is gone to take mouse peripheral blood, it is sterile to win mouse spleen simultaneously for separating serum Grinding；SP2/0 cells are mixed about 1 with Mouse spleen cells:5-1:10 ratio is mixed, and 800rpm centrifugations abandon supernatant in 4 minutes, Cell is mixed again, centrifuge tube is put into 40 DEG C of deionized water, 1mL polyethylene glycol cells were uniformly added into 1 minute and are melted Mixture, 1mL1640 cell culture fluids were added in 5 seconds, and 15mL1640 cell culture fluids are added, 90 seconds by slow-to-fast, Terminate fusion reaction；Incubation at room temperature 10 minutes, 800rmp centrifugations abandon supernatant in 4 minutes；With 1640/ containing Peritoneal Cells of Mice The cell of 20%FBS/HAT culture mediums suspension fusion is laid in 96 orifice plates, 37 DEG C, 5%CO2 environment cultures；
Step 3, the screening and cloning of hybridoma positive colony：Fused cell culture is entered after 14 days to positive cell strain Row screening, cell culture fluid is detected by rPPRV-HN-F albumen coated elisa plate with indirect elisa method；Select indirect elisa method The positive hole of detection, then with indirect immunofluorescene assay and the reactionogenicity of PPR virus；Choose Immunofluorescence test sun Property hole is subcloned；Positive monoclonal cell line is obtained with Endpoint Dilution Method；The positive hole of Immunofluorescence test is trained with 1640 Nutrient solution, which suspends, to be moved into 1.5mL centrifuge tubes, and cell counting count board adjusts the concentration of cell to the cell count in positive cell hole Cell is diluted to 1/100 μ L to 0.5-2/μ L, then with 1460/20%FBS/HT cell culture fluids, 96 hole cells are laid on In culture plate, per the μ L1460/20%FBS/HT cell culture fluids of hole 100；Finally, Peritoneal Cells of Mice is contained in addition 1460/20%FBS/HT nutrient solutions 100mL；After 14 days, positive cell strain is screened with same method, such as no positive monoclonal is thin Born of the same parents' strain, continuation is subcloned to positive hole until obtaining positive monoclonal cell line；It is pre- using limiting dilution assay screening secretion Determine the cell of monoclonal antibody specific；The positive of monoclonal antibody needed for being produced using indirect ELISA and immunofluorescence screening Hybridoma；Clonal expansion finally is carried out to required positive hybridoma cell；
Step 4, monoclonal antibody hypotype and titration：
Hypotype is detected according to the specification of monoclonal antibody hypotype detection kit；
Sample-adding：Positive monoclonal cell line cell conditioned medium is added in ELISA Plate sample well, per the μ L of hole 50, without being incubated；
Enzyme-added labeling antibody：1 × IgM+IgG-HRP of sheep anti mouse dilutions are added in ELISA Plate sample well, per the μ L of hole 50, Mix；
It is incubated：Shrouding film is capped, 1 hour is incubated at room temperature；
Washing：Liquid is removed, is washed 3 times, 2 minutes every time, dried with PBST；
Colour developing：The nitrite ion prepared is added into sample well, per the μ L of hole 100；
It is incubated：The ELISA Plate for having added nitrite ion is placed in light protected environment, room temperature 20 minutes；
Terminate：Terminate liquid is added, per the μ L of hole 100；
Read：Read OD450 or observe, color is most deep or OD value highests are corresponding hypotype；
As a result：The hypotype of 10E3 monoclonal antibodies is IgG2b；
Antibody titer measure is carried out to positive monoclonal cell line cell conditioned medium with indirect ELISA method, it is thin with SP2/0 Born of the same parents' supernatant is negative control+2SD as negative control, critical value, and potency is 1:800；
It is prepared by step 5, monoclonal antibody batch
Culture is enlarged to the positive monoclonal cell line of acquisition, cell and culture medium is collected, cell is noted with abdominal cavity The mode penetrated is injected into the culture of adult dams abdominal cavity, and the peritoneal fluid of mouse is collected after about 7 days.
The identification of epitope：
The epitope of prediction passes through biosynthesis, and identification meets requirement of experiment, solid phase carrier is used as using Aminated ELISA Plate Indirect ELISA method carry out；
Dilution：The deionized water synthesized and lyophilized epitope peptide is pressed through with height is diluted to 1 μ g/ μ L；
Epitope peptide is coated with：The epitope peptide diluted is coated on Aminated ELISA Plate, per the μ L coating buffers of hole 50, antigen Content is 5 μ g, and 4 DEG C overnight；
Board-washing：Liquid in enzyme mark hole is got rid of to the greatest extent as far as possible, about 250 μ LPBST are then added per hole, 3 minutes are stood, weight It is multiple three times, finally ELISA Plate is patted dry on gauze；
Closing：The μ L of 1 × confining liquid 150 are added into enzyme mark hole, 37 DEG C of incubations are after 1.5 hours, by enzyme mark hole Confining liquid is got rid of to the greatest extent as far as possible, standby after finally ELISA Plate is patted dry on gauze；
It is incubated primary antibody：Monoclonal antibody is pressed 1 with confining liquid:500 dilutions, add ELISA Plate per the μ L of hole 50, place 37 DEG C It is incubated 1 hour in incubator；
Board-washing：Ibid walk board-washing；
It is incubated secondary antibody：HRP is marked into anti-mouse IgG secondary antibodies by 1 with confining liquid:5 000 dilutions, add ELISA Plate per the μ of hole 50 L, places in 37 DEG C of incubators and is incubated 1 hour；
Board-washing：Ibid walk board-washing；
Colour developing：50 μ L TMB nitrite ions are added per hole, 37 DEG C of lucifuge is incubated 15 minutes；
Terminate：50 μ L terminate liquids, color development stopping are added per hole；
Read：ELISA Plate is detected at wavelength 450nm with end-point method, OD450nm absorbances are read.
By Fig. 1 it is obvious that VeroE6 cells be inoculated with PPR virus, detection monoclonal antibody 10E3 with The reactionogenicity of PPR virus and the specificity of antibody, as a result show, in monoclonal antibody 10E3 and Vero cells PPR virus reaction, show stronger green fluorescence (Fig. 1 left parts), there is no fluorescence with Vero cell effects (Fig. 1 right parts)；SP2/0 cell culture mediums do not react with the PPR virus in Vero cells, without fluorescence (Fig. 1 Center section).Show, the specific antibody of monoclonal antibody PPR virus.
By Fig. 2 it is obvious that 10E3 monoclonal antibodies and rPPRV-HN-F albumen have good reactionogenicity and Specificity.
1：There is single band in rPPRV-HN-F albumen swimming lane, and with it is expected consistent, illustrate monoclonal antibody tool There is preferable reactionogenicity；2：There is not band in empty carrier Host Strains lysate swimming lane, illustrates that monoclonal antibody has good Specificity.
By Fig. 3 it is obvious that blue portion is epitope H362 in figure, this it appears that should from 3D structures Epitope is located at the surface more prominent place of antigen, and this is the important original that the corresponding antibody of the epitope has sound response originality Cause.
By Fig. 4 it is obvious that detecting epitope H362 reactionogenicities, monoclonal antibody with indirect ELISA method OD450 values (the p that the OD450 values of group detection are extremely significantly detected with SP2/0 cell culture mediums group<0.00001) table, is shown Position has good reactionogenicity with monoclonal antibody.
The epitope peptide H362 identified by above scheme, is that PPR virus epiposition vaccine antigen and diagnostic reagent are anti- Research and development prepared by original provide theoretical foundation, and the preventing and treating to PPR virus is significant.
The present invention is not limited to above-mentioned preferred forms, and anyone can show that other are various under the enlightenment of the present invention The product of form, however, make any change in its shape or structure, it is every that there is skill identical or similar to the present application Art scheme, is within the scope of the present invention.

Claims (3)

1. a kind of PPR virus HN Protein Epitopes peptides, it is characterised in that：The amino acid sequence of the epitope peptide It is classified as H362:362EANWVVPSTDVRDL375。
2. a kind of PPR virus HN Protein Epitopes peptides H362 is in PPR virus according to claim 1 Application in epiposition vaccine antigen and the preparation of diagnostic reagent antigen.
3. a kind of determination of PPR virus HN Protein Epitopes, preparation method, its feature according to claim 1 It is：Methods described comprises the following steps：
Calculating simulation：
Step one, virtual HN albumen head 3D structures are built with molecular simulation software：Utilize Discovery Studio V4.5 couple The two target proteins --- street strain PPRV HN and vaccine strain PPRV HN albumen carry out Blast search；Wherein PPRV Hw, GenBank accession number is FJ905304；PPRV Hv, GenBank accession number：X74443；
Step 2, search pattern：PDB databases (www.pdb.org) are searched for, are found by testing the homologous protein parsed Structure, selection sequence identity is more than 30%, and the longer protein structure of aligned sequences is as formwork structure, carries out follow-up Calculating；
Step 3, adjusts sequence alignment：Choose after template protein, the space coordinate of albumen is obtained from PDB databases, will To template protein sequence be compared with target protein；
Step 4, modeling：Comparison result is submitted into server, speculated according to the similitude of the space coordinate of template protein and sequence Structure, target protein structure is obtained so as to calculate, i.e., each mould builds the different target protein structure of generation at least five, it is desirable to PDF The model for selecting DOPE Score minimum while Total Energy are relatively low is subsequently calculated；
Step 5, optimization：Using the CHARMm field of forces, first carry out Steepest Decent and optimize 5000 steps, then carry out Conjugate Gradient optimize 2000 steps；
Step 6, evaluation structure：The reasonability of protein structure is assessed using Laplace conformation figure analysis method, area is disapproved if being located at Non- glycine/proline proportion be no more than 5%, then illustrate that the protein structure of simulation is rational, can carry out follow-up Calculating analysis；
Prediction based on Immunoinformatics：
Step one, PPRV-HN protein B cell antigen epitopes are predicted：By the amino acid sequence IEDB of PPRV HN albumen, Immunomedicine Group and BepiPred Immunoinformatics analysis softwares are analyzed, and comprehensive analysis predicts its B cell Epitope；
Step 2, the synthesis of epitope：GeneScript companies are sent to use the epitope amino acid sequence of software prediction many Peptide synthesizer synthesize, after synthesis with high performance liquid chromatography (high performance liquid chromatography, HPLC) purity of analysis Peptide systhesis and the correctness of amino acid；
The preparation of monoclonal antibody：
Step one, animal is immunized：6-8 week old female Balb/c mouse are immunized with reactionogenicity purpose antigen rPPRV-HN-F, The subcutaneous 4 points of injections of nape part, immune protein sample size is 50 μ g/, and the μ L/ of volume 200 are immunized only；First immunisation is with equal volume Freund's complete adjuvant is emulsified, and secondary immunity and three times are immune with isometric incomplete Freund's adjuvant adjuvant emulsion；Exempted from every 14 days Once, the antibody after three times immune 14 days in mouse docking blood sampling detection serum treats that antibody titer is high by 1 to epidemic disease：It can enter after 10000 Row four is exempted from, and four exempt from without adjuvant, intraperitoneal injection；
Step 2, cell fusion：Mouse antibodies potency is higher than 1:10 000, the 4th immune mouse prepares mice spleen after three days Lymphocyte；After mouse anesthesia, eyeball is gone to take mouse peripheral blood, it is sterile to win mouse spleen and grind for separating serum； SP2/0 cells are mixed about 1 with Mouse spleen cells:5-1:10 ratio is mixed, and supernatant is abandoned in 800rpm centrifugations for 4 minutes, will be thin Born of the same parents are mixed again, and centrifuge tube is put into 40 DEG C of deionized water, and 1mL polyethylene glycol cell fusion agent was uniformly added into 1 minute, 1mL1640 cell culture fluids were added in 5 seconds, and add 15mL1640 cell culture fluids, are melted by slow-to-fast, termination within 90 seconds Close reaction；Incubation at room temperature 10 minutes, 800rmp centrifugations abandon supernatant in 4 minutes；With the 1640/20%FBS/ containing Peritoneal Cells of Mice The cell of HAT culture mediums suspension fusion is laid in 96 orifice plates, 37 DEG C, 5%CO2 environment cultures；
Step 3, the screening and cloning of hybridoma positive colony：Fused cell culture is sieved after 14 days to positive cell strain Choosing, cell culture fluid is detected by rPPRV-HN-F albumen coated elisa plate with indirect elisa method；Select indirect elisa method detection Positive hole, then with indirect immunofluorescene assay and the reactionogenicity of PPR virus；Choose the positive hole of Immunofluorescence test It is subcloned；Positive monoclonal cell line is obtained with Endpoint Dilution Method；By the positive hole 1640 culture medium of Immunofluorescence test Suspend and move into 1.5mL centrifuge tubes, the concentration of cell is adjusted to by cell counting count board to the cell count in positive cell hole 0.5-2/μ L, then cell is diluted to 1/100 μ L with 1460/20%FBS/HT cell culture fluids, it is laid on 96 hole cells trainings Support in plate, per the μ L1460/20%FBS/HT cell culture fluids of hole 100；Finally, 1460/ containing Peritoneal Cells of Mice is being added 20%FBS/HT nutrient solutions 100mL；After 14 days, positive cell strain, such as no positive monoclonal cell strain are screened with same method, Continuation is subcloned to positive hole until obtaining positive monoclonal cell line；It is predetermined special using limiting dilution assay screening secretion The cell of property monoclonal antibody；The positive hybridoma of monoclonal antibody needed for being produced using indirect ELISA and immunofluorescence screening Cell；Clonal expansion finally is carried out to required positive hybridoma cell；
Step 4, monoclonal antibody hypotype and titration：
Hypotype is detected according to the specification of monoclonal antibody hypotype detection kit；
Sample-adding：Positive monoclonal cell line cell conditioned medium is added in ELISA Plate sample well, per the μ L of hole 50, without being incubated；
Enzyme-added labeling antibody：1 × IgM+IgG-HRP of sheep anti mouse dilutions are added in ELISA Plate sample well, per the μ L of hole 50, mixed；
It is incubated：Shrouding film is capped, 1 hour is incubated at room temperature；
Washing：Liquid is removed, is washed 3 times, 2 minutes every time, dried with PBST；
Colour developing：The nitrite ion prepared is added into sample well, per the μ L of hole 100；
It is incubated：The ELISA Plate for having added nitrite ion is placed in light protected environment, room temperature 20 minutes；
Terminate：Terminate liquid is added, per the μ L of hole 100；
Read：Read OD450 or observe, color is most deep or OD value highests are corresponding hypotype；
As a result：The hypotype of 10E3 monoclonal antibodies is IgG2b；
Antibody titer measure is carried out to positive monoclonal cell line cell conditioned medium with indirect ELISA method, with SP2/0 cell conditioned mediums As negative control, critical value is negative control+2SD, and potency is 1:800；
It is prepared by step 5, monoclonal antibody batch：
Culture is enlarged to the positive monoclonal cell line of acquisition, collects cell and culture medium, by cell to be injected intraperitoneally Mode is injected into the culture of adult dams abdominal cavity, and the peritoneal fluid of mouse is collected after about 7 days；
The identification of epitope：
The epitope of prediction passes through biosynthesis, and identification meets requirement of experiment, using Aminated ELISA Plate as between solid phase carrier Connect ELISA method progress；
Dilution：The deionized water synthesized and lyophilized epitope peptide is pressed through with height is diluted to 1 μ g/ μ L；
Epitope peptide is coated with：The epitope peptide diluted is coated on Aminated ELISA Plate, per the μ L coating buffers of hole 50, the content of antigen For 5 μ g, 4 DEG C overnight；
Board-washing：Liquid in enzyme mark hole is got rid of to the greatest extent as far as possible, about 250 μ LPBST are then added per hole, 3 minutes are stood, three are repeated It is secondary, finally ELISA Plate is patted dry on gauze；
Closing：The μ L of 1 × confining liquid 150 are added into enzyme mark hole, 37 DEG C of incubations are after 1.5 hours, by the closing in enzyme mark hole Liquid is got rid of to the greatest extent as far as possible, standby after finally ELISA Plate is patted dry on gauze；
It is incubated primary antibody：Monoclonal antibody is pressed 1 with confining liquid:500 dilutions, add ELISA Plate per the μ L of hole 50, place 37 DEG C of incubators It is middle to be incubated 1 hour；
Board-washing：Ibid walk board-washing；
It is incubated secondary antibody：HRP is marked into anti-mouse IgG secondary antibodies by 1 with confining liquid:5 000 dilutions, add ELISA Plate per the μ L of hole 50, put Put in 37 DEG C of incubators and be incubated 1 hour；
Board-washing：Ibid walk board-washing；
Colour developing：50 μ L TMB nitrite ions are added per hole, 37 DEG C of lucifuge is incubated 15 minutes；
Terminate：50 μ L terminate liquids, color development stopping are added per hole；
Read：ELISA Plate is detected at wavelength 450nm with end-point method, OD450nm absorbances are read.
CN201710500780.XA 2017-06-27 2017-06-27 Peste des petits ruminants virus HN protein epitope peptide H362, determination method, preparation method and application thereof Active CN107216372B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710500780.XA CN107216372B (en) 2017-06-27 2017-06-27 Peste des petits ruminants virus HN protein epitope peptide H362, determination method, preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710500780.XA CN107216372B (en) 2017-06-27 2017-06-27 Peste des petits ruminants virus HN protein epitope peptide H362, determination method, preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN107216372A true CN107216372A (en) 2017-09-29
CN107216372B CN107216372B (en) 2020-09-04

Family

ID=59950963

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710500780.XA Active CN107216372B (en) 2017-06-27 2017-06-27 Peste des petits ruminants virus HN protein epitope peptide H362, determination method, preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN107216372B (en)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110346566A (en) * 2018-04-03 2019-10-18 苏州工业园区强东医药科技有限公司 PPR virus antibody assay kit
CN110498842A (en) * 2018-05-18 2019-11-26 中国兽医药品监察所 Peste des petits ruminants diagnostic kit
CN111378017A (en) * 2018-12-28 2020-07-07 浙江海隆生物科技有限公司 Subunit F protein of peste des petits ruminants virus and preparation method and application thereof
CN111378016A (en) * 2018-12-28 2020-07-07 浙江海隆生物科技有限公司 Subunit H protein of peste des petits ruminants virus, preparation method and application thereof
CN111790327A (en) * 2019-04-09 2020-10-20 中国科学院化学研究所 Molecular design method of ice control material
CN112213493A (en) * 2019-07-11 2021-01-12 中国兽医药品监察所 Peste des petits ruminants detection kit capable of distinguishing vaccine immunity from natural infection
CN113219172A (en) * 2021-04-25 2021-08-06 山东第一医科大学附属省立医院(山东省立医院) Method for detecting tumor antigen OVA12 serum antibody

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102559707A (en) * 2011-12-27 2012-07-11 中国农业科学院兰州兽医研究所 Peste des petits ruminants virus H and F protein antigenic epitope gene fragment, and expression and application thereof
WO2013142380A1 (en) * 2012-03-22 2013-09-26 The Regents Of The University Of California Oncovector nucleic acid molecules and methods of use

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102559707A (en) * 2011-12-27 2012-07-11 中国农业科学院兰州兽医研究所 Peste des petits ruminants virus H and F protein antigenic epitope gene fragment, and expression and application thereof
WO2013142380A1 (en) * 2012-03-22 2013-09-26 The Regents Of The University Of California Oncovector nucleic acid molecules and methods of use

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
梁忠祥: "小反刍兽疫多表位疫苗抗原的设计及其免疫效力评价", 《中国优秀硕士学位论文全文数据库农业科技辑》 *
石晓妮等: "小反刍兽疫病毒H 和F 蛋白多抗原表位的表达及其反应原性", 《中国兽医科学》 *
陈蕾: "小反刍兽疫病毒血凝素蛋白与宿主受体相互作用的研究", 《中国博士学位论文全文数据库农业科技辑》 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110346567A (en) * 2018-04-03 2019-10-18 苏州工业园区强东医药科技有限公司 PPR virus antibody assay kit
CN110346567B (en) * 2018-04-03 2022-08-12 苏州工业园区强东医药科技有限公司 Peste des petits ruminants virus antibody detection kit
CN110346566B (en) * 2018-04-03 2022-04-12 苏州工业园区强东医药科技有限公司 Peste des petits ruminants virus antibody detection kit
CN110346566A (en) * 2018-04-03 2019-10-18 苏州工业园区强东医药科技有限公司 PPR virus antibody assay kit
CN110498842B (en) * 2018-05-18 2021-01-26 中国兽医药品监察所 Peste des petits ruminants diagnostic kit
CN110498842A (en) * 2018-05-18 2019-11-26 中国兽医药品监察所 Peste des petits ruminants diagnostic kit
CN111378016B (en) * 2018-12-28 2022-02-11 浙江海隆生物科技有限公司 Subunit H protein of peste des petits ruminants virus, preparation method and application thereof
CN111378017B (en) * 2018-12-28 2022-02-11 浙江海隆生物科技有限公司 Subunit F protein of peste des petits ruminants virus and preparation method and application thereof
CN111378016A (en) * 2018-12-28 2020-07-07 浙江海隆生物科技有限公司 Subunit H protein of peste des petits ruminants virus, preparation method and application thereof
CN111378017A (en) * 2018-12-28 2020-07-07 浙江海隆生物科技有限公司 Subunit F protein of peste des petits ruminants virus and preparation method and application thereof
CN111790327A (en) * 2019-04-09 2020-10-20 中国科学院化学研究所 Molecular design method of ice control material
CN112213493A (en) * 2019-07-11 2021-01-12 中国兽医药品监察所 Peste des petits ruminants detection kit capable of distinguishing vaccine immunity from natural infection
CN112213493B (en) * 2019-07-11 2023-07-07 中国兽医药品监察所 Peste des petits ruminants detection kit capable of being used for distinguishing vaccine immunity from natural infection
CN113219172A (en) * 2021-04-25 2021-08-06 山东第一医科大学附属省立医院(山东省立医院) Method for detecting tumor antigen OVA12 serum antibody

Also Published As

Publication number Publication date
CN107216372B (en) 2020-09-04

Similar Documents

Publication Publication Date Title
CN107216372A (en) A kind of PPR virus HN Protein Epitopes peptide H362 and its determination, preparation method and application
CN111474345A (en) SARS-CoV-2 antibody detection method
ES2328536T3 (en) INTRACELLULAR PRODUCTION OF POLYPEPTIDE E2 TRUNCATED FROM HEPATITIS C.
CN109970852A (en) Secrete hybridoma cell strain and the application of rabies poison M protein monoclonal antibody
CN113049818A (en) Method and reagent for identifying mutant antigen
Zhang et al. Identification of linear B cell epitope on gB, gC, and gE proteins of porcine pseudorabies virus using monoclonal antibodies
CN108614121A (en) Bovine viral diarrhea virus E2 proteantigen multi-epitope fusogenic peptides and its preparation and application
CN107033226A (en) A kind of PPR virus F protein epitope peptide and its determination, preparation method and application
CN114874995A (en) Classical swine fever virus type 2E rns Monoclonal antibody hybridoma cell strain of protein and application
CN104744572B (en) Fowl and pig hepatitis E virus share antigen, monoclonal antibody and preparation method and application
CN113563461A (en) Competitive monoclonal antibody based on African swine fever virus CD2v protein, kit and application thereof
CN107033225B (en) Peste des petits ruminants virus HN protein epitope peptide and determination, preparation method and application thereof
CN111378628A (en) Hybridoma cell strain secreting mycobacterium tuberculosis ESAT6 protein specific antibody, antibody and application thereof
CN114807178B (en) African swine fever virus P72 protein C-terminal multi-epitope recombinant antigen and application thereof
CN116840475A (en) Application of sandwich blocking ELISA (enzyme-linked immunosorbent assay) reagent in preparation of foot-and-mouth disease virus antibody detection kit
CN109666071A (en) A kind of polyclonal antibody of Pasteurella albumen and the preparation of monoclonal antibody
CN107271667B (en) Blue tongue virus type specificity competitive ELISA detection kit and its application
CN106290862B (en) Mumps virus HN antigens and its purposes in detection material for anti parotitis antiviral antibody
CN110221080A (en) A kind of source of people norovirus immune colloid gold reagent box and cell strain
CN113980126B (en) Pasteurella multocida toxin monoclonal antibody and blocking ELISA kit thereof
CN110161246A (en) Distinguish the indirect competitive ELISA method and kit of newcastle disease virus sample particle vaccines immune serum and wild virus infection serum
CN116790509B (en) Monoclonal hybridoma cell strain secreting anti-porcine pseudorabies virus gB protein antibody and application thereof
CN117169499B (en) Double-antibody sandwich ELISA detection kit for Gatavirus and application thereof
CN110794148B (en) Double-antibody sandwich ELISA quantitative detection kit for pig inflammation small NLRP3 and application thereof
Shapouri et al. Production of monoclonal antibody against recombinant NS3 protein of bovine viral diarrhea virus (NADL strain)

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
GR01 Patent grant