CN107033226A - A kind of PPR virus F protein epitope peptide and its determination, preparation method and application - Google Patents

A kind of PPR virus F protein epitope peptide and its determination, preparation method and application Download PDF

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CN107033226A
CN107033226A CN201710502539.0A CN201710502539A CN107033226A CN 107033226 A CN107033226 A CN 107033226A CN 201710502539 A CN201710502539 A CN 201710502539A CN 107033226 A CN107033226 A CN 107033226A
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antibody
epitope
mouse
protein
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CN107033226B (en
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窦永喜
梁忠祥
朱学亮
蒙学莲
张志东
才学鹏
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Lanzhou Veterinary Research Institute of CAAS
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    • 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

Abstract

The present invention relates to a kind of PPR virus F protein epitope peptide, the amino acid sequence of epitope peptide is respectively：F176：176DYINNELVPSVHRMSCEL193, or/and F347：347MSPLLQECFRGSTKS361, F391：Or/and391CKCYTTETVINQDPDKLL408, or/and F417：417TVINQDPDKGPVGSREYPD435, F430：Or/and430SREYPDSVYLH440, or/and F502：502VSLGLVTLICCCKGRCRNK520；The present invention carries out B cell epitope to target protein and is predicted, and the different epitopes to prediction carry out artificial synthesized, detection and the reactionogenicity of the antibody of F protein, so as to identify the B cell epitope of PPRV F proteins respectively.

Description

A kind of PPR virus F protein epitope peptide and its determination, preparation method and Using
Technical field
The invention belongs to bioinformatics and immunological technique field, and in particular to a kind of PPR virus F protein Epitope peptide 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, F is the glycoprotein being embedded on PPRV cyst membranes, is constituted The fibre of virion surface is dashed forward, and HN albumen can be invaded with the SLAM acceptor interactions mediate retroviral on lymphocyte, still The phagocytic process of virus needs the help of F protein just to complete, and the cell that PPR virus can cause body strong is exempted from Epidemic disease and humoral immune response, and F protein is then main protective antigens；In addition, F protein is probably to have in Morbillivirus The main cause of cross protection characteristic, F protein target spot important in vaccine design and detection method are set up, therefore F protein Epitope collection of illustrative plates 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 F protein epitope peptide.
Another object of the present invention is to provide the PPR virus F protein epitope peptide in PPR disease Application in malicious epiposition vaccine antigen and diagnostic reagent antigen preparation.
Another object of the present invention is to provide determination, the preparation method of the PPR virus F protein epitope.
The technical solution adopted by the present invention is：A kind of PPR virus F protein epitope peptide, the epitope The amino acid sequence of peptide is：F176：176DYINNELVPSVHRMSCEL193, or/and F347：347MSPLLQECFRGSTKS361, Or/and F391：391CKCYTTETVINQDPDKLL408, or/and F417：417TVINQDPDKGPVGSREYPD435, or/and F430 ：430SREYPDSVYLH440, or/and F502：502VSLGLVTLICCCKGRCRNK520。
A kind of PPR virus F protein epitope peptide is tried in PPR virus epiposition vaccine antigen and diagnosis Application in the preparation of agent antigen.
A kind of determination of PPR virus F protein epitope, preparation method, methods described comprise the following steps：
Calculating simulation：
Step one, the 3D structures of virtual F protein are built with molecular simulation software：Using SWISS-MODEL to vaccine strain PPRV-F albumen carries out Blast search (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 20%, and the longer protein structure of aligned sequences is as formwork structure, carries out Follow-up calculating；
Step 3, 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 3 different target protein structures of generation；
Step 4, 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 F proteins, 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 antibody：
Step one, animal is immunized：With reactionogenicity purpose antigen rPPRV-HN-F and PPR virus glycoprotein (PPRV-Glycoprotein) 6-8 week old female Balb/c mouse, the subcutaneous 4 points of injections of nape part, immune protein sample is immunized Measure as 50 μ g/ only, the μ L/ of volume 200 are immunized only；First immunisation is emulsified with isometric Freund's complete adjuvant, secondary immunity and three times It is immunized with isometric incomplete Freund's adjuvant adjuvant emulsion；Every 14 days it is immune once, three times it is immune 14 days after mouse docking adopt The antibody that blood examination is surveyed in serum, treats that antibody titer is high by 1：Four can be carried out after 10000 to exempt from, four exempt from without adjuvant, intraperitoneal injection；
The absorbent cotton of ether complete wetting and mouse are put into anesthesia box simultaneously, treated by step 2, the purifying of polyclonal antibody When mouse is lost consciousness completely, win eyeball and take, after the completion of collection, the disconnected neck of mouse is put to death, whole blood puts 37 DEG C and is incubated 30 points Stayed overnight for 4 DEG C after clock, 4 DEG C, 1500rpm centrifuges 15 minutes, takes serum, -20 DEG C standby；
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.Multi-epitope ANTIGEN DESIGNThe：
Step one：Multi-epitope gene and induced expression are designed, epitope is arranged according to the order in native protein, epitope Between plus GS linkers, send Nanjing Jin Sirui companies synthetic gene fragment, be connected in pET30a carriers, carrier is transformed into In Host Strains, according to 37 DEG C of cultivation temperature, the final concentration of 1.0mM of derivant IPTG, ampicillin LB culture mediums, induction 7 is small Shi Jinhang induced expressions；
Step 2：The purifying of multi-epitope antigen,
Broken thalline：By thalline multigelation 3 times, thalline ultrasonication is resuspended with PBS；
Centrifugation：Broken 4 DEG C of 10 000rpm of thalline is centrifuged 20 minutes, precipitation and supernatant are collected respectively；
Preparation of samples：Using the precipitation that collection is walked in buffer solution I (pH=7.4) resuspension, fully mix, 8 000rpm, 4 DEG C Centrifugation 20 minutes, takes supernatant to treat loading with 0.45 μm of filter filtration treatment；
Balance Ni posts：First a small amount of deionized water is added in bottom of the pillar and exclude air, be reloaded into 2mL Ni-NAT fillers, will The pillar installed is washed with the deionized water of 5 times of volumes to remove ethanol, is finally put down with the buffer solution I (pH=7.4) of 5 times of volumes Weigh post material；
Loading：By the sample after filter filtration treatment, combined, stood with post material by several times, treat natural subsidence, collect the rapid liquid of stream 4 DEG C of environment are preserved；
Washing：Buffer solution I (pH=7.4) is added into pillar by several times, cumulative volume should be greater than 5 times of volumes of applied sample amount；Wash It is de-：Egg is eluted using the elution buffer containing 10mM, 20mM, 50mM, 100mM, 200mM, 300mM and 400mM imidazole concentration In vain, each wash-out concentration stands 15 minutes, collects each concentration eluent and is placed on ice；
Pillar is preserved：After the completion of elution step, pillar is cleaned with the buffer solution I (pH=7.4) of 5 times of post material volumes, is added Appropriate 20% ethanol, 4 DEG C of preservations；
Step 3：The identification of multi-epitope antigen,
Protein electrophoresis preparation of samples：The protein sample separated in 40 μ L albumen above-mentioned steps is taken in 1.5mL centrifuge tubes, then 10 μ L 5 × albumen sample-loading buffers are added, boils 10 minutes, is placed in 4 DEG C of refrigerator fast coolings；Loading and electrophoresis：Take 10 μ L in In 12%SDS-PAGE, 100mV electrophoresis；
Transferring film：By NC films cut to gel equivalent size, be placed in soaking 5 minutes in transfering buffering liquid, according to from negative pole Order to positive pole places thick filter paper, gel, NC films, thick filter paper respectively, notes to avoid producing bubble, wet turn of 200mA constant currents 100 minutes；
Closing：The NC films after transfer are taken out, are placed in appropriate 5% skimmed milk power, room temperature is closed two hours；
Plus primary antibody：The NC films for closing completion are placed in 5% BSA, the anti-His monoclonal antibodies (dilution ratio of mouse is added 1:2 500) (dilution ratio is 1 with sheep source PPR positive serum:250), 4 DEG C of refrigerator overnights are incubated；
Washing：After the completion of primary antibody is incubated, washing 3 times, every time 10 minutes is shaken with PBST；
Plus secondary antibody：NC films are placed in rabbit anti-mouse igg-HRP (1:5 000 dilution proportions) and the anti-sheep IgG-HRP (1 of donkey:25 000 dilution proportion) in secondary antibody, dilution is the PBST containing 5% skimmed milk power, and room temperature, which is shaken, to be incubated 1 hour；
Washing：Ibid step washing；
Colour developing：The NC films for washing completion are placed in gel imaging system, the super quick luminescent solution A and B of ECL is mixed, drops in NC It is incubated at room temperature 3 minutes on film；
Observation：The regulation different time for exposure, until obvious band is seen in cleaning；
Step 4：Immune mouse, is divided into 5 groups, A groups by small white mouse：PBS+ incomplete Freund's adjuvants, B groups：Vaccine, C groups： EHF1+ incomplete Freund's adjuvants, D groups：EHF2+ incomplete Freund's adjuvants, E groups：EHF3+ incomplete Freund's adjuvants.Every group 12 The μ L of volume every 200 are immunized in mouse, every μ L of immune protein 50.The hypodermic injection of 4 points of nape part, is immunized again after 21 days；
Step 5：Mouse detection of specific antibody, docking blood sampling in 0 day, 7 days, 14 days, 28 days and 35 days, 37 DEG C are incubated 30 Minute, 4 DEG C overnight, 3000rpm centrifuge 15 minutes collect serum, be stored in -80 DEG C it is standby.With coating PPRV totivirus it is indirect ELISA detects antibody level；
Step 6：Mouse T lymphocyte level detects that mouse CD3+CD4+T cells and CD3+CD8+T cells is immunized in detection Increment situation, using Flow Cytometry detect it is immune after 0 day, 14 days and 26 days periphery blood T cell, in the 35th day detection spleen T lymphocytes in dirty；Step is as follows：
Blood sampling：Mouse anticoagulation, 5 mouse of every group of collection, every 100 μ L are taken in docking；
Antibody incubation：Every of collection 100 μ L anticoagulations are divided into two parts, a copy of it adds APC Hamster Anti-Mouse CD3e antibody and each 2.5 μ L, FITC Rat Anti-Mouse of PE Rat Anti-Mouse CD4 antibody The μ L of CD8a antibody 1；Another adds APC Hamster IgG1, κ Isotype Control antibody, PE Rat IgG2a, κ Isotype Control antibody and FITC Rat IgG2a, κ each 2.5 μ L of Isotype Control antibody, after mixing, lucifuge 4 DEG C be incubated 15 minutes；
Splitting erythrocyte：1mL1 × erythrocyte cracked liquid is added, is mixed, lucifuge is incubated at room temperature 10 minutes；
Washing：1mL Hank ' S liquid is added, is mixed, 1 000g is centrifuged 5 minutes, is repeated 2 times；
Filtering：With 300 mesh nylon net filters after high pressure before loading；
Loading：Ready sample is placed on flow cytometer in sample rack, prepares to collect cell.
The beneficial effects of the invention are as follows：
PPR virus is inoculated with VeroE6 cells, the reaction of detection polyclonal antibody and PPR virus is former The specificity of property and antibody.As a result show, small in rPPRV-HN-F polyclonal antibodies (Figure 1A) and Vero cells ruminates beast Epidemic disease poison reaction, shows stronger green fluorescence, does not have fluorescence (Fig. 1 C) with Vero cell effects；Mouse negative serum not with PPR virus reaction (Figure 1B) in Vero cells, without fluorescence.Show, rPPRV-HN-F polyclonal antibodies are small anti- The specific antibody of hay epizootic disease virus；PPRV-Glycoprotein polyclonal antibodies (Fig. 2) are same with PPR virus to be had There are good reactionogenicity and specificity.
With rPPRV-HN-F (Fig. 3 A) and PPRV-Glycoprotein (figure of the Western-Blotting methods to acquisition 3B) polyclonal antibody has carried out the specific detection based on Prokaryotic expression, purification rPPRV-HN-F albumen, and empty carrier expression bacterium makees For 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, Show that two kinds of polyclonal antibodies and rPPRV-HN-F albumen have good reactionogenicity, and do not reacted with empty carrier expression bacterium With good specificity.
Candidate's epi-position and rPPRV-HN-F polyclonal antibodies (Fig. 4) and PPRV- are have detected using Aminated ELISA Plate The reactionogenicity of Glycoprotein (Fig. 5) polyclonal antibody, determine candidate's epi-position F176, F347, F391, H417, F430 and F502。
The head construction of the strain F proteins of PPR virus Nigeria 75/1 is predicted using SWISS-MODEL, is shown Show that F176, F347, F391, F417 and F430 are located at the surface (Fig. 6 blue portions) of F protein.
It is connected between three kinds of multi-epitope genes of synthesis in pET30a carriers, identifies that (Fig. 7) shows purpose bar by double digestion Band has been properly inserted on the corresponding position of carrier.Song Jinwei intelligence biotech firm carries out the result and composition sequence of plasmid order-checking Compare, find no catastrophe, show that correct sequence is inserted on the correct position of carrier.
Plasmid passes through induced expression, and purifying obtains the antigen protein (Fig. 8) of high-purity.
Western-Blotting specific detections are carried out to the fusion protein of expression using His tag antibodies.As a result show Show occur specific band in positive colony swimming lane, and there is no band (Fig. 9) in empty carrier Host Strains swimming lane, show place Main bacterium successful expression His fusion proteins, as destination protein.
Mouse peripheral blood antibody level is detected, the result based on totivirus shows (Figure 10) that each multi-epitope antigen can swash The antibody level of mouse is played, EHF1 protein groups effects are preferable.
To understand the increment change of T lymphocytes, the CD3 using Flow cytometry in mouse peripheral blood and spleen The increment situation of+CD4+T lymphocytes and CD3+CD8+T lymphocytes.To 0th day after immune, 14 days, 26 days it is different with 35 days The time point T level of lymphocyte is detected, is as a result shown, different groups in same time point CD3+CD4+T lymphocytes Level is different, but changes not significantly (Figure 11).But, the water of difference group different time points CD3+CD8+T lymphocytes Flat really different (Figure 11).Therefore, multi-epitope antigen can effectively excite the cellular immunity of mouse.
Brief description of the drawings
Fig. 1 be rPPRV-HN-F polyclonal antibodies be PPR virus specific antibody；
Fig. 2 PPRV-Glycoprotein polyclonal antibodies are same with PPR virus to have good reactionogenicity And specificity；
Fig. 3 Western Blotting detect polyclonal antibody and rPPRV-HN-F albumen have good reactionogenicity and Specificity；
Fig. 4 rPPRV-HN-F polyclonal antibodies screen F protein B cell epi-position storehouse result；
Fig. 5 PPRV-Glycoprotein polyclonal antibodies screen F protein B cell epi-position storehouse result；
Position of Fig. 6 F176, F347, F391, F417 and F430 epitope in F protein structure；
Fig. 7 recombinant plasmids pET30a-EHF1, pET30a-EHF2 and pET30a-EHF3 double digestion identification；
SDS-PAGE is analyzed after purification by Fig. 8 multi-epitope recombination antigens EHF1, EHF2 and EHF3；
Fig. 9 Western-Blotting detect His expressing fusion proteins；
Figure 10 is based on totivirus indirect elisa method and detects mouse antibodies level；
Figure 11 immunized mice T lymphocyte proliferations change.
Embodiment
The present invention is described in further details with reference to embodiment.
A kind of PPR virus F protein epitope peptide of the present invention, the amino acid sequence of its epitope peptide It is classified as：
F176：176DYINNELVPSVHRMSCEL193(SEQ ID NO.1)
Or/and F347：347MSPLLQECFRGSTKS361(SEQ ID NO.2)
Or/and F391：391CKCYTTETVINQDPDKLL408(SEQ ID NO.3)
Or/and F417：417TVINQDPDKGPVGSREYPD435(SEQ ID NO.4)
Or/and F430：430SREYPDSVYLH440(SEQ ID NO.5)
Or/and F502：502VSLGLVTLICCCKGRCRNK520(SEQ ID NO.6)。
The present invention carries out B cell epitope to target protein using panimmunity informatics software and is predicted, then to pre- The different epitopes of survey carry out respectively it is artificial synthesized, using indirect ELISA method carry out verify its reactionogenicity, different polypeptide bags By Aminated ELISA Plate, detection and the reactionogenicity of the antibody of F protein, so as to identify the B cell epitope of PPRV F proteins.
A kind of determination of PPR virus F protein epitope of the present invention, preparation method, methods described includes as follows Step：
Calculating simulation：
Step one, the 3D structures of virtual F protein are built with molecular simulation software：Using SWISS-MODEL to vaccine strain PPRV-F albumen carries out Blast search (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 20%, and the longer protein structure of aligned sequences is as formwork structure, carries out Follow-up calculating；
Step 3, 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 3 different target protein structures of generation；
Step 4, 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 F proteins, 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 antibody：
Step one, animal is immunized：With reactionogenicity purpose antigen rPPRV-HN-F and PPR virus glycoprotein (PPRV-Glycoprotein) 6-8 week old female Balb/c mouse, the subcutaneous 4 points of injections of nape part, immune protein sample is immunized Measure as 50 μ g/ only, the μ L/ of volume 200 are immunized only；First immunisation is emulsified with isometric Freund's complete adjuvant, secondary immunity and three times It is immunized with isometric incomplete Freund's adjuvant adjuvant emulsion；Every 14 days it is immune once, three times it is immune 14 days after mouse docking adopt The antibody that blood examination is surveyed in serum, treats that antibody titer is high by 1：Four can be carried out after 10000 to exempt from, four exempt from without adjuvant, intraperitoneal injection；
The absorbent cotton of ether complete wetting and mouse are put into anesthesia box simultaneously, treated by step 2, the purifying of polyclonal antibody When mouse is lost consciousness completely, win eyeball and take, after the completion of collection, the disconnected neck of mouse is put to death, whole blood puts 37 DEG C and is incubated 30 points Stayed overnight for 4 DEG C after clock, 4 DEG C, 1500rpm centrifuges 15 minutes, takes serum, -20 DEG C standby；
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；
Multi-epitope antigen is designed：
Step one：Multi-epitope gene and induced expression are designed, epitope is arranged according to the order in native protein, epitope Between plus GS linkers, send Nanjing Jin Sirui companies synthetic gene fragment, be connected in pET30a carriers.Carrier is transformed into In Host Strains, according to 37 DEG C of cultivation temperature, the final concentration of 1.0mM of derivant IPTG, ampicillin LB culture mediums, induction 7 is small Shi Jinhang induced expressions.
Step 2：The purifying of multi-epitope antigen,
Broken thalline：By thalline multigelation 3 times, thalline ultrasonication is resuspended with PBS；
Centrifugation：Broken 4 DEG C of 10 000rpm of thalline is centrifuged 20 minutes, precipitation and supernatant are collected respectively；
Preparation of samples：Using the precipitation that collection is walked in buffer solution I (pH=7.4) resuspension, fully mix, 8 000rpm, 4 DEG C Centrifugation 20 minutes, takes supernatant to treat loading with 0.45 μm of filter filtration treatment；
Balance Ni posts：First a small amount of deionized water is added in bottom of the pillar and exclude air, be reloaded into 2mL Ni-NAT fillers, will The pillar installed is washed with the deionized water of 5 times of volumes to remove ethanol, is finally put down with the buffer solution I (pH=7.4) of 5 times of volumes Weigh post material；
Loading：By the sample after filter filtration treatment, combined, stood with post material by several times, treat natural subsidence, collect the rapid liquid of stream 4 DEG C of environment are preserved；
Washing：Buffer solution I (pH=7.4) is added into pillar by several times, cumulative volume should be greater than 5 times of volumes of applied sample amount；
Elution：It is slow using the elution containing 10mM, 20mM, 50mM, 100mM, 200mM, 300mM and 400mM imidazole concentration Fliud flushing elutes albumen, and each wash-out concentration stands 15 minutes, collects each concentration eluent and is placed on ice；
Pillar is preserved：After the completion of elution step, pillar is cleaned with the buffer solution I (pH=7.4) of 5 times of post material volumes, is added Appropriate 20% ethanol, 4 DEG C of preservations.
Step 3：The identification of multi-epitope antigen,
Protein electrophoresis preparation of samples：The protein sample separated in 40 μ L albumen above-mentioned steps is taken in 1.5mL centrifuge tubes, then 10 μ L 5 × albumen sample-loading buffers are added, boils 10 minutes, is placed in 4 DEG C of refrigerator fast coolings；
Loading and electrophoresis：10 μ L are taken in 12%SDS-PAGE, 100mV electrophoresis；
Transferring film：By NC films cut to gel equivalent size, be placed in soaking 5 minutes in transfering buffering liquid, according to from negative pole Order to positive pole places thick filter paper, gel, NC films, thick filter paper respectively, notes to avoid producing bubble, wet turn of 200mA constant currents 100 minutes；
Closing：The NC films after transfer are taken out, are placed in appropriate 5% skimmed milk power, room temperature is closed two hours；
Plus primary antibody：The NC films for closing completion are placed in 5% BSA, the anti-His monoclonal antibodies (dilution ratio of mouse is added 1:2 500) (dilution ratio is 1 with sheep source PPR positive serum:250), 4 DEG C of refrigerator overnights are incubated；
Washing：After the completion of primary antibody is incubated, washing 3 times, every time 10 minutes is shaken with PBST；
Plus secondary antibody：NC films are placed in rabbit anti-mouse igg-HRP (1:5 000 dilution proportions) and the anti-sheep IgG-HRP (1 of donkey:25 000 dilution proportion) in secondary antibody, dilution is the PBST containing 5% skimmed milk power, and room temperature, which is shaken, to be incubated 1 hour；
Washing：Ibid step washing；
Colour developing：The NC films for washing completion are placed in gel imaging system, the super quick luminescent solution A and B of ECL is mixed, drops in NC It is incubated at room temperature 3 minutes on film；
Observation：The regulation different time for exposure, until obvious band is seen in cleaning.
Step 4：Immune mouse, is divided into 5 groups, A groups by small white mouse：PBS+ incomplete Freund's adjuvants, B groups：Vaccine, C groups： EHF1+ incomplete Freund's adjuvants, D groups：EHF2+ incomplete Freund's adjuvants, E groups：EHF3+ incomplete Freund's adjuvants, every group 12 The μ L of volume every 200 are immunized in mouse, every μ L of immune protein 50.The hypodermic injection of 4 points of nape part, is immunized again after 21 days；
Step 5：Mouse detection of specific antibody, docking blood sampling in 0 day, 7 days, 14 days, 28 days and 35 days, 37 DEG C are incubated 30 Minute, 4 DEG C overnight, 3000rpm centrifuge 15 minutes collect serum, be stored in -80 DEG C it is standby, with coating PPRV totivirus it is indirect ELISA detects antibody level；
Step 6：Mouse T lymphocyte level detects that mouse CD3+CD4+T cells and CD3+CD8+T cells is immunized in detection Increment situation, using Flow Cytometry detect it is immune after 0 day, 14 days and 26 days periphery blood T cell, in the 35th day detection spleen T lymphocytes in dirty, step is as follows：
Blood sampling：Mouse anticoagulation, 5 mouse of every group of collection, every 100 μ L are taken in docking；
Antibody incubation：Every of collection 100 μ L anticoagulations are divided into two parts, a copy of it adds APCHamster Anti-Mouse CD3e antibody and each 2.5 μ L, FITC Rat Anti-Mouse of PE Rat Anti-Mouse CD4 antibody The μ L of CD8a antibody 1；Another adds APC Hamster IgG1, κ Isotype Control antibody, PE Rat IgG2a, κ Isotype Control antibody and FITC Rat IgG2a, κ each 2.5 μ L of Isotype Control antibody, after mixing, lucifuge 4 DEG C be incubated 15 minutes；
Splitting erythrocyte：1mL1 × erythrocyte cracked liquid is added, is mixed, lucifuge is incubated at room temperature 10 minutes；
Washing：1mL Hank ' S liquid is added, is mixed, 1 000g is centrifuged 5 minutes, is repeated 2 times；
Filtering：With 300 mesh nylon net filters after high pressure before loading；
Loading：Ready sample is placed on flow cytometer in sample rack, prepares to collect cell.
By Fig. 1 and 2 it is obvious that VeroE6 cells be inoculated with PPR virus, detection polyclonal antibody with The reactionogenicity of PPR virus and the specificity of antibody.As a result show, rPPRV-HN-F polyclonal antibodies (Figure 1A) Reacted with the PPR virus in Vero cells, show stronger green fluorescence, there is no fluorescence with Vero cell effects (Fig. 1 C)；Mouse negative serum does not react (Figure 1B) with the PPR virus in Vero cells, without fluorescence.Show, RPPRV-HN-F polyclonal antibodies are the specific antibodies of PPR virus.PPRV-Glycoprotein polyclonal antibodies (Fig. 2) is same with PPR virus to have good reactionogenicity and specificity.
Fig. 1 and 2 indirect immunofluorescene assays polyclonal antibody and PPR virus have good reactionogenicity and Specificity.
A.VeroE6 cells shows go out plasomidum, and fluorescence intensity is brighter, and appear on the cell of lesion, illustrate antibody With good reactionogenicity.
There is not fluorescence in B.VeroE6 cells, illustrate that control antibodies are not reacted with virus.
There is not fluorescence in C.VeroE6 cells, show monoclonal antibody not with VeroE6 cell effects, illustrate monoclonal Antibody has good specificity.
Fig. 3 Western Blotting detect polyclonal antibody and rPPRV-HN-F albumen have good reactionogenicity and Specificity.
1 swimming lane is rPPRV-HN-F albumen in Fig. 3 A, an obvious band occurs, and 2 swimming lane empty carriers express bacterium, do not have There is band, illustrate that polyclonal antibody and rPPRV-HN-F albumen have good reactionogenicity and specificity；Same result goes out Now in figure 3b.
Fig. 4 rPPRV-HN-F polyclonal antibodies screen F protein B cell epi-position storehouse result
With based on the indirect ELISA method detection polyclonal antibody that amidized ELISA Plate is solid phase carrier and epitope peptide Reactionogenicity.It will become apparent from F176, F347, F391, F417 and F502 epitope peptide, rPPRV-HN-F polyclonal antibody groups OD450 values extremely significantly with control group OD450 values (p<0.00001), rPPRV-HN-F polyclonal antibody groups in F430 epitope peptides OD450 values extremely significantly with control group OD450 values (p<0.001), show F176, F347, F391, F417, F430 and F502 with RPPRV-HN-F polyclonal antibodies have good reactionogenicity, as effective epitope.
Fig. 5 PPRV-Glycoprotein polyclonal antibodies screen F protein B cell epi-position storehouse result
With based on the indirect ELISA method detection polyclonal antibody that amidized ELISA Plate is solid phase carrier and epitope peptide Reactionogenicity.It will become apparent from F502 epitope peptides, the OD450 values of PPRV-Glycoprotein polyclonal antibody groups are extremely notable With control group OD450 values (p<0.00001)；In F417 epitope peptides, the OD450 of PPRV-Glycoprotein polyclonal antibody groups Value extremely significantly with control group OD450 values (p<0.001)；In F430 epitope peptides, PPRV-Glycoprotein polyclonal antibody groups OD450 values significantly with control group OD450 values (p<0.05) F417, F430 and F502 epitope and PPRV-Glycoprotein, are shown Polyclonal antibody has good reactionogenicity, as effective epitope.
Position of Fig. 6 F176, F347, F391, F417 and F430 epitope in F protein structure
It is obvious that blue portion is epitope in figure, this it appears that the epitope is located at from 3D structures The surface of antigen more prominent place, this is the major reason that the corresponding antibody of epitope has sound response originality.
Fig. 7 recombinant plasmids pET30a-EHF1, pET30a-EHF2 and pET30a-EHF3 double digestion identification
M：DNA Marker；1：PET30a carrier double digestion products；2：PET30a-EHF1 carrier double digestion products；3： PET30a-EHF2 carrier double digestion products；4：PET30a-EHF3 carrier double digestion products
Will become apparent from the equal band of outlet two of pET30a-EHF1, pET30a-EHF2 and pET30a-EHF3, wherein one with PET30a is compareed always, and band in addition is consistent with EHF1, EHF2 and EHF3, shows that purpose band has been properly inserted load On the corresponding position of body.
SDS-PAGE is analyzed after purification by Fig. 8 multi-epitope recombination antigens EHF1, EHF2 and EHF3
M：Albumen Marker；1-3：Multi-epitope recombination antigen EHF1, EHF2 and EHF3
Multi-epitope recombination antigen is purified by nickel agarose Gel column, passes through the elution of different imidazole concentrations, destination protein Eluted from pillar, SDS-PAGE analysis results show that each swimming lane of multi-epitope recombination antigen, without obvious miscellaneous band, shows Obtain purer albumen.
Fig. 9 Western-Blotting detect His expressing fusion proteins
1：Empty carrier Host Strains are precipitated；2：PET30a-EHF3/BL21 (DE3) is precipitated；3：pET30a-EHF2/BL21 (DE3) precipitate；4：PET30a-EHF1/BL21 (DE3) is precipitated
In positive colony pET30a-EHF1/BL21 (DE3), pET30a-EHF2/BL21 (DE3) and pET30a-EHF3/ BL21 (DE3) precipitation swimming lane in there is specific band, respectively between 25kDa or so, 40-55kDa and 55-70kDa it Between, and there is no band in empty carrier Host Strains swimming lane, show Host Strains successful expression His fusion proteins, as destination protein.
Figure 10 is based on totivirus indirect elisa method and detects mouse antibodies level
Relatively low antibody level is all produced in the 7th day every group of mouse, in the 14th and 21 day every group of mouse antibodies level all Constantly raising, the 21st day secondary immunity, in the 28th and 35 day every group of mouse antibodies level in rise, PBS groups do not have all the time yet Antibody.But, compared to vaccine group, the antibody level of test group is relatively low.However, the level of EHF1 albumen was above epidemic disease at the 21st day Seedling group.Three groups of test groups are compared, and EHF1 protein groups remain higher antibody level；EHF2 protein groups are after secondary immunity Just significantly rise, suitable in the 35th day EHF1 protein groups antibody level；But EHF3 protein groups antibody level is minimum all the time.
Figure 11 immunized mice T lymphocyte proliferations change
14th day vaccine group, EHF1, EHF2 and EHF3 CD3+CD8+T cent lymphocytes are significantly higher than after immune PBS groups；26th day after immune, there is significant difference in the CD3+CD8+T cent lymphocytes of different experiments group, vaccine group, EHF1 groups and EHF2 groups are all remarkably higher than PBS groups, and EHF3 is extremely significantly higher than PBS groups；Vaccine group and EHF1 groups, EHF2 groups and EHF3 groups are compared, and EHF1 groups and EHF2 groups are significantly higher than vaccine group, and then pole is significantly higher than vaccine group to EHF3 groups；35th after immune My god, vaccine group, EHF1 groups, the CD3+CD8+T cent lymphocytes pole of EHF2 groups and EHF3 groups are significantly higher than PBS groups, EHF3 The CD3+CD8+T cent lymphocytes of group are then significantly higher than PBS groups, and the CD3+CD8+T lymphocyte percentages of EHF1 groups It is significantly higher than vaccine group than pole.
The epitope peptide identified by above scheme, is PPR virus epiposition vaccine antigen and diagnostic reagent antigen system Standby research and development 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.
SEQUENCE LISTING
<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
<120>A kind of PPR virus F protein epitope peptide and its determination, preparation method and application
<130> 2017
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> PRT
<213>PPR virus
<400> 1
DYINNELVPSVHRMSCEL
1 5 10 15
<210> 2
<211> 15
<212> PRT
<213>PPR virus
<400> 2
MSPLLQECFRGSTKS
1 5 10 15
<210> 3
<211> 18
<212> PRT
<213>PPR virus
<400> 3
CKCYTTETVINQDPDKLL
1 5 10 15
<210> 4
<211> 19
<212> PRT
<213>PPR virus
<400> 4
TVINQDPDKGPVGSREYPD
1 5 10 15
<210> 5
<211> 11
<212> PRT
<213>PPR virus
<400> 5
SREYPDSVYLH
1 5 10
<210> 6
<211> 19
<212> PRT
<213>PPR virus
<400> 6
VSLGLVTLICCCKGRCRNK
1 5 10 15

Claims (3)

1. a kind of PPR virus F protein epitope peptide, it is characterised in that：The amino acid sequence of the epitope peptide For：
F176：176DYINNELVPSVHRMSCEL193；
Or/and F347：347MSPLLQECFRGSTKS361；
Or/and F391：391CKCYTTETVINQDPDKLL408；
Or/and F417：417TVINQDPDKGPVGSREYPD435；
Or/and F430：430SREYPDSVYLH440；
Or/and F502：502VSLGLVTLICCCKGRCRNK520。
2. a kind of PPR virus F protein epitope peptide is in PPR virus epitope according to claim 1 Application in vaccine antigen and the preparation of diagnostic reagent antigen.
3. a kind of determination of PPR virus F protein epitope, preparation method, its feature according to claim 1 It is：Methods described comprises the following steps：
Calculating simulation：
Step one, the 3D structures of virtual F protein are built with molecular simulation software：Using SWISS-MODEL to vaccine strain PPRV-F Albumen carries out Blast search (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 20%, and the longer protein structure of aligned sequences is as formwork structure, carries out follow-up Calculating；
Step 3, 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 3 different target protein structures of generation；
Step 4, 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 antibody：
Step one, animal is immunized：With reactionogenicity purpose antigen rPPRV-HN-F and PPR virus glycoprotein (PPRV-Glycoprotein) 6-8 week old female Balb/c mouse, the subcutaneous 4 points of injections of nape part, immune protein sample is immunized Measure as 50 μ g/ only, the μ L/ of volume 200 are immunized only；First immunisation is emulsified with isometric Freund's complete adjuvant, secondary immunity and three times It is immune to be emulsified with isometric incomplete Freund's adjuvant；Every 14 days it is immune once, three times it is immune 14 days after mouse docking blood sampling inspection The antibody surveyed in serum, treats that antibody titer is high by 1：Four can be carried out after 10000 to exempt from, four exempt from without adjuvant, intraperitoneal injection；
The absorbent cotton of ether complete wetting and mouse are put into anesthesia box simultaneously, treat mouse by step 2, the purifying of polyclonal antibody When losing consciousness completely, win eyeball and take, after the completion of collection, by mouse break neck put to death, whole blood put 37 DEG C be incubated 30 minutes after 4 DEG C overnight, 4 DEG C, 1500rpm, centrifuge 15 minutes, take serum, -20 DEG C are standby；
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；
Multi-epitope antigen is designed：
Step one：Multi-epitope gene and induced expression are designed, epitope is arranged according to the order in native protein, between epitope Plus GS linkers, Nanjing Jin Sirui companies synthetic gene fragment is sent, is connected in pET30a carriers, carrier is transformed into host In bacterium, according to 37 DEG C of cultivation temperature, the final concentration of 1.0mM of derivant IPTG, ampicillin LB culture mediums, induction is entered for 7 hours Row induced expression；
Step 2：The purifying of multi-epitope antigen,
Broken thalline：By thalline multigelation 3 times, thalline ultrasonication is resuspended with PBS；
Centrifugation：Broken 4 DEG C of 10 000rpm of thalline is centrifuged 20 minutes, precipitation and supernatant are collected respectively；
Preparation of samples：Using the precipitation that collection is walked in pH=7.4 buffer solution I resuspensions, fully mix, 8 000rpm, 4 DEG C of centrifugations 20 minutes, supernatant is taken to treat loading with 0.45 μm of filter filtration treatment；
Balance Ni posts：First a small amount of deionized water is added in bottom of the pillar and exclude air, be reloaded into 2mL Ni-NAT fillers, will install Pillar washed with the deionized water of 5 times of volumes to remove ethanol, finally with the pH=7.4 of 5 times of volumes buffer solution I balance columns Material；
Loading：By the sample after filter filtration treatment, combined, stood with post material by several times, treat natural subsidence, collect rapid 4 DEG C of the liquid of stream Environment is preserved；
Washing：PH=7.4 buffer solution I is added into pillar by several times, cumulative volume should be greater than 5 times of volumes of applied sample amount；Elution：Make Albumen is eluted with the elution buffer containing 10mM, 20mM, 50mM, 100mM, 200mM, 300mM and 400mM imidazole concentration, often Individual wash-out concentration stands 15 minutes, collects each concentration eluent and is placed on ice；Pillar is preserved：After the completion of elution step, with 5 times of posts The pH=7.4 of material volume buffer solution I cleaning pillars, add appropriate 20% ethanol, 4 DEG C of preservations；
Step 3：The identification of multi-epitope antigen,
Protein electrophoresis preparation of samples：Take the protein sample separated in 40 μ L albumen above-mentioned steps in 1.5mL centrifuge tubes, add 10 μ L 5 × albumen sample-loading buffers, boil 10 minutes, are placed in 4 DEG C of refrigerator fast coolings；
Loading and electrophoresis：10 μ L are taken in 12%SDS-PAGE, 100mV electrophoresis；
Transferring film：By NC films cut to gel equivalent size, be placed in transfering buffering liquid soak 5 minutes, according to from negative pole to just The order of pole places thick filter paper, gel, NC films, thick filter paper respectively, notes to avoid producing bubble, wet turn 100 points of 200mA constant currents Clock；
Closing：The NC films after transfer are taken out, are placed in appropriate 5% skimmed milk power, room temperature is closed two hours；
Plus primary antibody：The NC films for closing completion are placed in 5% BSA, the anti-His monoclonal antibodies of mouse is added and presses 1:2 500 ratios Dilution and sheep source PPR positive serum press 1:250 dilution proportions, 4 DEG C of refrigerator overnights are incubated；
Washing：After the completion of primary antibody is incubated, washing 3 times, every time 10 minutes is shaken with PBST；
Plus secondary antibody：NC films are placed in rabbit anti-mouse igg-HRP by 1:5 000 dilution proportions and the anti-sheep IgG-HRP of donkey press 1:25000 ratios In example dilution secondary antibody, dilution is the PBST containing 5% skimmed milk power, and room temperature, which is shaken, to be incubated 1 hour；Washing：Ibid step washing；
Colour developing：The NC films for washing completion are placed in gel imaging system, the super quick luminescent solution A and B of ECL is mixed, drops on NC films Incubation at room temperature 3 minutes；
Observation：The regulation different time for exposure, until obvious band is seen in cleaning；
Step 4：Immune mouse, is divided into 5 groups, A groups by small white mouse：PBS+ incomplete Freund's adjuvants, B groups：Vaccine, C groups：EHF1+ Incomplete Freund's adjuvant, D groups：EHF2+ incomplete Freund's adjuvants, E groups：EHF3+ incomplete Freund's adjuvants.Every group of 12 mouse, Every μ L of immune protein 50, is immunized the μ L of volume every 200.The hypodermic injection of 4 points of nape part, is immunized again after 21 days；
Step 5：Mouse detection of specific antibody, docking blood sampling in 0 day, 7 days, 14 days, 28 days and 35 days, 37 DEG C are incubated 30 minutes, 4 DEG C overnight, 3000rpm centrifuge 15 minutes collect serum, be stored in -80 DEG C it is standby, with coating PPRV totivirus it is indirect enzyme-linked Immunization detects antibody level；
Step 6：Mouse T lymphocyte level detects that the increasing of mouse CD3+CD4+T cells and CD3+CD8+T cells is immunized in detection Value situation, immune rear 0 day, 14 days and 26 days periphery blood T cell is detected using Flow Cytometry, in the 35th day detection spleen T lymphocytes, step is as follows：
Blood sampling：Mouse anticoagulation, 5 mouse of every group of collection, every 100 μ L are taken in docking；
Antibody incubation：Every of collection 100 μ L anticoagulations are divided into two parts, a copy of it adds APC Hamster Anti-Mouse CD3e antibody and each 2.5 μ L, FITC Rat Anti-Mouse of PE Rat Anti-Mouse CD4 antibody The μ L of CD8a antibody 1；Another adds APC Hamster IgG1, κ Isotype Control antibody, PE Rat IgG2a, κ Isotype Control antibody and FITC Rat IgG2a, κ each 2.5 μ L of Isotype Control antibody, after mixing, lucifuge 4 DEG C be incubated 15 minutes；
Splitting erythrocyte：1mL1 × erythrocyte cracked liquid is added, is mixed, lucifuge is incubated at room temperature 10 minutes；
Washing：1mL Hank ' S liquid is added, is mixed, 1 000g is centrifuged 5 minutes, is repeated 2 times；
Filtering：With 300 mesh nylon net filters after high pressure before loading；
Loading：Ready sample is placed on flow cytometer in sample rack, prepares to collect cell.
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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
CN114242169A (en) * 2021-12-15 2022-03-25 河北省科学院应用数学研究所 Antigen epitope prediction method for B cells
CN114242169B (en) * 2021-12-15 2023-10-20 河北省科学院应用数学研究所 Antigen epitope prediction method for B cells
CN117074673A (en) * 2023-10-18 2023-11-17 北京华诺泰生物医药科技有限公司 Method for quantitatively detecting specific activity of recombinant herpes zoster vaccine or/and stock solution
CN117363582A (en) * 2023-12-06 2024-01-09 中国兽医药品监察所 Hybridoma cell strain secreting anti-peste des petits ruminants virus F protein monoclonal antibody, monoclonal antibody thereof and application
CN117363582B (en) * 2023-12-06 2024-03-01 中国兽医药品监察所 Hybridoma cell strain secreting anti-peste des petits ruminants virus F protein monoclonal antibody, monoclonal antibody thereof and application

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