CN107739402A - A kind of preparation and application of the epitope, antibody of CSFV Erns albumen - Google Patents
A kind of preparation and application of the epitope, antibody of CSFV Erns albumen Download PDFInfo
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- CN107739402A CN107739402A CN201710850113.4A CN201710850113A CN107739402A CN 107739402 A CN107739402 A CN 107739402A CN 201710850113 A CN201710850113 A CN 201710850113A CN 107739402 A CN107739402 A CN 107739402A
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Classifications
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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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- 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
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- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
- C12N2770/24322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
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- Molecular Biology (AREA)
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- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a kind of epitope of CSFV Erns albumen, the epitope is：Encode the small peptide of 1D5 epitopes, sequence such as SEQ ID NO.1：Shown in LATDTEL, 46 52AA are positioned at.The epitope can react with CSFV, without being reacted with pCold TF empty carriers, also not reacted with Vero cells.Application of the epitope in antibody against swine fever virus drug test reagent, the application allows antibody against swine fever virus diagnostic antigen general chemistry synthetic method to prepare, it is higher as diagnostic antigen, synthetic antigen simple process, purity compared to gene engineering expression albumen.
Description
Technical field
The invention belongs to biological technical field.It is more particularly to a kind of epitope, the antibody of CSFV Erns albumen
Preparation and application.
Background technology
CSF is a kind of highly contagious disease of the pig as caused by CSFV, and serious economic loss is caused to pig industry.
CSFV belongs to one of flaviviridae, pestivirus member.CSFV is the single strand plus RNA virus for having cyst membrane, and Genome Size is about
12.3kb, only containing a big Open reading frame (ORF).This ORF is translated into containing 3898 amino acid residues, molecular weight
About 438kDa polyprotein, and further it is processed into structural proteins and non-knot in the presence of virus and host cell proteins enzyme
Structure albumen, the coded sequence of its structural proteins and non-structural protein on viral RNA be Npro, C, Erns (E0), E1, E2, P7,
NS2-3、NS4A、NS4B、NS5A、NS5B.Wherein, except C, Erns, E1 and E2, in addition to structural proteins, remaining is non-structural protein
In vain.
Erns, it is a kind of membrane glycoprotein of virus, also referred to as gp44/48, is once called as E0.There are 9 possible glycosylation sites,
The peptide backbone molecular weight about 26KDa of glycosyl is removed, is made up of Glu268-Ala494 in virus O RF.Erns exists in infection cell
Accumulated in endoplasmic reticulum, and may be present in cell membrane surface or be secreted into extracellular.Erns is with the homologous of 97KDa in virion
Dimeric forms are present in cyst membrane surface.It is weak with virus envelope adhesion because its intramolecular lacks hydrophobic film anchorage zone, easily
Dissociate to get off from cyst membrane.Erns can induce the neutralizing antibody for producing swine fever, and immune swine, which can induce, to be produced to lethal dose CSFV's
Protective immunity.It is high due to encoding sequence preservative degree of the Erns nucleotide sequence in category internal ratio coding E2, therefore Erns can make
To prevent and treat a kind of target protein of swine fever.
In virus infection, it is mediate retroviral intrusion host cell that the acceptor on cell membrane is combined with viral ligand
Key factor, and virus can infection cell key.Therefore, the interaction between virus receptor and viral ligand is studied
One of hot issue as current viral pathogenesis Mechanism Study because using it is therein any one can be hindered as drug target
Disconnected virus and the combination of target cell, so as to suppress the infection of virus.On the research of CSFV parts, the participation of Erns albumen is had proven to
The early stage absorption of virus, the heterodimer that E1 and E2 albumen is formed can mediate CSFV to invade host cell, and this is different
Source dimer also acts as the effect for merging mammalian cell.But which amino acid sequence of these three albumen is made actually
Combined for viral ligand with virus receptor, there is presently no detailed report.
The content of the invention
The purpose of the present invention is to be the provision of the epitope of CSFV Erns albumen, and the epitope is：Compile
The small peptide of code 1D5 epitopes, sequence such as SEQ ID NO.1：Shown in LATDTEL, 46-52AA is positioned at.The epitope
It can react with CSFV, without being reacted with pCold-TF empty carriers, also not reacted with Vero cells.
A further object of the invention is to be the provision of a kind of epitope of CSFV Erns albumen in hog cholera
Application in malicious antibody drug detection reagent, the application allow antibody against swine fever virus diagnostic antigen general chemistry synthesis side
Prepared by method, higher as diagnostic antigen, synthetic antigen simple process, purity compared to gene engineering expression albumen.
Another object of the present invention is to be the provision of a kind of epitope simulative peptide of CSFV Erns albumen
Preparation method, this method advantage are that the epitope of swine fever Erns monoclonal antibodies 1D5 identifications is screened using phage random peptide library,
Different from original series but intimate comformational epitope antigen, i.e. mimic epitope antigen can be filtered out.
In order to realize above-mentioned purpose, the present invention realizes by following technical measures
A kind of preparation method of the epitope of CSFV Erns albumen, its step are：
A, amplification of the molecular biology to Erns Main Antigenic Regions domain gene and the structure of recombinant plasmid are utilized.
B, by the induced expression of recombinant protein and purifying,
C, mouse immune is carried out using purifying protein, obtains hybridoma and monoclonal antibody.
D, the stability and specific detection of monoclonal antibody.
E, the epitope of above-mentioned monoclonal antibody specific is obtained, and it is identified.
A kind of epitope of CSFV Erns albumen is preparing treatment or prevention CSFV ELSIA antibody drug inspections
The application surveyed in (kit), its step are：
1st, using the epitope of acquisition as referring to chemical synthesis antigen.
2nd, with synthetic antigen reagent preparation box key component antigen coated microplate.
3rd, reagent preparation box other components according to a conventional method.
4th, Antibody against rabies virus in serum is detected using kit.
The present invention compared with prior art, has advantages below and effect：
1st, epitope of the invention is by the specific monoclonal antibody screening for Classical Swine Fever Virus Shimen Strain, is had high
Specificity and stability.
2nd, compared with existing inactivation of viruses antigen or gene expression antigen technology, strain and poison are not related in production process
Strain, process safety is secure, and environment will not be polluted.
3rd, using the epitope antigenic synthetic peptide of acquisition compared with gene engineering expression antigen, with short production cycle, Yi Chun
Change and purity is higher.
4th, the present invention is adapted to large-scale clinical serum detection, and the reaction time is short, can go out result within 2 hours.
5th, kit of the present invention and other virus-positive serum no cross reactions, sensitiveness is high, and specificity is good.
6th, kit of the present invention is easy to operate, high with CSFV forward direction indirect hemagglutination diagnostic reagent box coincidence rate.
Brief description of the drawings
Fig. 1 be Erns recombinant proteins expression and purification, M：Protein molecular weight standard；1：The pCold- of IPTG inductions
Erns transformed bacterias lysate precipitates；2：The pCold-Erns transformed bacteria lysate supernatants of IPTG inductions；3：IPTG inductions
The full bacterium of pCold-Erns transformed bacterias；4：The pCold-TF transformed bacterias of IPTG inductions.
Fig. 2 is the specificity that IFA identifies Erns protein monoclonal antibodies.
Fig. 3 is that the Western blot of Erns albumen monoclonal antibodies are identified, 1：Marker；2：The pCold-Erns of IPTG inductions turns
Change the supernatant of bacterium lysate purifying；3：The pCold-TF transformed bacterias of IPTG inductions.
Fig. 4 is the qualification result of Erns-1D5 monoclonal antibody epitopes, wherein, (A) Erns-1D5 identification epitopes the 1st are taken turns；
(B) Erns-1D5 identifies that epitope the 2nd is taken turns (C) Erns-1D5 identification epitopes the 3rd and taken turns
Embodiment
Embodiment 1：Materials and methods
Strain, cell and experimental animal Classical Swine Fever Virus Shimen Strain, SP2/O myeloma cell, PK-15, Vero cells by
China Agriculture Academe Shanghai Veterinary Institute preserves.Cell is in 37 DEG C, 5%CO2With 10%FBS (FBS, Sigma,
Shanghai, China) under the conditions of cultivate.BALB/c female mices are purchased from Shanghai Si Laike experimental animals company.
Carrier, reagent and bacterial strain pCold-TF carriers, e. coli bl21 (DE3) are purchased from Takara (Shanghai) company；HRP
The mountain sheep anti-mouse igg of mountain sheep anti-mouse igg and the FITC mark of mark is purchased from Sigma (Shanghai) company；Endonuclease EcoR I
Purchased from NEB (Shanghai) company.
Embodiment 2：It is prepared by the amplification of Erns Main Antigenics, albumen
The amplification of Erns Main Antigenic Regions domain gene and the structure of recombinant plasmid
The CSFV Erns gene orders delivered according to NCBI, company (Jin Weizhi) optimization is sent to close the total length of Erns genes
Into, and Erns full length genes are connected to structure plasmid with pCold-TF carriers, after plasmid order-checking is correct, positive plasmid is named as
pCold-Erns。
The induced expression of recombinant protein and purifying
Recombinant plasmid pCold-Erns is transformed into BL21 (DE3) Escherichia coli, 37 DEG C of shaken cultivations to OD600 values reach
During to 0.6~0.8, final concentration of 1mM IPTG inductions are added, 16 DEG C vibrate 24 hours.Collect thalline and add PBS progress ultrasounds
Cracking, add after 5 × SDS buffer boil 10min and carry out SDS-PAGE electrophoresis, coomassie is carried out to SDS-PAGE protein adhesives
Light blue dyes, and observes protein expression situation, while sets empty carrier expression group as control.Afterwards by pCold-Erns translation tables
It is inoculated into up to bacterium in 1: 1000 ratio in LB culture mediums of the 4mL containing 100 μ g/mL Amp, 37 DEG C, 200r/min shaken cultivations 12h.
Then the bacterium solution of recovery is inoculated in LB nutrient solutions of the 100mL containing 100 μ g/mL Amp by 1: 100 again, 37 DEG C, 200r/min
Culture, stops when reaching 0.6~0.8 to OD600, adds final concentration of 1mmol/L IPTG, and after 16 DEG C induce 24h, centrifugation is received
Collect thalline.Thalline after expression carries out ultrasonication, and supernatant protein is collected after differential centrifugation.Carried out using Ni posts (Biotool)
Purifying, -80 DEG C are stored in after the albumen packing of purifying.
The expression bacterium E.coli BL21 (DE3) of pCold-Erns carriers conversion, after IPTG is induced, collect thalline ultrasound
Cracking carries out PAGE gel detection.As a result show, compared with vehicle Control group, pCold-Erns expression bacterium occur relatively
Molecular weight is about 90kDa (TF auxilins about 60KDa) purpose band, is consistent with recombinant protein size.Destination protein is main
All in lysate supernatant.Through Ni posts after purification, electrophoresis result display purifying destination protein purity is higher (Fig. 1) for recombinant protein.
Embodiment 3：The preparation of monoclonal antibody
Mouse immune
100 μ g purifying proteins and the not mixing and emulsifying of formula Freund's complete adjuvant 1: 1 are taken, are inoculated with 6 week old Healthy female BALB/c mouses,
Hereafter carried out second with equivalent purifying protein and incomplete Freund's adjuvant emulsification every 2 weeks and be immunized for the third time, third time is immune
Take a blood sample within the 10th day afterwards, determine ELISA antibody titers, mouse of the potency more than 1: 10000, pass through within the 3-4 days before cell fusion
The pure μ g of antigen 200 are injected intraperitoneally, carry out booster immunization.
McAbs preparation
1d takes Turnover of Mouse Peritoneal Macrophages to choose immunizing potency highest mouse afterwards as feeder cells and enter before fusion
Row splenocyte and SP2/O cell fusions.Hybridoma Cell Culture is being covered with 96 orifice plates of feeder cells, 37 DEG C, 5%
CO2 is cultivated under the conditions of HAT selective mediums.When hybridoma covers with 96 1/3~1/2 area of orifice plate, by indirect
ELISA and IFA method is detected to screen positive cell clone strain.By the positive cell clone strain of screening through 3~5 wheel cellses
After subclone, culture, liquid nitrogen cryopreservation are enlarged to the hybridoma of energy stably excreting antibody.The hybridoma of screening is thin
In the generation of born of the same parents' strain continuous passage 20, the generation cell supernatant of the 5th, 10,15 and 20 is taken respectively, detected with the method for indirect ELISA, reflect
Determine hybridoma secretory antibody stability.The hybridoma of liquid nitrogen cryopreservation is recovered, takes the cell supernatant of recovery cell,
Detected with indirect ELISA and IFA method, identify the stability of hybridoma secretory antibody.
Embodiment 4：The identification of monoclonal antibody
According to the sequence of the Erns genes of optimum synthesis, primer amplified Erns full length sequences are designed.
Sense primer Erns-F：5’-tcgagctcaagcttcgaattctgGAAAATATCACCCAGTGGAAT-3’(SEQ
ID NO.2)；
Anti-sense primer Erns-R：5’-gtaccgtcgactgcagaattTGCATAGGCGCCAAACCA-3’(SEQ ID
NO.3).Lower-case portion be with pEGFP-C3 carriers carry out homologous recombination homology arm sequence, amplified production 687bp.With
Primerstar HS (Takara) high-fidelity enzymatic amplification Erns genes.PCR amplification programs：95℃ 5min；98 DEG C of 10s, 58 DEG C
10s, 72 DEG C of 1min (35 circulations)；72℃ 10min.Method by PCR primer by homologous recombination (promise is only praised), with
The EcoR I positions restructuring of pEGFP-C3 vector multiple cloning sites, after plasmid order-checking is correct, positive plasmid is named as EGFP-
Erns。
Vero cells are layered in 6 orifice plates, when cell length is to 50%-80%, EGFP-Erns plasmids turned by 2ug/ holes
Contaminate (FUGENE, Promega) cell.After 24h, 6 orifice plates are closed into 1h with 5%BSA room temperatures, add what is collected after PBS board-washings
The hybridoma supernatant of Erns albumen, it is incubated at room temperature 1h.PBS is washed 3 times, lucifuge add FITC mark sheep anti-mouse igg (1:
800PBS dilutes) room temperature lucifuge incubation 1h.In fluorescence microscopy Microscopic observation fluorescence.
EGFP-Erns plasmid transfection vero cells, after 24h, fixed cell carries out indirect immunofluorescence assay, to detect list
Clonal antibody Erns-1D5 specific reaction.As a result show, monoclonal antibody Erns-1D5 can be anti-with EGFP-Erns albumen
Answer (Fig. 2), and also do not reacted with EGFP-C3 zero load precursor reactants with Vero cells neither.
Western blot detect monoclonal antibody
Vero cells are layered in 6 orifice plates, when cell length is to 50%-80%, EGFP-Erns plasmids turned by 2ug/ holes
Contaminate (FUGENE, Promega) cell.Cell pyrolysis liquid cell lysis is used after 24h, cell protein is collected and carries out SDS-PAGE electricity
Swimming.Use BoleSD cell system (BIO-RAD, USA) transfer NC films, 5% skimmed milk power room temperature envelope
2h is closed, with 4 DEG C of the hybridoma supernatant incubation 10h of 1: 500 dilution, after TBST washes film 3 times, 20min/ times.Add 1: 5000
The sheep anti-mouse igg of the HRP marks of dilution, reacts at room temperature 1h, after TBST washes film, by luminous, exposure, development, is swept after fixing
Trace designs piece.
PCold-Erns carriers conversion expression bacterium E.coli BL21 (DE3) after IPTG is induced, carry out SDS-PAGE,
Western blot methods are to detect monoclonal antibody Erns-1D5.As a result show, monoclonal antibody Erns-1D5 energy and Erns
Specific reaction occurs for albumen, in 90KDa specific bands, without being reacted (Fig. 3) with pCold-TF empty carriers.
Embodiment 5：The identification of E2 monoclonal antibody epitopes
According to gene order, 14 pairs of primers (table 1) are designed, primer is respectively provided with pCold-TF carrier EcoR I restriction enzyme sites
Upstream arm and downstream arm, Erns genes are truncated, the DNA fragmentation amplified is connected to pCold-TF prokaryotic expression carriers
On, after being sequenced correctly, it is transformed into BL21 (DE3) Escherichia coli and is expressed and induced, the albumen of induced expression is passed through
SDS-PAGE is analyzed, and Western blot identifications are carried out with monoclonal antibody.
The primer sequence of table 1
Note：Lower-case portion is the homology arm sequence that homologous recombination is carried out with pCold-TF；Upper-case portion is amplification ErnsFragment
Primer.
The Erns albumen of truncation is identified by monoclonal antibody Western blot, monoclonal antibody 1D5 strains are directed to
Epitope is positioned at 46-52AA (Fig. 4), amino acid sequence 46LATDTEL52, is positioned at 46-52AA.The epitope
It can react with CSFV, without being reacted with pCold-TF empty carriers, also not reacted with Vero cells.
Embodiment 6；A kind of epitope of CSFV Erns albumen treats or prevents CSFV ELSIA in preparation and resisted
Application in body drug test (kit), its step are：
A, prepared by the antigen coated microplate in CSFV ELISA antibody assay kits：
According to SEQ ID NO of the present invention：Sequence shown in 1, using chemical synthesis process conventional in the art, prepare pure
Small peptide of the degree more than 95%, this small peptide are to detect antigen.This antigen carbonate buffer solution is dissolved and is diluted to 2 μ g/mL,
Then it is added to by every μ L of hole 100 in 96 hole elisa Plates, 4 DEG C stand overnight, and make Antigen adsorption in ELISA Plate.Discard within second day
Liquid in hole, 150 μ L phosphate buffers (containing 0.5%BSA) are added per hole, puts in 37 DEG C of incubators 2 hours, discard liquid in hole
Body.Pat dry.
B, prepared by CSFV ELISA antibody assay kits other components：
Kit other components are also comprising enzyme marker, sample diluting liquid, positive control, negative control, nitrite ion A, aobvious
Color liquid B, cleaning solution and terminate liquid.Enzyme marker is goat-anti pig secondary antibody, and sample diluting liquid is phosphate buffer, and positive control is
The immune pig positive control serum of classical swine fever virus vaccine, negative control are the health pig feminine gender blood of not immune classical swine fever virus vaccine
Clearly.Cleaning solution is the PBS containing 0.05%Tween-20；Nitrite ion A is the lemon containing 50mg/mL carbamide peroxides
Phthalate buffer, nitrite ion B liquid are the citric acid/sodium citrate buffer solution containing 0.2mg/mL TMB pH5.0.Terminate liquid is
Containing 0.25% volume ratio hydrofluoric acid solution.
C, CSFV ELISA antibody assay kits operating procedure：
1) the pre-coated detection plate for having antigen is taken out from kit, by the serum to be checked diluted (1: 40 dilution) 100 μ
L is added in antigen coated microplate, while sets positive and negative control wells, respectively sets 2 holes, per the μ L of hole 100.
2) gently shake sample in even hole, puts 37 DEG C and incubates 60 minutes.The solution in plate hole is got rid of, adds the μ L/ of cleaning solution 200
Hole, board-washing 5 times, is patted dry on blotting paper for the last time.
3) per the enzyme-added μ L of label 100 in hole, 37 DEG C is put and is incubated 30 minutes.Washing 5 times, method is the same as step 2.
4) add each 50 μ L of nitrite ion A, nitrite ion B per hole, mix, room temperature (18~26 DEG C) lucifuge develops the color 10 minutes.Per hole
Add the μ L of terminate liquid 50, determined in 10 minutes with ELIASA per hole OD450nm readings.
Kit criterion of the present invention is：Experiment establishment condition is that the average OD450nm values of negative control hole and the positive are right
It is more than or equal to 0.5 according to the difference of the average OD450nm values in hole.S=sample well OD450nm values, N=negative control holes are averaged
OD450nm values.If S/N ratios are more than 2.1, sample is judged to the antibody against swine fever virus positive.If S/N ratios are less than or equal to 2.1, sample
Product are judged to antibody against swine fever virus feminine gender.
D, the application of CSFV ELSIA antibody assay kits：
1st, specific test detects CSFV, Pseudorabies virus, pig mouth with CSFV ELISA antibody assay kits
The standard positive serums such as fever aphthous (O-shaped), pig parvoviral, swine flu and porcine reproductive and respiratory syndrome and CSFV feminine gender blood
Clearly, in addition to the S/N values of CSFV standard positive serum are noticeably greater than 2.1, remaining serum S/N values are respectively less than 2.1, meet feminine gender
The criterion of serum, show that the specificity of this method is good (being shown in Table 2, wherein institute's column of figure is OD450nm values).
The serological specificity of table 2 detects
2nd, the detection of sensitiveness detects the CSFV positive serum of different dilution factors with kit, can from table 3
Even if going out the CSFV positive serum of 1280 times of dilutions, still test positive, illustrates that the sensitiveness of kit is very high.
The serum sensitive of table 3 detects
3rd, repeated experiment
The ELISA conditions established using the condition after optimization are detected, the between-group variation coefficient of acquisition 1.19%~
Between 6.34%；Between-group variation coefficient is obtained between 1.98%~6.14%, shows that the ELISA established has good repetition
Property.
To sum up, the ELISA kit that the pig plague virus specific epitope foundation of gained is prepared using the present invention detects swine fever
Virus has the advantages of high specificity, reproducible, high sensitivity, the detection available for clinical CSFV sample.
It should be appreciated that although above present invention is made with a general description of the specific embodiments detailed
Description, but on the basis of the present invention, some modifications can be carried out to it or are improved, this is to those skilled in the art
Obviously.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belonging to the present invention will
Seek the scope of protection.
Claims (8)
1. a kind of epitope of CSFV Erns albumen, the epitope can react with CSFV, without with pCold-
TF empty carriers react, and are not also reacted with Vero cells.
2. a kind of epitope of CSFV Erns albumen as claimed in claim 1, the epitope sequence such as SEQ
ID NO.1：Shown in LATDTEL, 46-52AA is positioned at.
3. a kind of antibody preparation, the antibody preparation includes specificity for the CSFV Erns described in claim 1 or 2
The antibody of the epitope of albumen, the antibody is polyclonal or monoclonal；Or the preparation includes the fragment of the antibody.
4. the antibody preparation described in claim 3, the antibody preparation can effectively treat or prevent CSFV.
5. one kind prepares sero-fast method, methods described is dynamic including the epitope described in claim 1 or 2 is administered to
Thing host is included in the antiserum of caused antibody in the animal reservoir to produce antibody and recovery in the animal reservoir.
6. a kind of antigen composition, the antigen composition includes at least one antigen, wherein at least one antigen includes
At least part albumen or polypeptide and at least part albumen or polypeptide of CSFV Erns albumen include at least one swine fever
The epitope of viral Erns albumen or antigenic determinant.
7. antigen composition as claimed in claim 6, the epitope of described CSFV Erns albumen, the antigen table
Bit sequence such as SEQ ID NO.1：Shown in LATDTEL, 46-52AA is positioned at.
8. the antibody described in a kind of epitope of CSFV Erns albumen described in claim 1 or 2, claim 3 or 4
Antigen composition described in preparation, claim 6,7 is preparing treatment or prevention CSFV ELSIA antibody drug detection reagents
Application in box.
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| TWI811793B (en) * | 2021-09-15 | 2023-08-11 | 國立屏東科技大學 | Soluble erns recombinant protein of classical swine fever virus, method of making the same, kit and method of identifying classical swine fever virus infection using the same |
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|---|---|---|---|---|
| CN106188250A (en) * | 2016-03-25 | 2016-12-07 | 武汉科前生物股份有限公司 | The epitope simulative peptide of a kind of CSFV E 2 protein and preparation method and application |
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| CN106188250A (en) * | 2016-03-25 | 2016-12-07 | 武汉科前生物股份有限公司 | The epitope simulative peptide of a kind of CSFV E 2 protein and preparation method and application |
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| ANDREAS CHRISTMANN等: "Epitope mapping and affinity purification of monospecific antibodies by Escherichia coli cell surface display of gene-derived random peptide libraries", 《JOURNAL OF IMMUNOLOGICAL METHODS》 * |
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| TWI811793B (en) * | 2021-09-15 | 2023-08-11 | 國立屏東科技大學 | Soluble erns recombinant protein of classical swine fever virus, method of making the same, kit and method of identifying classical swine fever virus infection using the same |
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