CN109627293A - Seneca Valley virus structural protein antigen epitope polypeptide and its application - Google Patents
Seneca Valley virus structural protein antigen epitope polypeptide and its application Download PDFInfo
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Abstract
The invention discloses Seneca Valley virus structural protein antigen epitope polypeptide and its applications.The polypeptide is polypeptide shown in sequence 4 in polypeptide shown in sequence 3 in polypeptide shown in sequence 2 in polypeptide shown in sequence 1 in sequence table, sequence table, sequence table or sequence table.Seneca Valley virus structural protein antigen epitope polypeptide of the invention is used to prepare kit and uses chemical synthesis Antigenic Peptide coated elisa plate, antigen dosage is few, sensitivity and specificity are high, can efficiently detect whether the structural proteins antibody that there is infection Seneca Valley virus.Antigen epitope polypeptide sensibility of the present invention is high, specificity is good and operation is convenient, has good market prospects.
Description
The application is that application No. is " 201710688944.6 ", entitled " Seneca Valley virus structural proteins antibody
The divisional application of the patent application of enzyme-linked immunologic detecting kit "
Technical field
The invention belongs to technical field of biological, more particularly it relates to a kind of Seneca Valley virus structure egg
White antibody ELISA immunity detection reagent.
Background technique
Seneca Valley virus (SenecaValleyVirus, SVV), also known as SenecavirusA (SVA) are that pig is primary
The main pathogen of property blister sore (Swineidiopathicvesiculardisease, SIVD).After SVV infects swinery, although not
It will cause larger politics, economic loss, but caused blister venereal disease becomes, with foot and mouth disease virus, swine pox, vesiculovirus mouth
Lesion caused by inflammation, pig vesicle etc. is similar, and certain difficulty is caused to Clinical differential diagnosis.Currently, North America, South American region
And the report that Australia has swinery that SIVD occurs.There is SVV several times and infect swinery and go out in China in 2015 and Brazil
Existing serious clinical and dead symptom, causes serious economic loss, China was also isolated to SVV in 2016 for the first time.
SVV is the unique member of Picornaviridae (Picornaviridae) Sai Nika Tobamovirus (Senecavirus),
SVV is sub-thread, normal chain, the RNA virus of non-segmented negative.It is carried out by 12 representative Tobamovirus viruses to Picornaviridae
Whole genome sequence compares discovery, and Sai Nika Tobamovirus and myocarditis virus category genetic affinity are nearest.SVV is diameter about 27nm's
RNA virus of the icosahedron without cyst membrane single-stranded positive non-segmented negative.Viral genome contains 7280 nucleotide, and an opening is read
Frame contains 6543 nucleotide, encodes the polyprotein of 2181 amino acid, can be divided into 12 polypeptides, constitute standard
Picornaviridae L-4-3-4 mode.
As all Picornaviridae members, P1 polypeptide is cracked into VP0, VP3 and VP1 by HRV 3CP, constitutes virus
Nucleocapsid.Mature VP0 is cracked to form VP2 and VP4.Virus VP 1~VP4 protein subunit structural conservation, may be thin with virus
Born of the same parents' preferendum is related.The interaction of VP2 and VP4 some regions participates in packaged nucleic acid.
Since China was isolated to SVV in 2016 for the first time, the current method in relation to SVV antibody test is still in research rank
Section, the relevant research of SVV antibody detection method mainly have indirect ELISA and competitive ELISA.
Summary of the invention
The purpose of the present invention is to provide Seneca Valley virus structural protein antigen epitope polypeptide and its preparing enzyme-linked exempt from
Application in epidemic disease detection kit.
The Seneca Valley virus structural protein antigen epitope polypeptide is polypeptide, sequence table shown in sequence 1 in sequence table
Polypeptide shown in middle sequence 2, polypeptide shown in sequence 4 in polypeptide shown in sequence 3 or sequence table in sequence table.
The present invention also provides Seneca Valley virus structural protein antigen epitope polypeptides to monitor whether infection Sai Nika in preparation
Application in the enzyme-linked immunologic detecting kit of paddy virus.
Wherein, the kit is for detecting in Swine serum whether contain Seneca Valley virus structural proteins antibody.
In order to achieve the above object, it is more to screen the Seneca Valley virus epitope being had excellent performance first by the present invention
Peptide combinations, Seneca Valley virus structural protein antigen epitope polypeptide composition provided by the invention are sequence table in sequence table
One or more of polypeptide shown in sequence 4 in polypeptide shown in sequence 3 or sequence table in middle sequence 2, sequence table
Any combination.When the peptide composition is in polypeptide shown in polypeptide shown in polypeptide shown in sequence 2, sequence 3 and sequence 4
Two kinds when, the mass ratioes of two kinds of polypeptides is (0.5~1.5): (0.5~1.5)；Preferably, their mass ratio is 1:1；When
When the peptide composition is polypeptide shown in sequence 2, polypeptide shown in polypeptide and sequence 4 shown in sequence 3, the quality of polypeptide
Than for (0.5~1.5): (0.5~1.5): (0.5~1.5)；Preferably, their mass ratio is 1:1:1；
Seneca Valley virus structural proteins antibody ELISA immunity detection reagent of the invention, including enzyme-linked reaction plate, sun
Property control serum, negative control sera and ELIAS secondary antibody；Wherein the enzyme-linked reaction plate is coated with Seneca Valley virus structure egg
White antigen epitope polypeptide composition.
The enzyme-linked reaction plate is detachable 96 hole elisa Plates；The Seneca Valley virus structural protein antigen epitope polypeptide
Polypeptide in composition, which is that chemistry is artificial synthesized, to be obtained.
The best preparation method and condition of the enzyme-linked reaction plate are by the Seneca Valley virus structural protein antigen table
Position peptide composition is dissolved in the carbonate solution of pH 9.6, is then added to 96 hole polystyrene enzyme-linked reaction plates, every hole 200ng is more
Peptide is placed 8~12 hours at 2~8 DEG C, combines polypeptide antigen sufficiently with enzyme-linked reaction plate, is then added according to 300 holes μ l/
PBS buffer solution containing 1% (g/ml) bovine serum albumin(BSA) (BSA) pH7.4,37 DEG C Seal treatment 2~3 hours, after drying, to
It is sealed for 4 DEG C after enzyme-linked reaction plate is dry.
The positive control serum is the Swine serum that acquires after the Seneca Valley virus artificial challenge；The negative control
Serum is the Swine serum of no-special pathogen (SPF).
The ELIAS secondary antibody is horseradish peroxidase-labeled rabbit-anti pig IgG antibody.
The kit further includes substrate solution A, substrate solution B and terminate liquid, and the substrate solution A is containing 0.06% (g/ml) mistake
The citrate phosphate buffer of urea hydrogen is aoxidized, the substrate solution B is the tetramethyl biphenyl amine aqueous solution of 0.2mg/ml, when use
The two is mixed with the ratio of 1:1.The terminate liquid is the sulfuric acid solution of 2mol/L.
The kit further includes sample diluting liquid and 20 times of concentrated cleaning solutions；Sample diluting liquid is to contain 0.5% (g/
100ml) 0.01M of casein, the phosphate buffer that pH value is 7.4；Concentrated cleaning solution be containing concentration be 0.8%~
The 0.01M of the Tween-20 of 1.2% (ml/ml), the phosphate buffer that pH value is 7.4.
The detection program of kit of the present invention are as follows:
1, it balances: kit being taken out from cold storage environment, it is spare to set equilibrium at room temperature 30min；The preceding mixing of liquid reagent.
2, match liquid: the 20 times of dilutions of concentrated cleaning solution distilled water or deionized water are obtained into washing buffer；
3, set: 2 negative control holes and 2 Positive control wells, remaining is sample to be tested hole.
4, sample pre-dilution to be measured: use sample diluting liquid by measuring samples serum, negative control sera, positive control blood
Clearly according to the dilution proportion of 1:20.
5, be loaded: each hole is respectively by presetting plus the 100 diluted samples to be tested of μ l.Being loaded process time span should be as far as possible
It is short.
6, incubate: concussion mixes, and sets in 37 DEG C of incubators, reacts 30min.
7, board-washing: discarding reaction solution, and every hole adds the washing buffer after 300 μ l dilution, impregnates 15s, get rid of abandoning washing lotion, continuously
It is patted dry after board-washing 4 times.
8, enzyme: each hole adds 100 μ l horseradish peroxidase-labeled rabbit-anti pig IgG antibody.
9, it incubates: setting 37 DEG C of incubators, react 30min.
10, board-washing: discarding reaction solution, and the 300 μ l of washing buffer after dilution is added in every hole, impregnates 15s, gets rid of abandoning washing
Liquid pats dry after continuous board-washing 4 times.
11, substrate solution A and substrate solution B mixed in equal amounts (are substrate work by the 100 μ l substrate working solutions of every hole addition that develop the color
Liquid, matching while using), concussion mixes, and sets in 37 DEG C of incubators, is protected from light 15min.
12, colour developing 50 μ l of terminate liquid is added in every hole, and oscillation, which mixes, terminates reaction.
13, the OD in every hole is measured450nm(OD should be read to value in 15min by adding the reaction plate of terminate liquid450nmValue).
The judgement of testing result:
1, negative control OD450nmAverage value should≤0.15, otherwise in vain.
2, each detected value of positive control should be between 1.0~2.5, otherwise in vain.
3, the calculating of critical value: critical value=0.17 × positive control OD450nmIt is worth average value.
Serum to be checked measures OD450nmValue >=critical value person is judged to the positive；Serum to be checked measures OD450nmValue < critical value person sentences
For feminine gender.
Mentioned reagent box of the invention can be used for detecting Seneca Valley virus structural proteins antibody, to judge tested animal
With the presence or absence of the Seneca Valley virus structural proteins antibody generated after infection.
Application in the kit that preparation detects whether infection Seneca Valley virus disease also belongs to protection model of the invention
It encloses；
The positive effect of the present invention is: the present invention is using bioinformatics method to Seneca Valley virus structural proteins
Epitope carries out Accurate Analysis, and suitable ELISA detection is filtered out from the Main Antigenic on VP1, VP2, VP3 albumen and is used
Peptide fragment.The peptide fragment has concentrated epitope, has the advantages that sensitivity height, high specificity.
Meanwhile being used to be coated with the preparation of enzyme reaction plate using advanced technology for solid phase synthesis of peptide synthetic polypeptide antigen.
In addition, the envelope antigen as used in kit is chemically synthesized polypeptide, foreign protein, purity is high, into one are free of
Step improves the efficiency of detection Seneca Valley virus structural proteins antibody, to judge whether tested animal infects Sai Nika paddy disease
Poison.
In short, Antigenic Peptide coating of this kit using chemical synthesis Structural protein VP1, VP2, VP3 major antigenic sites
Enzyme-linked reaction plate, antigen dosage is few, high sensitivity, high specificity, after Seneca Valley virus virus infection can be effectively detected
The structural proteins antibody of generation, to judge whether tested animal infects Seneca Valley virus virus.The experimental results showed that the present invention
Kit it is reproducible, high specificity, high sensitivity.The needs of different levels personnel are able to satisfy, before having a vast market
Scape and good economical, societal benefits.
Whether Seneca Valley virus structural proteins enzyme-linked immunologic detecting kit according to the present invention is for detecting animal
Seneca Valley virus is infected, the foundation of China's Seneca Valley virus prevention and control system is conducive to.
Specific embodiment
Method in following embodiments is unless otherwise instructed conventional method.
The preparation of embodiment 1, Seneca Valley virus structural proteins antibody ELISA immunity detection reagent envelope antigen
This test is using bioinformatics method to the major antigen table of Seneca Valley virus Structural protein VP1, VP2, VP3
Position carries out Accurate Analysis, filters out suitable peptide fragment, is respectively synthesized out four peptides with full-automatic polypeptide synthetic instrument, sequence is respectively
In sequence table shown in sequence 1, sequence 2, sequence 3 and sequence 4, the more full envelope antigen of the update of purity about 80% is made, it can
The main Neutralization and crystallization for covering Seneca Valley virus virus, improves the recall rate of antibody positive.Polypeptide synthesis method can be
Conventional method, the present invention synthesize four polypeptides of the invention, the coating antigen as kit of the present invention with the following method.
Applied Biosystem full-automatic polypeptide synthetic instrument (model 433A) system can be used in envelope antigen of the invention
It is standby.With Merrifield solid-phase synthesis, using Fmoc (9-fluorenylmethyloxycarbonyl, 9- fluorenes first
Oxygen carbonyl) modification amino acid, use Rink Amide MBHA resin as solid phase carrier.Production process includes polypeptide antigen
Synthesis in solid state, polypeptide cleavage and identification, five parts of antigen purification, freeze-drying and preservation.It is illustrated individually below:
One, envelope antigen synthesis in solid state
1, the preparation of synthetic agent
Synthesize envelope antigen amino acid sequence such as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID
Shown in NO:4.
Prepare the amino acid (purchased from NOVA company) of suitable Fmoc modification according to envelope antigen sequence and synthesis scale,
It is added into corresponding Cartridge.Equally synthesis scale claims resin 5g as required, is put into reaction chamber, upper and lower lid is tightened,
Labelling records the title of synthesized peptide, lot number, the weight of the TARE of reaction chamber and alleged resin.Reaction chamber is packed into and is synthesized
Instrument.Prepare suitable synthetic agent include 100% NMP, 3% AIM (acyl imidazoles), 35% PIP (piperidines), 100%
MeOH (methanol) etc. be placed into corresponding reagent bottle.
2, the detection of synthesizer state
It checks 433A Peptide systhesis instrument whether normal operation, after booting, runs Run Self Test program, instrument is certainly
Whether normal examine indices.In addition check whether nitrogen is sufficient, whether normal (the normal gauge pressure of 433A of system gauge pressure
10.2psi).The performance of reply instrument is had gained some understanding before synthesis, so to be measured to the flow velocity of every kind of synthetic agent.433A
Synthesizer: Flow Rate1-18 is sent to synthesizer, Main Menu-Module Test-is selected to look for by Prer or next
Module A, ModuleD, ModuleI, ModuleI, Module A)-measured or observed by more by Start-, if
Flow is improper, then adjusts lower valve pressure, until reaching requirement (specific testing requirements see the table below 1).
1. Peptide synthesizer flow rate detection standard scale of table
| Reagent | Bottle number | Module | Critical field |
| 35%Piperidine | 1 | A | 1.0~1.2ml |
| 3%AIM | 4 | D | 1.0~1.2ml |
| 100%MeOH | 9 | I | 3.5~4.0ml |
| DIC | 8 | I | 0.45~0.55g |
| 100%NMP | 10 | A | 2.6~2.8ml |
3, envelope antigen synthesis starts
The amino acid sequence that will be synthesized in the program of 433A synthesizer sends Std Fmoc 1.0Sol DIC90 to conjunction
On Cheng Yi.The sequence of File-New-Sequence- Edit and Compose peptide saves.Whether File-New-Run checks Chemistry
For Std Fmoc 1.0Sol DIC 90；Whether Sequence is to be deposited name；Set Cycles；It saves.It is finally sent to close
On Cheng Yi.
Main Menu-Cycle Monitor-begin, brings into operation.
4, envelope antigen synthesis carries out
The removing of Fmoc group, the electron attraction of the fluorenes ring system of Fmoc group make 9-H have acidity, are easily removed compared with weak base
It goes, when reaction is eliminated to form hexichol fluorenes alkene with piperidines (PIP) attack 9-H, β, it is easy to be formed by second level cyclammonium attack stable
Addition product.After the removing of Fmov group, "-NH2 " group is exposed to carry out synthetic reaction.Then the next of activation is added
In the amino acid and I-hydroxybenzotriazole (1-hydroxybenzotriazole, HOBT) to reactor of Fmoc radical protection.
Such as above-mentioned polypeptide sequence, synthesis when is since C-terminal to N-terminal, according to specific sequence, successively constantly
It repeats synthesis step (synthesizer is automatically performed by program, specific circulation step such as the following table 2).Period observe and record reagent dosage and
Operating condition.
2. envelope antigen of table synthesizes circulation step
5, envelope antigen synthesis terminates
Synthesizer will be automatically stopped after envelope antigen synthesizes, and peptide resin (peptide is additionally attached on resin now) base
This washes clean.Then reactor is removed from Peptide synthesizer, then after washing peptide resin 3 times with 100% methanol, in draught cupboard
Then polypeptide resin is fully transferred in the polyethylene bottle of brown by interior drying, be put into -20 DEG C of refrigerators, and sealed membrane sealing is standby
With.
Two, the cracking and identification of envelope antigen
1, the cracking of polypeptide antigen
It through the obtained polypeptide of above-mentioned reaction is chemically bound together with solid phase carrier, it is necessary to by specific
The acidolysis of organic acid polypeptide is separated with solid phase carrier.Also the guarantor on each amino acid functional group is eliminated while acidolysis
Protect base.Steps are as follows:
The polypeptide resin (referring to that peptide is additionally attached on resin) that synthesis is taken out out of refrigerator, is put into the round-bottomed flask of a 2L
It is interior, the tripropyl of 90ml trifluoroacetic acid (Trifluoroacetic acid, TFA), 10ml is added into flask in draught cupboard
Flask, is then steadily placed on magnetic stirring apparatus by silane (TIS) and magnetic stick, and persistently stirring 1h extremely reacts at room temperature
Completely.After reaction, the TFA in 30~120min removing crude product is persistently evaporated using the Rotary Evaporators with cold-trap.So
The crude product of polypeptide antigen is cleaned multiple times with dimethylformamide (DMF) afterwards, finally by the resin mixed sand core funnel
It filters out, both obtains envelope antigen.
2, the identification of envelope antigen
Polypeptide antigen is high with substance assistant laser desorpted winged examination time mass spectrum method (MODAL-TOF) and reverse phase after synthesizing
Pressure liquid chromatography (RP-HPLC) carries out qualitative and quantitative analysis, and synthesized peptide is identified with common amino acid analysis.
3, envelope antigen purifies
Polypeptide antigen after cyclisation is carried out ultrafiltration using circulating tangential flow filtration film packet (to be produced with PALL company
The circulating tangential flow filtration film packet of Tangential Flow Device and peristaltic pump matched with its), polypeptide antigen is as big
Molecule cannot be by the filter membrane of certain pore size, and the small molecule that synthesis process early period and later period cyclization are formed or introduced is miscellaneous
Matter can then pass through filter membrane.Then passing through aperture again is 0.2 μm of filter degerming, and last acquired solution is dispensed into aseptic plastic
It is labelled in bottle.Title, number, product batch number, concentration, date of manufacture, pot-life and the preservation of polypeptide are indicated on label
Condition, after packing, be stored in -20 DEG C or -40 DEG C it is spare.
4, envelope antigen is freeze-dried
For the ease of long-term preservation and transport, need for envelope antigen to be freeze-dried to obtain the more of solid state
Peptide.The envelope antigen freezed in advance is placed on the freeze drier of Labconco and is dried, solid state is obtained
Envelope antigen.It is labelled after packaging.Title, number, product batch number, the concentration, date of manufacture, preservation of polypeptide are indicated on label
Time limit and preservation condition.
The preparation of embodiment 2, Seneca Valley virus structural proteins antibody ELISA immunity detection reagent
Seneca Valley virus structural proteins antibody ELISA immunity detection reagent includes:
(1) it is coated with the removable polystyrene enzyme-linked reaction plate in 96 holes of Seneca Valley virus antigen；2 × 96 holes.
(2) positive control serum: being the positive to acquire Swine serum after Seneca Valley virus artificial challenge, as kit
Control serum (1 pipe, 1.5ml/ pipe).
(3) negative control sera: being the Swine serum of no-special pathogen (SPF), the negative control sera as kit
(1 pipe, 1.5ml/ pipe).
(4) ELIAS secondary antibody: being with horseradish peroxidase-labeled rabbit-anti pig IgG (purchased from sigma company, article No. A5670)
It is made after carrying out 1:30000 dilution as stoste, 2 bottles (12ml/ bottles).
(5) sample diluting liquid: for the 0.01M containing 0.5% (g/100ml) casein, the phosphate-buffered that pH value is 7.4
Liquid, 1 bottle (24ml/ bottles).
(6) substrate solution A: for the citrate phosphate buffer (1 bottle, 12ml/ bottles) of the hydrogen peroxide urea containing 0.6mg/ml
(7) substrate solution B: for tetramethyl benzidine (TMB) solution (1 bottle, 12ml/ bottles) of 0.2mg/ml.
(8) terminate liquid: the sulfuric acid solution (1 bottle, 12ml/ bottles) of 2mol/L.
(9) 20 times of concentrated cleaning solutions: for 0.01M, pH containing the Tween-20 that concentration is 0.8%~1.2% (ml/ml)
The phosphate buffer (50ml/ bottles, 2 bottles) that value is 7.4.
As needed, serum can also dilutes plate (2 pieces, 96 holes/block) in kit, the dilution for blood serum sample.
Wherein, be coated with the removable polystyrene enzyme-linked reaction plate in 96 holes of Seneca Valley virus antigen the preparation method comprises the following steps:
1. polypeptide antigen prepared by embodiment 1 to be dissolved in the carbonate solution of pH 9.6, it is then added to 96 hole polystyrene integrated enzyme reactions
Plate, (wherein polypeptide shown in sequence 1 is 50ng to every hole 200ng polypeptide in sequence table, and polypeptide shown in sequence 2 is in sequence table
50ng, polypeptide shown in sequence 3 is 50ng in sequence table, and polypeptide shown in sequence 4 is 50ng in sequence table), 8 are placed at 2~8 DEG C
It~12 hours, combines polypeptide antigen sufficiently with enzyme-linked reaction plate, is then added according to 300 holes μ l/ and contains 1% (g/ml) ox blood
The PBS buffer solution of pure albumen (BSA) pH7.4,37 DEG C Seal treatment 2~3 hours, after drying, 4 after enzyme-linked reaction plate is dry
It DEG C is sealed.
The sensitivity tests of embodiment 3, Seneca Valley virus structural proteins antibody ELISA immunity detection reagent
One, the application method of Seneca Valley virus structural proteins antibody ELISA immunity detection reagent
1, it balances: kit being taken out from cold storage environment, it is spare to set equilibrium at room temperature 30min；The preceding mixing of liquid reagent.
2, match liquid: the 20 times of dilutions of 20 times of concentrated cleaning solution distilled water or deionized water are obtained into washing buffer；
3, set: 2 negative control holes and 2 Positive control wells, remaining is sample to be tested hole.
4, sample pre-dilution to be measured: use sample diluting liquid by measuring samples serum, negative control sera, positive control blood
Clearly according to the dilution proportion of 1:20.
5, be loaded: each hole is respectively by presetting plus the 100 diluted samples to be tested of μ l.Being loaded process time span should be as far as possible
It is short.
6, incubate: concussion mixes, and sets in 37 DEG C of incubators, reacts 30min.
7, board-washing: discarding reaction solution, and every hole adds 300 μ l washing buffers, impregnates 15s, gets rid of abandoning washing lotion, continuous board-washing 4 times
After pat dry.
8, enzyme: each hole adds 100 μ l horseradish peroxidase-labeled rabbit-anti pig IgG antibody.
9, it incubates: setting 37 DEG C of incubators, react 30min.
10, board-washing: discarding reaction solution, and 300 μ l of washing buffer is added in every hole, impregnates 15s, gets rid of and abandons cleaning solution buffer,
It is patted dry after continuous board-washing 4 times.
11, substrate solution A and substrate solution B mixed in equal amounts (are substrate work by the 100 μ l substrate working solutions of every hole addition that develop the color
Liquid, matching while using), concussion mixes, and sets in 37 DEG C of incubators, is protected from light 15min.
12, colour developing 50 μ l of terminate liquid is added in every hole, and oscillation, which mixes, terminates reaction.
13, the OD in every hole is measured450nm(OD should be read to value in 15min by adding the reaction plate of terminate liquid450nmValue).
The judgement of testing result:
1, negative control OD450nmAverage value should≤0.15, otherwise in vain.
2, each detected value of positive control should be between 1.0~2.5, otherwise in vain.
3, the calculating of critical value: critical value=0.17 × positive control OD450nmIt is worth average value.
Serum to be checked measures OD450nmValue >=critical value person is judged to the positive；Serum to be checked measures OD450nmValue < critical value person sentences
For feminine gender.
Two, to the sensitivity tests of known positive serum
It is tried using three batches of Seneca Valley virus structural proteins antibody ELISA immune detections of the method preparation according to embodiment 2
Agent box (batch ZM2017001, ZM2017002, ZM2017003), respectively in accordance with above-mentioned Seneca Valley virus structural proteins antibody
Enzyme-linked immunologic detecting kit application method to 35 parts of Swine serum of Seneca Valley virus infection, (face by Seneca Valley virus infection
The Swine serum that different time acquires after bed symptom) sensitivity tests is carried out, experimental result is shown in Table 3, Sai Nika paddy disease of the invention
Malicious structural proteins antibody ELISA immunity detection reagent detects 34 parts altogether, has 1 part to be not detected, the results showed that this kit is to 35
The sensibility of positive serum known to part is 97.1%.
The sensitivity Detection result of 3. Seneca Valley virus structural proteins antibody ELISA immunity detection reagent of table
| Kit lot number | Recall rate | Sensibility |
| ZM2017001 | 34/35 | 97.1% |
| ZM2017002 | 34/35 | 97.1% |
| ZM2017003 | 34/35 | 97.1% |
Three, lowest detection limitation test
3 parts of Swine serum for choosing Seneca Valley virus structural proteins antibody positive carry out doubling dilution 1:20,1:40~1:
320, the three batches of Seneca Valley virus structural proteins antibody ELISA immunity detection reagent (batches prepared using embodiment 2
ZM2017001, ZM2017002, ZM2017003), it is tried according to above-mentioned Seneca Valley virus structural proteins antibody ELISA immune detection
Agent box application method detects doubling dilution Swine serum, the results showed that, kit of the present invention can detecte 1:160 times
Diluted positive serum, wherein table 4 is the experimental result of one of batch.In table 4, positive control: being with Sai Nika paddy disease
Swine serum is acquired after malicious artificial challenge, the positive control serum (1 pipe, 1.5ml/ pipe) as kit.Negative control: being no spy
Determine pathogen (SPF) Swine serum, the negative control sera (1 pipe, 1.5ml/ pipe) as kit.Critical value (Cut-off value)
=0.17 × positive control OD450nmIt is worth average value.
The lowest detection limitation testing inspection knot of 4 Seneca Valley virus structural proteins antibody ELISA immunity detection reagent of table
Fruit
The specific test of embodiment 4, Seneca Valley virus structural proteins antibody ELISA immunity detection reagent
Using three batches of kits in embodiment 2 according to Seneca Valley virus structural proteins antibody described in embodiment 3
The application method of enzyme-linked immunologic detecting kit is to 50 parts of healthy Swine serum (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd.
There is provided), (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd. mentions O-shaped (FMD-O) positive serum of 2 parts of swine foot-and-mouth disease virus
For), 2 parts of swine foot-and-mouth disease virus A type (FMD-A) positive serums (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd.'s offer), 2
Part swine pox positive serum (SVD) (Zhongmu Industry Co., Ltd's offer), 2 parts of pig vesicular stomatitis virus (VSV) sun
Property serum (Zhongmu Industry Co., Ltd's offer), is detected respectively.
The specific detection result such as following table (table 5) of kit is shown, is shown to the testing result of 50 parts of healthy Swine serums,
The specificity of ZM2017001 kit is that the specificity of 100.0%, ZM2017002 kit is 100.0%, ZM2017003 examination
The specificity of agent box is 100.0%.To O-shaped (FMD-O) positive serum of 2 parts of swine foot-and-mouth disease virus, 2 parts of swine foot-and-mouth disease virus A types
(FMD-A) inspection of positive serum, 2 parts of swine pox (SVD) positive serums, 2 parts of pig vesicular stomatitis virus (VSV) positive serums
It surveys result and is illustrated as feminine gender, therefore the specificity that three kits detect the Antigen positive hybridomas serum of this 8 parts of related diseases is
100%.
5 Seneca Valley virus structural proteins antibody ELISA immunity detection reagent specific detection result of table
The coincidence rate test of embodiment 5, Seneca Valley virus structural proteins antibody ELISA immunity detection reagent
One, virus neutralization tests (VNT) method
The generally acknowledged detection method of Seneca Valley virus is virus neutralization tests (VNT) at present, therefore with this kit with disease
Malicious neutralization test carries out coincidence rate test.
1. material
1.1 infection Swine serums, 24 parts (serum number I1~I24), to there are clinical symptoms after Seneca Valley virus infection
The Swine serum acquired afterwards is inactivated through 56 DEG C of 30min, is provided by Zhongmu Industry Co., Ltd.
1.2 healthy Swine serums, 24 parts (serum number N1~N24), inactivate, You Zhongmu industry share has through 56 DEG C of 30min
Limit company provides.
1.3 tests are the PK15 cell toxicant of Seneca Valley virus with virus (SVV virus), adjust its malicious valence extremely
400TCID50/ 0.1ml is placed in -20 DEG C and saves for use.
2. method virus neutralization tests method is as follows: after inactivating blood serum, carrying out doubling dilution (25 μ l/ with DMEM culture medium
Hole), each dilution does 3 repetitions；Isometric (25 hole μ l/) 100TCID is added in every hole50SVV viral (except control), set
It is incubated for 1 hour in 37 DEG C；Then every hole, which is added, uses the diluted 100 μ l PK15 cell (2~3 × 10 of DMEM culture medium4), it is placed in
37 DEG C, 5%CO2Incubator culture observed cytopathy (CPE) after 72 hours.The last one repressed dilution (90 of CPE
~100% is suppressed) it is considered as virus neutralization tests (VNT) potency, potency >=1:64 is judged to the positive.
Two, coincidence rate test result:
The Seneca Valley virus structural proteins antibody ELISA immunity detection reagent and virus prepared using embodiment 2 is neutralized
It tests (VNT) and detects 24 parts of Seneca Valley virus Swine serums and 24 parts of healthy serum respectively.
Seneca Valley virus structural proteins antibody ELISA immunity detection reagent infects 24 parts the testing result of Swine serum
6 are shown in Table, the sensibility of kit of the present invention is 95.8%, and the sensibility of virus neutralization tests is 87.5%, is infected at 24 parts
In Swine serum, consistent two methods testing result is 22 parts.Therefore, the coincidence rate of kit of the present invention and virus neutralization tests
It is 91.7%, kit sensibility with higher of the present invention.The coincidence rate of both tests to healthy Swine serum is 100%,
Testing result is negative (being shown in Table 6).
Seneca Valley virus structural proteins antibody ELISA immunity detection reagent and virus neutralization tests detect altogether 48 parts
Swine serum, wherein 46 parts of Swine serum two methods testing results are consistent, 2 parts of Swine serum testing results are variant, and coincidence rate is
95.8%.
6 Seneca Valley virus structural proteins antibody ELISA immunity detection reagent of table and virus neutralization tests method are to infection pig
The testing result of serum and healthy Swine serum
Specific description of embodiments of the present invention above is not intended to limit the present invention, and those skilled in the art can be according to this
Invention is variously modified or deforms, and as long as it does not depart from the spirit of the invention, should belong to the model of appended claims of the present invention
It encloses.
Sequence table
<110>Zhongmu Industry Co., Ltd
<120>Seneca Valley virus structural protein antigen epitope polypeptide and its application
<130> WHOI190003
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 37
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 1
Ser Asp Leu Glu Val Thr Val Val Ser Leu Glu Pro Asp Leu Glu Phe
1 5 10 15
Ala Val Gly Trp Phe Pro Ser Gly Ser Glu Tyr Gln Ala Ser Ser Phe
20 25 30
Val Tyr Asp Gln Leu
35
<210> 2
<211> 36
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 2
Thr Lys Ser Asp Pro Pro Ser Ser Ser Thr Asp Gln Pro Thr Thr Thr
1 5 10 15
Phe Thr Ala Ile Asp Arg Trp Tyr Thr Gly Arg Leu Asn Ser Trp Thr
20 25 30
Lys Ala Val Lys
35
<210> 3
<211> 38
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 3
Pro Tyr Ile Gly Glu Thr Pro Thr Gln Ser Ser Glu Thr Gln Asn Ser
1 5 10 15
Trp Thr Leu Leu Val Met Val Leu Val Pro Leu Asp Tyr Lys Glu Gly
20 25 30
Ala Thr Thr Asp Pro Glu
35
<210> 4
<211> 34
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 4
Thr Leu Ser Glu Ala Met Gln Cys Thr Tyr Ser Ile Trp Asp Ile Gly
1 5 10 15
Leu Asn Ser Ser Trp Thr Phe Val Val Pro Tyr Ile Ser Pro Ser Asp
20 25 30
Tyr Arg
Claims (10)
1. Seneca Valley virus structural protein antigen epitope polypeptide is polypeptide shown in sequence 1 in sequence table, sequence in sequence table
Polypeptide shown in sequence 4 in polypeptide shown in sequence 3 or sequence table in polypeptide, sequence table shown in 2.
2. Seneca Valley virus structural protein antigen epitope polypeptide composition is polypeptide, sequence table shown in sequence 2 in sequence table
Any combination of one or more of polypeptide shown in sequence 4 in polypeptide shown in middle sequence 3 or sequence table.
3. Seneca Valley virus structural protein antigen epitope polypeptide composition according to claim 1, it is characterised in that: work as institute
When to state peptide composition be polypeptide shown in sequence 2, two kinds in polypeptide shown in polypeptide and sequence 4 shown in sequence 3, two kinds
The mass ratio of polypeptide is (0.5~1.5): (0.5~1.5)；Preferably, their mass ratio is 1:1；When the peptide composition
When for three kinds in polypeptide shown in polypeptide shown in polypeptide shown in sequence 2, sequence 3 and sequence 4, the matter of any three kinds of polypeptides
Amount is than being (0.5~1.5): (0.5~1.5): (0.5~1.5)；Preferably, their mass ratio is 1:1:1.
4. a kind of Seneca Valley virus structural proteins antibody ELISA immunity detection reagent, including enzyme-linked reaction plate, positive control
Wherein the enzyme-linked reaction plate is coated with Sai Nika paddy described in claim 2 or 3 for serum, negative control sera and ELIAS secondary antibody
Virus structural protein antigen epitope polypeptide composition.
5. enzyme-linked immunologic detecting kit according to claim 4, it is characterised in that: the enzyme-linked reaction plate is detachable
96 hole elisa Plates；Each polypeptide is that chemistry is artificial synthesized in the Seneca Valley virus structural protein antigen epitope polypeptide composition
It obtains.
6. enzyme-linked immunologic detecting kit according to claim 4, it is characterised in that: the acquisition side of the enzyme-linked reaction plate
Method is the pH 9.6 that Seneca Valley virus structural protein antigen epitope polypeptide composition as claimed in claim 1 or 2 is dissolved in 100 μ l
Carbonate solution, be then added to 96 hole polystyrene enzyme-linked reaction plates, every hole 200ng Seneca Valley virus structural protein antigen
Epitope polypeptide composition is placed 8~12 hours at 2~8 DEG C, makes Seneca Valley virus structural protein antigen epitope polypeptide composition
It is sufficiently combined with enzyme-linked reaction plate, it is slow that the PBS containing 0.01g/ml bovine serum albumin(BSA) pH7.4 then is added according to 300 holes μ l/
Fliud flushing, 37 DEG C Seal treatment 2~3 hours, after drying, be sealed for 4 DEG C after enzyme-linked reaction plate is dry.
7. enzyme-linked immunologic detecting kit according to claim 4, it is characterised in that:
The positive control serum is the Swine serum that acquires after the Seneca Valley virus artificial challenge；The negative control sera
For the Swine serum of no-special pathogen.
8. enzyme-linked immunologic detecting kit according to claim 4, it is characterised in that: the ELIAS secondary antibody is horseradish peroxide
Compound enzyme marks rabbit-anti pig IgG antibody.
9. enzyme-linked immunologic detecting kit according to claim 4, it is characterised in that: the kit further includes substrate solution
A, substrate solution B and terminate liquid, the substrate solution A are the citrate phosphate buffer of the hydrogen peroxide urea containing 0.0006g/ml,
The substrate solution B is the tetramethyl biphenyl amine aqueous solution of 0.2mg/ml, is mixed both when use with the ratio of 1:1；The terminate liquid
For the sulfuric acid solution of 2mol/L；
The kit further includes sample diluting liquid and 20 times of concentrated cleaning solutions；Sample diluting liquid is to contain 0.00005g/ml junket
The 0.01mol/L of albumen, the phosphate buffer that pH value is 7.4；Concentrated cleaning solution be containing concentration expressed in percentage by volume be 0.8%~
The 0.01mol/L of 1.2% Tween-20, the phosphate buffer that pH value is 7.4.
10. Seneca Valley virus structural protein antigen epitope polypeptide described in claim 1 monitors whether infection plug Buddhist nun in preparation
Block the application in the enzyme-linked immunologic detecting kit of paddy virus.
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| CN201910028355.4A CN109627293B (en) | 2017-08-13 | 2017-08-13 | Seikaga valley virus structural protein epitope polypeptide and application thereof |
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Cited By (3)
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| CN107253978A (en) * | 2017-08-13 | 2017-10-17 | 中牧实业股份有限公司 | Sai Nika paddy virus structural protein antibody ELISA immunity detection reagents |
| CN110196325A (en) * | 2019-06-24 | 2019-09-03 | 中国动物疫病预防控制中心(农业农村部屠宰技术中心) | Senecan virus diagnostic kit and test paper |
| CN110229218A (en) * | 2019-06-24 | 2019-09-13 | 中国动物疫病预防控制中心(农业农村部屠宰技术中心) | Detect the reagent and its polypeptide used of Senecan antiviral antibody |
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| CN109627292B (en) * | 2018-01-03 | 2021-02-09 | 中牧实业股份有限公司 | Porcine circovirus type 3 epitope polypeptide and application thereof |
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| CN108761074A (en) * | 2018-05-23 | 2018-11-06 | 中国农业科学院兰州兽医研究所 | Senecan virus ELISA antibody assay kit and preparation method, application |
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| CN107253978A (en) * | 2017-08-13 | 2017-10-17 | 中牧实业股份有限公司 | Sai Nika paddy virus structural protein antibody ELISA immunity detection reagents |
| CN107253978B (en) * | 2017-08-13 | 2020-11-20 | 中牧实业股份有限公司 | Enzyme-linked immunoassay kit for structural protein antibody of Seneca valley virus |
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| CN112433049A (en) * | 2019-06-24 | 2021-03-02 | 中国动物疫病预防控制中心(农业农村部屠宰技术中心) | Reagent for detecting Seneca virus antibody and application thereof |
| CN112433049B (en) * | 2019-06-24 | 2023-08-18 | 中国动物疫病预防控制中心(农业农村部屠宰技术中心) | Reagent for detecting sai card virus antibody and application thereof |
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| CN109627293B (en) | 2021-02-09 |
| CN107253978A (en) | 2017-10-17 |
| CN107253978B (en) | 2020-11-20 |
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