CN109627292A - 3 type antigen epitope polypeptide of pig circular ring virus and its application - Google Patents
3 type antigen epitope polypeptide of pig circular ring virus and its application Download PDFInfo
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Abstract
The invention discloses 3 type antigen epitope polypeptide of pig circular ring virus and its applications.The antigen epitope polypeptide be in sequence table in polypeptide, sequence table shown in sequence 1 in polypeptide shown in sequence 2 or sequence table one or more of polypeptide shown in sequence 3 any combination.Antigen epitope polypeptide of the invention can be used for indirect ELISA, use chemical synthesis Antigenic Peptide coated elisa plate, antigen dosage is few, sensitivity and specificity are high, it can efficiently detect whether that there are 3 type antibody of pig circular ring virus, with sensibility height, specificity is good and operation is convenient, has good market prospects.
Description
The application is that application No. is " 201810004832.9 ", entitled " 3 type ELISA antibody of pig circular ring virus inspections
The divisional application of the patent application of test agent box "
Technical field
The invention belongs to technical field of biological, more particularly it relates to a kind of 3 type ELISA of pig circular ring virus
Antibody assay kit.
Background technique
Pig circular ring virus (Porcine circovirus, PCV) is the smallest animal virus having now been found that, is annulus
One of the important member of Viraceae Circovirus.According to its antigenicity, nucleotide sequence and pathogenic, two can be divided into
Genotype (genotype): 1 type and 2 types, i.e. PCV1 and PCV2.Wherein PCV1 no pathogenicity, is widely present in pig body and pig
In the continuous cell line of source, and PCV2 is then one of pathogen very harmful to world's pig breeding industry at present, to the aquaculture in the whole world
It has brought tremendous economic losses, pmws (Postweaning multisystemic can be caused
Wasting syndrome, PMWS), piglet congenital tremors, pigskin inflammation nephrotic syndrome, porcine respiratory syndrome, sow it is numerous
Grow obstacle and intestines problem.
It is 17~20nm that the virion of PCV maturation, which is assembled into a diameter by 60 nucleocapsid protein matter (Cap) subunits,
Regular dodecahedron spheric granules, viral genome package wherein, encodes two main open reading frame (Open
Reading frame, ORFs): cap and rep (duplication relevant enzyme).Cap protein is its unique structural proteins and major antigen,
It can induce body and generate effective immunoprotection reaction.
In October, 2016, Kansas state university, the U.S. and University of California almost report a new PCV genotype simultaneously,
Referred to as PCV3.The virus is isolated from the sow or piglet for occur illness, while detecting PCV2 as feminine gender, and gene order is divided
Analysis display, PCV3 genome include 2000 bases, and Cap protein is made of 214 amino acid residues, and fewer than PCV2 Cap protein 19
~20 amino acid.
Currently, China is successively in relation to the report of PCV3, respectively Guangdong, Fujian, Henan, Jiangxi, Chongqing, Anhui,
The ground such as Hubei, Liaoning detect that PCV3 exists using the detection method of PCR, therefore research and develop easy to operate, specific good, sensibility
Height, can large-scale promotion PCV3 Serum Antibody Detection kit become detection PCV3 antibody one of reliable approach.
Summary of the invention
The purpose of the present invention is to provide 3 type antigen epitope polypeptide of pig circular ring virus and its applications.
3 type antigen epitope polypeptide of pig circular ring virus is polypeptide shown in sequence 1 in sequence table, sequence 2 or sequence in sequence table
Polypeptide shown in sequence 3 in list.
The application is that the 3 type antigen epitope polypeptide of pig circular ring virus is anti-in preparation detection 3 type of pig circular ring virus
Application in the indirect ELISA testing kit of body.
Wherein, for detecting the indirect ELISA testing kit of 3 type antibody of pig circular ring virus, which utilizes pig circle
3 type antigen epitope polypeptide of circovirus virus establishes a species specificity, sensibility and reproducible indirect as envelope antigen
ELISA method, for detecting in Swine serum whether contain 3 type antibody of pig circular ring virus.
In order to achieve the above object, the present invention screens the 3 type epitope of pig circular ring virus being had excellent performance first
Peptide composition, 3 type antigen epitope polypeptide composition of pig circular ring virus provided by the invention, for shown in sequence 2 in sequence table
Any combination of one or more of polypeptide shown in sequence 3 in polypeptide or sequence table.When the peptide composition is
When two kinds in polypeptide shown in polypeptide shown in sequence 2 or sequence 3, the mass ratio of two kinds of polypeptides is (0.5~1.5): (0.5
~1.5)；Preferably, their mass ratio is 1:1.
Wherein, 3 type antigen epitope polypeptide of pig circular ring virus is also protection scope of the present invention, 3 type of pig circular ring virus
Antigen epitope polypeptide is polypeptide shown in sequence 3 in sequence 2 in polypeptide shown in sequence 1 in sequence table, sequence table or sequence table.
3 type ELISA antibody assay kit of pig circular ring virus of the invention, including enzyme-linked reaction plate, positive control serum,
Negative control sera and ELIAS secondary antibody, wherein the enzyme-linked reaction plate is coated with the combination of 3 type antigen epitope polypeptide of pig circular ring virus
Object.
The enzyme-linked reaction plate is detachable 96 hole elisa Plates；The 3 type antigen epitope polypeptide composition of pig circular ring virus
In polypeptide be that chemistry artificial synthesized obtains.
The best preparation method and condition of the enzyme-linked reaction plate are by the 3 type antigen epitope polypeptide group of pig circular ring virus
The carbonate solution that object is dissolved in pH 9.6 is closed, is then added to 96 hole polystyrene enzyme-linked reaction plates, every hole 150ng polypeptide is (wherein
Polypeptide shown in sequence 1 is 50ng in sequence table, and polypeptide shown in sequence 2 is 50ng in sequence table, in sequence table shown in sequence 3
Polypeptide be 50ng), place 8~12 hours at 2~8 DEG C, combine polypeptide antigen sufficiently with enzyme-linked reaction plate, then according to
The PBS buffer solution for containing 1% (g/ml) bovine serum albumin(BSA) (BSA) pH7.4 is added in 300 holes μ l/, and 37 DEG C of Seal treatments 2~3 are small
When, after drying, it is sealed for 4 DEG C after enzyme-linked reaction plate is dry.
The positive control serum is the Swine serum acquired after the 3 type inactivation of viruses of pig circular ring virus is immune；The yin
Property control serum be no-special pathogen (SPF) Swine serum, i.e., without the Swine serum of pig circular ring virus, including such as without pig circular ring virus 2
The healthy Swine serum of poison.
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 peroxidating containing 0.6mg/ml
The citrate phosphate buffer of urea hydrogen, the substrate solution B is the tetramethyl biphenyl amine aqueous solution of 0.2mg/ml, both when use
It is mixed with 1: 1 ratio.The terminate liquid is the sulfuric acid solution of 2mol/L.
The kit further includes sample diluting liquid and concentrated cleaning solution (20 times)；Sample diluting liquid is to contain 0.5% (g/
Ml) 0.01M of casein, the phosphate buffer that pH value is 7.4；It is 0.8%~1.2% that concentrated cleaning solution, which is containing concentration,
(ml/ml) 0.01M of Tween-20, 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=0.18 × 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 3 type antibody of pig circular ring virus, to judge that tested animal whether there is
The 3 type antibody of pig circular ring virus generated after infection.
Application in the kit that preparation detects whether infection 3 type of pig circular ring 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 3 type Cap protein of pig circular ring virus
Epitope carries out the peptide fragment that Accurate Analysis filters out suitable ELISA detection.The peptide fragment has concentrated epitope, has sensitive
Property high, high specificity the advantages of.
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 3 type antibody of pig circular ring virus, to judge whether tested animal infects 3 type of pig circular ring virus.
In short, this kit is coated with using the Antigenic Peptide of 3 type Cap protein major antigenic sites of chemical synthesis pig circular ring virus
Enzyme-linked reaction plate, antigen dosage is few, high sensitivity, high specificity, generates after the infection of 3 type of pig circular ring virus can be effectively detected
Cap protein antibody, to judge whether tested animal infects 3 type of pig circular ring virus.The experimental results showed that kit of the invention
It is reproducible, high specificity, high sensitivity.The needs of different levels personnel are able to satisfy, are had a vast market foreground and well
Economical, societal benefits.
Whether 3 type ELISA diagnostic kit of pig circular ring virus according to the present invention is for detecting animal
3 type of pig circular ring virus is infected, quick diagnosis effectively can be carried out to epidemic disease caused by 3 type of pig circular ring virus.
Specific embodiment
Method in following embodiments is unless otherwise instructed conventional method.
The preparation of embodiment 1,3 type ELISA antibody assay kit envelope antigen of pig circular ring virus
This test is carried out accurate using Main Antigenic of the bioinformatics method to 3 type Cap protein of pig circular ring virus
Analysis, filters out suitable peptide fragment, is respectively synthesized out three peptides with full-automatic polypeptide synthetic instrument, sequence is respectively sequence in sequence table
Shown in column 1, sequence 2 and sequence 3, the more full envelope antigen of the update of purity about 80% is made, 3 type of pig circular ring virus can be covered
Main Neutralization and crystallization, improve the recall rate of antibody positive.Polypeptide synthesis method can be conventional method, and the present invention is using such as
Lower method synthesizes three polypeptides of the invention, the coating antigen as kit of the present invention.
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 SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 in envelope antigen amino acid sequence such as sequence table
It is shown.
SEQ ID NO:1:MRHRAIFRRRPRPRRRRRHRRRYARRRLF；
SEQ ID NO:2:LQDDPYAESSTRKVMTSKKKHSRYFT；
SEQ ID NO:3:EKTGMTDFYGTKEVWIRYKSVL.
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 2,3 type ELISA antibody assay kit of pig circular ring virus
3 type ELISA antibody assay kit of pig circular ring virus includes:
(1) it is coated with the removable polystyrene enzyme-linked reaction plate in 96 holes of 3 type antigen of pig circular ring virus；2 × 96 holes.
(2) positive control serum: being the Swine serum to acquire after 3 type inactivation of virus virus immunity of pig circular ring virus, as
The positive control serum (1 pipe, 1.5ml/ pipe) of kit.
(3) negative control sera: being no-special pathogen (SPF) Swine serum, the negative control sera (1 as kit
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 3 type antigen of pig circular ring virus 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, and every hole 150ng polypeptide (when using single peptide as envelope antigen, polypeptide shown in sequence 1, polypeptide or sequence shown in sequence 2
Polypeptide shown in 3 is respectively 150ng；When mixed with polypeptide shown in polypeptide shown in polypeptide shown in sequence 1, sequence 2 and sequence 3
When cooperation is envelope antigen, polypeptide shown in sequence 1 is 50ng in sequence table, and polypeptide shown in sequence 2 is 50ng in sequence table,
Polypeptide shown in sequence 3 is 50ng in sequence table), it places 8~12 hours at 2~8 DEG C, makes polypeptide antigen and enzyme-linked reaction plate
It sufficiently combines, the PBS buffer solution for containing 1% (g/ml) bovine serum albumin(BSA) (BSA) pH7.4 is then added according to 300 holes μ l/,
37 DEG C Seal treatment 2~3 hours, after drying, be sealed for 4 DEG C after enzyme-linked reaction plate is dry.
The sensitivity tests of embodiment 3,3 type ELISA antibody assay kit of pig circular ring virus
One, the application method of 3 type ELISA antibody assay kit of pig circular ring virus
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=0.18 × 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
Using the 3 type ELISA antibody assay kit of pig circular ring virus of the method preparation according to embodiment 2 (respectively with sequence
Polypeptide shown in column 1, polypeptide or hybrid peptide shown in polypeptide, sequence 3 shown in sequence 2 are as envelope antigen), respectively in accordance with upper
Stating 3 type ELISA antibody assay kit application method of pig circular ring virus (has the infection of 3 type of pig circular ring virus for 35 parts of Swine serum
PDNS and miscarriage symptom, pathological material of disease are positive animal through RT-PCR amplification PCV3-Cap Identification of Fusion Protein) sensitivity tests is carried out, it is real
It tests and the results are shown in Table 3, the kit using sequence 1 as envelope antigen is 85.7% to the sensibility of positive serum known to 35 parts, with
Sequence 2 is 71.4% to the sensibility of positive serum known to 35 parts as the kit of envelope antigen, anti-using sequence 3 as coating
Former kit is 80.0% to the sensibility of positive serum known to 35 parts, to mix 3 peptides as the kit of envelope antigen
Sensibility to positive serum known to 35 parts is 94.3%.
The sensitivity Detection result of 3. pig circular ring virus of table, 3 type ELISA antibody assay kit
| Kit lot number | Recall rate | Sensibility |
| The single peptide of sequence 1 | 30/35 | 85.7% |
| The single peptide of sequence 2 | 25/35 | 71.4% |
| The single peptide of sequence 3 | 28/35 | 80.0% |
| 3 peptide hybrid peptides | 33/35 | 94.3% |
Three, lowest detection limitation test
1 part of Swine serum for choosing 3 type antibody positive of pig circular ring virus carries out doubling dilution 1:20,1:40~1:320, makes
The 3 type ELISA antibody assay kit of pig circular ring virus prepared with embodiment 2 is (respectively with polypeptide shown in sequence 1,2 institute of sequence
Polypeptide shown in the polypeptide that shows, sequence 3 or hybrid peptide are as envelope antigen), according to above-mentioned 3 type ELISA antibody of pig circular ring virus
Detection kit application method detects doubling dilution Swine serum, the results showed that, the examination prepared with polypeptide shown in sequence 1
Agent box can detecte 1:160 times of diluted positive serum, can detecte with kit prepared by polypeptide shown in sequence 2 to 1:
40 times of diluted positive serums can detecte 1:160 times of diluted positive blood with kit prepared by polypeptide shown in sequence 3
Clearly, 1:320 times of diluted positive serum can detecte with kit prepared by hybrid peptide.In table 4, positive control: being with pig
The Swine serum acquired after 3 type inactivation of virus virus immunity of circovirus, positive control serum (1 pipe, 1.5ml/ as kit
Pipe).Negative control: being no-special pathogen (SPF) Swine serum, negative control sera (1 pipe, the 1.5ml/ as kit
Pipe).Critical value (Cut-off value)=0.18 × positive control OD450nmIt is worth average value.
The lowest detection limitation testing inspection result of 4. pig circular ring virus of table, 3 type ELISA antibody assay kit
The specific test of embodiment 4,3 type ELISA antibody assay kit of pig circular ring virus
Using the kit in embodiment 2 (respectively shown in polypeptide shown in polypeptide shown in sequence 1, sequence 2, sequence 3
Polypeptide or hybrid peptide as envelope antigen) according to the type ELISA antibody test reagent of pig circular ring virus 3 described in embodiment 3
The application method of box is to 50 parts of healthy Swine serums (Zhongmu Industry Co., Ltd's offer), 2 parts of porcine circovirus 2 type positive bloods
Clearly (Zhongmu Industry Co., Ltd's offer), (Zhongmu Industry Co., Ltd mentions 2 parts of swine foot-and-mouth disease virus positive serums
For), 2 parts of swine fever positive serums (being purchased from China Veterinery Drug Inspection Office), 2 parts of porcine reproductive and respiratory syndrome positive serums (in
Industry Co., Ltd's offer is provided), it 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,
With the kit specificity of the preparation of polypeptide shown in sequence 1 for 100.0%, with the kit specificity of the preparation of polypeptide shown in sequence 2
It is 100.0%, it is special with kit prepared by hybrid peptide with the kit specificity of the preparation of polypeptide shown in sequence 3 for 100.0%
Property is 100.0%.To 2 parts of 2 type positive serums (PCV2) of pig annulus, 2 parts of Schweineseuche positive serums (FMDV), 2 parts of swine fever sun
Property serum (HC), 2 parts of porcine reproductive and respiratory syndrome positive serums (PRRS) testing result, all kit test results are equal
For feminine gender, therefore the specificity that all kits detect the Antigen positive hybridomas serum of this 8 parts of related diseases is 100%.
5 pig circular ring virus of table, 3 type ELISA antibody assay kit specific detection result
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>3 type antigen epitope polypeptide of pig circular ring virus and its application
<130> WHOI190002
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 29
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 1
Met Arg His Arg Ala Ile Phe Arg Arg Arg Pro Arg Pro Arg Arg Arg
1 5 10 15
Arg Arg His Arg Arg Arg Tyr Ala Arg Arg Arg Leu Phe
20 25
<210> 2
<211> 26
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 2
Leu Gln Asp Asp Pro Tyr Ala Glu Ser Ser Thr Arg Lys Val Met Thr
1 5 10 15
Ser Lys Lys Lys His Ser Arg Tyr Phe Thr
20 25
<210> 3
<211> 22
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 3
Glu Lys Thr Gly Met Thr Asp Phe Tyr Gly Thr Lys Glu Val Trp Ile
1 5 10 15
Arg Tyr Lys Ser Val Leu
20
Claims (10)
1. 3 type antigen epitope polypeptide of pig circular ring virus is polypeptide shown in sequence 1 in sequence table, sequence 2 or sequence in sequence table
Polypeptide shown in sequence 3 in table.
2. 3 type antigen epitope polypeptide of pig circular ring virus described in claim 1 is detected in preparation between 3 type antibody of pig circular ring virus
Connect the application in ELISA detection kit.
3. 3 type antigen epitope polypeptide composition of pig circular ring virus is more shown in sequence 3 in sequence 2 in sequence table and sequence table
Any combination of one or more of peptide.
4. 3 type antigen epitope polypeptide composition of pig circular ring virus according to claim 1, which is characterized in that when described more
When peptide is two kinds in polypeptide shown in sequence 2 and sequence 3, the mass ratio of two kinds of polypeptides is (0.5~1.5): (0.5~1.5)；
Preferably, their mass ratio is 1:1.
5. a kind of 3 type ELISA antibody assay kit of pig circular ring virus, which is characterized in that the kit includes pig annulus
Viral 3 type antigen epitope polypeptide compositions as the enzyme-linked reaction plate of antigen coat, positive control serum, negative control sera and
ELIAS secondary antibody；The 3 type antigen epitope polypeptide composition of pig circular ring virus is claim 2 or pig as claimed in claim 3 circle
3 type antigen epitope polypeptide composition of circovirus virus.
6. kit according to claim 5, which is characterized in that the preparation method of the enzyme-linked reaction plate is to want right
3 type antigen epitope polypeptide composition of pig circular ring virus described in asking 1 or 2 is dissolved in the carbonate solution of the pH 9.6 of 100 μ l, then
It is added to 96 hole polystyrene enzyme-linked reaction plates, every 3 type antigen epitope polypeptide composition of hole 150ng pig circular ring virus, at 2~8 DEG C
Place 8~12 hours, combine 3 type antigen epitope polypeptide composition of pig circular ring virus sufficiently with enzyme-linked reaction plate, then according to
300 holes μ l/ be added the PBS buffer solution containing 0.01g/ml bovine serum albumin(BSA) pH7.4,37 DEG C Seal treatment 2~3 hours, get rid of
After dry, it is sealed for 4 DEG C after enzyme-linked reaction plate is dry.
7. kit according to claim 5, which is characterized in that the positive control serum is with 3 type of pig circular ring virus
The Swine serum acquired after inactivation of virus virus immunity.
8. kit according to claim 5, which is characterized in that the negative control sera is the pig without pig circular ring virus
Serum.
9. kit according to claim 5, which is characterized in that the ELIAS secondary antibody is horseradish peroxidase-labeled rabbit
Anti- pig IgG antibody.
10. kit according to claim 5, which is characterized in that the kit further include substrate solution A, substrate solution B and
Terminate liquid, the substrate solution A are the citrate phosphate buffer of the hydrogen peroxide urea containing 0.6mg/ml, and the substrate solution B is
The tetramethyl biphenyl amine aqueous solution of 0.2mg/ml is mixed both when use with 1: 1 ratio；The terminate liquid is the sulfuric acid of 2mol/L
Solution；The kit further includes sample diluting liquid and concentrated cleaning solution；Sample diluting liquid is to contain 0.5% (g/ml) casein
0.01mol/L, pH value be 7.4 phosphate buffer；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.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110283235A (en) * | 2019-07-18 | 2019-09-27 | 华南农业大学 | PCV3 Cap protein B cell linear epitope polypeptide and its screening and application |
| CN112946262A (en) * | 2021-01-26 | 2021-06-11 | 石河子大学 | PCV3 double-antigen sandwich ELISA antibody detection kit and application thereof |
| CN114805501A (en) * | 2022-03-24 | 2022-07-29 | 中牧实业股份有限公司 | Porcine circovirus type 2 antigen composition and application thereof in antigen quantification method |
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| CN109678935A (en) * | 2018-12-13 | 2019-04-26 | 广西壮族自治区兽医研究所 | PCV3Cap proteantigen polypeptide, the polyclonal antibody of anti-PCV3 Cap protein and its application |
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| CN112946262A (en) * | 2021-01-26 | 2021-06-11 | 石河子大学 | PCV3 double-antigen sandwich ELISA antibody detection kit and application thereof |
| CN114805501A (en) * | 2022-03-24 | 2022-07-29 | 中牧实业股份有限公司 | Porcine circovirus type 2 antigen composition and application thereof in antigen quantification method |
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| CN108196065A (en) | 2018-06-22 |
| CN108196065B (en) | 2020-06-09 |
| CN109627292B (en) | 2021-02-09 |
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