CN108872574A - A kind of chemical luminescence immune analysis reagent box for Seneca Valley virus Structural protein VP1 antibody test - Google Patents
A kind of chemical luminescence immune analysis reagent box for Seneca Valley virus Structural protein VP1 antibody test Download PDFInfo
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Abstract
The invention discloses a kind of chemical luminescence immune analysis reagent boxes for Seneca Valley virus Structural protein VP1 antibody test.The kit includes chemiluminescence immune assay plate, positive control serum, negative control sera, enzyme labelled antibody, Chemoluminescent substrate, luminescence enhancer and the concentrated cleaning solution of Seneca Valley virus Structural protein VP1 antigen coat.The amino acid sequence of the Seneca Valley virus Structural protein VP1 is as shown in SEQ ID NO.2.Detection kit of the invention uses the Structural protein VP1 antigen of prokaryotic expression, and protein yield is high, and antigen purity is determined using enzyme-catalyzed chemical luminescence reaction system as a result, improving detection sensitivity up to 90% or more.Detection kit of the present invention has the advantages that easy to operate, detection time is short, and sensibility is high, high specificity, can be used for the Seneca Valley virus detection and epidemiological survey of a large amount of samples, has good market prospects.
Description
Technical field
The present invention relates to a kind of chemiluminescence immune assay examinations for Seneca Valley virus Structural protein VP1 antibody test
Agent box further relates to the application method of the kit.The invention belongs to technical field of virus detection.
Background technique
Pig Sai Nika paddy disease is that a boar caused by Seneca Valley virus (Seneca Valley virus, SVV) is primary
Property blister sore.The disease mainly causes pig mouth and nose mucous membrane and hoof bubble and ulcer occur.2008 and 2012, the U.S. added and takes
Big that some areas is waited to be diagnosed to be SVV, in September, 2015 in morbidity swinery, there is cyllopodia, bubble and companion in certain pig farm swinery of the U.S.
With newborn piglet death, PCR detection exclude foot and mouth disease virus (FMDV), Porcine epidemic diarrhea virus (PEDV), porcine rotavirus,
The infection of transmissible gastro-enteritis virus (TGEV) and pig breathing and breeding syndrome viral (PRRSV), serum, the skin of morbid pig
Skin, excrement, hoof coronary band PCR testing result be shown as SVV the positive, be finally diagnosed as Seneca Valley virus infection.Together
Year, blister disease occurs in succession for Guangdong Province of China pig farm, and sow fever anorexia, mouth and nose and hoof bubble occur, propagate speed
Degree is fast, and is feminine gender through detection FMD, SVD, VS testing result, SVV is shown as positive, then with delivery room piglet acute death
One plant of Seneca Valley virus strain (CH-01-2015) is isolated, this is that first Chinese report has pig to infect SVV.Although pig plug
Buddhist nun, which blocks paddy disease, not will cause heavy economic losses identical with foot and mouth disease virus, but the disease also have for newborn piglet it is higher
Lethality, clinical symptoms and aftosa, swine pox, vesicular stomatitis etc. are highly similar, bring to identifying and distinguishing the disease
Certain difficulty, currently, there is no effective vaccine and diagnostic reagent for prevention and control SVV.Therefore, develop corresponding vaccine and
Diagnostic reagent is of great significance to prevent and diagnose pig Sai Nika paddy disease.
SVV full-length genome about 7.2kb, including 5 '-U TR, 3 '-UTR, 1 open reading frame (ORF) and 3 ' ends
Poly (A) tail, studies have shown that the RNA of SVV encodes the polyprotein of 1 a length of 2181 amino acid, which can be into one
One-step hydrolysis is leader protein L, structural proteins P1, and non-structural protein P2 and P3, P1 are further had albumen in P3 precursor protein
The 3C of enzymatic activity and some possible cell restriction endonuclease cuttings are decomposed into tri- kinds of structural proteins of VP0, VP1 and VP3, and wherein VP1 is
The main component for inducing neutralizing antibody, is the key structure albumen of antigenic variation, has important antigen site.
Chemiluminescence immunoassay technology (chemiluminescence immunoassay, CLIA) is that will have Gao Ling
The chemical luminescent detecting technology of sensitivity is combined with the immune response of high specific, for various antigens, haptens, antibody, is swashed
The detection and analysis technology of element, enzyme, fatty acid, vitamin and drug etc..It is to exempt from analysis, fluoroimmunoassay after radioimmunology analysis, enzyme
With the newest immunoassay to grow up after time-resolved fluoroimmunoassay.Chemiluminescence immune assay it is quick
Perception and signal-to-noise ratio are high, and background fluorescence signal is low, and the analyte of extremely low concentration can be detected in the very wide range of linearity, can
By extensively being applied many advantages, such as luminometer device quantitative analysis test sample in each field of life science, such as detect
AIDS virus, hepatitis type B virus, detection tumor marker etc., current application of the technology in the diagnosis of Sai Nika paddy disease
It has not been reported.
The present invention is using Seneca Valley virus Structural protein VP1 as candidate gene, and clone obtains the VP1 gene of SVV, with big
It is expressed in enterobacteria and purifies VP1 albumen as envelope antigen and opened in the diagnosis of CLIA technical application to pig Sai Nika paddy disease
Sent out it is a kind of for detecting the chemical luminescence immune analysis reagent box of pig Seneca Valley virus Structural protein VP1 antibody, for from now on
The possibility epidemic situation for coping with SVV, make a good technical reserve be of great significance in advance.
Summary of the invention
The purpose of the present invention is to provide a kind of detection time is short, specificity is good, high sensitivity for Sai Nika paddy disease
The chemical luminescence immune analysis reagent box of malicious Structural protein VP1 antibody test, it is intended to solve to there is no at present in above-mentioned background technique
Effectively, the problem of quickly diagnostic reagent is used for the prevention and control of SVV carries out technology to cope with the possibility epidemic situation of SVV in advance from now on
Deposit.
To achieve the goals above, present invention employs following technological means：
A kind of chemiluminescence immune assay reagent for Seneca Valley virus Structural protein VP1 antibody test of the invention
Box, the kit include chemiluminescence immune assay plate, the negative control of Seneca Valley virus Structural protein VP1 antigen coat
Serum, positive control serum, sample diluting liquid, enzyme labelled antibody, Chemoluminescent substrate, luminescence enhancer and thickening and washing
Liquid.
Wherein, it is preferred that the Seneca Valley virus Structural protein VP1 antigen is obtained through prokaryotic expression system, amino
Acid sequence is as shown in SEQ ID NO.2.
Wherein, it is preferred that the purifying for the Seneca Valley virus Structural protein VP1 for obtaining prokaryotic expression produces
Object is diluted to 2.5 μ g/ml, is coated with chemiluminescence immune assay plate according to 100 holes μ l/, 4 DEG C of refrigerator coatings are overnight.
Wherein, it is preferred that the positive control serum is 14 days after Seneca Valley virus infects high infection titers
Swine serum；The negative control sera is the healthy Swine serum that any vaccine was not immunized.
Wherein, it is preferred that the enzyme labelled antibody is the rabbit-anti pig IgG secondary antibody of horseradish peroxidase-labeled.
Wherein, it is preferred that the sample diluting liquid is trehalose containing 1w/v%, 0.01w/v% thimerosal, 0.01v/
The 0.01M phosphate solution of v%Tween20 and 0.5w/v% casein.
Wherein, it is preferred that the Chemoluminescent substrate is with Tris-HCl buffer containing luminol and right
The mixed liquor of iodophenol, the luminescence enhancer are with Tris-HCl buffer containing hydrogen peroxide and Tween20
Mixed liquor.
It is furthermore preferred that the Chemoluminescent substrate is to contain 0.1mmol/ with 0.05M Tris-HCl buffer
To the mixed liquor of iodophenol, the luminescence enhancer is to be matched with 0.05MTris-HCl buffer by L luminol and 0.1mmol/L
The mixed liquor of the Tween20 of the hydrogen peroxide containing 7.5mmol/L, 0.005v/v% made.
Wherein, it is preferred that the concentrated cleaning solution is 10 × PBST solution, i.e., containing 0.5v/v%Tween20's
0.1mol/L PBS solution, pH 7.4 dilute 10 times when use.
Further, anti-the invention also provides Seneca Valley virus Structural protein VP1 is carried out using the kit
The method that physical examination is surveyed, follows the steps below：
(1) Sample Dilution
By sample to be tested, positive control serum and negative control sera sample diluting liquid according to volume ratio 1：32 carry out
Dilution；
(2) board-washing
The chemiluminescence immune assay plate that Seneca Valley virus Structural protein VP1 antigen coat is taken out from 4 DEG C of refrigerators, beats
Packaging bag is opened, takes out chemiluminescence immune assay plate, with cleaning solution board-washing 3 times diluted, blotting paper is patted dry, each 3min；
(3) it is loaded
Sample to be tested, positive control serum and negative control sera after dilution is separately added into chemiluminescence immunoassay point
It analyses in plate, every hole 100 μ L, 37 DEG C of incubation 5min；
(4) it washs
Chemiluminescence immune assay plate is taken out, blood serum sample is got rid of, is rinsed 4 times with cleaning solution, blotting paper pats dry；
(5) enzyme labelled antibody is added
The rabbit-anti pig IgG secondary antibody of the horseradish peroxidase-labeled diluted, 100 holes μ L/, 37 DEG C of incubation 5min are added；
(6) add substrate：
Enzyme labelled antibody is got rid of, is rinsed 4 times with cleaning solution, blotting paper pats dry, chemiluminescent substrate and luminescence enhancer is added,
It mixes, is protected from light and stands 5min；
(7) luminous value, calculating and judgement are measured：
In measuring luminous value on chemical illumination immunity analysis instrument device, all samples to be tested are indicated with percent positive
(positive percent, PP), reduction formula is as follows：
PP=(sample to be tested luminous value-negative control sera average irradiance) × 100%/(positive control serum is average
Luminous value-negative control sera average irradiance)
Critical value is set to 6.14%, and the positive is determined as if calculated result >=6.14%, otherwise is feminine gender.
Compared to the prior art, the beneficial effects of the present invention are：
(1) detection kit of the present invention is by Seneca Valley virus Structural protein VP1 in E.Coli high efficient expression, and albumen produces
Amount is high, and antigen purity is up to 90% or more；
(2) this kit is determined using enzyme-catalyzed chemical luminescence reaction system as a result, improving detection sensitivity；
(3) chemiluminescence immune analysis method currently about the diagnosis of Sai Nika paddy disease has not been reported, present invention detection
Kit has the advantages that easy to operate, detection time is short, and sensibility is high, high specificity, can be used for a large amount of sample detections and
Epidemiological survey.
Detailed description of the invention
Fig. 1 is VP1 protein expression result provided in an embodiment of the present invention；
In figure：Swimming lane M：Protein Marker (116.0/66.2/45.0/35.0/25.0/18.4/14.4kDa)；
Swimming lane 1：The broken supernatant protein of inducing expression；Swimming lane 2：The broken insoluble protein of inducing expression；
Fig. 2 is renaturation inclusion body protein result provided in an embodiment of the present invention；
In figure：Swimming lane M：Protein Marker (116.0/66.2/45.0/35.0/25.0/18.4/14.4kDa)；
Swimming lane 1：Renaturation inclusion body protein；
Fig. 3 is purification renaturation albumen result provided in an embodiment of the present invention.
In figure：Swimming lane M：Protein Marker (116.0/66.2/45.0/35.0/25.0/18.4/14.4kDa)；
Swimming lane 1：Elute albumen.
Fig. 4 is the ROC analysis curve of VP1-CLIA provided in an embodiment of the present invention.
Specific embodiment
The present invention will be further described combined with specific embodiments below, but following embodiments will not limit in any way
Protection scope of the present invention.
Embodiment 1 is used for the chemical luminescence immune analysis reagent box of Seneca Valley virus Structural protein VP1 antibody test
Preparation and assembling
(1) preparation of kit：
1, the expression of Seneca Valley virus Structural protein VP1 antigen：
The encoding gene 792bp (shown in SEQ ID NO.1) of Seneca Valley virus VP1 albumen is cloned into pET-30a (+)
Prokaryotic expression carrier, after digestion, sequencing are correct, positive plasmid is converted to BL21DE3plysS bacterial strain, and card receives resistance LB plate
Monoclonal is screened, 37 DEG C, LB culture medium shakes bacterium and stays overnight；Bacterium solution (v/v) 1 by volume will be stayed overnight:100 are forwarded to fresh LB culture
Base, 37 DEG C, 200rpm shakes 3h and reaches 0.6 or so to OD600 value, and the IPTG that final concentration 1mmol/L is added continues to shake bacterium 3h,
Bacterium solution is collected by centrifugation in 6000rpm.Cultivation temperature is reduced to 30 DEG C；IPTG inducer is added to final concentration 0.5mM, 30 DEG C are continued to shake
Swing culture 3-4h；8000rpm is centrifuged 3min and collects thallus, is resuspended in 50mL pre-cooling NTA-0 buffer, ice bath 30min；Ultrasound
Broken thallus, 4 DEG C of centrifugation 50min of 16000rpm collect supernatant and precipitating；A small amount of supernatant and precipitating is taken to carry out SDS-PAGE
Detection, protein expression result are as shown in Figure 1.Remaining supernatant and precipitating be placed in 4 DEG C it is spare.
2, inclusion body protein purifying, renaturation：
DTT to final concentration 1mM is added with the resuspension of 50mL NTA-0 buffer in precipitating, and ultrasound promotes foreign protein dissolution, centrifugation
Precipitating is collected, supreme clear bright in triplicate, precipitating is resuspended with PBS, and supernatant is removed in ultrasound, centrifugation, is forgiven with the resuspension of 6M guanidine hydrochloride
Body adds DTT to final concentration 5mM；37 DEG C of concussion 3h are all dissolved to inclusion body, and supernatant is removed in centrifugation.Use protein renaturation liquid by egg again
White low temperature dialysis renaturation is added dropwise to 200mL renaturation solution with the 3M guanidine hydrochloride diluted protein solution of 2 times of volumes at 4 DEG C
(pH8.0) in, rotational speed regulation to maximum, stirring for 24 hours, takes protein solution in bag filter, slow with PBS after being concentrated with PEG60000
Fliud flushing dialysed overnight, the inclusion body protein SDS-PAGE testing result after renaturation are as shown in Figure 2.Albumen anion after renaturation
Exchange column Ni-NTA is purified again, collects albumen.The protein SDS-PAGE testing result of purifying is as shown in figure 3, its amino acid sequence
Column are as shown in SEQ ID NO.2.
3, the selection of antigen coat and best peridium concentration：
Purified product through the prokaryotic expression carries out a series of 5 μ of dilutions with pH=9.6 carbonate buffer solution
G/mL, 2.5 μ g/mL, 1.25 μ g/mL, 0.625 μ g/mL, 0.312 μ g/mL, each two column of concentration coating, 100 holes μ L/ are added to 96
On the chemiluminescence immune assay plate of hole, 4 DEG C of coatings are overnight.The result shows that envelope antigen concentration be 2.5 μ g/mL when P/N value most
Greatly, the luminous value ratio about 160 with feminine gender positive at this time, it is thus determined that the concentration is best peridium concentration.
4, the closing of chemiluminescence immune assay plate：
Above-mentioned chemiluminescence immune assay plate PBST (the 0.01mol/L PBS solution containing 0.05v/v%Tween20, pH
7.4) it washes 4 times, 100 μ l confining liquids (thimerosal of 5w/v% skimmed milk power and 0.01w/v%), 37 DEG C of placement 2h are added in every hole.
Discarding cleaning solution, PBST is washed 3 times, the chemiluminescence immune assay plate of pre-coated antigen is obtained, using aluminium foil bag vacuum seal,
4 DEG C of preservations.
5, the preparation of standard serum：
Susceptible piglet (the pig Seneca Valley virus serum of health of 70 ages in days is inoculated with according to immune programme with Seneca Valley virus
Neutralize antibody titers are not higher than 1:4, pig Seneca Valley virus, porcine reproductive and respiratory syndrome virus, swine foot-and-mouth disease virus antigen
It is negative), the Swine serum before acquisition inoculation is aseptic subpackaged as negative control sera by the batch sample, after in addition collecting inoculation
14 days serum, the batch sample is aseptic subpackaged as positive control serum.Standard serum is diluted with sample diluting liquid, dilution
Ratio selects 1 respectively:8,1:16,1:32,1:64,1:128, result such as the following table 1 institute of luminous value is read using Chemiluminescence Apparatus
Show.The result shows that when positive control serum sample diluting liquid presses 1:When 32 dilution, P/N value is maximum, therefore the dilution is made
For best serum dilution.
The sample diluting liquid is：Trehalose containing 1w/v%, 0.01w/v% thimerosal, 0.01v/v%Tween20 and
The 0.01M phosphate solution of 0.5w/v% casein.
The chemiluminescence readings of the different serum dilutions of table 1
6, enzyme labelled antibody (10 ×)
The rabbit-anti pig IgG secondary antibody of horseradish peroxidase-labeled, 10 times of uses of dilution when use.
7、PBST(10×)
0.1mol/L PBS solution containing 0.5v/v%Tween20, pH 7.4.10 times of uses of dilution when use.
8, Chemoluminescent substrate
Iodophenol is mixed with the luminol containing 0.1mmol/L and 0.1mmol/L of 0.05M Tris-HCl buffer
Close liquid.
9, luminescence enhancer
It is 0.005% with the hydrogen peroxide containing 7.5mmol/L of 0.05M Tris-HCl buffer, volume fraction
The mixed liquor of Tween20.
(2) assembling and preservation of kit：
1, the assembling of kit：
According to kit contents listed by table 2, kit is assembled, is saved after composition loaded on 4 DEG C.
2 pig Seneca Valley virus chemical luminescence immune analysis reagent box content of table
2, kit specification (detection method)
(1) it dilutes：PBST (10 ×) washing lotion is diluted to 250ml with aqua sterilisa, with sample diluting liquid by enzyme labelled antibody (10
×) it is diluted to 5ml.
(2) board-washing：It opens the package, the chemiluminescence immune assay plate of pre-coated antigen is taken out, with the PBST diluted
Board-washing 3 times, blotting paper pats dry, each 3min.
(3) it is loaded：By test serum sample sample diluting liquid with 1：32 times of dilutions, are added to instead according to every hole 100ul
It answers in plate, Seneca Valley virus positive control serum and negative control sera is added, with 1:32 times of dilutions, 100 holes μ L/, this is right
Two repetitions are done according to serum.37 DEG C of effect 5min.
(4) it washs：Blood serum sample is got rid of, is rinsed 4 times with PBST, blotting paper pats dry；
(5) enzyme labeling antibody：The Ig G secondary antibody of the HRP- rabbit-anti pig diluted, 100 holes μ L/, 37 DEG C of effect 5min are added.
(6) add substrate：Enzyme labelled antibody is got rid of, is rinsed 4 times with PBST, blotting paper pats dry.Chemiluminescent substrate (50 μ are added
The hole L/) and luminescence enhancer (100 hole μ L/).It mixes, is protected from light and stands 5min.
(7) luminous value, calculating and judgement are measured：
In measuring luminous value on chemical illumination immunity analysis instrument device, all samples to be tested are indicated with percent positive
(positive percent, PP), reduction formula is as follows：
PP=(sample to be tested luminous value-negative control sera average irradiance) × 100%/(positive control serum is average
Luminous value-negative control sera average irradiance).
Critical value is set to 6.14%, and the positive is determined as if calculated result >=6.14%, otherwise is feminine gender.
Embodiment 2 is used for the chemical luminescence immune analysis reagent box of Seneca Valley virus Structural protein VP1 antibody test
Sensibility, specificity experiments
1, the dilution of known background serum：
(1) 30 parts of blood serum samples are shared from clinically healthy pig, these pigs did not infect Seneca Valley virus, this
A little serum are used to evaluate the specificity of Seneca Valley virus structural proteins antibody chemical luminescence detection method.
(2) pig blisters positive serum, swine fever virus positive serum and each 30 parts of -2 type positive serum of pig circular ring virus, this
A little serum are used to evaluate the specificity of Seneca Valley virus structural proteins antibody chemical luminescence detection method.
(3) 80 parts of blood serum samples are shared from the pig for experimentally attacking Seneca Valley virus.These pigs are carrying out attacking poison
Do not carry out before any immune, and these blood serum samples are the blood serum samples collected after attacking poison the 14th day.These serum
For evaluating the sensibility of Seneca Valley virus structural proteins antibody chemical luminescence detection method.
The above serum is diluted with sample diluting liquid, dilution ratio 1:32, the sample diluting liquid is：Seaweed containing 1w/v%
The 0.01M phosphate solution of sugar, 0.01w/v% thimerosal, 0.01v/v%Tween20 and 0.5w/v% casein.
2, chemiluminescence immune assay：
Kit is prepared using embodiment 1 and detects above-mentioned blood serum sample, method is the same as embodiment 1.
3, the determination of critical value：
All samples to be tested indicate (positive percent, PP) with percent positive, and reduction formula is as follows：
PP=(test sample luminous value-standard female sample average luminous value) × 100%/(standard positive sample average
Luminous value-standard female sample average luminous value).
ROC curve analysis is done after obtaining the PP value of known background serum.Judge optimal critical value, diagnostic sensitivity, examine
Disconnected specificity.Interpretation of result：For this detection method, there is optimal diagnosis result when critical value is set to 6.14%.
4, sensitivity experiments：
Kit is prepared using embodiment 1 and detects 80 parts of blood serum samples, these samples are received after attacking poison 14 days
The blood serum sample of collection, these pigs do not carry out any immune before carrying out attacking poison, and detection method is the same as embodiment 1.Testing result table
The bright above sample VP1 antibody has very high positive rate (96.25%).Illustrate that kit of the invention has preferable sensibility, ties
Fruit is as shown in table 3.
The sensitivity tests of 3 chemical luminescence immune analysis reagent box of table
5, specificity experiments：
Kit is prepared using embodiment 1 and has detected pig blisters positive serum, swine fever virus positive serum and pig
- 2 type positive serum of circovirus and each 30 parts of pig Seneca Valley virus negative serum, detection method is the same as embodiment 1.These blood
The clear specificity for being used to evaluate Seneca Valley virus Structural protein VP1 antibody chemical luminescence immune assay method.Detect pig blister
The reaction of sick positive serum, swine fever virus positive serum, -2 type of pig circular ring virus and pig Seneca Valley virus negative serum is
Negative findings show that kit of the invention has good specificity, fundamentally ensure that testing result as a result such as the following table 4
Accurate and reliability.
4 ELISA kit specific test of table
The above serum Preliminary detection the result shows that：The albumen of selection has good sensibility and special as diagnostic antigen
Property, the ROC analysis curve of VP1-CLIA is as shown in Figure 4.
6, repeated identification
5 clear serum of background are respectively done into 3 repetitions on same chemiluminescence immune assay plate, calculates and becomes between its batch
Different coefficient (CV).Simultaneously by this 5 clear serum of background on 3 different luminescence immunoassay plates and the different time
It does and repeats three times, calculate its variation within batch coefficient (CV).
Repeated result is (table 5) as shown in the table, and the variation within batch coefficient of the serum of 5 groups of difference infection titers is
1.86%-8.45%.Interassay coefficient of variation is 1.64%-12.31%.Therefore batch between with batch in the coefficient of variation all less than
15%, it was demonstrated that this method has repeatability well.
5 repeatability identification of table
7, the identification of stability
In order to measure the storage life of Seneca Valley virus structural proteins antibody chemical luminescence immune assay plate, by what is be coated with
Reaction plate is placed in 37 DEG C of environment lower 15 days.Then Seneca Valley virus positive serum is measured.As a result Seneca Valley virus positive blood
Clear luminous value is 2650,000, meets the standard that experiment is set up, and the PP value of standard female serum is not above 1%.Therefore
The coating plate has good stability.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
<120>A kind of chemical luminescence immune analysis reagent box for Seneca Valley virus Structural protein VP1 antibody test
<160>2
<170>Patent-In 3.5
<210>1
<211>792
<212>DNA
<213>Seneca Valley virus
<400>1
tccaccgaca acgccgagac tggggttatt gaggcaggta acactgacac cgatttctct 60
ggtgaactgg cggctcctgg ctctaaccat actaatgtca aattcctgtt tgatcgatcc 120
cgactactga atgtaattaa ggtgctggag aaggacgccg tcttcccccg tcctttcccc 180
acagcaacag gtgcacagca ggacgatggt tacttttgtc tcctaacacc ccgcccaaca 240
gtcgcttccc gacccgccac tcgtttcggc ctgtacgtca acccgtctga cagtggcgtt 300
ctcgccaaca cttcactgga tttcaatttt tacagtttgg cctgtttcac ttactttaga 360
tcagaccttg aagtcacggt ggtctcacta gagccagatt tggaattcgc cgtggggtgg 420
ttcccctctg gcagtgagta tcaggcttct agctttgtct acgaccaact gcatgtaccc 480
taccacttca ctgggcgcac tccccgcgct ttcaccagca agggtggaaa ggtatctttc 540
gtgctccctt ggaactctgt ctcttccgtg cttcccgtgc gctggggggg cgcttccaag 600
ctttcttctg ccacgcgggg tctgccggct catgctgact gggggaccat ttacgccttt 660
atcccccgcc ctaacgagaa gaaaagcacc gctgtaaaac acgtggcggt gtacgttcgg 720
tacaagaacg cgcgtgcctg gtgccccagc atgcttccct ttcgcagcta caagcagaag 780
atgctgatgc aa 792
<210>2
<211>264
<212>PRT
<213>Seneca Valley virus
<400>2
Ser Thr Asp Asn Ala Glu Thr Gly Val Ile Glu Ala Gly Asn Thr 15
Asp Thr Asp Phe Ser Gly Glu Leu Ala Ala Pro Gly Ser Asn His 30
Thr Asn Val Lys Phe Leu Phe Asp Arg Ser Arg Leu Leu Asn Val 45
Ile Lys Val Leu Glu Lys Asp Ala Val Phe Pro Arg Pro Phe Pro 60
Thr Ala Thr Gly Ala Gln Gln Asp Asp Gly Tyr Phe Cys Leu Leu 75
Thr Pro Arg Pro Thr Val Ala Ser Arg Pro Ala Thr Arg Phe Gly 90
Leu Tyr Val Asn Pro Ser Asp Ser Gly Val Leu Ala Asn Thr Ser 105
Leu Asp Phe Asn Phe Tyr Ser Leu Ala Cys Phe Thr Tyr Phe Arg 120
Ser Asp Leu Glu Val Thr Val Val Ser Leu Glu Pro Asp Leu Glu 135
Phe Ala Val Gly Trp Phe Pro Ser Gly Ser Glu Tyr Gln Ala Ser 150
Ser Phe Val Tyr Asp Gln Leu His Val Pro Tyr His Phe Thr Gly 165
Arg Thr Pro Arg Ala Phe Thr Ser Lys Gly Gly Lys Val Ser Phe 180
Val Leu Pro Trp Asn Ser Val Ser Ser Val Leu Pro Val Arg Trp 195
Gly Gly Ala Ser Lys Leu Ser Ser Ala Thr Arg Gly Leu Pro Ala 210
His Ala Asp Trp Gly Thr Ile Tyr Ala Phe Ile Pro Arg Pro Asn 225
Glu Lys Lys Ser Thr Ala Val Lys His Val Ala Val Tyr Val Arg 240
Tyr Lys Asn Ala Arg Ala Trp Cys Pro Ser Met Leu Pro Phe Arg 255
Ser Tyr Lys Gln Lys Met Leu Met Gln 264
Claims (10)
1. a kind of chemical luminescence immune analysis reagent box for Seneca Valley virus Structural protein VP1 antibody test, feature
It is, the kit includes the chemiluminescence immune assay plate, negative right of Seneca Valley virus Structural protein VP1 antigen coat
According to serum, positive control serum, sample diluting liquid, enzyme labelled antibody, Chemoluminescent substrate, luminescence enhancer and thickening and washing
Liquid.
2. chemical luminescence immune analysis reagent box as described in claim 1, which is characterized in that the Seneca Valley virus structure
Albumen VP1 antigen is obtained through prokaryotic expression system, and amino acid sequence is as shown in SEQ ID NO.2.
3. chemical luminescence immune analysis reagent box as described in claim 1, which is characterized in that obtain prokaryotic expression
The purified product of the Seneca Valley virus Structural protein VP1 obtained, is diluted to 2.5 μ g/ml, is coated with chemiluminescence according to 100 holes μ l/
Immunoassay plate, 4 DEG C of refrigerator coatings are overnight.
4. chemical luminescence immune analysis reagent box as described in claim 1, which is characterized in that the positive control serum be through
The Swine serum of 14 days high infection titers after Seneca Valley virus infection；The negative control sera is that any vaccine was not immunized
Healthy Swine serum.
5. chemical luminescence immune analysis reagent box as described in claim 1, which is characterized in that the enzyme labelled antibody is horseradish
The rabbit-anti pig IgG secondary antibody of peroxidase labelling.
6. chemical luminescence immune analysis reagent box as described in claim 1, which is characterized in that the sample diluting liquid be containing
1w/v% trehalose, 0.01w/v% thimerosal, the 0.01M phosphate of 0.01v/v%Tween20 and 0.5w/v% casein are molten
Liquid.
7. chemical luminescence immune analysis reagent box as described in claim 1, which is characterized in that the Chemoluminescent substrate
For with Tris-HCl buffer, containing luminol and to the mixed liquor of iodophenol, the luminescence enhancer is to use Tris-
The mixed liquor containing hydrogen peroxide and Tween20 of HCl buffer.
8. chemical luminescence immune analysis reagent box as described in claim 1, which is characterized in that the Chemoluminescent substrate
For the mixed liquor with the luminol containing 0.1mmol/L of 0.05M Tris-HCl buffer and 0.1mmol/L to iodophenol, institute
The luminescence enhancer stated is hydrogen peroxide containing 7.5mmol/L, the 0.005v/v% with 0.05M Tris-HCl buffer
The mixed liquor of Tween20.
9. chemical luminescence immune analysis reagent box as described in claim 1, which is characterized in that the concentrated cleaning solution is 10
× PBST solution, i.e. the 0.1mol/L PBS solution containing 0.5v/v%Tween20, pH 7.4 dilute 10 times when use.
10. chemical luminescence immune analysis reagent box as described in claim 1, which is characterized in that using the kit into
When row Seneca Valley virus Structural protein VP1 antibody test, follow the steps below：
(1) Sample Dilution
By sample to be tested, positive control serum and negative control sera sample diluting liquid according to volume ratio 1：32 progress are dilute
It releases；
(2) board-washing
The chemiluminescence immune assay plate that Seneca Valley virus Structural protein VP1 antigen coat is taken out from 4 DEG C of refrigerators, opens packet
Chemiluminescence immune assay plate is taken out in pack, and with cleaning solution board-washing 3 times diluted, blotting paper is patted dry, each 3min；
(3) it is loaded
Sample to be tested, positive control serum and negative control sera after dilution is separately added into chemiluminescence immune assay plate
In, every hole 100 μ L, 37 DEG C of incubation 5min；
(4) it washs
Chemiluminescence immune assay plate is taken out, blood serum sample is got rid of, is rinsed 4 times with cleaning solution, blotting paper pats dry；
(5) enzyme labelled antibody is added
The rabbit-anti pig IgG secondary antibody of the horseradish peroxidase-labeled diluted, 100 holes μ L/, 37 DEG C of incubation 5min are added；
(6) add substrate：
Enzyme labelled antibody is got rid of, is rinsed 4 times with cleaning solution, blotting paper pats dry, and chemiluminescent substrate and luminescence enhancer is added, and mixes
It is even, it is protected from light and stands 5min；
(7) luminous value, calculating and judgement are measured：
In measuring luminous value on chemical illumination immunity analysis instrument device, all samples to be tested indicate (positive with percent positive
Percent, PP), reduction formula is as follows：
PP=(sample to be tested luminous value-negative control sera average irradiance) × 100%/(positive control serum average luminescence
Value-negative control sera average irradiance)
Critical value is set to 6.14%, and the positive is determined as if calculated result >=6.14%, otherwise is feminine gender.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810763876.XA CN108872574A (en) | 2018-07-12 | 2018-07-12 | A kind of chemical luminescence immune analysis reagent box for Seneca Valley virus Structural protein VP1 antibody test |
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|---|---|---|---|
| CN201810763876.XA CN108872574A (en) | 2018-07-12 | 2018-07-12 | A kind of chemical luminescence immune analysis reagent box for Seneca Valley virus Structural protein VP1 antibody test |
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ID=64301460
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|---|---|---|---|
| CN201810763876.XA Pending CN108872574A (en) | 2018-07-12 | 2018-07-12 | A kind of chemical luminescence immune analysis reagent box for Seneca Valley virus Structural protein VP1 antibody test |
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| CN114675024A (en) * | 2022-03-03 | 2022-06-28 | 中国农业科学院兰州兽医研究所 | Porcine Seneca virus A neutralizing antibody, competitive ELISA detection kit and detection method |
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Application publication date: 20181123 |