CN108872576A - A kind of chemical luminescence immune analysis reagent box for Seneca Valley virus nonstructural protein 3A BC antibody test - Google Patents
A kind of chemical luminescence immune analysis reagent box for Seneca Valley virus nonstructural protein 3A BC 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 nonstructural protein 3A BC antibody test.The kit includes chemiluminescence immune assay plate, positive control serum, negative control sera, enzyme labelled antibody, sample diluting liquid, Chemoluminescent substrate, luminescence enhancer and the concentrated cleaning solution of Seneca Valley virus nonstructural protein 3A BC antigen coat.The amino acid sequence of the Seneca Valley virus nonstructural protein 3A BC is as shown in SEQ ID NO.2.Kit of the present invention uses the nonstructural protein 3A BC antigen coat reaction plate of prokaryotic expression, antigen dosage is few, it can efficiently detect with the presence or absence of Seneca Valley virus in Swine serum, and not reacted with pig blisters poison, swine fever virus and -2 type of pig circular ring virus.Determined using enzyme-catalyzed chemical luminescence reaction system as a result, improving detection sensitivity.Kit specificity of the present invention is good, sensitive, efficient, has good market prospects.
Description
Technical field
The present invention relates to a kind of chemiluminescence immunoassays for Seneca Valley virus nonstructural protein 3A BC antibody test point
Kit is analysed, the application method of the kit is further related to.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
Antidiastole 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.For pig plug Buddhist nun
For the diagnosis for blocking paddy disease, detects non-structural protein or its antibody can effectively distinguish vaccine immunity animal and wild virus infection is dynamic
Object.
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..Currently the chemiluminescence about the diagnosis of pig Sai Nika paddy disease is exempted from
Epidemic disease analysis method has not been reported.
The present invention is using Seneca Valley virus nonstructural protein 3A BC as candidate gene, and clone obtains 3ABC gene, with big
It is expressed in enterobacteria and purifies 3ABC albumen as envelope antigen, establish the chemistry hair of the rapid differential diagnosis of Seneca Valley virus
Light immunoassay kits is maked a good technical reserve in advance for the possibility epidemic situation for coping with Seneca Valley virus from now on important meaning
Justice.
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 nonstructural protein 3A BC antibody test.It aims to solve the problem that in above-mentioned background technique, at present
The problem of there is no prevention and control of effective, the quick antidiastole reagent for Seneca Valley virus, for Buddhist nun is filled in reply in advance from now on
The possibility epidemic situation of card paddy virus make a good technical reserve.
To achieve the goals above, present invention employs following technological means：
The present invention provides a kind of chemiluminescence immunoassay for Seneca Valley virus nonstructural protein 3A BC antibody test point
Kit is analysed, the kit includes the chemiluminescence immune assay of Seneca Valley virus nonstructural protein 3A BC antigen coat
Plate, positive control serum, negative control sera, enzyme labelled antibody, sample diluting liquid, Chemoluminescent substrate, luminescence enhancer with
And concentrated cleaning solution.
Wherein, it is preferred that the Seneca Valley virus nonstructural protein 3A BC antigen is obtained through prokaryotic expression system, ammonia
Base acid sequence is as shown in SEQ ID NO.2.
Wherein, it is preferred that the chemiluminescence immunoassay of the Seneca Valley virus nonstructural protein 3A BC antigen coat point
Analysis plate is prepared in accordance with the following methods：
(1) preparation and coating of envelope antigen
The recombinant protein inclusion body that prokaryotic expression obtains purifies to obtain Sai Nika paddy disease through renaturation and Ni-NTA
Malicious nonstructural protein 3A BC, amino acid sequence are diluted to 2.5 μ with carbonate buffer solution when coating as shown in SEQ ID NO.2
G/ml, according in the coating to chemiluminescence immune assay plate of 100 holes μ l/, 4 DEG C of refrigerators are stood overnight；
(2) closing and preservation of chemiluminescence immune assay plate
Chemiluminescence immune assay plate with PBST washing 3 times, every hole be added 100 μ l contain 5w/v% skimmed milk power and
The confining liquid of 0.01w/v% thimerosal, 37 DEG C of closing 2h discard confining liquid, and PBST is washed 3 times, using aluminium foil bag vacuum seal,
4 DEG C of preservations.
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/L with 0.05M Tris-HCl buffer
To the mixed liquor of iodophenol, the luminescence enhancer is to be matched with 0.05M Tris-HCl buffer by luminol and 0.1mmol/L
The mixed liquor of the hydrogen peroxide containing 7.5mmol/L of system, the Tween20 that volume fraction is 0.005%.
Wherein, it is preferred that the concentrated cleaning solution is 10 × PBST solution, i.e., containing 0.5v/v%Tween20's
0.1mol/LPBS solution, pH 7.4 dilute 10 times when use.
Further, the invention also provides carry out Seneca Valley virus nonstructural protein 3A BC using the kit
The method of antibody test, 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 of Seneca Valley virus nonstructural protein 3A BC antigen coat is taken out from 4 DEG C of refrigerators
Plate opens aluminium foil bag, takes out chemiluminescence immune assay plate, with cleaning solution board-washing 3 times diluted, blotting paper is patted dry, every time
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 work 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 7.27%, and the positive is determined as if calculated result >=7.27%, 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 nonstructural protein 3A BC in E.Coli high efficient expression, egg
White yield 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 3ABC 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
Road 1：Host strain whole bacterial protein；Swimming lane 2：IPTG induces whole bacterial protein；
Fig. 2 is the bacterial cell disruption supernatant and precipitating SDS-PAGE electrophoresis of expression 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
Road 1：The broken supernatant protein of inducing expression；Swimming lane 2：The broken insoluble protein of inducing expression；
Fig. 3 is purification of recombinant proteins 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
Road 1：Elute albumen.
Fig. 4 is the ROC analysis curve of 3ABC-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 nonstructural protein 3A BC antibody test
Preparation and assembling
(1) preparation of kit：
1, the expression of Seneca Valley virus nonstructural protein 3A BC antigen：
The encoding gene 969bp (shown in SEQ ID NO.1) of Seneca Valley virus nonstructural protein 3A BC is cloned into
PET-30a (+) prokaryotic expression carrier, after digestion, sequencing are correct, positive plasmid is converted to BL21DE3plysS bacterial strain, blocks that
Chloramphenicol resistance LB plate screening monoclonal, 37 DEG C, LB culture medium shakes bacterium and stays overnight；Bacterium solution (v/v) 1 by volume will be stayed overnight:100 turns
It is connected to fresh LB, 37 DEG C, 200rpm shakes 3h and reaches 0.6 or so to OD600 value, and the IPTG of final concentration 1mmol/L is added
Continue 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 of continuation shake culture 3-4h；8000rpm is centrifuged 3min and collects thallus, is resuspended in 50mL pre-cooling NTA-0 buffer
In, ice bath 30min；Ultrasonication thallus, 4 DEG C of centrifugation 50min of 16000rpm collect supernatant and precipitating, take a small amount of supernatant and
Precipitating carries out SDS-PAGE detection, and protein expression result is as shown in Figure 1.The bacterial cell disruption supernatant and precipitating SDS-PAGE electricity of expression
Swimming figure is as shown in Figure 2.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,
Ultrasound promotes foreign protein dissolution, and precipitating is collected by centrifugation, supreme clear bright in triplicate, and precipitating is resuspended with PBS, and ultrasound, centrifugation is gone
Supernatant is resuspended inclusion body with 6M guanidine hydrochloride, adds DTT to final concentration 5mM；37 DEG C of concussion 3h are all dissolved to inclusion body, and centrifugation is gone
Supernatant.Again with protein renaturation liquid by albumen low temperature dialysis renaturation, with the 3M guanidine hydrochloride diluted protein solution of 2 times of volumes, at 4 DEG C
It being added dropwise in 200mL renaturation solution (pH8.0), rotational speed regulation to maximum, stirring for 24 hours, takes protein solution in bag filter, with
With PBS buffer solution dialysed overnight after PEG60000 concentration.Albumen after renaturation is purified again with anion-exchange column Ni-NTA, is received
Collect albumen.Protein purification result as shown in figure 3, Seneca Valley virus nonstructural protein 3A BC antigen amino acid sequence such as SEQ
Shown in 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/LPBS 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 closing 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 serum dilution 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 the casein of 0.5w/v%.
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/LPBS 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.
The content of 2 Seneca Valley virus chemical luminescence immune analysis reagent box 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：Aluminium foil bag is opened, 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, every hole 100ul are added to reaction plate
In, Seneca Valley virus positive control serum and negative control sera is added, with 1:32 times of dilutions, 100 holes μ L/, the control blood
Two repetitions are done clearly.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 work 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 A (50 μ are added
The hole L/) and luminescence enhancer B (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 7.27%, and the positive is determined as if calculated result >=7.27%, otherwise is feminine gender.
Embodiment 2 is used for the chemical luminescence immune analysis reagent box of Seneca Valley virus nonstructural protein 3A BC 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 non-structural protein 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 nonstructural protein 3A BC antibody chemical luminescence detection method.
(3) 80 parts of blood serum samples are shared from the pig for experimentally attacking SVV.These pigs do not carry out before carrying out attacking poison
It is any immune, and these blood serum samples are the blood serum samples collected after attacking poison.These serum are for evaluating Sai Nika paddy disease
The sensibility of malicious nonstructural protein 3A BC 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=(sample to be tested luminous value-negative control sera average irradiance) × 100%/(positive control serum is average
Luminous value-negative control sera average irradiance).
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 7.27%.
4, sensitivity experiments：
Kit is prepared using embodiment 1 and detects 80 parts of blood serum samples, these samples are the blood collected after attacking poison
Final proof product, these pigs do not carried out before carrying out attacking poison it is any immune, by calculating the sensibility of positive rate analysis method,
Detection method is the same as embodiment 1.Testing result shows that the above sample 3ABC antibody has very high positive rate (97.5%).Illustrate this hair
Bright kit has preferable sensibility, and the results are 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 nonstructural protein 3A BC antibody chemical luminescence immune assay method.Detect pig water
Blister disease positive serum, swine fever virus positive serum, the reaction of -2 type of pig circular ring virus and pig Seneca Valley virus negative serum are equal
Show that kit of the invention has good specificity as a result such as the following table 4 for negative findings, fundamentally ensure that detection knot
The accurate and reliability of fruit.The above serum Preliminary detection the result shows that：The Seneca Valley virus nonstructural protein 3A BC egg of selection
It is white to be used as diagnostic antigen that there is good sensibility and specificity.The ROC analysis curve of 3ABC-CLIA is as shown in Figure 4.
Table 4ELISA kit specific test
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
1.83%-9.82%.Interassay coefficient of variation is 1.77%-13.15%.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 non-structural protein antibody chemical luminescence immune assay plate, will be coated with
Reaction plate be placed in 37 DEG C of environment lower 15 days.Then Seneca Valley virus positive serum is measured.As a result Seneca Valley virus is positive
The luminous value of serum is 2950,000, meets the standard that experiment is set up, and the PP value of standard female serum is not above 1%.Cause
This coating plate has good stability.
The above serum Preliminary detection the result shows that：The albumen of selection has good sensibility and special as diagnostic antigen
Property, shows that detection kit detection time of the present invention is short, reproducible, sensibility is high, high specificity.
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 nonstructural protein 3A BC antibody test
<160>2
<170>Patent-In 3.5
<210>1
<211>969
<212>DNA
<213>Seneca Valley virus
<400>1
agccccaacg agaatgatga cacccccgtc gacgaggcgt tgggtagagt tctctccccc 60
gctgcggtcg atgaggcgct tgtcgacctc actccagagg ccgacccggt tggccgtttg 120
gctattcttg ccaagctagg tcttgcccta gctgcggtca cccctggtct gataatcttg 180
gcagtgggac tctacaggta cttctctggc tctgatgcag accaagaaga aacagaaagt 240
gagggatctg tcaaggcacc caggagcgaa aatgcttatg acggcccgaa gaaaaactct 300
aagccccctg gagcactctc tctcatggaa atgcaacagc ccaacgtgga catgggcttt 360
gaggctgcgg tcgctaagaa agtggtcgtc cccattacct tcatggttcc caacagacct 420
tctgggctta cacagtccgc tcttctggtg accggccgga ccttcctaat caatgaacat 480
acatggtcca atccctcctg gaccagcttc acaatccgcg gtgaggtaca cactcgtgat 540
gagcccttcc aaacggttca tttcactcac cacggtattc ccacagatct gatgatggta 600
cgtctcggac cgggcaattc tttccctaac aatctagaca agtttggact tgaccagatg 660
ccggcacgca actcccgtgt ggttggcgtt tcgtccagtt acggaaactt cttcttctct 720
ggaaatttcc tcggatttgt tgattccatc acctctgaac aaggaactta cgcaagactc 780
tttaggtaca gggtgacgac ctacaaagga tggtgcggct cggccctggt ctgtgaggcc 840
ggtggcgtcc gacgcatcat tggcctgcat tctgctggcg ccgccggtat cggcgccggg 900
acctatatct caaaattagg actaatcaaa gccctgaaac acctcggtga acctttggcc 960
acaatgcaa 969
<210>2
<211>323
<212>PRT
<213>Seneca Valley virus
<400>2
Ser Pro Asn Glu Asn Asp Asp Thr Pro Val Asp Glu Ala Leu Gly 15
Arg Val Leu Ser Pro Ala Ala Val Asp Glu Ala Leu Val Asp Leu 30
Thr Pro Glu Ala Asp Pro Val Gly Arg Leu Ala Ile Leu Ala Lys 45
Leu Gly Leu Ala Leu Ala Ala Val Thr Pro Gly Leu Ile Ile Leu 60
Ala Val Gly Leu Tyr Arg Tyr Phe Ser Gly Ser Asp Ala Asp Gln 75
Glu Glu Thr Glu Ser Glu Gly Ser Val Lys Ala Pro Arg Ser Glu 90
Asn Ala Tyr Asp Gly Pro Lys Lys Asn Ser Lys Pro Pro Gly Ala 105
Leu Ser Leu Met Glu Met Gln Gln Pro Asn Val Asp Met Gly Phe 120
Glu Ala Ala Val Ala Lys Lys Val Val Val Pro Ile Thr Phe Met 135
Val Pro Asn Arg Pro Ser Gly Leu Thr Gln Ser Ala Leu Leu Val 150
Thr Gly Arg Thr Phe Leu Ile Asn Glu His Thr Trp Ser Asn Pro 165
Ser Trp Thr Ser Phe Thr Ile Arg Gly Glu Val His Thr Arg Asp 180
Glu Pro Phe Gln Thr Val His Phe Thr His His Gly Ile Pro Thr 195
Asp Leu Met Met Val Arg Leu Gly Pro Gly Asn Ser Phe Pro Asn 210
Asn Leu Asp Lys Phe Gly Leu Asp Gln Met Pro Ala Arg Asn Ser 225
Arg Val Val Gly Val Ser Ser Ser Tyr Gly Asn Phe Phe Phe Ser 240
Gly Asn Phe Leu Gly Phe Val Asp Ser Ile Thr Ser Glu Gln Gly 255
Thr Tyr Ala Arg Leu Phe Arg Tyr Arg Val Thr Thr Tyr Lys Gly 270
Trp Cys Gly Ser Ala Leu Val Cys Glu Ala Gly Gly Val Arg Arg 285
Ile Ile Gly Leu His Ser Ala Gly Ala Ala Gly Ile Gly Ala Gly 300
Thr Tyr Ile Ser Lys Leu Gly Leu Ile Lys Ala Leu Lys His Leu 315
Gly Glu Pro Leu Ala Thr Met Gln 323
Claims (10)
1. a kind of chemical luminescence immune analysis reagent box for Seneca Valley virus nonstructural protein 3A BC antibody test, special
Sign is, the kit include Seneca Valley virus nonstructural protein 3A BC antigen coat chemiluminescence immune assay plate,
Positive control serum, negative control sera, enzyme labelled antibody, sample diluting liquid, Chemoluminescent substrate, luminescence enhancer and dense
Contracting cleaning solution.
2. chemical luminescence immune analysis reagent box as described in claim 1, which is characterized in that the non-knot of Seneca Valley virus
Structure albumen 3ABC 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 the Seneca Valley virus is non-
The chemiluminescence immune assay plate of structural proteins 3ABC antigen coat is prepared in accordance with the following methods：
(1) preparation and coating of envelope antigen
The recombinant protein inclusion body that prokaryotic expression obtains purifies to obtain Seneca Valley virus non-through renaturation and Ni-NTA
Structural proteins 3ABC, amino acid sequence are diluted to 2.5 μ g/ with carbonate buffer solution when coating as shown in SEQ ID NO.2
Ml, according in the coating to chemiluminescence immune assay plate of 100 holes μ l/, 4 DEG C of refrigerators are stood overnight；
(2) closing and preservation of chemiluminescence immune assay plate
With PBST washing 3 times, every hole is added 100 μ l and contains 5w/v% skimmed milk power and 0.01w/ chemiluminescence immune assay plate
The confining liquid of v% thimerosal, 37 DEG C of closing 2h discard confining liquid, and PBST is washed 3 times, using aluminium foil bag vacuum seal, 4 DEG C of guarantors
It deposits.
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 nonstructural protein 3A BC 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 nonstructural protein 3A BC antigen coat is taken out from 4 DEG C of refrigerators, beats
Aluminium foil 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 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 work 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 7.27%, and the positive is determined as if calculated result >=7.27%, otherwise is feminine gender.
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| 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 |
| CN110221064A (en) * | 2019-05-23 | 2019-09-10 | 南京农业大学 | Pig Seneca Valley virus ELISA antibody detection method and application |
| CN111273036A (en) * | 2020-03-06 | 2020-06-12 | 中国农业科学院兰州兽医研究所 | Monoclonal antibody competition chemiluminescence kit for detecting foot-and-mouth disease non-structural protein antibody |
| CN111944764A (en) * | 2020-08-31 | 2020-11-17 | 信阳农林学院 | Cell line for expressing suijiancai valley virus protein, construction method and application |
| CN112745387A (en) * | 2019-10-31 | 2021-05-04 | 洛阳普泰生物技术有限公司 | Anti-porcine seneca valley virus monoclonal antibody and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110221064A (en) * | 2019-05-23 | 2019-09-10 | 南京农业大学 | Pig Seneca Valley virus ELISA antibody detection method and application |
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| CN111273036A (en) * | 2020-03-06 | 2020-06-12 | 中国农业科学院兰州兽医研究所 | Monoclonal antibody competition chemiluminescence kit for detecting foot-and-mouth disease non-structural protein antibody |
| CN111273036B (en) * | 2020-03-06 | 2021-06-15 | 中国农业科学院兰州兽医研究所 | Monoclonal antibody competition chemiluminescence kit for detecting foot-and-mouth disease non-structural protein antibody |
| CN111944764A (en) * | 2020-08-31 | 2020-11-17 | 信阳农林学院 | Cell line for expressing suijiancai valley virus protein, construction method and application |
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