CN109187952A - One boar atypia pestivirus ELISA antibody assay kit - Google Patents
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Abstract
The object of the present invention is to provide a boar atypia pestivirus APPV antibody test ELISA kit and the production methods of a boar atypia seasonal febrile diseases toxalbumin, can efficiently express pig atypia seasonal febrile diseases toxalbumin.Present invention firstly provides the genes of a boar atypia seasonal febrile diseases toxalbumin, can be classified as SEQ ID NO.2 in E. coli pig atypia seasonal febrile diseases toxalbumin, the nucleotides sequence of the gene;Another aspect of the present invention provides a boar atypia pestivirus ELISA antibody assay kit, includes Bacillus coli expression, and amino acid sequence is elisa plate of the pig atypia pestivirus E2 albumen of SEQ ID NO:5 as antigen coat.The present invention provides the methods of a large amount of preparation pig atypia pestivirus E2 albumen, and it provides using pig atypia pestivirus E2 albumen as the ELISA antibody assay kit of antigen, kit sensitivity with higher, specificity, repeatability, can be with large-scale promotion application.
Description
Technical field
The invention belongs to vaccine detection technique fields, and in particular to boar atypia pestivirus (APPV) antibody test
ELISA kit.
Background technique
Pig atypia pestivirus (APPV) is a kind of extremely strong RNA virus of variability, can cause piglet congenital tremors,
" Shiver disease " being clinically commonly called as is more common in the first tire sow litter, is spy with bilateral skeletal muscle clonic contraction
Sign, when serious symptom can lead to pig walking, suck the breast it is difficult, often because colostrum insufficiency of intake and it is hungry due to it is dead.The virus can induce
The mixed infection and secondary infection of a variety of viruses, bacterium, cause huge economic loss to global pig breeding industry.This disease is ground
Study carefully, it is especially just of increasing concern to the research of its vaccine.With the development of China's pig breeding industry, the first-born sow newborn piglet
Harm by pig atypia pestivirus infection is got worse, therefore researches and develops a kind of fast and accurately APPV Serum Antibody Detection
The demand of method is more urgent.
Currently, presently mainly passing through epidemiology, clinical symptoms and pathology dissect clinically for the detection of APPV
Tentative diagnosis is made in variation, but APPV and CSFV etc. is difficult to make a definite diagnosis by means such as simple clinical examination, pathology dissects.Cause
This, making a definite diagnosis for the disease also needs to be accompanied by laboratory diagnosis.However laboratory diagnosis such as indirect immunofluorescence, polymerase chain
The methods of reaction, virus purification culture, there is detection, time-consuming, costly, process is complicated, needs specific instrument and equipment,
Large-scale promotion difficulty is higher.Therefore, research and development are easy to operate, sensibility is high, the detection kit of high specificity is detection APPV
Necessary means.
APPV E2 membrane glycoprotein is the major structural protein of swine fever virus, is swine fever virus virulence and host range
Major determinant is located at virion outer surface, has transmembrane region, be the master for establishing swine fever virus Serology test
Want antigen.Therefore, CSFV E 2 protein is the important protein molecular for developing swine fever virus new generation vaccine and diagnostic reagent.For
Wider to utilize pig atypia seasonal febrile diseases toxalbumin, researcher is by pig atypia pestivirus E2 protein gene cloning to large intestine
It is expressed in bacillus.But pig atypia pestivirus E2 albumen expression efficiency in prokaryotic cell is very low, makes E2 albumen as inspection
Antigen application range is surveyed to be restricted.
Summary of the invention
The object of the present invention is to provide boar atypia pestivirus (APPV) antibody test ELISA kit, Yi Jiyi
The production method of boar atypia seasonal febrile diseases toxalbumin can efficiently express pig atypia seasonal febrile diseases toxalbumin, to make up existing
The deficiency of technology.
Present invention firstly provides the genes of a boar atypia seasonal febrile diseases toxalbumin, can be in E. coli pig
Atypia seasonal febrile diseases toxalbumin, the nucleotides sequence of the gene are classified as SEQ ID NO.2;
AATGGTAGTTATAACATAGTGAGGCAGGCCAGAGACGAAGTA AGCCCGTCGACAGGGTGTAAAGAAG
GATATCCCTTCCTGTTTT CTGGAGAAAGATCCGACACCTCATGTCTAAAGCCTCCATCCAC TAGTTGGGTCCGT
CCAGTAAAAATGGATGAGGTATCTGTGGC AGATAGCTTCGCCCATGGTGTAGACAAGGCGATCATCCTGATT CG TAAAGGTGCGTCTGGTATCATCAACTTTCTGGACACCATTGGTCGTTGGCTGCCAGTGGCTGAAGCCGCCATCGTA
CCATATTG TGAAACTTATACTGTAACCGGTATGTACGTTCATGTGAAGCAT TGCCTGCCGAAGGGTCTGCCAAA
GCATTCTAAAATCATTTCCC CAACCATGATCTACCTGGGTGAAGGTGACCCAGCCCATAACAT CCAGCATCTGT
TTGGCTCTGGCATCGCAAAGTGGGTTCTGGTTCTGCTGGGTGTTCTGGGTGAGTGGTATGGTGAGCTGGCCTCCAC CATCTATCTGCTGCTGGAGTACGGTACTGAATGGCTGGAACA TGAATCTCTGGTTACCGAAGGTCTGATCCCGGG
CATCAACATC ACCGTAGAGCTGCCGGCTTCTCACACCGTACCAGGTTGGGTGT GGGTTGCTGGCCAGTGGATCT
GCGTGAAGCCAGATTGGTGGCCGACCCAGATTTGGATTGAAACCATCGTGACCGAGGTATGGCAT ATCCTGAAAA TCCTGGCATCTGCTCTGGTAAACATCGTTACTG CATTCGTAAACCTCGAGCTGGTGTACCTGGTTATCATCCTGG TTAAAATCTCTAAGGGTAACCTGATCGGTGCTGTACTGTGGTGC CTGCTGCTGTCTGGTGCTGAGGGTTCTTGCC
ACCGTCGTCAAG ACTATTACAACATCCAACTGGTTGTTGAGGAAAAAACCGGTATCGAGAAGCGTTCTATCATTG
GCAAGTGGACTGTGATCACTCGT GAAGGCCGTGAACCACGTCTGATGGAGCAGATCAACATGGTGTCTAACGAGACCCTGTCTGAAACTTACTGC
For expanding the amplimer of gene after above-mentioned mutation, sequence information is as follows:
Upstream primer F1:5 '-CGGATCCCGTCCAGTAAAAATGGATG-3’
(BamHI)(SEQ ID NO.3);
Downstream primer R1:5 '-CGGAATTCGCAGTAAGTT TCAGACAGGG-3’
(EcoRI)(SEQ ID NO.4)。
Another aspect of the present invention provides a boar atypia pestivirus ELISA antibody assay kit, includes large intestine
Bacillus expression, amino acid sequence is pig atypia pestivirus (APPV) E2 albumen of SEQ ID NO:5 as antigen coat
Elisa plate, positive control serum, negative control sera, sample diluting liquid, enzyme label conjugate, cleaning solution, substrate solution A,
Substrate solution B and terminate liquid;
Wherein positive control serum is made of APPV vaccine inoculation health pig;
Negative control sera is the serum of health pig;
The sample diluting liquid is the diluted bacillus coli BL21 of PBS solution with 0.01M pH7.2
Supernatant solution after the thallus ultrasound of (E.coli BL21), protein concentration 1.2mg/ml;
The enzyme label conjugate is that the goat-anti pig antibody enzyme labelled antibody dilution of horseradish peroxidase-labeled is dilute
(example: 10 μ l enzyme labelled antibodies, enzyme labeling antibody dilution 120ml are taken) made of releasing 12000 times.
The cleaning solution is by 14.9g KH2PO4, 79.1g K2HPO4·3H2O, 50.0ml Tritonx-100,
1.0g NaN3, 8.0g NaCl is dissolved in 500ml water, adjusts pH 7.3, is finally settled to 1L.
The substrate solution A is by 4.2g citric acid, 13.6g sodium acetate, and 0.5g peroxide crosses urea and is dissolved in 100ml
In ionized water, it is settled to 1L, adjusts pH value to made of 5.0;
Substrate solution B is that 1g TMB is dissolved in 50ml dimethyl sulfoxide, 100ml formaldehyde is added after dissolution,
16.0g polyvinylpyrrolidone is settled to 1L with deionized water after dissolution and completes preparation.
The terminate liquid is the sulfuric acid solution of 2mol/L.
Pig atypia pestivirus (APPV) E2 albumen used in the present invention, preparation method are as follows:
The E2 protein coding gene that nucleotides sequence is classified as SEQ ID NO.2 is integrated on pET28a carrier and is built into weight
Recombinant plasmid is imported competent escherichia coli cell and carries out inducing expression, isolated and purified and had by His nickel column by group plasmid
The E2 albumen of His label,
The wherein preparation method of elisa plate is by pig atypia pestivirus (APPV) the E2 proteantigen pH of purifying
9.5, the carbonate buffer solution of 0.05mol/L is diluted to 25 μ g/ml, is added in the coating plate of 96 holes, 4 DEG C of mistakes are set in 100 holes μ l/
Night after washing 3 times with cleaning solution, is added the pH 7.2PBS of 0.01mol/L containing 1%BSA closing overnight, gets rid of deblocking liquid, set
Under room epidemic disaster 40%, the preparation of completion in 24-48 hours is spontaneously dried.
The present invention provides the methods of a large amount of preparation pig atypia pestivirus (APPV) E2 albumen, and provide non-with pig
Typical pestivirus (APPV) E2 albumen be antigen ELISA antibody assay kit, kit sensitivity with higher,
Specificity, repeatability, can be with large-scale promotion application.
Detailed description of the invention
Fig. 1: mutation front and back gene expression protein electrophoresis figure;Wherein M: albumen Marker;1: former sequence expresses protein purification
Electrophoresis;2: sequence table reaches protein purification electrophoresis after mutation;
Fig. 2: recombinant plasmid pET28a-E2 double digestion electrophoretogram;Wherein, M is DNA Marker;1 identifies for double digestion
Figure;
Fig. 3: pig atypia pestivirus E2 Identification of Fusion Protein electrophoretogram;Wherein, M is Protein Marker;1 is expression bacterium
Supernatant after the cracking of E2-pET28a thallus ultrasound;2 precipitate to express after bacterium E2-pET28a thallus ultrasound cracks;
Fig. 4: pig atypia pestivirus E2 protein purification electrophoretogram;Wherein, M is Protein Marker;1 is after purification
E2 albumen;
Fig. 5: pig atypia pestivirus E2 albumen Western blot electrophoretogram.
Specific embodiment
Technology used in following embodiment is unless stated otherwise known to those skilled in the art normal
Rule technology;Used instrument and equipment, reagent etc., it is the research and technology of this field that only this specification, which illustrates,
What personnel can be obtained by public approach.
The preparation method of 1 pig atypia pestivirus E2 albumen of embodiment
1. the building of recombinant expression plasmid
Applicant is to the pig atypia seasonal febrile diseases virus gene (KY612413SEQ ID NO:1) delivered in Genbank:
AATGGTAGTTATAACATAGTGAGGCAGGCCAGAGACGAAGTAA GCCCGTCGACAGGGTGTAAAGAAG
GATATCCCTTCCTGTTTTCT GGAGAAAGATCCGACACCTCATGTCTAAAGCCTCCATCCACTA GTTGGGTCAGA
CCAGTAAAAATGGATGAGGTATCAGTGGCAGA TAGCTTCGCCCATGGAGTAGACAAGGCGATAATATTAATTAGAA
AAGGGGCGTCAGGAATCATCAACTTTTTAGACACCATTGGGAG GTGGCTGCCAGTGGCTGAAGCCGCCATAGTAC
CATATTGTGAA ACTTATACTGTAACAGGGATGTACGTTCATGTGAAGCATTGCCT CCCTAAGGGGCTACCAAAG
CATTCAAAAATAATTTCCCCAACA ATGATATACCTGGGGGAAGGTGACCCAGCCCATAATATCCAGCA TTTGTT
TGGCTCAGGCATAGCAAAGTGGGTCCTAGTCTTACTTG GTGTCTTGGGTGAGTGGTATGGGGAGTTGGCCTCCAC
GATATAT CTGCTACTGGAGTACGGGACTGAATGGTTGGAACATGAAAGTC TAGTCACGGAAGGGTTGATCCCTG
GCATCAATATCACAGTAGA GCTCCCCGCTAGTCACACGGTACCAGGTTGGGTGTGGGTCGCT GGCCAGTGGATA
TGCGTGAAGCCAGATTGGTGGCCTACACAGA TTTGGATTGAAACCATAGTGACAGAGGTATGGCATATACTGAAA
ATATTGGCATCAGCTTTGGTAAATATAGTCACTGCATTCGTAAAC CTAGAGTTAGTCTATCTGGTCATAATACTA
GTCAAAATATCAAA GGGGAACTTGATAGGTGCTGTATTGTGGTGCCTCTTGCTGTCAG GAGCTGAGGGGTCATG
CCACAGAAGACAAGACTATTACAACAT CCAACTGGTCGTCGAGGAAAAAACAGGAATAGAGAAGCGATC TATAA
TTGGCAAGTGGACTGTGATAACTAGGGAAGGCAGAGAA CCAAGATTAATGGAGCAGATAAATATGGTGTCGAATG
AGACCCT ATCAGAAACTTACTGC
According to the preference of e. coli codon, former sequence is mutated under the premise of not changing amino acid sequence,
The nucleotide sequence that solution expression with high efficiency can be carried out in Escherichia coli is obtained by screening, and external artificial synthesized
New pig atypia pestivirus gene nucleotide series (SEQ ID NO.2),
The amplimer of gene after above-mentioned modification is expanded, sequence information is as follows:
Upstream primer F1:5 '-CGGATCCCGTCCAGTAAAAATGGATG-3’
(BamHI)(SEQ ID NO.3);
Downstream primer R1:5 '-CGGAATTCGCAGTAAGTTTCAGACAGGG-3’
(EcoRI)(SEQ ID NO.4)。
The amino acid sequence of the E2 albumen of its coding of amplified production is SEQ ID NO.5.
RPVKMDEVSVADSFAHGVDKAIILIRKGASGIINFLDTIGRWLP VAEAAIVPYCETYTVTGMYVHVK
HCLPKGLPKHSKIISPTMIYLGE GDPAHNIQHLFGSGIAKWVLVLLGVLGEWYGELASTIYLLLEYGTE WLEHE
SLVTEGLIPGINITVELPASHTVPGWVWVAGQWICVKPDW WPTQIWIETIVTEVWHILKILASALVNIVTAFVNL
ELVYLVIILVKISK GNLIGAVLWCLLLSGAEGSCHRRQDYYNIQLVVEEKTGIEKRSIIGK
WTVITREGREPRLMEQINMVSNETLSETYC
2. the amplification and recycling of pig atypia seasonal febrile diseases virus gene
Using the gene of synthesis as template, PCR amplification is carried out.After reaction by PCR product through 1% Ago-Gel electricity
Swimming analysis amplification, ultraviolet light irradiation incision take the Ago-Gel containing target fragment, use DNA plastic recovery kit
Target fragment is recycled.BamHI and EcoRI is used to carry out respectively the PCR product being recovered to and carrier pET28a double
Digestion, reaction system difference are as follows: target fragment recycling: 10 11 10 μ of μ L, 10 × K of μ L, EcoRI of μ L, BamHI of target fragment
18 μ L of L, ddH2O, 40 μ L of total volume.Carrier segments recycling: 511 10 μ L of μ L, 10 × K of μ L, EcoRI of μ L, BamHI of carrier,
23 μ L of ddH2O, 40 μ L of total volume.37 DEG C of digestion 3h.Target fragment and carrier are returned using DNA plastic recovery kit
It receives.Under the action of T4 ligase, 4 DEG C of connections overnight.It takes 10 μ L connection products to convert into Top10 Escherichia coli, is coated on
Block on the LB solid plate of that resistance, overnight incubation.Picking single bacterium colony is inoculated in the liquid LB culture medium of that resistance of card
In, plasmid is extracted after shaking bacterium overnight, double digestion identification is carried out, sees Fig. 2.
3. the inducing expression and purifying of recombinant expression protein
Inducing expression: the recombinant expression plasmid E2-pET28a of building is converted into BL21 Escherichia coli, is inoculated in and contains
In the LB liquid medium for having that antibiotic of card, after 37 DEG C of 200rpm shake culture 8h, it is inoculated in the anti-containing that is blocked of 200ml
In the LB liquid medium of raw element, 37 DEG C of 200rpm shake culture 3h or so reach 0.6 or so to OD value, are added thereto
0.05 ‰ IPTG, 37 DEG C of inducing expression 8h.It taking out 4 DEG C of 8000rpm/min of bacterium solution and is centrifuged 7min, careful supernatant of drawing discards,
Thallus is resuspended using 15mL imidazoles binding soln, ultrasonic treatment thallus, condition is work 4s, stops 2s, altogether ultrasound
30min, whole operation carry out on ice.4 DEG C of 1000rpm/min of ultrasonic lysate are centrifuged 15min after ultrasound, separation it is upper
Cleer and peaceful precipitating carries out SDS-PAGE analysis.
Purifying: His binding resin is filled into protein purification column, using combining imidazole buffer to balance pillar, then
Cracking supernatant is slowly added into pillar, is eluted using His elution buffer, eluent is collected and carries out SDS-PAGE inspection
The result of purifying.
4. the measurement of purifying protein concentration
Sequence table reaches albumen sample after measuring former sequence expression protein sample concentration and mutation respectively using Brandford method
Product concentration, respectively 0.231mg/ml, 1.216mg/ml;The expressing quantity of sequence obviously raises after mutation.Former sequence expression
Sequence table is as shown in Figure 1 up to protein purification sample progress SDS-PAGE after protein purification sample and mutation.
The foundation of 2 pig atypia pestivirus ELISA antibody detection method of embodiment
(1) foundation of APPV ELISA antibody detection method
The best antigen coat concentration, serum dilution to be checked, ELIAS secondary antibody of E2 albumen are determined most using square matrix titration
Good dilution respectively walks optimum reacting time.
It is coated with the preparation of plate: by Recombinant Swine atypia pestivirus (APPV) the E2 proteantigen pH 9.5 of purifying
0.05mol/L carbonate buffer solution is diluted to 25 μ g/ml, is added into 96 holes coating plate, and 100 holes μ l/ are set 4 DEG C overnight, used
After cleaning solution washs 3 times, 1%BSA 0.01mol/L pH 7.2PBS closing is added overnight, gets rid of deblocking liquid, room temperature, humidity
40% hereinafter, spontaneously dry 24-48 hours, packaging.
The preparation of the enzyme labelled antibody (No. 1 liquid) of working concentration: the horseradish peroxidase-labeled for taking BETHYL company to produce
12000 times of goat-anti pig antibody enzyme labelled antibody diluted (example: take 10 μ l enzyme labelled antibodies, enzyme labeling antibody dilution
120ml)。
The preparation of cleaning solution (No. 2 liquid): being by 14.9g KH2PO4, 79.1g K2HPO43H2O, 50.0ml
Tritonx-100,1.0g NaN3, 8.0g NaCl is dissolved in 500ml water, adjusts pH 7.3, is finally settled to 1L.
The preparation of substrate developing solution: by 4.2g citric acid, 13.6g sodium acetate, 0.5g urea peroxide is dissolved in 100ml and goes
In ionized water, it is settled to 1L, adjusts pH value to 5.0, as substrate A liquid (No. 3 liquid);It is sub- that 1g TMB is dissolved in 50ml dimethyl
100ml formaldehyde is added in sulfone, after dissolution, 16.0g polyvinylpyrrolidone is settled to 1L with deionized water after dissolution, i.e.,
For substrate B liquid (No. 4 liquid)
The preparation of sample diluting liquid (No. 5 liquid): from picking bacillus coli BL21 (E.coli on LB culture plate
BL21) single colonie is transferred in the test tube of the culture solution containing LB, 37 DEG C of overnight incubations, preparation saturation bacterium solution.Saturation bacterium solution is turned
It is connected in the LB culture medium of 250ml, inoculum concentration about 5%, cultivates 8 hours.6000 revs/min are centrifuged 15 minutes.Thallus is dissolved in
15mlPBS.The bacterium solution of dissolution is thawed, ultrasonic treatment.6000 revs/min are set, is centrifuged 15 minutes, supernatant is collected.With BCA egg
White reagent box for detecting content measures protein content.Then broken bacterium solution is diluted to total protein content with 0.01M pH7.2PBS
For 1.2mg/ml.
The preparation of terminate liquid (No. 6 liquid): 2mol/L sulfuric acid solution is prepared.
The preparation of positive control serum and negative control sera:
Positive control serum: two week old piglets, every leg muscle are inoculated with Recombinant Swine atypia pestivirus inactivated vaccine
Vaccinate 1ml.One exempts from 14 days afterwards, carries out two with same vaccine same dose and exempts from, and two exempt to take a blood sample for latter 21 days, measures APPV
ELISA antibody.When ELISA detects OD450nmWhen value is not less than 1.50, blood sampling separates serum.
Negative control sera: two week old sodium selenites (APPV-ELISA negative antibody), blood sampling separate serum.
(2) application method of atypia seasonal febrile diseases viral disease ELISA antibody assay kit:
1, operating procedure
(1) each reagent is taken out from refrigerator, is restored to room temperature (20~25 DEG C).
(2) serum to be checked is done into 1:400 dilution with serum dilution and (5 μ l+95 μ l sample diluting liquid of serum is taken, after mixing
Take 6 μ l+114 μ l sample diluting liquids).
(3) plus 100 μ l are not required to diluted negative serum into the hole A1 and A2.
(4) plus 100 μ l are not required to diluted positive serum into the hole A3 and A4.
(5) plus 100 μ l serum dilutions are into the hole A5, A6, are blank control.
(6) blood serum sample for adding 100 μ l to dilute is into coating plate hole.
(7) ELISA Plate 37 DEG C are mounted to incubate 30 minutes.
(8) it washs: cleaning solution 2 is added into coating plate hole and drips (or 50ul), is washed 5 times, is being absorbed water repeatedly with distilled water
It is patted dry on paper.
(9) enzyme label conjugate 2 is added into coating plate hole and drips (or 100ul), 37 DEG C are placed 30 minutes.
(10) it washs: repeating step (8)
(11) it develops the color: substrate solution A 2 is added and drips, substrate solution B 2 drips (or 50ul), gently shakes up, 37 DEG C of reactions 10
Minute.
(12) every hole adds the only whole drop of liquid 2 (or 100ul) termination reaction.
(13) ELISA Plate is placed in microplate reader, reads the absorbance value under 450nm wavelength.
2, calculation method
3, sample to be tested positive judgment criteria
Criterion positive control serum OD450nm> 0.8, negative control sera OD450nm< 0.20, blank well OD450<
0.10, experiment is set up, otherwise in vain.
Calculation formula: S/N value=sample to be examined OD450nm/ negative control OD450nmMean value
Criterion:
S/N >=2.5 are the positive;
2.0 < S/N < 2.5 are suspicious;
S/N≤2.0 are feminine gender.
Two, the kit forms constructed
ELISA Plate: coating APPV-E2 recombinant protein, peridium concentration are 25 μ g/ml, every 100 μ L of hole;
Sample diluting liquid: self-control sample diluting liquid
Cleaning solution: PBST
ELIAS secondary antibody: the goat-anti pig IgG of HRP label
Substrate developing solution: 3.3 ' -5,5 '-tetramethyl biphenyl amine aqueous solutions (TMB)
Terminate liquid: the H of 2mol/L2SO4
Positive criteria product: known APPV positive serum
Negative standards' product: known APPV negative serum
Three, APPV ELISA antibody assay kit operating procedure
Measuring samples are diluted with 400 times of sample diluting liquid, every 100 μ L of hole, if positive, negative and blank control, as
37 DEG C are incubated for 30 minutes, and cleaning solution 2 is added into coating plate hole and drips (or 50ul), is washed 5 times, is being absorbed water repeatedly with distilled water
It is patted dry on paper;100 μ L of ELIAS secondary antibody is added in every hole, and 37 DEG C are incubated for 30 minutes, be added into coating plate hole the drop of cleaning solution 2 (or
50ul), it is washed with distilled water 5 times, is patted dry on blotting paper repeatedly;Each 100 μ L of substrate developing solution A, substrate colour developing is added in every hole
Each 50 μ L of liquid B, 37 DEG C are protected from light display 10min, and terminate liquid, every 100 μ L of hole is added.Measure OD450nm。
Result judgement: as standard positive serum OD450Value >=0.8 nm when standard female serum OD value≤0.2, illustrates to try
It tests effectively;When S/N value >=2.5, it is judged to the positive;It is judged to when 2.0 < S/P < 2.5 suspicious;Feminine gender is judged to when S/P value≤2.0.
The specificity of 3 atypia seasonal febrile diseases viral disease ELISA antibody assay kit of embodiment, sensitivity tests
(1) specific test: the APPV ELISA antibody assay kit that the present invention develops is detection kit counterweight
Group pig atypia pestivirus specific detection, with 3 batches of kits detect respectively pig atypia pestivirus, porcine circovirus 2 type,
10 kinds of positive serums such as porcine reproductive and respiratory syndrome virus, swine fever, pseudorabies (by being made after the susceptible piglet of vaccine immunity)
It is detected, as a result other positive serum testing results are feminine gender in addition to APPV positive serum, and see Table 1 for details, shows kit
With good specificity.
Table 1:3 batches of kits detect specific test result
Note: 1=porcine reproductive and respiratory syndrome positive serum;2=swine fever positive serum;3=pseudorabies positive blood
Clearly;4=Schweineseuche positive serum;5=pig vesicular stomatitis positive serum;6=atrophic rhinitis positive serum;7=pig
Pleuropneumonia positive serum;8=haemophilus parasuis positive serum;9=pig toxoplasma positive serum;10=swine C.psittaci sun
Property serum;11,12=APPV positive serum;13,14=APPV negative serum.
(2) sensitivity is tested: the APPV ELISA antibody assay kit that the present invention develops is to 10 parts of APPV E2 albumen
35 days immune serums and the doubtful affected animal serum of 15 parts of clinics are detected after immune, and test result shows development
APPV ELISA antibody assay kit sensibility with higher, as a result see Table 2 for details.No. 1-10 is Post-immunisation serum, 11-
25 be clinical doubtful affected animal serum.
Table 2: kit sensitivity tests result
Note: the criterion of 1.APPV ELISA antibody assay kit are as follows: when S/N value >=2.5, be judged to the positive;2.0
It is judged to when < S/P < 2.5 suspicious;Feminine gender is judged to when S/P value≤2.0.(3) repetitive test: 4 parts of APPV sun are chosen in this research
Property serum, every part of repetition does 4 holes, is coated under the ELISA Plate similarity condition of batch and detect 3 times with three respectively, and measurement makes a variation
Coefficient.Test result shows the coefficient of variation < 6% of repeated testing result.Illustrate that this law has repeatability well.Knot
See Table 3 for details for fruit.
Table 3: repetitive test result
4 kit forms of embodiment
ELISA Plate: coating APPV-E2 recombinant protein, peridium concentration are 25 μ g/ml, every 100 μ L of hole;
Dilution: self-control dilution
Cleaning solution: PBST
ELIAS secondary antibody: the goat-anti pig IgG of HRP label
Substrate developing solution: 3.3 ' -5,5 '-tetramethyl biphenyl amine aqueous solutions (TMB)
Terminate liquid: the H2SO4 of 2mol/L
Positive criteria product: known APPV positive serum
Negative standards' product: known APPV negative serum
APPV ELISA antibody assay kit operating procedure
Measuring samples are diluted with 400 times of Sample dilution, every 100 μ L of hole, if positive, negative and blank control, as
37 DEG C of incubation 1h, cleaning solution wash 3 times, each 5min, drying;ELIAS secondary antibody 100 μ L, 37 DEG C of incubation 1h, washing is added in every hole
Liquid washs 3 times, each 5min, drying;Each 50 μ L of substrate developing solution A, B respectively is added in every hole, is protected from light display 10min, is added
Terminate liquid, every 50 μ L of hole.Measure OD450nm.
Result judgement: when value >=1.0 standard positive serum OD450nm, when standard female serum OD value≤0.2, illustrate to try
It tests effectively;When S/N value >=2.5, it is judged to the positive;It is judged to when 2.0 < S/P < 2.5 suspicious;Feminine gender is judged to when S/P value≤2.0.
Sequence table
<110>YEBIO Bioengineering Co., Ltd of Qingdao
<120>one boar atypia pestivirus ELISA antibody assay kits
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1059
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
aatggtagtt ataacatagt gaggcaggcc agagacgaag taagcccgtc gacagggtgt 60
aaagaaggat atcccttcct gttttctgga gaaagatccg acacctcatg tctaaagcct 120
ccatccacta gttgggtcag accagtaaaa atggatgagg tatcagtggc agatagcttc 180
gcccatggag tagacaaggc gataatatta attagaaaag gggcgtcagg aatcatcaac 240
tttttagaca ccattgggag gtggctgcca gtggctgaag ccgccatagt accatattgt 300
gaaacttata ctgtaacagg gatgtacgtt catgtgaagc attgcctccc taaggggcta 360
ccaaagcatt caaaaataat ttccccaaca atgatatacc tgggggaagg tgacccagcc 420
cataatatcc agcatttgtt tggctcaggc atagcaaagt gggtcctagt cttacttggt 480
gtcttgggtg agtggtatgg ggagttggcc tccacgatat atctgctact ggagtacggg 540
actgaatggt tggaacatga aagtctagtc acggaagggt tgatccctgg catcaatatc 600
acagtagagc tccccgctag tcacacggta ccaggttggg tgtgggtcgc tggccagtgg 660
atatgcgtga agccagattg gtggcctaca cagatttgga ttgaaaccat agtgacagag 720
gtatggcata tactgaaaat attggcatca gctttggtaa atatagtcac tgcattcgta 780
aacctagagt tagtctatct ggtcataata ctagtcaaaa tatcaaaggg gaacttgata 840
ggtgctgtat tgtggtgcct cttgctgtca ggagctgagg ggtcatgcca cagaagacaa 900
gactattaca acatccaact ggtcgtcgag gaaaaaacag gaatagagaa gcgatctata 960
attggcaagt ggactgtgat aactagggaa ggcagagaac caagattaat ggagcagata 1020
aatatggtgt cgaatgagac cctatcagaa acttactgc 1059
<210> 2
<211> 1059
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
aatggtagtt ataacatagt gaggcaggcc agagacgaag taagcccgtc gacagggtgt 60
aaagaaggat atcccttcct gttttctgga gaaagatccg acacctcatg tctaaagcct 120
ccatccacta gttgggtccg tccagtaaaa atggatgagg tatctgtggc agatagcttc 180
gcccatggtg tagacaaggc gatcatcctg attcgtaaag gtgcgtctgg tatcatcaac 240
tttctggaca ccattggtcg ttggctgcca gtggctgaag ccgccatcgt accatattgt 300
gaaacttata ctgtaaccgg tatgtacgtt catgtgaagc attgcctgcc gaagggtctg 360
ccaaagcatt ctaaaatcat ttccccaacc atgatctacc tgggtgaagg tgacccagcc 420
cataacatcc agcatctgtt tggctctggc atcgcaaagt gggttctggt tctgctgggt 480
gttctgggtg agtggtatgg tgagctggcc tccaccatct atctgctgct ggagtacggt 540
actgaatggc tggaacatga atctctggtt accgaaggtc tgatcccggg catcaacatc 600
accgtagagc tgccggcttc tcacaccgta ccaggttggg tgtgggttgc tggccagtgg 660
atctgcgtga agccagattg gtggccgacc cagatttgga ttgaaaccat cgtgaccgag 720
gtatggcata tcctgaaaat cctggcatct gctctggtaa acatcgttac tgcattcgta 780
aacctcgagc tggtgtacct ggttatcatc ctggttaaaa tctctaaggg taacctgatc 840
ggtgctgtac tgtggtgcct gctgctgtct ggtgctgagg gttcttgcca ccgtcgtcaa 900
gactattaca acatccaact ggttgttgag gaaaaaaccg gtatcgagaa gcgttctatc 960
attggcaagt ggactgtgat cactcgtgaa ggccgtgaac cacgtctgat ggagcagatc 1020
aacatggtgt ctaacgagac cctgtctgaa acttactgc 1059
<210> 3
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
cggatcccgt ccagtaaaaa tggatg 26
<210> 4
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
cggaattcgc agtaagtttc agacaggg 28
<210> 5
<211> 307
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 5
Arg Pro Val Lys Met Asp Glu Val Ser Val Ala Asp Ser Phe Ala His
1 5 10 15
Gly Val Asp Lys Ala Ile Ile Leu Ile Arg Lys Gly Ala Ser Gly Ile
20 25 30
Ile Asn Phe Leu Asp Thr Ile Gly Arg Trp Leu Pro Val Ala Glu Ala
35 40 45
Ala Ile Val Pro Tyr Cys Glu Thr Tyr Thr Val Thr Gly Met Tyr Val
50 55 60
His Val Lys His Cys Leu Pro Lys Gly Leu Pro Lys His Ser Lys Ile
65 70 75 80
Ile Ser Pro Thr Met Ile Tyr Leu Gly Glu Gly Asp Pro Ala His Asn
85 90 95
Ile Gln His Leu Phe Gly Ser Gly Ile Ala Lys Trp Val Leu Val Leu
100 105 110
Leu Gly Val Leu Gly Glu Trp Tyr Gly Glu Leu Ala Ser Thr Ile Tyr
115 120 125
Leu Leu Leu Glu Tyr Gly Thr Glu Trp Leu Glu His Glu Ser Leu Val
130 135 140
Thr Glu Gly Leu Ile Pro Gly Ile Asn Ile Thr Val Glu Leu Pro Ala
145 150 155 160
Ser His Thr Val Pro Gly Trp Val Trp Val Ala Gly Gln Trp Ile Cys
165 170 175
Val Lys Pro Asp Trp Trp Pro Thr Gln Ile Trp Ile Glu Thr Ile Val
180 185 190
Thr Glu Val Trp His Ile Leu Lys Ile Leu Ala Ser Ala Leu Val Asn
195 200 205
Ile Val Thr Ala Phe Val Asn Leu Glu Leu Val Tyr Leu Val Ile Ile
210 215 220
Leu Val Lys Ile Ser Lys Gly Asn Leu Ile Gly Ala Val Leu Trp Cys
225 230 235 240
Leu Leu Leu Ser Gly Ala Glu Gly Ser Cys His Arg Arg Gln Asp Tyr
245 250 255
Tyr Asn Ile Gln Leu Val Val Glu Glu Lys Thr Gly Ile Glu Lys Arg
260 265 270
Ser Ile Ile Gly Lys Trp Thr Val Ile Thr Arg Glu Gly Arg Glu Pro
275 280 285
Arg Leu Met Glu Gln Ile Asn Met Val Ser Asn Glu Thr Leu Ser Glu
290 295 300
Thr Tyr Cys
305
Claims (10)
1. the gene of a boar atypia seasonal febrile diseases toxalbumin, which is characterized in that the nucleotides sequence of the gene is classified as SEQ ID
NO.2。
2. a kind of for expanding the amplimer pair of gene described in claim 1, which is characterized in that the primer pair it is upper
The sequence for swimming primer is SEQ ID NO.3;The sequence of downstream primer is SEQ ID NO.4.
3. a boar atypia pestivirus ELISA antibody assay kit, which is characterized in that the kit includes amino
Acid sequence be SEQ ID NO:1 pig atypia pestivirus E2 albumen as the elisa plate of antigen coat, positive control serum,
Negative control sera, sample diluting liquid, enzyme label conjugate, cleaning solution, substrate solution A, substrate solution B and terminate liquid;
Wherein positive control serum is made of APPV vaccine inoculation health pig;
Negative control sera is the serum of health pig;
The E2 protein coding gene that nucleotides sequence is classified as SEQ ID NO.2 is integrated on pET28a carrier by the E2 albumen
It is built into recombinant plasmid, recombinant plasmid is imported into competent escherichia coli cell and carries out inducing expression, is separated by His nickel column pure
Change preparation.
4. kit as claimed in claim 3, which is characterized in that the sample diluting liquid is the PBS with 0.01M pH7.2
Supernatant solution after the thallus ultrasound of the diluted bacillus coli BL21 of solution.
5. kit as claimed in claim 3, which is characterized in that the enzyme label conjugate is horseradish peroxidase mark
The goat-anti pig antibody of note is made of 12000 times of enzyme labelled antibody diluted.
6. kit as claimed in claim 3, which is characterized in that the cleaning solution is by 14.9g KH2PO4, 79.1g
K2HPO4·3H2O, 50.0ml Tritonx-100,1.0g NaN3, 8.0g NaCl is dissolved in 500ml water, adjusts pH 7.3, finally
It is settled to 1L.
7. kit as claimed in claim 3, which is characterized in that the substrate solution A is by 4.2g citric acid, 13.6g
Sodium acetate, 0.5g peroxide are crossed urea and are dissolved in 100ml deionized water, are settled to 1L, adjust pH value to made of 5.0.
8. kit as claimed in claim 3, which is characterized in that the substrate solution B is that 1g TMB is dissolved in 50ml
100ml formaldehyde is added in dimethyl sulfoxide, after dissolution, 16.0g polyvinylpyrrolidone is settled to after dissolution with deionized water
1L completes preparation.
9. kit as claimed in claim 3, which is characterized in that the terminate liquid is the sulfuric acid solution of 2mol/L.
10. kit as claimed in claim 3, which is characterized in that the preparation method of the elisa plate will purify
Pig atypia pestivirus E2 proteantigen is diluted to 25 μ g/ml with the carbonate buffer solution of pH 9.5,0.05mol/L, is added 96
Hole is coated in plate, 100 holes μ l/, sets 4 DEG C overnight, and after washing 3 times with cleaning solution, the pH of 0.01mol/L containing 1%BSA is added
7.2PBS closing overnight, is got rid of deblocking liquid, is placed under room epidemic disaster 40%, and the preparation of completion in 24-48 hours is spontaneously dried.
Priority Applications (1)
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CN201811075364.0A CN109187952A (en) | 2018-09-14 | 2018-09-14 | One boar atypia pestivirus ELISA antibody assay kit |
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CN201811075364.0A CN109187952A (en) | 2018-09-14 | 2018-09-14 | One boar atypia pestivirus ELISA antibody assay kit |
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Family
ID=64911324
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CN201811075364.0A Withdrawn CN109187952A (en) | 2018-09-14 | 2018-09-14 | One boar atypia pestivirus ELISA antibody assay kit |
Country Status (1)
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CN (1) | CN109187952A (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110873792A (en) * | 2019-11-29 | 2020-03-10 | 江苏省农业科学院 | African swine fever virus antibody detection kit |
CN112285347A (en) * | 2020-09-30 | 2021-01-29 | 西北农林科技大学 | ELISA detection kit for pathogen antibody in porcine serum sample |
-
2018
- 2018-09-14 CN CN201811075364.0A patent/CN109187952A/en not_active Withdrawn
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110873792A (en) * | 2019-11-29 | 2020-03-10 | 江苏省农业科学院 | African swine fever virus antibody detection kit |
CN110873792B (en) * | 2019-11-29 | 2022-11-29 | 江苏省农业科学院 | African swine fever virus antibody detection kit |
CN112285347A (en) * | 2020-09-30 | 2021-01-29 | 西北农林科技大学 | ELISA detection kit for pathogen antibody in porcine serum sample |
CN112285347B (en) * | 2020-09-30 | 2023-12-22 | 西北农林科技大学 | ELISA detection kit for pathogenic antibodies in pig serum sample |
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