CN109187952A - One boar atypia pestivirus ELISA antibody assay kit - Google Patents

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

One boar atypia pestivirus ELISA antibody assay kit

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 OD_450nmWhen 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 OD_450nm> 0.8, negative control sera OD_450nm< 0.20, blank well OD₄₅₀< 0.10, experiment is set up, otherwise in vain.

Calculation formula: S/N value=sample to be examined OD_450nm/ negative control OD_450nmMean 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)

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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

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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.