CN104087609B - A kind of universal DHAV antibody ELISA detection kit - Google Patents
A kind of universal DHAV antibody ELISA detection kit Download PDFInfo
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Abstract
The invention discloses a kind of universal DHAV antibody ELISA detection kit.This test kit contains with the duck hepatitis A virus (HAV) DHAV 1VP3 coated elisa plate of 3VP1 recombiant protein.This recombiant protein is to obtain with following method: using DHAV 1 and DHAV 3 as material, respectively DHAV 1VP3 gene and DHAV 1VP1 gene expanded, clone and obtain recombiant plasmid pMD 1VP3 and pMD 3VP1, then orienting series be inserted into pET 32a (+) expression vector, screening obtains recombinant expression plasmid pET 1VP3 3VP1, through ITPG abduction delivering, purification, it is thus achieved that DHAV 1VP3 3VP1 recombiant protein.1 and DHAV 3 two kind of antibody horizontal of DHAV can be made an appraisal by this test kit one-time detection, with low cost, easy and simple to handle, quick, is suitable for the detection of batch sample.
Description
Technical field
The present invention relates to a kind of universal DHAV antibody assay kit, for DHAV 1 type
(DHAV-1) the quick detection of and 3 types (DHAV-3) antibody, belongs to biological technical field.
Background technology
Duck viral hepatitis is the one lethal infectious diseases acute, high of harm duckling, have that morbidity is anxious, the course of disease is short, propagation is fast,
Case fatality rate high, duckling within main infringement 3 week old, serious threat duck culturing industry develops in a healthy way.Its cause of disease is duck A type liver
Scorching virus (Duck hepatitis A virus, DHAV), the fowl hepatitis virus belonging to Picornaviridae belongs to, due to DHAV
There is no cross reaction between significant difference, and veriform DHAV between sequence, again DHAV is divided into DHAV-1, DHAV-2
And DHAV-3, its respectively corresponding Gene A type (serum 1 type), gene Type B (Taiwan is novel) and gene C type (Korea S
Novel).Up to now, domestic popular predominantly DHAV-1 and DHAV-3 strain.In addition to Taiwan, inland there is not yet DHAV-2
The report of strain.Along with duck culturing industry constantly develops to industrialization, intensive and scale direction, the tradition that many areas are carried out
Active immunity or passive immunity failure phenomenon that duck liver is scorching frequently occur, and the prevention and control scorching to duck liver propose new challenge.At present
DHAV-1 vaccine has got the Green Light code, and DHAV-3 vaccine is still in development process.In view of current DHAV-1 and DHAV-3
Harm be on the rise, the exploitation of multivalence Seedling is extremely urgent.
After vaccine development success, face again and how to be tracked monitoring to immune antibody？Traditional serum neutralization test cycle is long,
Waste time and energy, be unfavorable for the quick detection of batch sample.ELISA method based on totivirus foundation is more tired because of DHV purification ratio
Difficult and limit the popularization and application of the method, the ELISA side that the major antigen albumen utilizing escherichia coli expression reported is set up
Method is the DHAV for one of them serotype, when evaluating for polyvalent vaccine, need to carry out respectively DHAV-1 and
The detection of DHAV-3 antibody, complex operation, cost is double.Have not yet to see for DHAV-1 and DHAV-3 antibody general
The report of type ELISA detection method.In picornavirus, VP1 and VP3 albumen as main host's protected protein,
Encode main antigen site and have in main type specificity and site, being the main component determining virus antigenicity, be mesh
The focus gene of front research.Therefore, utilize technique for gene engineering to develop and be applicable to DHAV-1 and DHAV-3 antibody test
Universal ELISA detection kit special, sensitive, that be suitable for basic unit's application, duck hepatitis A current for China is sick
Quick diagnosis and effective prevention and control of poison are significant.
Summary of the invention
It is an object of the invention to overcome the deficiency of tradition duck liver inflammation context of detection, DHAV-1 and DHAV-3 two kinds can be carried out simultaneously
The evaluation of antibody horizontal.The present invention provides a kind of universal ELISA detection kit for DHAV-1 and DHAV-3 antibody,
This test kit is with low cost, easy and simple to handle, quick, is particularly suited for the antibody detection of batch sample after vaccine immunity, significantly carries
The high scorching serodiagnostic speed of duck liver.
It is an object of the invention to be achieved through the following technical solutions: general DHAV 1 type VP3 and 3 types
The preparation of VP1 (DHAV-1VP3-3VP1) recombiant protein, comprises the following steps:
1) using DHAV-1 virus as material, by RT-PCR method, its VP3 gene (SEQ-1) is expanded, gram
Grand obtain recombiant plasmid pMD-1VP3；Expand the upstream and downstream primer of VP3 gene respectively:
Positive strand primer (forward primer): 5'-AAGGGATCCGGAAAGAGAAARCCACG-3'(SEQ-5)(BamH I)
Negative strand primer (downstream primer): 5'-ATCGTCGACCTGATYATTGGTTGCCAT-3'(SEQ-6)(Sal I)
Amplified fragments size is 711bp；
2) using DHAV-3 virus as material, by RT-PCR method, its VP1 gene (SEQ-2) is expanded, gram
Grand obtain recombiant plasmid pMD-3VP1；Expand the upstream and downstream primer of VP1 gene respectively:
Positive strand primer (forward primer): 5'-ATTGTCGACGGTGATTCCAATCAGCTTG-3'(SEQ-7)(Sal I)
Negative strand primer (downstream primer): 5'-AGTCTCGAGTTCAATYTCCARATGGAG-3'(SEQ-8)(Xhol I)
Amplified fragments size is 720bp；
3) with BamH I and Sal I simultaneously to recombiant plasmid pMD-1VP3 and pET-32a (+) carrier carries out enzyme action, reclaims purpose
Fragment, 16 DEG C connect overnight, convert DH5 α competent cell, extract plasmid, are just identifying through BamH I and Sal I double digestion
Positive recombiant plasmid pET-1VP3 is obtained after really；With Sal I and Xhol I simultaneously to recombiant plasmid pMD-3VP1 and pET-1VP3
Carrying out enzyme action, reclaim purpose fragment, 16 DEG C connect overnight, convert BL21 (DE3) competent cell, extract plasmid, through Sal I
The correct positive recombinant expression plasmid pET-1VP3-3VP1 of rear acquisition is identified with Xhol I double digestion；
4) by positive recombinant expression plasmid bacterium in 37 DEG C of cultivations, when A600 value reaches 0.4～0.6, IPTG is added to the denseest
Spend and carry out abduction delivering for 0.8mmol/L, the thalline of 5h after collection abduction delivering, ultrasonic disruption, take after being centrifuged and precipitate into one
Walk isolated and purified, obtain purer purpose recombiant protein.
The technical scheme is that a kind of universal DHAV antibody ELISA detection kit, it is characterized in that,
Including with the coated elisa plate of DHAV-1VP3-3VP1 recombiant protein.
The optimum preparating condition of elisa plate is: be coated liquid with the 0.05M Tris-HCL buffer of pH8.5, will
DHAV-1VP3-3VP1 recombiant protein is diluted to 10 μ g/mL, adds in ELISA Sptting plate by 100 μ L/ holes, 37 DEG C of effects 2
Hour, 4 DEG C are coated overnight, pat dry, and close 2 hours with 1% bovine serum albumin (BSA) 37 DEG C, with containing 0.05% tween
The PBS washing of-20pH7.4, pats dry, and adds 20% sucrose phosphate buffer room temperature and protects 3 hours, treats that it is dried
Load in the packaging bag containing desiccant and preserve.
The further technical scheme of the present invention is: a kind of universal DHAV antibody ELISA detection kit, including
Following components: with the coated elisa plate of DHAV-1VP3-3VP1 recombiant protein: 5 pieces；Sample diluting liquid: 200mL；10
× concentrated cleaning solution: 400mL (dilutes with front 1:10)；Enzyme conjugates working solution: 50mL；Nitrite ion: 100mL；Stop buffer:
60mL；Positive control: 2mL；Negative control: 2mL.
In above-mentioned universal DHAV antibody ELISA detection kit, sample diluting liquid is containing 0.05% tween 20
The phosphate buffer of 0.01M pH7.4；10 × concentration washing liquid is that the phosphate of the 0.1M pH7.4 containing 0.5% tween 20 delays
Rush liquid；Enzyme conjugates working solution is the HRP-goat-anti duck IgG that KPL company of the U.S. produces；Nitrite ion is 0.2mg/mL tetramethyl
Benzidine (TMB) solution and the citrate-phosphate salt buffer containing 0.5 ‰ hydrogen peroxide ureas, the two volume ratio is 1:1；
Stop buffer is 0.31% hydrofluoric acid solution；Positive control is the positive serum obtained through DHAV-1VP3-3VP1 recombiant protein immunity
(OD650nm>=1.0), the mycillin of 1000U/mL, aseptic filtration are added；Negative control is the negative serum obtained through screening
(OD650nm≤ 0.25), the mycillin of 1000U/mL, aseptic filtration are added.
Above-mentioned universal DHAV antibody ELISA detection kit using method: by serum sample diluting liquid to be checked
Make 1:200 dilution, add in antibody test plate by 100 μ L/ holes, set negative control, positive control simultaneously, hatch 45min for 37 DEG C；
Discarding the liquid in reacting hole, every hole adds cleaning mixture 350 μ L, washs 3～5 times, every minor tick 1min, pats dry；Every hole adds 100 μ L
Enzyme conjugates working solution, hatch 45min for 37 DEG C；Wash 3～5 times, every minor tick 1min, pat dry；It is sequentially added into 100 μ L
Nitrite ion, 37 DEG C of lucifuges hatch 15min；Add 50 μ L stop buffers, under 650nm wavelength, measure each hole absorbance A by microplate reader
Value, readings calculates and result of determination.The criterion of its detection sample is: with sample OD to be checked650nmValue and negative control
OD650nmThe ratio (P/N) of value is more than or equal to 2.1, and sample OD to be checked650nmValue is judged to the positive more than 0.381.
The present invention has the advantage that
1. the universal DHAV antibody ELISA detection kit that the present invention sets up, one-time detection can be right
Two kinds of antibody horizontals of DHAV-1 and DHAV-3 are made an appraisal, and when relatively DHAV-1 and DHAV-3 detects respectively, cost drops significantly
Low.This test kit is easy and simple to handle, quick, is suitable for the detection of batch sample, substantially increases the scorching serodiagnostic speed of duck liver.
2. the present invention is prepared from based on the DHAV-1VP3-3VP1 recombiant protein of gene engineering expression, and recombiant protein is non-
Totivirus antigen, safety is good, without unrelated foreign protein, only with DHAV-1 and DHAV-3 positive serum specific bond, no
With the positive serum generation cross reaction of other virus, having good antigenicity, the detection kit therefore invented has very
High specificity and sensitivity.
Accompanying drawing explanation
Fig. 1 is the electrophoresis picture of DHAV-1VP3 gene amplification, wherein M:DNA Marker DL2000,1:DHAV-1VP3
Gene.
Fig. 2 is the electrophoresis picture of DHAV-3VP1 gene amplification, wherein M:DNA Marker DL2000,1:DHAV-3VP1
Gene.
Fig. 3 is the enzyme action qualification figure that DHAV-1VP3 gene is cloned into the recombiant plasmid pMD-1VP3 that pMD18-T carrier obtains
Sheet, wherein M1:DNA Marker DL15000, M2:DNA Marker DL2000,1: recombiant plasmid pMD-1VP3
Endonuclease bamhi.
Fig. 4 is the enzyme action qualification figure that DHAV-3VP1 gene is cloned into the recombiant plasmid pMD-3VP1 that pMD18-T carrier obtains
Sheet, wherein M1:DNA Marker DL15000, M2:DNA Marker DL2000,1: recombiant plasmid pMD-3VP1
Endonuclease bamhi.
Fig. 5 be DHAV-1VP3 with DHAV-3VP1 gene tandem be inserted into pET-32a (+) recombiant plasmid that obtains of carrier
Enzyme action qualification picture, wherein the M1:DNA Marker DL15000 of pET-1VP3-3VP1, M2:DNA Marker DL2000,
1: the endonuclease bamhi of recombiant plasmid pET-1VP3-3VP1.
Fig. 6 is that the SDS-PAGE of induction expression protein analyzes picture, wherein M: albumen Marker, 1～4 recombiant plasmid
PET-1VP3-3VP1 abduction delivering 2h respectively, the supernatant of 3h, 4h, 5h, 5～8: recombiant plasmid pET-1VP3-3VP1 is respectively
Abduction delivering 2h, the precipitation of 3h, 4h, 5h.
Fig. 7 is the Western blotting analysis picture that DHAV-1VP3-3VP1 expression product reacts with DHAV-1 positive serum,
Wherein M: pre-dyed albumen Marker, 1: induction expression protein.
Fig. 8 is the Western blotting analysis picture that DHAV-1VP3-3VP1 expression product reacts with DHAV-3 positive serum,
Wherein M: pre-dyed albumen Marker, 1: induction expression protein.
Fig. 9 is the purification picture of recombiant protein, wherein M: albumen Marker, 1: the recombiant protein of purification, 2: unpurified
Recombiant protein.
Detailed description of the invention
The amplification of 1.DHAV-1VP3 gene and DHAV-3VP1 gene and the structure of expression vector
According to DHAV-1VP3 gene order (SEQ-1) listed in GenBank and DHAV-3VP1 gene order
(SEQ-2), design two is to primer: P1 forward primer: 5'-AAGGGATCCGGAAAGAGAAARCCACG-3'
(SEQ-5)；P1 downstream primer: 5'-ATCGTCGACCTGATYATTGGTTGCCAT-3'(SEQ-6).P2 draws upstream
Thing: 5'-ATTGTCGACGGTGATTCCAATCAGCTTG-3'(SEQ-7)；P2 downstream primer: 5'
-AGTCTCGAGTTCAATYTCCARATGGAG-3'(SEQ-8).Trizol reagent is utilized to extract DHAV-1 respectively
With DHAV-3RNA template, utilize P1 and P2 primer to carrying out RT-PCR reaction respectively.RT reaction system (20 μ L):
25mmol/L Mg2+1.2 μ L, 10mmol/L dNTP2.0 μ L, 5 × RT buffer 4.0 μ L, downstream primer 1.0 μ L, RNA
Enzyme inhibitor 0.5 μ L, RNA template 10.3 μ L.Being placed in PCR amplification instrument to react, reverse transcription 1 μ L adds in 42 DEG C time
Enter；Response procedures: 70 DEG C of 5min, 42 DEG C of 30min, 95 DEG C of 2min.PCR reaction system (50 μ L): ddH2O38.1μL、
10 × buffer 4.0 μ L, 25mmol/L Mg2+1.4 μ L, 5U/ μ L rTaqDNA polymerase 0.5 μ L, forward primer 1.0 μ L, RT
Product 5.0 μ L.It is placed in PCR amplification instrument to react, response procedures: 95 DEG C of 5min denaturations；94 DEG C of degeneration 30sec, 50 DEG C
Annealing 30sec, 72 DEG C of extensions 30sec, totally 30 circulations；10min is extended after 72 DEG C；4 DEG C of preservations.PCR primer is 1%
Electrophoresis in agarose gel, to determine product size (as depicted in figs. 1 and 2).
The DHAV-1VP3 gene amplified is cloned in pMD18-T carrier, identify through BamH I and Sal I double digestion,
The named pMD-1VP3 of the positive colony (as shown in Figure 3) checking order correct.The DHAV-3VP1 gene amplified is cloned into
In pMD18-T carrier, identify through Sal I and Xhol I double digestion, the named pMD-3VP1 of correct positive colony that checks order (as
Shown in Fig. 4).With BamH I and Sal I simultaneously to recombiant plasmid pMD-1VP3 and pET-32a (+) carrier carries out enzyme action, reclaim
Purpose fragment, 16 DEG C connect overnight, convert DH5 α competent cell, extract plasmid, reflect through BamH I and Sal I double digestion
The fixed correct positive recombiant plasmid pET-1VP3 of rear acquisition.With Sal I and Xhol I simultaneously to recombiant plasmid pMD-3VP1 and
PET-1VP3 carries out enzyme action, reclaims purpose fragment, and 16 DEG C connect overnight, convert BL21 (DE3) competent cell, extract matter
Grain, obtains positive recombinant expression plasmid pET-1VP3-3VP1 (as shown in Figure 5) after Sal I and Xhol I double digestion identify correctly.
2. the abduction delivering of recombinant expression plasmid
By the positive recombinant expression plasmid bacterium of acquisition in 37 DEG C of cultivations, when A600 value reaches 0.4～0.6, add IPTG extremely
Final concentration of 0.8mmol/L, carries out abduction delivering.Collecting the thalline that different induction time is expressed, ultrasonic disruption, after being centrifuged
Take cleer and peaceful precipitation respectively and carry out SDS-PAGE electrophoresis, and expression product is carried out Western blotting analysis, an anti-difference
With chicken anti-DHAV-1 and DHAV-3 positive serum, the sheep anti-chicken IgG of two anti-horseradish peroxidase (HRP) labellings, DAB
Colour developing.Result shows that recombiant protein is present in thalline with insoluble inclusion bodies, and molecular weight is about 74ku, with expected results
It is consistent, maximum (as shown in Figure 6) with 5h expression after induction.Western blotting analyzes discovery, the recombiant protein energy of expression
Enough react with DHAV-1 and DHAV-3 positive serum, there is good immunologic competence (as shown in Figure 7 and Figure 8).
3. the purification of recombiant protein
Select KCL development process: the recombinant bacterium crushed is carried out SDS-PAGE electrophoresis, and electrophoresis takes off gel after terminating, and first uses
Distilled water washs, and develop the color in the KCL solution of the 250mmol/L being then immersed in 4 DEG C of pre-coolings 5-10min, finally washes with distilled water
Wash.Destination protein band is cut, PBS wash 3 times, smash carefully with Glass rod, multigelation 3 times, 8000r/min from
Heart 10min, draws supernatant, and SDS-PAGE electrophoresis carries out Purity.Result only has the destination protein band of a 74ku,
Show that KCL development process obtains purer destination protein (as shown in Figure 9).
4. the preparation of sample diluting liquid, cleaning mixture, stop buffer
Sample diluting liquid is the 0.01M pH7.4 phosphate buffer (KH containing 0.05% tween 202PO40.2g,
NaHPO4·12H2O2.9g, NaCl8g, be settled to 1000mL, then add 0.5mL tween 20)；10 × concentrated cleaning solution is for containing
The 0.1M pH7.4 phosphate buffer (KH of 0.5% tween 202PO42g, NaHPO4·12H2O29g, NaCl80g, fixed
Hold to 1000mL, then add 5mL tween 20)；Stop buffer is that 0.31% hydrofluoric acid solution (takes Fluohydric acid. 0.31ml, distilled water
It is settled to 100mL).
5. positive control and the preparation of negative control
The positive serum sample diluting liquid that DHAV-1VP3-3VP1 recombiant protein immunity obtains is made 1:200 dilution
(OD650nm>=1.0), the mycillin of addition 1000U/mL, aseptic filtration, indirect as universal anti-HAV
Positive control in ELISA detection kit；Negative serum (the OD that screening is obtained650nm≤ 0.25), 1000U/mL is added
Mycillin, aseptic filtration, as the negative control in universal anti-HAV indirect ELISA testing kit.
6. the preparation of nitrite ion
Nitrite ion: weigh 200mg tetramethyl benzidine (TMB), after dissolving with 100mL dehydrated alcohol or DMSO, with double
Steam water and be settled to 1000mL；Weigh 21g citric acid (C6H8O7·H2O), 28.2g disodium hydrogen phosphate,anhydrous (Na2HPO4),
6.4mL0.75% hydrogen peroxide urea, distilled water is settled to 1000mL, adjusts pH value 4.5～5.0；The volume ratio of the two is 1:1.
7. the determination of the indirect ELISA reaction condition of detection DHAV-1 and DHAV-3 antibody
Antigen and the determination of serum best effort concentration: use square formation test to determine.With being coated buffer by DHAV-1VP3-3VP1
Recombiant protein makees the serial dilutions such as 1:10,1:20,1:40,1:80, is coated ELISA Sptting plate, 100 μ L/ holes；Anti-DHAV-1
1:50,1:100,1:200,1:400,1:800 is made respectively with positive serum and the negative serum sample diluting liquid of DHAV-3,
The serial dilutions such as 1:1600,1:3200；Carry out indirect ELISA mensuration；Nitrite ion develops the color, and stop buffer terminates reaction；Measure light
The OD value of wavelength 650nm.Take positive serum OD650nmAbout 1.0, negative serum OD650nmAbout 0.3, and positive serum
OD650nm/ negative serum OD650nmI.e. P/N value more than 2.1 antigen concentration and serum dilution be best effort concentration, result
As shown in table 1, result shows that serum optimum dilution degree is 1:200, antigen optium concentration 10 μ g/mL.
Table 1 antigen and the determination (OD of serum best effort concentration450nmValue)
8. result criterion
By clear for 30 parts of Sanguis Anas domestica without DHAV-1 and DHAV-3 antibody of collection, under best operating condition, carry out indirect ELISA
Measure, to determine Sanguis Anas domestica its absorption value scope when infecting clearly without DHAV-1 and DHAV-3, it is thus determined that with sample OD to be checked650nmValue and negative OD650nmThe ratio of value
(P/N) more than or equal to 2.1, and sample OD to be checked650nmValue is judged to the positive more than 0.381.
The preparation of the most universal DHAV antibody test elisa plate
It is coated liquid with the 0.05M Tris-HCL buffer of pH8.5, DHAV-1VP3-3VP1 recombiant protein is diluted to
10 μ g/mL, add in ELISA Sptting plate by 100 μ L/ holes, and 37 DEG C act on 2 hours, and 4 DEG C are coated overnight, pat dry, with 1%
Bovine serum albumin (BSA) 37 DEG C is closed 2 hours, washs with the PBS containing 0.05% tween 20 pH7.4, pats dry, then add
Enter 20% sucrose phosphate buffer room temperature to protect 3 hours, treat that it loads in the packaging bag containing desiccant after drying standby.
The determination of 10.ELISA operation sequence
Operate by optimal conditions determined above, i.e. obtain the process optimization program of this method:
Serum sample diluting liquid to be checked being made 1:200 dilution, adds in antibody test plate by 100 μ L/ holes, to set negative right simultaneously
According to, positive control, hatch 45min for 37 DEG C；Discarding the liquid in reacting hole, every hole adds cleaning mixture 350 μ L, washs 3～5 times,
Every minor tick 1min, pats dry；Every hole adds the enzyme conjugates working solution of 100 μ L, hatches 45min for 37 DEG C；Wash 3～5 times, often
Minor tick 1min, pats dry；Adding 100 μ L nitrite ions, 37 DEG C of lucifuges hatch 15min；Add 50 μ L stop buffers, use microplate reader
Measuring each hole absorbance A value under 650nm wavelength, readings calculates and result of determination.
Embodiment:
One, universal duck hepatitis A virus (HAV) antibody ELISA detection kit, including following components:
1) elisa plate bar (96 hole): 5 pieces
2) 10 × concentrated cleaning solution: 400mL (dilutes with front 1:10)
3) sample diluting liquid: 200mL
4) goat-anti duck ELIAS secondary antibody (enzyme conjugates working solution): 50mL
5) nitrite ion: 100mL
6) stop buffer: 60mL
7) positive serum controls (+): 2mL
8) negative serum control (-): 2mL
Two, operating procedure:
1, serum sample diluting liquid to be checked is made 1:200 dilution, adds in antibody test plate by 100 μ L/ holes, set feminine gender simultaneously
Serum control, positive serum controls, hatch 45min for 37 DEG C；
2, discarding the liquid in reacting hole, every hole adds cleaning mixture 350 μ L, washs 3～5 times, every minor tick 1min, pats dry；
3, every hole adds the enzyme conjugates working solution of 100 μ L, hatches 45min for 37 DEG C；
4, step 2 is repeated；
5, adding 100 μ L nitrite ions, 37 DEG C of lucifuges hatch 15min；
6, adding 50 μ L stop buffers, measure each hole absorbance A value by microplate reader under 650nm wavelength, readings calculates and judges knot
Really.
Three, application
1, specific test
1.1 cross matchings: with DHAV-1VP3-3VP1 recombiant protein set up indirect ELISA testing kit detect respectively duck pestilence,
Positive serum and the feminine genders such as duck reovirus disease, duck newcastle, duck influenza, duck hepatitis A virus (HAV) 1 type, duck hepatitis A virus (HAV) 3 type
Serum, the repetition of 4, every sample, carry out cross reactivity mensuration, testing result display duck hepatitis A virus (HAV) 1 type, duck hepatitis A virus (HAV)
3 type positive serums are positive findings, and remaining is feminine gender, show this detection kit and duck pestilence, duck reovirus disease,
The no cross reactions such as duck newcastle and duck influenza；
1.2 prevent test: by DHAV-1VP3-3VP1 positive serum respectively with isopyknic DHAV-1 (200TCID50/0.2ml)
With DHAV-3 (200TCID50/ 0.2ml) and duck plague virus (200TCID50/ 0.2ml) mixing, room temperature effect 30min,
Serum after processing and the serum not making any process are carried out preventing test determination, result to show by optimum reaction condition respectively
DHAV-1VP3-3VP1 positive serum can only be prevented by DHAV-1 and DHAV-3 specificity, and can not be prevented by duck plague virus.
2, sensitivity tests
Each 5 parts of DHAV-1 and DHAV-3 positive serum starts doubling dilution from 1:2 respectively, and remaining condition presses optimum response bar
Part carries out ELISA detection, surveys its end point titres with DHAV-1 and DHAV-3 virus neutralization tests (VN) respectively
Fixed.Result shows, the sensitivity of ELISA detection kit will be apparently higher than neutralization test (as shown in table 2 and table 3), ELISA
Test kit is respectively 0.993 and 0.984 with the correlation coefficient of DHAV-1 and DHAV-3 neutralization test.
Table 2 sensitivity tests (DHAV-1 positive serum)
Table 3 sensitivity tests (DHAV-3 positive serum)
3, replica test
By twice coated ELISA Plate, detect 5 parts of DHAV-1 positive serums, 5 parts of DHAV-3 positive serums and 5 parts of negative blood
Clearly, each sample duplicate detection 5 times, measure its coefficient of variation CV% (CV=S.D./X × 100%, S.D.: standard deviation, X:
Arithmetic mean of instantaneous value).Result shows that the coefficient of variation is 5.42%, minimum 1.2% to the maximum.15 parts of serum coefficient of variation are the least,
There is preferable repeatability.
4, clinical practice
The sample of clinical practice inspection is test specimen and part submitted sample.First detect with serum neutralization test, distinguish
DHAV-1 and DHAV-3 positive serum and common negative serum, then detect by universal ELISA detection kit, than
The relatively coincidence rate of two kinds of methods.Wherein neutralization test detection DHAV-1 positive serum 30 parts, positive rate is 55.56%,
ELISA detection kit detection DHAV-1 positive serum is 32 parts, and positive rate is 59.26%, meeting of two kinds of methods
Rate is 96.3%；Neutralization test detection DHAV-3 positive serum 38 parts, positive rate is 61.29%, and ELISA detects examination
Agent box detection DHAV-3 positive serum is 41 parts, and positive rate is 66.13%, the coincidence rate of two kinds of methods be 96.77% (as
Shown in table 4).
Table 4ELISA detection kit compares with neutralization test
Note: detection coincidence rate=(common number positive+common negative number)/gross sample number × 100%
Claims (8)
1. a DHAV DHAV-1VP3-3VP1 recombiant protein, is characterized in that, following method prepare:
1) using DHAV-1 virus as material, by RT-PCR method, its VP3 gene expanded, clone and obtain recombiant plasmid pMD-1VP3；
2) using DHAV-3 virus as material, by RT-PCR method, its VP1 gene expanded, clone and obtain recombiant plasmid pMD-3VP1；
3) withBamH I andSalI simultaneously to recombiant plasmid pMD-1VP3 and pET-32a (+) carrier carries out enzyme action, reclaims purpose fragment, 16 DEG C connect overnight, convert DH5 α competent cell, extract plasmid, warpBamH I andSalI double digestion identifies the correct positive recombiant plasmid pET-1VP3 of rear acquisition；WithSalI andXhoL I carries out enzyme action simultaneously to recombiant plasmid pMD-3VP1 and pET-1VP3, reclaims purpose fragment, and 16 DEG C connect overnight, convert BL21 (DE3) competent cell, extract plasmid, warpSalI andXhoL I double digestion identifies the correct positive recombinant expression plasmid pET-1VP3-3VP1 of rear acquisition；
4) by the positive plasmid bacterium that obtains in 37 DEG C of cultivation, when A600 value reaches 0.4～0.6, add IPTG to final concentration of 0.8mmol/L and carry out abduction delivering, collect the thalline of 5h, ultrasonic disruption after abduction delivering, take after being centrifuged precipitate the most isolated and purified.
2. DHAV DHAV-1VP3-3VP1 recombiant protein as claimed in claim 1, is characterized in that, the upstream and downstream primer of described amplification VP3 gene respectively:
Forward primer: 5'-AAGGGATCCGGAAAGAGAAARCCACG-3',
Downstream primer: 5'-ATCGTCGACCTGATYATTGGTTGCCAT-3'；
Amplified fragments size is 711bp.
3. DHAV DHAV-1VP3-3VP1 recombiant protein as claimed in claim 1, is characterized in that, the upstream and downstream primer of described amplification VP1 gene respectively:
Forward primer: 5'-ATTGTCGACGGTGATTCCAATCAGCTTG-3',
Downstream primer: 5'-AGTCTCGAGTTCAATYTCCARATGGAG-3'；
Amplified fragments size is 720bp.
4. a universal DHAV antibody ELISA detection kit, is characterized in that, including with the coated elisa plate of DHAV-1VP3-3VP1 recombiant protein described in any one in claim 1-3.
Universal DHAV antibody ELISA detection kit the most as claimed in claim 4; it is characterized in that; the preparation method of described elisa plate is: be coated liquid with the 0.05M Tris-HCL buffer of pH8.5; DHAV-1VP3-3VP1 recombiant protein is diluted to 10 μ g/mL; add in ELISA Sptting plate by 100 μ L/ holes; 37 DEG C act on 2 hours; 4 DEG C are coated overnight; pat dry; close 2 hours with 1% bovine serum albumin 37 DEG C; wash with the PBS containing 0.05% tween 20 pH7.4, pat dry, add 20% sucrose phosphate buffer room temperature and protect 3 hours.
6. the universal DHAV antibody ELISA detection kit as described in claim 4 or 5, is characterized in that, also includes sample diluting liquid, 10 × concentrated cleaning solution, enzyme conjugates working solution, nitrite ion, stop buffer, positive control and negative control.
Universal DHAV antibody ELISA detection kit the most as claimed in claim 6, is characterized in that, described sample diluting liquid is the phosphate buffer of the 0.01M pH7.4 containing 0.05% tween 20；Described 10 × concentrated cleaning solution is the phosphate buffer of the 0.1M pH7.4 containing 0.5% tween 20；Described enzyme conjugates working solution is HRP-goat-anti duck IgG；Described nitrite ion is 0.2mg/mL tetramethyl biphenyl amine aqueous solution and the citrate-phosphate salt buffer containing 0.5 ‰ hydrogen peroxide ureas, and the two volume ratio is 1:1；Described stop buffer is 0.31% hydrofluoric acid solution；Described positive control is the positive serum obtained through DHAV-1VP3-3VP1 recombiant protein immunity, its OD650nm>=1.0, add the mycillin of 1000U/mL, aseptic filtration；Described negative control is the negative serum obtained through screening, its OD650nm≤ 0.25, add the mycillin of 1000U/mL, aseptic filtration.
Universal DHAV antibody ELISA detection kit the most as claimed in claim 7, is characterized in that, including following components: with the coated elisa plate of DHAV-1VP3-3VP1 recombiant protein: 5 pieces；Sample diluting liquid: 200mL；10 × concentrated cleaning solution: 400mL；Enzyme conjugates working solution: 50mL；Nitrite ion: 100mL；Stop buffer: 60mL；Positive control: 2mL；Negative control: 2mL.
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| CN106370854A (en) * | 2016-08-17 | 2017-02-01 | 中国科学院微生物研究所 | Duck hepatitis A virus 1 antibody detection method and special-purpose kit thereof |
| CN106556693A (en) * | 2016-11-30 | 2017-04-05 | 吉林省畜牧兽医科学研究院 | A kind of toxoplasma series connection multi-epitope gene ELISA detection kit |
| CN108169496B (en) * | 2018-03-14 | 2022-06-28 | 山东省农业科学院家禽研究所 | DHAV-3 type polypeptide indirect ELISA antibody detection kit and application thereof |
| CN108445208B (en) * | 2018-03-14 | 2022-06-28 | 山东省农业科学院家禽研究所 | Universal DHAV polypeptide indirect ELISA antibody detection kit and application thereof |
| CN110095607B (en) * | 2019-04-16 | 2021-08-10 | 东北农业大学 | Universal indirect ELISA kit for detecting 1-type and 3-type duck hepatitis A virus serum antibodies and application thereof |
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| CN102127531A (en) * | 2010-12-22 | 2011-07-20 | 山东省农业科学院家禽研究所 | Korean novel duck hepatitis viral antibody ELISA (Enzyme-Linked Immunosorbent Assay) detection kit |
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