CN106188249B - For detecting the antigen and method and kit of PEDV variation strain antibody - Google Patents
For detecting the antigen and method and kit of PEDV variation strain antibody Download PDFInfo
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Abstract
The present invention relates to the kit of antigen, indirect ELISA for detecting Porcine epidemic diarrhea virus (PEDV) variation strain antibody and the indirect ELISA methods of detection Porcine epidemic diarrhea virus variation strain antibody.The antigen includes the major antigen of PEDV S1, can be with the PEDV of specific detection Beijing area prevalence variation strain antibody using the antigen protein.
Description
Technical field
The present invention relates to animal epidemic detection fields, and in particular to for detect PEDV variation strain antibody antigen and its
ELISA kit and detection method.
Background technique
Pig epidemic diarrhea (porcine epidemic diarrhea, PED) virosis is flowed by the pig of coronavirus genus
Row diarrhea virus (porcine epidemic diarrhea virus, PEDV) causes, and main clinic symptoms are suckling pigs
Vomiting very serious and diarrhea, finally dehydration is dead, and it is a kind of extremely strong intestines problem of infection of pig that morbidity is anxious.Hair earliest
Now in Britain in 1971, then rapidly diffuse into all over the world.Start within 1976, the ground such as China Guangdong, Shanghai report this
The generation of disease.All pigs no matter kind and size it is susceptible, sow, wean pig, growing and fattening pigs, the equal infectious of feeder pig, 1 week
Suckling pig infectious age reaches 90%-100% in age.The disease has serious harmfulness, causes to world live pig market
Immeasurable loss.Belgian virologist Pensaert was found by experiment that and confirmed in 1977, although the disease with
TGEV is extremely similar in epidemiology, clinical symptoms or even pathological change, but the two has no serological cross reaction, and belongs to
In new PEDV member.
Vaccine inoculation is the important means of its prevention, and China starts the dyad inactivated vaccine using PEDV and TGEV from nineteen ninety-five
And Attenuate vaccine.The generation of the disease is effectively controlled whithin a period of time, but the disease appeared in immune swinery from 2006.
Since 2010, which starts to break out with unprecedented situation, successively in China's Mainland, the U.S., Canada, Japan, TaiWan, China
The ground such as area and Mexico occur, and the arch-criminal of -2014 years 2010 pig farm diarrhoeal diseases in China is PEDV.Current prevalence poison
Pnca gene sequence variations are big, lead to the relatively Big mutation rate of amino acid, cause vaccine clinical effect unobvious.PED in addition to lethality not
It is disconnected increase it is outer, epidemic season, duration and infection scope also with different, and swinery morbidity more frequency is previously reported
It is numerous, it brings and seriously threatens and huge challenge to world's pig breeding industry.
Long 28Kb of the nucleic acid of PEDV or so, positive chain RNA are not segmented linearly and have infectivity.4 kinds of structure eggs of main code
It is white: nucleocapsid N, fine prominent S, membranelle E and film sugar M, 3 kinds of non-structural albumen: Pol (1a/1b) and ORF3,5'-3' sequence are as follows:
5'-Pol(1a/1b)-S-ORF3-E-M-N-3'.Porcine epidemic diarrhea virus is divided into G1 and G2 two ratios according to S gene by expert
Biggish gene group, research find G1 gene group there are different degrees of base insertion or missing the phenomenon that (mainly with
Gene insertion is common)；And G2 gene group is primarily present the point mutation phenomenon of base deletion.By establishing S phylogenetic trees, send out
Existing G2 branch is mainly the strain of the live vaccines such as CV777, DR13 and the getting up early separation strains of PEDV, and G1 branch is new PEDV popular
Strain, such as the strain of South Korea and U.S.'s prevalence.PEDV S gene is immunodominance gene, using S gene as diagnostic techniques research and
In the vaccine of candidate gene, good and specific very strong immunogenicity is shown.Some researches show that the S proteins of anti-PEDV
Antibody, do not reacted with TGEV S protein, PEDV S protein can be work perfectly well as ELISA detection target protein.
ELISA method has many advantages, such as high sensitivity, reproducible, high specificity, and is suitble to the inspection of high-volume sample
It surveys.The method can be not only used for the antigen in measurement excrement, it can also be used to the antibody in serum is measured, it is wide by researcher and clinic at present
General use is used by many provincial, and municipal level animal epidemic prevention and control mechanism laboratories or international trade specified value diagnoses
One of method.
Summary of the invention
The object of the present invention is to provide for detect Porcine epidemic diarrhea virus (PEDV) variation strain antibody antigen, indirectly
ELISA kit and the indirect ELISA method for detecting Porcine epidemic diarrhea virus antibody.Utilize antigen and method of the invention
It can be with the PEDV of specific detection Beijing area prevalence variation strain antibody with kit.
An aspect of of the present present invention provides the antigen for detecting Porcine epidemic diarrhea virus (PEDV) variant, amino acid
Sequence is as follows:
PQDVTRCSANTNFRRFFSKFNVQAPAVVVLGGYLPIGENQGVNSTWYCAGQHPTASGVHGIFVSHIRG
GHGFEIGISQEPFDPSGYQLYLHKATNGNTNATARLRICQFPSIKTLGPTANNDVTTGRNCLFNKAIPAHMSEHSV
VGITWDNDRVTVFSDKIYYFYFKNDWSRVATKCYNSGGCAMQYVYEPTYYMLNVTSAGEDGISYQPCTANCIGYSA
NVFATEPNGHIPEGFSFNNWFLLSNDSTLVHGKVVSNQ(SEQ ID NO:1)。
In some embodiments, the antigen is recombination.In a further embodiment, the antigen is by big
The recombinant polypeptide of enterobacteria expression.
Another aspect of the present invention provides the nucleic acid sequence encoding of above-mentioned antigen.It is preferred that the nucleic acid sequence is as follows
(774bp):
CCACAAGATGTCACTAGGTGCTCAGCTAACACTAATTTTAGGCGGTTCTTTTCAAAATTTAATGTTCA
GGCGCCTGCAGTTGTTGTACTGGGCGGTTATCTACCTATTGGTGAAAACCAGGGTGTCAATTCAACTTGGTACTGT
GCTGGCCAACATCCAACTGCTAGTGGCGTTCATGGTATCTTTGTTAGCCATATTAGAGGTGGTCATGGCTTTGAGA
TTGGCATTTCGCAAGAGCCTTTTGACCCTAGTGGTTACCAGCTTTATTTACATAAGGCTACTAACGGTAACACTAA
TGCTACTGCGCGACTGCGCATTTGCCAGTTTCCTAGCATTAAAACATTGGGCCCCACTGCTAATAATGATGTTACA
ACAGGTCGTAATTGCCTATTTAACAAAGCCATCCCAGCTCATATGAGTGAACATAGTGTTGTCGGCATAACATGGG
ATAATGATCGTGTCACTGTCTTCTCTGACAAGATCTATTATTTTTATTTTAAAAATGATTGGTCCCGTGTTGCGAC
AAAGTGTTACAACAGTGGAGGTTGTGCTATGCAATATGTTTATGAACCCACCTATTACATGCTTAATGTTACTAGT
GCTGGTGAGGATGGTATTTCTTATCAACCCTGTACAGCTAATTGCATTGGTTATTCTGCCAATGTATTTGCTACTG
AGCCCAATGGCCACATACCAGAAGGTTTTAGTTTTAATAATTGGTTTCTTTTGTCCAATGATTCCACTTTGGTGCA
TGGTAAAGTGGTTTCCAACCAA(SEQ ID NO:2)。
Another aspect of the present invention is provided for detecting the indirect of Porcine epidemic diarrhea virus (PEDV) variation strain antibody
ELISA kit, it includes above-mentioned antigens.
In a further embodiment, one kind needed for further including indirect ELISA detection method in the kit
Or a variety of other reagents.
In preferred embodiments, one or more other reagents can be selected from: 96 orifice plates, coating buffer, washing lotion,
Confining liquid, ELIAS secondary antibody, color developing agent, terminate liquid, positive control, negative control and other required reagents.
Wherein, coating buffer can be the 0.05M carbonate buffer solution of pH9.6, wherein containing Na2CO31.59g/L
NaHCO3 2.93g/L)。
Wherein, washing lotion can contain NaCl 8g/L, KCl 0.2g/L, Na2HPO41.42g/L KH2PO4 0.27g/L、
0.05%Tween-20, pH7.2.
Wherein, confining liquid can contain NaCl 8g/L, KCl 0.2g/L, Na2HPO41.42g/L KH2PO4 0.27g/
L, 10% fetal calf serum.
Wherein, developing solution may include A liquid and B liquid, wherein A liquid contain sodium acetate 27.2g/L, citric acid 27.2g/L,
30%H2O20.6ml/L, B liquid contain EDTA-Na 0.4g/L, citric acid 1.9g/L, glycerol 100ml/L, TMB 0.4g/L.
Wherein, terminate liquid can contain the 108.5ml concentrated sulfuric acid/L, and the concentrated sulfuric acid is added dropwise in distilled water and is prepared.
It is anti-that another aspect of the present invention provides detection Porcine epidemic diarrhea virus variant in non-diagnostic purpose test sample
The method of body, which is characterized in that using above-mentioned antigen by detecting Porcine Epidemic Diarrhea in indirect ELISA method test sample
Poison variation strain antibody.
Another aspect of the present invention provides above-mentioned antigen, the nucleic acid sequence of the above-mentioned antigen of coding or mentioned reagent box and is preparing
For the application in the reagent for the strain antibody that made a variation by Porcine epidemic diarrhea virus in indirect ELISA method test sample.
In preferred embodiments, described " indirect ELISA method " or " indirect ELISA detection method " include following step
It is rapid: (1) with the antigen coat solid support of purifying；(2) under conditions of being suitable for forming antigenantibody complex, make
The solid support and sample to be tested for being fixed with above-mentioned antigen incubate；(3) cleaning removes unbonded antibody；(4) it is added through marking
The secondary antibody of note, incubates under conditions of being suitable for forming antigenantibody complex；(5) cleaning removes unbonded secondary antibody；
(6) antigenantibody complex in Incubation mixtures is detected.
In certain embodiments, above-mentioned steps (6) " antigenantibody complex in detection Incubation mixtures "
It is the OD value that 450nm wavelength is measured after developing the color.
In preferred embodiments, become by detecting Porcine epidemic diarrhea virus in indirect ELISA method test sample
The following steps are included: (1) antigen coat liquid is added and purifying antigen 69ng is coated with, then 37 DEG C of 1hr are washed different strain antibody
It washs；(2) confining liquid containing 10% fetal calf serum is added to be closed, 37 DEG C of 2hr are washed out；(3) addition is diluted with confining liquid
40 times of measuring samples；(4) 37 DEG C of incubation 30min, are washed out；(5) addition dilutes 25000 times of enzyme mark two with confining liquid
It is anti-；(6) 37 DEG C of incubation 30min, are washed out；(7) color developing agent is added, 37 DEG C are protected from light colour developing 20min；(8) terminate liquid is added；
(9) every hole OD450 value is detected.
In a more preferred embodiment, by detecting Porcine epidemic diarrhea virus in indirect ELISA method test sample
Variation strain antibody the following steps are included: (1) be added antigen coat liquid 100 μ l and purifying antigen 69ng be coated with, 37 DEG C of 1hr,
It is washed out；(2) 10% fetal calf serum, 200 μ l is added to be closed, 37 DEG C of 2hr are washed out；(3) it is added dilute with confining liquid
Release 40 times of 100 μ l of measuring samples；(4) 37 DEG C of incubation 30min, are washed out；(5) addition dilutes 25000 times with confining liquid
100 μ l of ELIAS secondary antibody；(6) 37 DEG C of incubation 30min, are washed out；(7) 100 μ l of color developing agent is added, 37 DEG C are protected from light colour developing 20min,
Wherein color developing agent is that A liquid and B liquid equivalent mix；(8) 50 μ l of terminate liquid is added；(9) in 15min, every hole OD450 value is detected.
In certain embodiments, if the OD450 average value in measuring samples hole is more than or equal to 0.1781, to sample
Product are the positive.If the OD450 average value in measuring samples hole is less than or equal to 0.1524, measuring samples are feminine gender.
Wherein, the solid support can be organic or inorganic polymer.
Wherein, the secondary antibody can be anti-igg antibody, anti-IgM and/or anti-IgA antibody, and use radioactive isotope
Label, or be marked with horseradish peroxidase, alkaline phosphatase, beta galactosidase or acetylcholinesterase.
The sample refers to blood, serum, blood plasma, urine, saliva, body fluid and other secretion or row from subject
Let out object and tissue or cell extract.In preferred embodiments, the sample is serum.
In preferred embodiments, the subject is pig.
The present invention, by screening, obtains one kind based on the S gene of the PEDV variant of current Beijing area prevalence
The antigen protein of major antigenic sites comprising S1, can be with the PEDV of specific detection Beijing area prevalence using the antigen protein
Variant S protein antibody.It is high, fast and convenient that the present invention also establishes a kind of high specificity, sensibility using the antigen protein
The indirect ELISA antibody detection method of PEDV.The detection method can be with more efficiently immunologic surveillance swinery antibody level and prison
PEDV epidemic situation is controlled, can be used for clinical expansion, is relatively specific for the detection that farm, base carries out batch sample, and is the fast of PED
Speed diagnosis, epidemiological survey and prevention and control provide theoretical foundation and technical support.
The advantages of method and kit of detection PEDV antiviral antibody of the invention, is as follows:
(1) easy to operate: to detect the S protein antibody of currently a popular Porcine epidemic diarrhea virus using indirect method, operate
Step is few, and routine experimentation personnel can grasp operating method, easy to spread.
(2) can be used to diagnose pig whether infect PEDV or detect after pig immune vaccine for S protein antibody water
Flat, specificity is high, and detection effect is accurate.
Detailed description of the invention
Fig. 1 is the PCR amplification result of PEDV S1seg.Wherein Marker is 2kd DNA, and 1,2,3 be PEDV S1seg
PCR product.
Fig. 2 is the PCR digestion purification result of expression vector pET-32a and PEDV S1seg.Wherein Marker is 2kd
DNA, 1 is EcoR I, the pET32a carrier of III digestion of Hind after purification, and 2 be EcoR I, the PEDV of III digestion of Hind after purification
S1seg PCR product.
Fig. 3 is the bacterium colony PCR result containing recombinant expression plasmid pET-32a/PEDV S1seg.Wherein Marker is 2kd
DNA, 1-10 are the PCR products of 10 BL21 bacterium colonies containing pET-32a/PEDV S1seg.
Fig. 4 is preliminary expression and the soluble analysis of S1seg albumen.Wherein Marker is 14-100kDa protein；1
It is the full bacterium of empty carrier PET32a/BL21 not induced；2 be the full bacterium of empty carrier of 18 DEG C of 180rpm 18h induction；3 do not induce
The full bacterium of recombinant bacterium；4 be the full bacterium of recombinant bacterium of 18 DEG C of 180rpm 18h induction；5 be on the recombinant bacterium of 18 DEG C of 180rpm 18h induction
Clearly；6 be the recombinant bacterium precipitating (inclusion body) of 18 DEG C of 180rpm 18h inductions.
Fig. 5 is the SDS-PAGE electrophoresis after recombinant protein purification.Wherein Marker is 14-100KDa protein；1,2
It is albumen after purification.
Fig. 6 is the Western-blot of His monoclonal antibody detection recombinant protein.Wherein 1 is expression of recombinant plasmid albumen；2 be empty
Plasmid control.
Fig. 7 is the Western-blot of Swine serum identification S1seg albumen.Wherein 1,2 be positive serum detection；3 be negative
Serum detection.
Specific embodiment
Unless otherwise indicated, there is term used herein the those of ordinary skill of relevant art to be generally understood
Meaning.
Term used in the present invention " S protein antibody " refers to animal in infection Porcine epidemic diarrhea virus or Pigs Inoculated
After epidemic diarrhea virus vaccine, what is generated in vivo can identify the antibody of S protein.
Term used in the present invention " sample " refers to blood, serum, blood plasma, urine, saliva, body fluid from subject
With other secretion or excreta and tissue or cell extract.
Term used in the present invention " purifying " refers to the polypeptide separated from natural surroundings or recombinant production source.Purifying
Polypeptide can be by chromatographic technique obtain purified polypeptide.
Heretofore described "comprising", " containing " mean including but not limited to, or substantially by ... form, or
By ... it forms.
In order to make the objectives, technical solutions and advantages of the present invention clearer, simultaneously reference combined with specific embodiments below
Attached drawing, the present invention is described in more detail.It should be understood that these descriptions are merely illustrative, and it is not intended to limit the present invention
Range.In addition, in the following description, descriptions of well-known structures and technologies are omitted, to avoid this hair is unnecessarily obscured
Bright concept.
The amplification of embodiment 1:PEDV S1 (19-277aa) (hereinafter referred to as S1seg) gene
Morbidity chitterlings (morbid pig is from Beijing area) is taken, Trizol method extracts RNA, and reverse transcription is at cDNA, and -20 DEG C
It saves backup.
Using the cDNA as template, the nucleic acid sequence of antigen is obtained by PCR method.
Design primer sequence P1 and P2, and introduce EcolR I, Hind III digestion site respectively in upstream and downstream primer, in advance
Estimating primer size is 774bp.Primer sequence is as shown in table 1, the synthesis of You Shenggong bioengineering Co., Ltd.
1 amplimer of table
| Primer | Sequence | Restriction enzyme site |
| P1 | GGAATTCCCACAAGATGTCACCAGGT | EcolR I |
| P2 | CAAGCTTGTTGGTTGGAAACCACCTT | Hind III |
PCR reaction system is 20 μ l, in which:
Add rear brief centrifugation 8s, reaction condition are as follows: 94 DEG C of 4min initial denaturations；Into circulation: 94 DEG C of 30s, 57 DEG C of 30s,
72 DEG C of 1min, 30 circulations；Finally extend: 72 DEG C of 10min.The electrophoresis under 180 volts of voltage, it is electric in 1% Ago-Gel
Between 750-1000bp as a result, there is specific band, as shown in Figure 1 in swimming observation.
Embodiment 2: prokaryotic expression plasmid pET-32a/PEDV S1seg building and identification
Use the PCR product and pET-32a plasmid of restriction enzyme EcoR I, Hind III double digestion S1seg gene
(5900bp), blend compounds QIAquick Gel Extraction Kit purify digestion products, and electrophoresis observation is as a result, such as Fig. 2 institute in 1% Ago-Gel
Show, between 750-1000bp and 5000bp appears above specific band, and expected in the same size.
It is attached with T4DNA ligase, obtains recombinant plasmid pET-32a/PEDV S1seg, recombinant plasmid transformed is entered
It is proliferated in e. coli bl21 competent cell, by the BL21 coated plate culture of conversion, to doubtful bacterium colony, the half of picking colony is straight
It connects and does PCR detection, screen positive bacterium colony, as a result as shown in Figure 3.For being accredited as positive bacterium colony person, remaining bacterium is shaken into bacterium culture,
Directly bacterium solution is sent to be sequenced.
Sequencing result is as follows:
Nucleic acid sequence:
CCACAAGATGTCACTAGGTGCTCAGCTAACACTAATTTTAGGCGGTTCTTTTCAAAATTTAATGTTCA
GGCGCCTGCAGTTGTTGTACTGGGCGGTTATCTACCTATTGGTGAAAACCAGGGTGTCAATTCAACTTGGTACTGT
GCTGGCCAACATCCAACTGCTAGTGGCGTTCATGGTATCTTTGTTAGCCATATTAGAGGTGGTCATGGCTTTGAGA
TTGGCATTTCGCAAGAGCCTTTTGACCCTAGTGGTTACCAGCTTTATTTACATAAGGCTACTAACGGTAACACTAA
TGCTACTGCGCGACTGCGCATTTGCCAGTTTCCTAGCATTAAAACATTGGGCCCCACTGCTAATAATGATGTTACA
ACAGGTCGTAATTGCCTATTTAACAAAGCCATCCCAGCTCATATGAGTGAACATAGTGTTGTCGGCATAACATGGG
ATAATGATCGTGTCACTGTCTTCTCTGACAAGATCTATTATTTTTATTTTAAAAATGATTGGTCCCGTGTTGCGAC
AAAGTGTTACAACAGTGGAGGTTGTGCTATGCAATATGTTTATGAACCCACCTATTACATGCTTAATGTTACTAGT
GCTGGTGAGGATGGTATTTCTTATCAACCCTGTACAGCTAATTGCATTGGTTATTCTGCCAATGTATTTGCTACTG
AGCCCAATGGCCACATACCAGAAGGTTTTAGTTTTAATAATTGGTTTCTTTTGTCCAATGATTCCACTTTGGTGCA
TGGTAAAGTGGTTTCCAACCAA。
Corresponding amino acid sequence:
PQDVTRCSANTNFRRFFSKFNVQAPAVVVLGGYLPIGENQGVNSTWYCAGQHPTASGVHGIFVSHIRG
GHGFEIGISQEPFDPSGYQLYLHKATNGNTNATARLRICQFPSIKTLGPTANNDVTTGRNCLFNKAIPAHMSEHSV
VGITWDNDRVTVFSDKIYYFYFKNDWSRVATKCYNSGGCAMQYVYEPTYYMLNVTSAGEDGISYQPCTANCIGYSA
NVFATEPNGHIPEGFSFNNWFLLSNDSTLVHGKVVSNQ。
The expression and identification of embodiment 3:PEDV S1seg albumen
The positive bacterium colony bacterium for choosing the bigger S1seg of pET-32a/PEDV containing recombinant plasmid is inoculated in 200ml and ammonia benzyl is added
LB culture medium in, under conditions of 37 DEG C of 200r/min of shaking table shaking culture 12h, in this, as seed liquor.By the ratio of 1:50
Example, inoculation seed liquor is into the conical flask containing 50mL LB (benzyl containing ammonia) fluid nutrient medium, and 37 DEG C are shaken bacterium, until OD600 reaches
Between 0.6~0.8, add IPTG to final concentration 1mmol/L, at 18 DEG C, 37 DEG C of 180rpm shake Fiber differentiation 18h, 12000r/
Min is centrifuged 1min, outwells supernatant.Precipitating is washed 3 times using PBS, suitable PBS is added, precipitating is resuspended.It is cracked through ultrasound, 4
DEG C 10000g/min is centrifuged 10min, and in the ratio of 1:4, supernatant precipitating is done SDS-PAGE identification respectively.
As shown in figure 4, albumen, the S1seg recombinant protein that SDS-PAGE is tentatively expressed as the result is shown is with insoluble inclusion body
Form is present in precipitating, in supernatant almost without.
It is purified, is operated to specifications, step is such as using the inclusion body protein purification kit that health is ShiJi Co., Ltd
Under:
The 500ml recombinant bacterial strain culture of inducing expression is taken, 4 DEG C of 10000r are centrifuged 5min, and 1- is added in every 100mg thallus
5ml bacterial lysate cracks thallus using Ultrasonic Cell Disruptor.After 4 DEG C of centrifugation 30min of 10000r, separation supernatant precipitating is received
Collection precipitating.Precipitating is resuspended in Binding Buffer, is mixed as far as possible, ice bath 1h dissolves inclusion body.10000g centrifugation
20min, 0.45 μm of filter filter supernatant.Protein solution is added on pillar with liquid-transfering gun, flow velocity can not be too fast, keeps 1 as far as possible
Hour trickle is 10 times of column volumes, recycling efflux to clean centrifuge tube.Use the Binding of 15 times of column volumes
Buffer rinses pillar, puts rubbish bottle below, washes away foreign protein.It is eluted using Elution Buffer, collects eluting peak.Elution
Afterwards, pillar successively is washed using the deionized water of 10 times of column volumes, then is balanced with 20% ethyl alcohol of 3 times of column volumes, in 4 DEG C of conditions
Under save backup.
SDS-PAGE is carried out after recombinant protein purification, as a result as shown in Figure 5.
Using His monoclonal antibody, PEDV positive pig serum as primary antibody, be Western blot, find His monoclonal antibody and PEDV
The mostly anti-albumen that can identify recombinant expression well of the serum of positive pig, as shown in Figure 6 and Figure 7.
Embodiment 4: for the ELISA detection kit and its application method of Porcine epidemic diarrhea virus S antibody
Kit forms:
Coating buffer: pH9.6 0.05M carbonate buffer solution (Na2CO31.59g NaHCO32.93g adds dH2O is extremely
1000ml)；
PBS: claim NaCl 8g, KCl 0.2g, Na2HPO41.42g KH2PO40.27g adds dH into 1L beaker2O is extremely
1000ml；
Washing lotion: PBST solution (contains 0.05%Tween-20, the PBS of pH7.2)
Confining liquid: fetal calf serum 10ml is dissolved in 100ml PBS；
Developing solution: A liquid (sodium acetate 13.6g, citric acid 13.6g, 30%H2O20.3ml adds dH2O to 500ml)；B liquid
(EDTA-Na 0.2g, citric acid 0.95g, glycerol 50ml, TMB0.2g add water to 500ml)；
Terminate liquid: the 21.7ml concentrated sulfuric acid is added dropwise in 178.3ml distilled water；
96 orifice plates are purchased from Costar company, and ELIAS secondary antibody is the IgG label HRP antibody of the anti-pig of goat, public purchased from EARTYOX
Department.
Detecting step is as follows:
(1) it is coated with: 100 μ l of antigen coat liquid and antigen 69ng, 37 DEG C of 1hr being added in every hole of 96 orifice plates.By liquid
It all outwells, patting removal makes remaining liq.It is washed with 200 μ l of washing lotion, 3 times, stands 3min every time.
(2) it closes: 200 μ l, 37 DEG C of 2hr, 200 μ l of the washing lotion washing of addition confining liquid, 3 times, each 3min.
(3) it is loaded: adding positive and negative to compare each 100 μ l (diluting 40 times with confining liquid) in positive and negative control wells respectively, to
Inspection the 1st hole of hole adds 100 μ l of serum to be checked (with 40 times of confining liquid dilutions), and metapore does doubling dilution to serum to be checked, not touch
Bottom hole and hole wall, light shake mix.
(4) it incubating: using 37 degree, 30min of sealing plate film sealing plate postposition, 200 μ l of washing lotion is washed, and 3 times, each 3min.
(5) add ELIAS secondary antibody: every hole adds 100 μ l of ELIAS secondary antibody (with 25000 times of confining liquid dilutions).
(6) it incubating: using 37 degree, 30min of sealing plate film sealing plate postposition, 200 μ l of washing lotion is washed, and 4 times, each 3min.
(7) develop the color: color developing agent A and color developing agent B equivalent mix, and every 100 μ l of hole, 37 DEG C are protected from light colour developing 20min.
(8) terminate: every hole adds 50 μ l of terminate liquid.
(9) it measures: in 15min, with microplate reader in the every hole OD value of 450nm wavelength detecting.
Each step is groped through condition, final to determine:
The best package amount of antigen is in 96 orifice plates, and every hole 69ng is coated the diluted S1seg albumen of buffer, best to be coated with
Condition be 37 DEG C 1 hour, best sealing condition be 10%FBS, 37 DEG C 2 hours, serum optimum dilution degree be 1:40, primary antibody be incubated for
37 DEG C of 30min of time, secondary antibody optimum dilution degree and action time are respectively 1:25000 and 37 DEG C of 30min, and best developing time is
37 DEG C are protected from light colour developing 20min.
Embodiment 5: kit performance test
1. the determination of positive and negative serum critical value:
20 parts of pig negative serums are detected with the ELISA method established in embodiment 4, every part of serum repeats two holes, according to
According to Principle of Statistics, when OD450 >=X+3SD, be determined as the positive, OD450≤X+2SD is judged to feminine gender, as a result among the two when
It is judged to doubt infection.Show that 20 parts of negative serums are averaged OD450 value (X)=0.10075, standard deviation (SD)=0.0257.According to public affairs
Formula, positive serum OD450 >=X+3SD=0.10075+3 × 0.0257=0.1781.Negative serum OD450≤X+2SD=
0.10075+2 × 0.0257=0.1524 is suspicious therebetween.
2. specific detection:
The positive serum for having detected CSFV, PRV, PRRSV, PCV and TGEV is feminine gender, and only PEDV positive serum is in
Existing positive virus similar with other clinical symptoms has no crossover phenomenon, illustrates that the ELISA method has high degree of specificity, number
According to as shown in table 1:
1 different virus serum cross-over experiment result of table
| Serum | PEDV(+) | PEDV(-) | PRRSV | PRV | TGEV | PCV | CSFV |
| OD450 | 0.902 | 0.142 | 0.19 | 0.138 | 0.12 | 0.179 | 0.17 |
| 0.822 | 0.105 | 0.086 | 0.105 | 0.085 | 0.14 | 0.113 | |
| 0.66 | 0.09 | 0.06 | 0.1 | 0.16 | 0.109 | 0.104 | |
| Average value | 0.795 | 0.112 | 0.112 | 0.114 | 0.121 | 0.143 | 0.129 |
| Result judgement | + | - | - | - | - | - | - |
3. sensitivity Detection:
Doubling dilution has been done to 5 parts of positive serums, as shown in table 2, has been determined under 1:800 diluting condition, serum is all in sun
Property, the ELISA method sensibility for illustrating that this test is established is higher.
2 different multiples dilute serum reaction result of table
4. repetitive test:
It is repeated in batch:
As shown in table 3, the albumen of same time purifying, is coated with 96 plates, does 4 repetitions respectively with 10 parts of serum and detects, becomes
For different coefficient all 10% hereinafter, explanation is small using same a collection of antigen coat elisa plate testing result degree of variation, repeatability is good.
3 batches, table interior repetition experimental results
It is repeated between batch:
As shown in table 4, the antigen coat elisa plate purified using different time, 10 parts of serum have been done 4 repetitions and detected,
The coefficient of variation is both less than 10%, illustrates the antigen using different time preparation purifying, testing result fluctuation is little, the coefficient of variation
Small, repeatability is good.
Experimental result is repeated between 4 batches, table
Clinical sample detects compared with commercially available related kit:
100 parts of Swine serums from Beijing difference pig farm of this laboratory preservation are had detected with mentioned reagent box, it is positive
Rate is 87%.ELISA method established by the present invention and commercially available PEDV antibody assay kit (detection is directed to M protein antibodies) into
Row relatively (table 5) is and the S egg because this method is the detection for S protein antibody it is found that the method for the present invention positive rate is higher
White is the main surface immunogen of PEDV, and the antibody of generation has a protective effect to host, and commercial reagent box spininess to M albumen or
The antibody of N protein, M albumen and N protein are viral internal antigen, activate the antibody of generation and S protein antibody variant, and to machine
The effect of body unprotect.Therefore, the method that the detection method that this test is established is better than commercial reagent box.
5 clinical sample of table detects and compared with kit
| Positive serum | Negative serum | It is suspicious | |
| Indirect ELISA method | 87 | 3 | 10 |
| ELISA kit | 64 | 36 | 0 |
Conclusion:
The kit obtained using above-mentioned experimental procedure, can be used for diagnosing whether pig infects anomaly PEDV or detection
Antibody level after pig immune vaccine.
It should be understood that above-mentioned specific embodiment of the invention is used only for exemplary illustration or explains of the invention
Principle, but not to limit the present invention.Therefore, that is done without departing from the spirit and scope of the present invention is any
Modification, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.In addition, appended claims purport of the present invention
Covering the whole variations fallen into attached claim scope and boundary or this range and the equivalent form on boundary and is repairing
Change example.
Claims (10)
1. the antigen for detecting Porcine epidemic diarrhea virus variation strain antibody, amino acid sequence is as shown in SEQ ID NO:1.
2. encoding the nucleic acid of the antigen of claim 1, sequence is as shown in SEQ ID NO:2.
3. the indirect ELISA reagent kit for detecting Porcine epidemic diarrhea virus variation strain antibody, it includes the anti-of claim 1
It is former.
4. kit according to claim 3 further includes one or more examinations needed for indirect ELISA detection method
Agent.
5. kit according to claim 4, wherein one or more reagents are selected from: 96 orifice plates, coating buffer, washing lotion, envelope
Close liquid, ELIAS secondary antibody, color developing agent, terminate liquid, positive control, negative control and other required reagents.
6. a kind of method of Porcine epidemic diarrhea virus variation strain antibody in non-diagnostic purpose test sample, which is characterized in that make
Pass through Porcine epidemic diarrhea virus variation strain antibody in indirect ELISA method test sample with the antigen of claim 1.
7. method according to claim 6, wherein the indirect ELISA method is the following steps are included: (1) uses the antigen packet of purifying
By solid support；(2) under conditions of being suitable for forming antigenantibody complex, make the solid phase for being fixed with above-mentioned antigen
Support and sample to be tested incubate；(3) cleaning removes unbonded antibody；(4) labeled secondary antibody is added, is being suitable for forming
It is incubated under conditions of antigenantibody complex；(5) cleaning removes unbonded secondary antibody；(6) it detects in Incubation mixtures
Antigenantibody complex.
8. method according to claim 7, wherein the antigen using claim 1 passes through in indirect ELISA method test sample
Porcine epidemic diarrhea virus antibody the following steps are included:
(1) antigen coat liquid is added and purifying antigen 69ng is coated with, 37 DEG C of 1hr are washed out；(2) it is added and contains 10% tire
The confining liquid of cow's serum is closed, and 37 DEG C of 2hr are washed out；(3) addition dilutes 40 times of measuring samples with confining liquid；(4)
37 DEG C of incubation 30min, are washed out；(5) addition dilutes 25000 times of ELIAS secondary antibody with confining liquid；(6) 37 DEG C of incubation 30min,
It is washed out；(7) color developing agent is added, 37 DEG C are protected from light colour developing 20min；(8) terminate liquid is added；(9) every hole OD450 value is detected.
9. method according to claim 8, wherein if the OD450 average value in hole to be checked is more than or equal to 0.1781, to sample
Product are the positive, if the OD450 average value in hole to be checked is less than or equal to 0.1524, measuring samples are feminine gender.
10. the nucleic acid sequence of antigen according to claim 1, claim 2 or the kit of claim 3 are in preparation for leading to
Cross the application in indirect ELISA method test sample in the reagent of Porcine epidemic diarrhea virus variation strain antibody.
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Inventor after: Ruan Wenke Inventor after: Guo Jiaojiao Inventor after: Ruan Yingyu Inventor after: Zhang Jianjun Inventor after: Gu Xuejia Inventor after: Xu Shuang Inventor before: Ruan Wenke |
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