CN109232720A - A kind of O-shaped virus sIgA antibody ELISA detection kit of aftosa and its application - Google Patents
A kind of O-shaped virus sIgA antibody ELISA detection kit of aftosa and its application Download PDFInfo
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Abstract
The invention discloses the ELISA detection kits and its application of a kind of O-shaped virus sIgA antibody of aftosa.The kit includes the coated enzyme reaction plate of the O-shaped viral broad spectrum multi-epitope recombination antigen of aftosa, 100 × concentration enzyme labelled antibody, enzyme labelled antibody dilution, sample diluting liquid, concentrated cleaning solution, developing solution, terminate liquid, positive control sample and negative control sample.The O-shaped viral broad spectrum multi-epitope recombination antigen of the aftosa is made of the main neutrality epitope that 98 3 sinotype (Cathay), Pan Asia type (PanAsia), Burma O-shaped viruses of pedigree aftosa represent strain, therefore the sensitivity and specificity of kit detection are improved, and is suitable for the detection of the different O-shaped virus infections of aftosa.Kit provided by the invention is adapted to detect for pig, ox, the sIgA antibody in three kinds of susceptible animal mucosal secretions of sheep, has great importance for the propagation and infection that prevent and treat the O-shaped virus of aftosa.
Description
Technical field
The present invention relates to a kind of O-shaped virus sIgA antibody indirect ELISA detection kit of aftosa and its detection methods.This
Invention belongs to technical field of biological.
Background technique
Aftosa (Foot-and-Mouth Disease, FMD) is caused by foot and mouth disease virus with the mouth of infected animal, hoof
There is the communicable disease that blister disease is characterized in portion.Foot and mouth disease virus (Foot-and-Mouth Disease Virus,
FMDV) belong to Picornaviridae, viral center is a single stranded RNA.According to serotype characteristic can be divided into A type, O-shaped, c-type,
SAT1 type, SAT2 type, SAT3 type (i.e. 1,2,3 type of South Africa) and 7 serotypes of Asial (Type Asia 1), it is several between different serotypes
There is no immune protective efficiency, the animal for having infected One serotype FMDV still can infect the FMDV of another serotype and fall ill.
MDV nucleic acid molecules are the RNA molecule of single-stranded positive, and about 8500 bases of full-length genome contain only a big open reading
Frame, the polyprotein progressive cracking for expressing generation generate the structural proteins (VP1, VP2, VP3, VP4) and non-structural protein of virus
(Lab, 2A, 2B, 2C, 3A, 3B, 3C, 3D), structural proteins assembling generate the capsid structure of virus, and non-structural protein then participates in disease
Poison and the interaction of host, the reproduction process of the transcription and translation mechanism for inhibiting host cell, participation virus.Viral capsid proteins
It is the main immunogens that induction generates neutralizing antibody, there are multiple conforma-tional with linear neutralizing epitope on structural proteins.
Early stage shows at least there are 5 functions in O-shaped FMDV by monoclonal antibody and the sequence analysis and research of immunologic escape mutant strain
Independent neutrality epitope, the section for forming antigen site mainly includes the G-H ring and its C-terminal amino acid of VP1, VP2
31,70~73,75 and 77 amino acids, 43 and 44 site amino acids of VP1B-C ring；58 amino acids of VP3, VP1149
The participations such as amino acids constitute five neutralizing epitopes.This 5 neutralizing epitopes are linear list in addition to the site 1 being made of VP1G-H ring
Position is outer, remaining is comformational epitope.The amino acid of the G-H ring of VP1 is very in four kinds of Structural protein VP1s of FMDV, VP2, VP3, VP4
It is easy variation, but arginine-glycine-aspartic acid therein sour (Arg-Gly-Asp (RGD)) is highly conserved.G-H ring across
Degree is about 20 amino acid residues of the position 140-160 of VP1.There is only main antigen site (141- on VP1 albumen
160aa, 200-213aa and 21-40aa), and host's recognition site containing FMD virus.Therefore the regional gene structure is selected
The albumen for building expression vector expression and purification can be used as the good alternative envelope antigen for preparing antibody assay kit.
It is not related to live virus by multi-epitope antigen production process prepared by prokaryotic expression system, there is no the wind of scattered poison
Danger；Both epitope sequences can be adjusted according to the variation of popular strain at any time, it is possible to use different antigen sequences increases the wide of antigen
Spectrality.The present invention for sinotype (Cathay), Pan Asia, the aftosa of 98 3 pedigrees of Burma is O-shaped represents strain O/Akesu/
58 (NCBI accession number: AF511039.1), O/TAW/81/97 (NCBI accession number: AJ296321.1), O/Tibet/99 (NCBI
Accession number: AJ318830.1), O/Mya98 (NCBI accession number: AJ303521.1), using bioinformatics method to this 4 plants of poison
Strain VP1 structural proteins carry out Accurate Analysis, filter out dominant antigen epitope, are purified to obtain aftosa with prokaryotic expression
The O-shaped main neutrality epitope area for representing strain prepares and updates more full envelope antigen, establishes the O-shaped disease of aftosa
Malicious sIgA antibody indirect ELISA detection method and its detection kit.
The main effects factor of the sIgA antibody as mucosa-immune, is widely present in the juices such as colostrum, tear, saliva
In, it not only can be in first time and viral, but also further activation system can be immunoreacted by way of mucous membrane, into
And prevent the intrusion of virus.Its major function has: suppress and stick, be immunized exclude, dissolution of bacteria, neutralize virus, mediate antibody according to
Bad cell-mediated cytotoxic effect (ADCC, antibody-dependent cell-mediated
Cytotoxicity), anti-inflammatory promotes native factor effect and adjusts mucosa-immune reaction.Foot-and-mouth disease virus resistant sIgA antibody
Be generate the mark of specificity in respiratory tract and alimentary canal first after mouth disease virus infection or vaccine mucosal immune, while
The effect of mucosa-immune vaccine is reacted.There are no the reagents that can effectively detect foot and mouth disease virus sIgA antibody currently on the market
Box is developed in a kind of detection mucosal secretions in view of going deep into recent years to mucosa-immune mechanism and mucosa-immune vaccine research
The ELISA kit of foot and mouth disease virus sIgA antibody level can more accurately react mouth disease virus infection and mucous membrane is exempted from
Epidemic disease state, while foot and mouth disease virus sIgA Antibody dynamics changing rule is monitored using this method, it is aftosa mucosa-immune vaccine
Theoretical and experimental basis is provided with mating detection method is established.
Summary of the invention
The purpose of the present invention is to above-mentioned defects in the prior art, provide a kind of O-shaped virus sIgA of aftosa
The detection kit and its method of antibody.
To achieve the goals above, technical solution provided by the invention are as follows:
The present invention is with DNAStar software to for sinotype (Cathay), Pan Asia, the aftosa of 98 3 pedigrees of Burma
It is O-shaped represent strain O/Akesu/58 (NCBI accession number: AF511039.1), O/TAW/81/97 (NCBI accession number:
AJ296321.1), O/Tibet/99 (NCBI accession number: AJ318830.1), O/Mya98 (NCBI accession number: AJ303521.1),
Accurate Analysis is carried out to this 4 plants of strain VP1 structural proteins using bioinformatics method, filters out main neutrality antigen table
Position, designs the series sequence of antigen epitope genes are as follows: (O/Akesu/58) VP1 132-160-GGGS-VP1 193-213-
GGGS-(O/TAW/81/97)VP1 132-160-GGGS-VP1193-213-GGGS-(O/Tibet/99)VP1132-160-
GGGS-VP1 193-213-GGGS- (O/Mya98) VP1 132-160-GGGS-VP1 193-213-6 × His, between each epitope
It is connected with GGGS, finally plus 6 His-Tag sequences, is conducive to the purifying for expressing albumen.Designed sequence transfers to Nanjing
The optimization and synthesis of GenScript company progress coded sequence.
A kind of O-shaped viral broad spectrum multi-epitope recombination antigen of aftosa that the present invention obtains, the O-shaped virus of the aftosa are wide
Spectrum multi-epitope recombination antigen includes that the O-shaped virus of aftosa represents strain O/Akesu/58, O/TAW/81/97, O/Tibet/99, O/
132-160 in the VP1 albumen of Mya98,193-213 main neutrality epitope sequences, the mouth hoof
The amino acid sequence of the O-shaped viral broad spectrum multi-epitope recombination antigen of epidemic disease is as shown in SEQ ID NO.2.
The nucleotide sequence of the O-shaped viral broad spectrum multi-epitope recombination antigen of the coding aftosa is also in protection of the invention
Within the scope of, it is preferred that the nucleotide sequence is as shown in SEQ ID NO.1.
Further, the invention also provides the O-shaped viral broad spectrum multi-epitope recombination antigens of the aftosa examines in preparation
Survey the purposes in the O-shaped virus sIgA antibody reagent of aftosa.Preferably, the reagent is kit.
Further, described the invention proposes a kind of O-shaped virus sIgA antibody ELISA detection kit of aftosa
Kit includes that the coated ELISA Plate of the O-shaped viral broad spectrum multi-epitope recombination antigen of the aftosa, 100 × concentration enzyme mark are anti-
Body, enzyme labelled antibody dilution, sample diluting liquid, concentrated cleaning solution, developing solution, terminate liquid, positive control sample and negative control
Sample.
Wherein, it is preferred that the O-shaped viral broad spectrum multi-epitope recombination antigen of the aftosa is obtained through prokaryotic expression
?.
Wherein, it is preferred that the O-shaped viral coated ELISA Plate of broad spectrum multi-epitope recombination antigen of the aftosa is according to following
Method is prepared:
(1) preparation and coating of envelope antigen
The recombinant protein inclusion body that prokaryotic expression obtains purifies to obtain the O-shaped disease of aftosa through renaturation and Ni-NTA
Malicious broad spectrum multi-epitope recombination antigen, amino acid sequence is as shown in SEQ ID NO.2, with the carbonate buffer of pH9.6 when coating
Liquid is diluted to 3 μ g/ml, and according to 100 hole μ l/ coated elisa plates, 4 DEG C of refrigerators are stood overnight；
(2) closing and preservation of ELISA Plate
It is coated with overnight ELISA Plate and washs 4 times with PBST and pat dry, every hole adds 10mM PBS of the 100 μ l containing 0.5w/v%BSA
Confining liquid, 37 DEG C of placement 2h discard confining liquid, and PBST is washed 3 times, obtain the ELISA Plate of pre-coated antigen, every hole adds 100 μ l to contain
The ELISA Plate stabilizer of 6v/v% horse serum, is placed at room temperature for 30min, discards naturally dry after liquid, with aluminium foil bag vacuum seal,
4 DEG C of preservations；
Wherein, the ELISA Plate stabilizer is to be added to 5g BSA, 10g sucrose, 20g trehalose
In 1000ml0.01mol/LPBS (pH7.4) gently concussion to dissolve, add 0.05w/v%Procline300, be stored in 4 DEG C it is standby
With.
Wherein, it is preferred that the positive control sample is that the acquisition O-shaped virus of aftosa attacks 7-21d pig, ox and sheep after poison
Nose swab sample, through mixing, a series of sample that detection OD450 value is 1.5 ± 0.05 after dilutions；The negative sample isFMDV NS kit detect foot and mouth disease virus non-structural protein antibody be feminine gender, foot-and-mouth disease a type, it is O-shaped,
AsiaI Liquid-phase blocking ELISA kit detect serum antibody titer < 1/4, FMDV specific PCR detection antigen be negative pig,
The nose swab of ox and sheep detects the sample that OD450 value is 0.1 ± 0.05 after being mixed, being diluted, aseptic subpackaged spare.
Wherein, it is preferred that described 100 × concentration enzyme labelled antibody is that horseradish peroxidase (HRP) marks the anti-pig of mouse, ox
Or the monoclonal antibody of sheep IgA, in use, with being used after 100 times of enzyme labelled antibody dilution dilutions, the enzyme labelled antibody dilution
Liquid is containing 1v/v% glycerol, 0.5w/v% bovine serum albumin(BSA), 1w/v% casein and 0.05w/v%Procline300
0.01mol/LPBS buffer, pH7.4.
Wherein, it is preferred that the sample diluting liquid is NaCl containing 0.5M, 2.68mM KCl, 2.79mM KH2PO4、
8.1mM Na2HPO4, 5g/L casein, 0.05%Tween20 and 10mM PBS mixed solution；The developing solution is aobvious for TMB
Color liquid, the terminate liquid are 1moL/L H2SO4Solution；The concentrated cleaning solution is 10 × PBST solution, that is, contains 0.5v/
The 0.1mol/L PBS solution of v%Tween20, pH7.6 dilute 10 times when use.
Wherein, it is preferred that when virus sIgA antibody test O-shaped using kit of the present invention progress aftosa, press
It is carried out according to following steps:
(1) Sample Dilution
By sample to be tested, positive control and negative control, with sample diluting liquid, 1:2 is diluted by volume；
(2) board-washing
The O-shaped virus of aftosa is taken out from 4 DEG C of refrigerators represents main neutrality epitope (TB/O) the albumen coating of strain
ELISA Plate, open aluminium foil bag, take out ELISA Plate, with cleaning solution board-washing 3 times diluted, blotting paper is patted dry, each 3min；
(3) it is loaded
Sample to be tested, positive control and negative control after dilution is separately added into ELISA Plate, 100 μ L of every hole, 37
DEG C be incubated for 45min；
(4) it washs
ELISA Plate is taken out, sample is got rid of, is rinsed 4 times with cleaning solution, blotting paper pats dry；
(5) enzyme labelled antibody is added
The anti-pig of mouse, ox or the sheep IgA monoclonal antibody for the horseradish peroxidase-labeled that addition has diluted, 100 holes μ L/,
37 DEG C of work 30min；
(6) add substrate:
Enzyme labelled antibody is got rid of, is rinsed 4 times with cleaning solution, blotting paper pats dry, and 37 DEG C of colour developing 15min of developing solution are added, and takes out
Terminate liquid is added immediately afterwards；
(7) result judgement
OD450nm numerical value is read in microplate reader, the OD450nm value of sample to be tested is average more than or equal to negative control sample
2.5 times of value are judged to the positive, and 2.5 times less than negative control sample average value are judged to feminine gender.
Technical key point of the present invention are as follows:
1, using the O-shaped viral broad spectrum multi-epitope recombination antigen of the aftosa of prokaryotic expression as envelope antigen, with
The monoclonal antibody of the anti-pig of mouse, ox or sheep IgA that enzyme marks are secondary antibody, after measuring samples are reacted with envelope antigen, pass through enzyme mark
Remember the further reaction of antibody, is finally compared with reference positive nose swab and negative nose swab, to evaluate foot and mouth disease virus sIgA
Antibody level.
2, envelope antigen be purified after Escherichia coli pET-30a (+) prokaryotic expression represent strain containing aftosa is O-shaped
The recombinant protein of main neutrality epitope, the antigen is by sinotype (Cathay), Pan Asia type (PanAsia), Burma 98 3
A O-shaped virus of pedigree aftosa represents the main neutrality epitope composition of strain, and the host containing foot and mouth disease virus
Recognition site (tri- peptide motif of RGD) has broad spectrum activity.
3, secondary antibody is the monoclonal of the anti-pig of mouse, ox or the sheep IgA that are marked with horseradish peroxidase (HRP) labelling kit
Antibody.
4, with reference to positive sample be laboratory attack the pig of 7-21 days infection aftosas after poison, the nose swab of ox and sheep it is mixed
Close object；Negative sample be throughIt is yin that FMDV NS kit, which detects foot and mouth disease virus non-structural protein antibody,
Property, A type, O-shaped, detection antibody titer < 1/4, the FMDV specific PCR detection of AsiaI aftosa Liquid-phase blocking ELISA kit is anti-
It originally was the mixture of the nose swab of negative pig, ox and sheep.
Compared to the prior art, the invention has the benefit that
1) the invention discloses a kind of method and its detection kit for detecting the O-shaped virus sIgA antibody of aftosa, pass through
Envelope antigen, the anti-pig of mouse of HRP label, ox, sheep IgA monoclonal antibody, provide a kind of evaluation aftosa mucosa-immune and imitate
Effective ways of fruit, and provide a kind of new method for the early diagnosis of infection of foot-and-mouth disease, can fast and accurately detect pig,
Foot and mouth disease A-type virus sIgA antibody in ox, sheep nose swab；
2) since popular malicious mutation occurrence frequency is higher and higher in recent years for foot and mouth disease virus, the present invention passes through prokaryotic expression
System expression 4 plants of aftosas O-shaped dominant antigen epitope for representing strain is as envelope antigen, and the antigen is by sinotype
(Cathay), 98 3 Pan Asia type (PanAsia), Burma O-shaped viruses of pedigree aftosa represent the main neutrality antigen of strain
Epitope composition, to prepare envelope antigen new, with broad spectrum activity, improves kit to the detection energy of existing strain
Power；
3) acquisition of sample is easy to operate, and manpower consumption is small, to animal stress be small；
4) this method is easy to operate, and required time is short, can be used for the detection of a large amount of samples and commenting for mucosa-immune effect
Valence.
Detailed description of the invention
Fig. 1 is EB/O protein SDS-PAGE figure provided in an embodiment of the present invention；
In figure: swimming lane M1: Protein Marker；Swimming lane 1:BSA albumen；Swimming lane 2:EB/O albumen；
Fig. 2 is purifying protein result provided in an embodiment of the present invention.
In figure: swimming lane M: Protein Marker；Swimming lane 1,2: elution albumen.
Specific embodiment
The present invention will be further described combined with specific embodiments below, but following embodiments will not limit in any way
Protection scope of the present invention.
The preparation and assembling of the O-shaped virus sIgA antibody ELISA detection kit of 1 aftosa of embodiment
(1) preparation of kit:
1, the O-shaped virus of aftosa represents the design of the main neutrality epitope (EB/O) of strain:
Foot-and-mouth disease VP1 is the dominant antigen of virus, and the natural VP1 albumen either isolated and purified is still
Recombination expression product can induce body to generate protectiveness neutralizing antibody, have type specificity.VP 1 Gene of Foot-and-Mouth Disease virus overall length
It is made of 639 nucleotide, one albumen with 213 amino acid of coding, Main Antigenic concentrates on 140-160
The amino acid and 200-213 amino acid sections of position.The present invention is with DNAStar biosoftware to 4 plants of mouths of isolated in China
The O-shaped virus of fever aphthous represent strain O/Akesu/58 (NCBI accession number: AF511039.1), O/TAW/81/97 (NCBI accession number:
AJ296321.1), O/Tibet/99 (NCBI accession number: AJ318830.1), O/Mya98 (NCBI accession number: AJ303521.1)
VP1 gene sequencing, it is determined that neutrality epitope 132-160,193-213 amino acid sections.Design antigen
The series sequence of epitope are as follows:
(O/Akesu/58)VP1 132-160-GGGS-VP1 193-213-GGGS-(O/TAW/81/97)VP1 132-
160-GGGS-VP1193-213-GGGS-(O/Tibet/99)VP1132-160-GGGS-VP1 193-213-GGGS-(O/
Mya98) VP1 132-160-GGGS-VP1 193-213-6 × His is connected between each epitope with GGGS, finally plus 6 histidines
Sequence label is conducive to the purifying for expressing albumen.
The nucleotide sequence such as SEQ ID No.1 institute of main neutrality epitope (EB/O) antigen of the coding finally obtained
Show, for amino acid sequence as shown in SEQ ID No.2, multi-epitope gene sequence entrusts Nanjing Jin Sirui biotechnology company to close
At.
2, the expression of main neutrality epitope (EB/O) albumen of the O-shaped virus of aftosa:
By main neutrality epitope (EB/O) the encoding gene 705bp of the O-shaped virus of aftosa (shown in SEQ ID NO.1)
It is cloned into pET-30a (+) prokaryotic expression carrier, after digestion, sequencing are correct, positive plasmid is converted to BL21DE3plysS bacterium
Strain, kalamycin resistance TB plate screening monoclonal, 37 DEG C, TB culture medium shakes bacterium and stays overnight；Bacterium solution will be stayed overnight by volume (v/v)
1:100 is forwarded to fresh TB culture medium, and 37 DEG C, 200rpm shakes 3h and reaches 4 or so to OD600 value, is added final concentration 1mmol/L's
IPTG continues to shake bacterium 3h, and bacterium solution is collected by centrifugation in 6000rpm.Cultivation temperature is reduced to 30 DEG C；IPTG inducer is added to final concentration
0.5mM, 30 DEG C of continuation shake culture 3-4h；8000rpm is centrifuged 3min and collects thallus, is resuspended in 50mL pre-cooling NTA-0 buffer
In, ice bath 30min；Ultrasonication thallus, 4 DEG C of centrifugation 50min of 16000rpm collect supernatant and precipitating；Take a small amount of supernatant and
Precipitating carries out SDS-PAGE detection, and protein expression result is as shown in Figure 1.Remaining supernatant and precipitating be placed in 4 DEG C it is spare.
3, protein purification, renaturation:
DTT to final concentration 1mM is added with the resuspension of 50mL NTA-0 buffer in precipitating, and ultrasound promotes foreign protein dissolution, centrifugation
Precipitating is collected, supreme clear bright in triplicate, precipitating is resuspended with PBS, and supernatant is removed in ultrasound, centrifugation, is forgiven with the resuspension of 6M guanidine hydrochloride
Body adds DTT to final concentration 5mM；37 DEG C of concussion 3h are all dissolved to inclusion body, and supernatant is removed in centrifugation.Use protein renaturation liquid by egg again
White low temperature dialysis renaturation is added dropwise to 200mL renaturation solution with the 3M guanidine hydrochloride diluted protein solution of 2 times of volumes at 4 DEG C
(pH8.0) in, rotational speed regulation to maximum, stirring for 24 hours, takes protein solution in bag filter, slow with PBS after being concentrated with PEG60000
Fliud flushing dialysed overnight.Albumen after renaturation is purified again with anion-exchange column Ni-NTA, collects albumen.Protein purification result is such as
Shown in Fig. 2.
4, the preparation and the selection of best peridium concentration of envelope antigen:
Purifying protein is serially diluted into 6 μ g/mL, 3 μ g/mL, 1.5 μ g/mL, 0.75 μ g/ with carbonate buffer solution (pH9.6)
ML, 0.38 μ g/mL, 0.19 μ g/mL, each two column of concentration coating are added in 96 hole elisa Plates, 4 DEG C of coatings according to 100 holes μ L/
Overnight.The result shows that positive value OD450 value is greater than 1.0 when envelope antigen concentration is 3 μ g/mL, and the value of P/N is maximum, therefore really
The fixed concentration is best peridium concentration (table 1).
1 Checkerboard titration antigen working concentration (OD450nm) of table
| Peridium concentration (μ g/mL) | 6.00 | 3.00 | 1.50 | 0.75 | 0.38 | 0.19 |
| Positive sample (P) | 1.122 | 1.084 | 0.916 | 0.746 | 0.653 | 0.554 |
| Negative sample (N) | 0.138 | 0.098 | 0.092 | 0.087 | 0.079 | 0.073 |
| P/N | 8.130 | 11.06 | 9.956 | 8.575 | 8.266 | 7.589 |
5, the closing of ELISA Plate:
Above-mentioned ELISA Plate washes 4 times with PBST (the 0.01mol/L PBS solution containing 0.5v/v%Tween20, pH 7.6), often
Hole adds 10mM PBS confining liquid of the 100 μ l containing 0.5w/v%BSA, 37 DEG C of 2 h of placement.Confining liquid is discarded, PBST is washed 3 times, obtained
To the ELISA Plate of pre-coated antigen, every hole adds the ELISA Plate stabilizer of 100 μ l horse serums containing 6v/v%, is placed at room temperature for 30min, abandons
Fall naturally dry after liquid, with aluminium foil bag vacuum seal, 4 DEG C of preservations；
Wherein, the ELISA Plate stabilizer is to be added to 5g BSA, 10g sucrose, 20g trehalose
In 1000ml0.01mol/L PBS (pH7.4) gently concussion to dissolve, add 0.05w/v%Procline300, be stored in 4 DEG C it is standby
With.
6, the preparation of standard positive and negative nose swab:
The positive control sample is the nose swab sample for acquiring the O-shaped virus of aftosa and attacking 7-21d pig, ox and sheep after poison
Product, a series of sample that detection OD450 value is 1.5 ± 0.05 after mixing, dilutions；The negative control sample isFMDV NS kit detect foot and mouth disease virus non-structural protein antibody be feminine gender, foot-and-mouth disease a type, it is O-shaped,
AsiaI Liquid-phase blocking ELISA kit detect serum antibody titer < 1/4, FMDV specific PCR detection antigen be negative pig,
The nose swab of ox and sheep detects the sample that OD450 value is 0.1 ± 0.05 after being mixed, being diluted, aseptic subpackaged spare.
7, enzyme labelled antibody (10 ×)
The anti-pig of the mouse of horseradish peroxidase-labeled, ox or sheep IgA secondary antibody, 10 times of uses of dilution when use.
8, sample diluting liquid
Sample diluting liquid is NaCl containing 0.5M, 2.68mM KCl, 2.79mM KH2PO4、8.1mM Na2HPO4, 5g/L junket
The mixed solution of albumen, 0.05%v/vTween20 and 10mM PBS.
9、PBST(10×)
0.1mol/LPBS solution containing 0.5v/v%Tween20, pH 7.6.10 times of uses of dilution when use.
10, developing solution and terminate liquid
Developing solution is TMB developing solution, terminate liquid 1moL/LH2SO4Solution.
(2) assembling and preservation of kit
According to kit contents listed by table 2, kit is assembled, is saved after composition loaded on 4 DEG C.
2 ELISA detection kit content of table
(3) application method (detection method) of kit
(1) it dilutes: PBST (10 ×) washing lotion being diluted to 250ml with aqua sterilisa, with sample diluting liquid by enzyme labelled antibody (10
×) it is diluted to 5ml.
(2) board-washing: opening the package, and takes out the ELISA Plate of pre-coated antigen, with PBST board-washing 4 times diluted, water suction
Paper pats dry, each 3min.
(3) it is loaded: sample is diluted with 1:2 times, every hole 100ul is added in reaction plate, and positive control and feminine gender is added
Control, 100 holes μ L/, the positive control, negative control do two repetitions respectively.37 DEG C of incubation 45min.
(4) enzyme labeling antibody: getting rid of sample, is rinsed 4 times with PBST, and blotting paper pats dry, and the horseradish peroxide diluted is added
The anti-pig of mouse, ox or the sheep IgA secondary antibody of compound enzyme label, 100 holes μ L/, 37 DEG C of incubation 30min.
(5) add substrate: getting rid of enzyme labelled antibody, rinsed 4 times with PBST, blotting paper pats dry.37 DEG C of TMB developing solution incubations are added
Then the reaction terminating liquid in 100 holes μ l/ is added in 15min.
(6) OD is read in microplate reader450Numerical value.
OD is read in microplate reader450Numerical value, the OD of sample to be tested450nmValue is more than or equal to negative control sera average value
2.5 times are judged to the positive, and 2.5 times less than negative control sera average value are judged to feminine gender.
The sensibility of the O-shaped virus sIgA antibody ELISA detection kit of 2 aftosa of embodiment, specificity experiments
1, sensitivity experiments:
Kit is prepared using embodiment 1 and detects 100 parts of nose swab samples, these samples are that O/ is respectively adopted
Akesu/58, O/TAW/81/97, O/Tibet/99, O/Mya98 test the nose swab sample for attacking the morbid pig collected after poison, often
Kind of Virus Sample is 25, does not carry out before carrying out attacking poison any immune, and detection method is the same as embodiment 1.It is positive by calculating
Recall rate analyzes the sensibility of this method.ELISA testing result shows 93 parts of the above Sample Positive, 7 parts negative, positive rate
It is 93%.Illustrate that kit of the invention has preferable sensibility, and is able to detect the sense of the O-shaped virus of different aftosas
Dye.The results are shown in Table 3.
3 ELISA kit sensitivity tests of table
| Detection method | Sample number (part) | Number positive (part) | Negative number (part) | Positive rate (%) |
| Indirect ELISA | 100 | 93 | 7 | 93% |
2, specificity experiments:
Kit is prepared using embodiment 1 and has detected Porcine epidemic diarrhea virus (PEDV) infection piglet intestinal mucosa punching
Wash Samples, porcine reproductive and respiratory syndrome virus (PRRSV) antibody positive sample, pig circular ring virus (PCV) specificity IgA are anti-
Body positive sample, swine fever virus (CSFV) Specific IgA antibody positive sample increase for A type, O-shaped FMDV cross-infection situation
Add 90 parts of A type FMDV infection hog snout swab samples to be detected, using A type FMDV positive sample as positive control, analyzes the party
The specificity of method.ELISA testing result shows that 90 parts of A type FMDV infection hog snout swab samples are 2 parts positive, suspicious 10 parts, negative
78 parts, coincidence rate 86.7%.And with Porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV),
Pig circular ring virus (PCV), swine fever virus IgA antibody all without cross reaction, ensure that the diagnostic kit while having good
Good sensibility and specificity, fundamentally ensure that the accuracy and reliability of result.
3, repeated experiment
The positive schneiderian membrane swab sample and 1 part of negative schneiderian membrane swab sample for choosing 6 parts of different antibodies levels, using same
One batch antigen coat elisa plate carries out repeating to test in 4 times batches in different time, and statistic mixed-state is as a result, variation within batch coefficient
Less than 9% (1.41%~8.64%).Show the antigen coat prepared with a schneiderian membrane swab sample in same batch
Coefficient of variation very little in elisa plate has good repeatability.Using the antigen coat elisa plate of 4 batches preparation same
Time carries out repeating to test between criticizing, and statistic mixed-state is as a result, interassay coefficient of variation less than 8% (2.28%~7.13%), shows together
A schneiderian membrane swab sample coefficient of variation also very little in the elisa plate of antigen coat prepared by different batches, illustrates this hair
The indirect ELISA method of bright foundation has repeated well (table 4).
4 repeated experiment of table
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
<120>a kind of O-shaped virus sIgA antibody ELISA detection kit of aftosa and its application
<160>2
<170>Patent-In 3.5
<210>1
<211>705
<212>DNA
<213> artificial sequence
<400>1
atgggaagct gcaggtacag caacagtaac gtgagcaacg tgagtggtga tctccaagtg 60
ttggctcaga aggcggcgag agcgctgcct ggaggaggaa gcgccatcca cccgagtgag 120
gctagacaca agcagaagat tgtggcaccc gcaaaacagc ttttgggagg aggaagcggg 180
agcagtaagt acggtgacac cagcactaac aacgtgagag gtgaccttca agtgttagct 240
cagaaggcag aaagaactct gcctggagga ggaagcgcca ttcaaccgag tgacgctaga 300
cacaagcaga ggattgtggc acccgcaaaa cagcttctgg gaggaggaag cgggaactgc 360
aagtatggcg agagccccgt gaccaatgtg agaggtgacc tgcaagtatt ggcccagaag 420
gcggcaagaa cgctgcctgg aggaggaagc gctattcacc cgagcgaagc tagacacaaa 480
caaaagattg tggcgcctgt gaaacagctt ttgggaggag gaagcgggaa ctgcaagtac 540
gccgagggct cactgaccaa cgtgagaggt gatctccagg tgctggctca gaaggcggcg 600
aggccgctgc ctggaggagg aagcgctgtc catccggatg aggctagaca caaacagaaa 660
atagtggcac ctgtgaagca gtccttgcac caccaccacc accac 705
<210>2
<211>235
<212>PRT
<213>artificial sequence
<400>2
Met Gly Ser Cys Arg Tyr Ser Asn Ser Asn Val Ser Asn Val Ser Gly Asp Leu Gln Val 20
Leu Ala Gln Lys Ala Ala Arg Ala Leu Pro Gly Gly Gly Ser Ala Ile His Pro Ser Glu 40
Ala Arg His Lys Gln Lys Ile Val Ala Pro Ala Lys Gln Leu Leu Gly Gly Gly Ser Gly 60
Ser Ser Lys Tyr Gly Asp Thr Ser Thr Asn Asn Val Arg Gly Asp Leu Gln Val Leu Ala 80
Gln Lys Ala Glu Arg Thr Leu Pro Gly Gly Gly Ser Ala Ile Gln Pro Ser Asp Ala Arg 100
His Lys Gln Arg Ile Val Ala Pro Ala Lys Gln Leu Leu Gly Gly Gly Ser Gly Asn Cys 120
Lys Tyr Gly Glu Ser Pro Val Thr Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys 140
Ala Ala Arg Thr Leu Pro Gly Gly Gly Ser Ala Ile His Pro Ser Glu Ala Arg His Lys 160
Gln Lys Ile Val Ala Pro Val Lys Gln Leu Leu Gly Gly Gly Ser Gly Asn Cys Lys Tyr 180
Ala Glu Gly Ser Leu Thr Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala 200
Arg Pro Leu Pro Gly Gly Gly Ser Ala Val His Pro Asp Glu Ala Arg His Lys Gln Lys 220
Ile Val Ala Pro Val Lys Gln Ser Leu His His His His His His 235
Claims (10)
1. a kind of O-shaped viral broad spectrum multi-epitope recombination antigen of aftosa, which is characterized in that the O-shaped viral wide spectrum of the aftosa
Multi-epitope recombination antigen includes that the O-shaped virus of aftosa represents strain O/Akesu/58, O/TAW/81/97, O/Tibet/99, O/
132-160 in the VP1 albumen of Mya98,193-213 main neutrality epitope sequences, the mouth hoof
The amino acid sequence of the O-shaped viral broad spectrum multi-epitope recombination antigen of epidemic disease is as shown in SEQ ID NO.2.
2. encoding the nucleotide sequence of the O-shaped viral broad spectrum multi-epitope recombination antigen of aftosa described in claim 1, it is preferred that
The nucleotide sequence is as shown in SEQ ID NO.1.
3. the O-shaped viral broad spectrum multi-epitope recombination antigen of aftosa described in claim 1 detects the O-shaped virus of aftosa in preparation
Purposes in sIgA antibody reagent.
4. a kind of O-shaped virus sIgA antibody ELISA detection kit of aftosa, which is characterized in that the kit includes right
It is required that the coated ELISA Plate of the O-shaped virus broad spectrum multi-epitope recombination antigen of aftosa described in 1,100 × concentration enzyme labelled antibody, enzyme mark
Antibody diluent, sample diluting liquid, concentrated cleaning solution, developing solution, terminate liquid, positive control sample and negative control sample.
5. the O-shaped virus sIgA antibody ELISA detection kit of aftosa as claimed in claim 4, which is characterized in that the mouth
The O-shaped viral broad spectrum multi-epitope recombination antigen of fever aphthous is obtained through prokaryotic expression.
6. the O-shaped virus sIgA antibody ELISA detection kit of aftosa as claimed in claim 4, which is characterized in that described
The O-shaped viral coated ELISA Plate of broad spectrum multi-epitope recombination antigen of aftosa is prepared in accordance with the following methods:
(1) preparation and coating of envelope antigen
The recombinant protein inclusion body that prokaryotic expression obtains purifies to obtain the O-shaped virus of aftosa extensively through renaturation and Ni-NTA
Multi-epitope recombination antigen is composed, for amino acid sequence as shown in SEQ ID NO.2, when coating is dilute with the carbonate buffer solution of pH9.6
It releases to 3 μ g/ml, according to 100 hole μ l/ coated elisa plates, 4 DEG C of refrigerators are stood overnight；
(2) closing and preservation of ELISA Plate
It is coated with overnight ELISA Plate and washs 4 times with PBST and pat dry, every hole adds 10mM PBS closing of the 100 μ l containing 0.5w/v%BSA
Liquid, 37 DEG C of placement 2h discard confining liquid, and PBST is washed 3 times, obtain the ELISA Plate of pre-coated antigen, and every hole adds 100 μ l containing 6v/
The ELISA Plate stabilizer of v% horse serum, is placed at room temperature for 30min, discards naturally dry after liquid, with aluminium foil bag vacuum seal, 4 DEG C
It saves；
Wherein, the ELISA Plate stabilizer is that 5g BSA, 10g sucrose, 20g trehalose are added to 1000ml 0.01mol/
In LPBS (pH7.4) gently concussion to dissolve, add 0.05w/v%Procline300, be stored in 4 DEG C it is spare.
7. the O-shaped virus sIgA antibody ELISA detection kit of aftosa as claimed in claim 4, which is characterized in that the sun
Property control sample be to acquire the O-shaped virus of aftosa to attack the nose swab sample of 7-21d pig, ox and sheep after poison, through mixing, a series of
The sample that OD450 value is 1.5 ± 0.05 is detected after dilution；The negative sample isFMDV NS kit
Detecting foot and mouth disease virus non-structural protein antibody is feminine gender, foot-and-mouth disease a type, O-shaped, AsiaI Liquid-phase blocking ELISA kit detection
Serum antibody titer < 1/4, FMDV specific PCR detects the nose swab that antigen is negative pig, ox and sheep, is mixed, is diluted
The sample that detection OD450 value is 0.1 ± 0.05 afterwards, it is aseptic subpackaged spare.
8. the O-shaped virus sIgA antibody ELISA detection kit of aftosa as claimed in claim 4, which is characterized in that described
100 × concentration enzyme labelled antibody is the monoclonal antibody that horseradish peroxidase (HRP) marks the anti-pig of mouse, ox or sheep IgA, is used
When, with using after 100 times of enzyme labelled antibody dilution dilutions, the enzyme labelled antibody dilution is containing 1v/v% glycerol, 0.5w/
The 0.01mol/LPBS buffer of v% bovine serum albumin(BSA), 1w/v% casein and 0.05w/v%Procline300,
pH7.4。
9. the O-shaped virus sIgA antibody ELISA detection kit of aftosa as claimed in claim 4, which is characterized in that described
Sample diluting liquid is NaCl containing 0.5M, 2.68mM KCl, 2.79mM KH2PO4、8.1mM Na2HPO4, 5g/L casein,
The mixed solution of 0.05%Tween20 and 10mM PBS；The developing solution is TMB developing solution, and the terminate liquid is 1moL/
L H2SO4Solution；The concentrated cleaning solution is 10 × PBST solution, i.e. the 0.1mol/L PBS containing 0.5v/v%Tween20
Solution, pH 7.6 dilute 10 times when use.
10. the O-shaped virus sIgA antibody ELISA detection kit of aftosa as described in claim 1, which is characterized in that utilize
When the kit carries out aftosa O-shaped virus sIgA antibody test, follow the steps below:
(1) Sample Dilution
By sample to be tested, positive control and negative control, with sample diluting liquid, 1:2 is diluted by volume；
(2) board-washing
The O-shaped virus of aftosa is taken out from 4 DEG C of refrigerators represents the coated enzyme of main neutrality epitope (TB/O) albumen of strain
Target opens aluminium foil bag, takes out ELISA Plate, with cleaning solution board-washing 3 times diluted, blotting paper is patted dry, each 3min；
(3) it is loaded
Sample to be tested, positive control and negative control after dilution is separately added into ELISA Plate, every 100 μ L of hole, 37 DEG C incubate
Educate 45min；
(4) it washs
ELISA Plate is taken out, sample is got rid of, is rinsed 4 times with cleaning solution, blotting paper pats dry；
(5) enzyme labelled antibody is added
The anti-pig of mouse, ox or the sheep IgA monoclonal antibody for the horseradish peroxidase-labeled that addition has diluted, 100 holes μ L/, 37 DEG C
Work 30min；
(6) add substrate:
Enzyme labelled antibody is got rid of, is rinsed 4 times with cleaning solution, blotting paper pats dry, and 37 DEG C of colour developing 15min of developing solution are added, and stands after taking-up
Terminate liquid is added；
(7) result judgement
OD450nm numerical value is read in microplate reader, the OD450nm value of sample to be tested is more than or equal to negative control sample average value
2.5 times are judged to the positive, and 2.5 times less than negative control sample average value are judged to feminine gender.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109655612A (en) * | 2019-01-31 | 2019-04-19 | 中国农业科学院兰州兽医研究所 | A kind of detection kit and detection method of Asia1 type foot and mouth disease virus |
| CN110642945A (en) * | 2019-10-25 | 2020-01-03 | 中国农业科学院兰州兽医研究所 | General type foot-and-mouth disease virus structural protein antibody and blocking ELISA detection kit thereof |
| CN111574596A (en) * | 2020-05-25 | 2020-08-25 | 中国农业科学院兰州兽医研究所 | Construction method of constitutive secretory expression O-type FMDV recombinant antigen epitope genetic engineering CHO cell line |
| CN113267621A (en) * | 2021-05-14 | 2021-08-17 | 北京金诺百泰生物技术有限公司 | Stabilizer for ELISA kit coated plate, preparation method of stabilizer, kit coated plate and kit |
| CN114705857A (en) * | 2022-05-16 | 2022-07-05 | 北京亿森宝生物科技有限公司 | Microporous plate type chemiluminescence detection kit for pig foot-and-mouth disease virus O-type and A-type antibodies and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999066954A1 (en) * | 1998-06-20 | 1999-12-29 | United Biomedical Inc. | Synthetic peptide vaccines for foot-and-mouth disease |
| CN103897065A (en) * | 2014-03-13 | 2014-07-02 | 中国农业科学院兰州兽医研究所 | Bovini Asia 1/O type foot-and-mouth disease bivalent multi-epitope vaccine and preparation method and application thereof |
-
2018
- 2018-09-13 CN CN201811068030.0A patent/CN109232720B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999066954A1 (en) * | 1998-06-20 | 1999-12-29 | United Biomedical Inc. | Synthetic peptide vaccines for foot-and-mouth disease |
| CN103897065A (en) * | 2014-03-13 | 2014-07-02 | 中国农业科学院兰州兽医研究所 | Bovini Asia 1/O type foot-and-mouth disease bivalent multi-epitope vaccine and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| SM SABBIR ALAM 等: "Antigenic heterogeneity of capsid protein", 《ADVANCES AND APPLICATIONS IN BIOINFORMATICS AND CHEMISTRY》 * |
| 张中旺 等: "抗原表位的研究方法及口蹄疫病毒抗原表位的研究进展", 《微生物学报》 * |
Cited By (5)
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|---|---|---|---|---|
| CN109655612A (en) * | 2019-01-31 | 2019-04-19 | 中国农业科学院兰州兽医研究所 | A kind of detection kit and detection method of Asia1 type foot and mouth disease virus |
| CN110642945A (en) * | 2019-10-25 | 2020-01-03 | 中国农业科学院兰州兽医研究所 | General type foot-and-mouth disease virus structural protein antibody and blocking ELISA detection kit thereof |
| CN111574596A (en) * | 2020-05-25 | 2020-08-25 | 中国农业科学院兰州兽医研究所 | Construction method of constitutive secretory expression O-type FMDV recombinant antigen epitope genetic engineering CHO cell line |
| CN113267621A (en) * | 2021-05-14 | 2021-08-17 | 北京金诺百泰生物技术有限公司 | Stabilizer for ELISA kit coated plate, preparation method of stabilizer, kit coated plate and kit |
| CN114705857A (en) * | 2022-05-16 | 2022-07-05 | 北京亿森宝生物科技有限公司 | Microporous plate type chemiluminescence detection kit for pig foot-and-mouth disease virus O-type and A-type antibodies and application thereof |
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