CN109856396A - Detect enzyme linked immunological kit and its application of mouth disease virus infection antibody - Google Patents

Abstract

The invention discloses the enzyme linked immunological kit of detection mouth disease virus infection antibody and its applications.The kit includes envelope antigen, the envelope antigen be following any protein: R1) amino acid sequence be SEQ ID No.2 protein, R2) amino acid sequence is the 166-282 protein of SEQ ID No.2, R3) in R1) or R2) shown in protein c-terminus or/and having with R1) of obtaining of aminoterminal fusion tag albumen or R2) identical active soluble fusion protein.The kit sensibility is high, accuracy is high, it is easy to operate, quick, it can be as identification aftosa wild virus infection and vaccine immunity antibody assay kit, this method is suitable for base's Veterinary office at different levels and Entry-Exit Inspection and Quarantine Bureau to the rapid, high volume selective mechanisms of aftosa wild virus infection serum antibody, provides important technical for the prevention and control and purification of aftosa.

Description

Detect enzyme linked immunological kit and its application of mouth disease virus infection antibody

Technical field

The present invention relates to the enzyme linked immunological kit of detection mouth disease virus infection antibody and its applications.

Background technique

Aftosa (Foot and Mouth Disease, FMD) is by foot and mouth disease virus (Foot and Mouth Disease Virus, FMDV) caused by artiodactyl is acute, hot, high degree in contact infects sexually transmitted disease.It is prevented and treated in aftosa Major measure in work is that susceptible animal is immunized with inactivated foot-and-mouth disease vaccine.But this measure while also counterpart hoof Animal is immunized in epidemic disease and infection animal antidiastole brings difficulty.Therefore, carry out the antidiastole technical research of aftosa pre- Prevent, control, put out and purify the disease to have very important significance.Virus isolation techniques are the gold marks of diagnosis FMD as defined in OIE Standard, although this method is more accurate, the time for needing to expend about several days could obtain as a result, and operating technology it is complicated, it is raw Object safety requirements is high, is not suitable for base animal doctor quick diagnosis demand.Reverse transcription PCR and real-time quantitative PCR are widely used for Detect FMDV.But both methods needs professional to carry out in the laboratory of profession, also diagnoses FMD to base testing staff Bring certain difficulty.At present for the serological diagnostic method of FMDV wild virus infection, most commonly used is base In the elisa technique of FMDV non-structural protein (Non Structure Protein) 3ABC.But 3ABC albumen is due to itself The factors such as amino acid structure feature and sequence are longer, eukaryotic system expression is more difficult, and albumen is essentially after prokaryotic expression Inclusion body structure.Because the expression product in inclusion body does not have biological activity, thus carries out denaturation and renaturation process. The denaturation of albumen and renaturation are an extremely complex processes, and the denaturing conditions of different albumen are different, and renaturation yield is often difficult to mention It is high.This is the active main restricting factor of limited antigen.

Summary of the invention

A technical problem to be solved by this invention is how to improve the spirit of the serology antibody test of foot and mouth disease virus Sensitivity, and reduce omission factor and false positive rate, thus it is sensitiveer, more accurately diagnose aftosa.

In order to solve the above-mentioned technical problems, the present invention provides application of the protein in reagent preparation box.

For protein provided by the present invention in the application in reagent preparation box, the kit can be aftosa diagnosis examination Agent box or the kit for detecting antibodies against foot-and-mouth disease virus, the protein are R1), R2) or protein R3):

R1) amino acid sequence is the protein of SEQ ID No.2,

R2) amino acid sequence is the 166-282 protein of SEQ ID No.2,

R3) in R1) or R2) shown in protein c-terminus or/and aminoterminal fusion tag albumen obtain have with R1) or R2) identical active soluble fusion protein.

In above-mentioned application, the label protein (protein-tag) refers to using DNA extracorporeal recombination, with purpose egg A kind of polypeptide or albumen of Bai Yiqi amalgamation and expression, in order to the expression of destination protein, detection, tracer and/or purifying.It is described Label protein can be Flag label protein, His label protein, MBP label protein, HA label protein, myc label protein, GST mark Sign albumen and/or SUMO label protein etc..

In above-mentioned application, R1) protein name be known as rtagP3AB1B2, R2) protein name be known as rP3AB1B2.SEQ ID No.2 is made of 290 amino acid residues.

In above-mentioned application, the protein can be prepared according to the method included the following steps: make the coding of the protein Gene is expressed to obtain the protein in biology, and the biology can be microorganism, plant or non-human animal.

In above-mentioned application, so that the encoding gene of the protein is carried out expression in biology may include by the protein Encoding gene imports recipient microorganism, obtains the recombinant microorganism for expressing the protein, cultivates the recombinant microorganism, expresses Obtain the protein.

Any one of in above-mentioned application, the recipient microorganism can be C1)-C4):

C1) prokaryotic micro-organisms,

C2) gramnegative bacterium,

C3) Escherichia bacteria,

C4) e. coli bl21 (DE3).

In above-mentioned application, the encoding gene of the protein can be following 1) -4) in any DNA molecular:

1) coded sequence is the DNA molecular of SEQ ID No.1,

2) nucleotide sequence is the DNA molecular of SEQ ID No.1,

3) coded sequence is the DNA molecular of the 496-846 nucleotide of SEQ ID No.1,

4) nucleotide sequence is the DNA molecular of the 496-846 nucleotide of SEQ ID No.1.

In above-mentioned application, the recombinant microorganism can obtain for pET32a-3AB1B2 is imported e. coli bl21 (DE3) Express amino acid sequence be SEQ ID No.2 protein recombinant microorganism, the recombinant microorganism is named as BL21 (DE3) it is 490-852 of SEQ ID No.1 that/pET32a-3AB1B2, the pET32a-3AB1B2, which are with nucleotide sequence, DNA replacement pET32a (+) BamH I and XhoI recognition site between segment, keep pET32a (+) other sequences it is constant, Obtained recombinant expression carrier.

In above-mentioned application, the expression can be inducing expression.

In above-mentioned application, the inducing expression be with the IPTG of 0.75mM 16 DEG C induction 13-16 hours or 13 hours.

In order to solve the above-mentioned technical problem, the present invention also provides a kind of kits.

Kit provided by the present invention is the enzyme linked immunological kit or detection antibodies against foot-and-mouth disease virus for diagnosing aftosa Enzyme linked immunological kit, the kit includes envelope antigen, the envelope antigen be the protein.

The kit further includes other reagents, such as ELIAS secondary antibody needed for carrying out ELISA.

Application of the mentioned reagent box in preparation aftosa monitoring reagent box also belongs to protection scope of the present invention.

The present invention links together nonstructural protein 3A segment, 3B1 and the 3B2 of FMDV by link peptide to obtain fusion egg RP3AB1B2 gene is inserted between BamH I and the XhoI recognition site of pET32a (+) and obtains to express by white rP3AB1B2 The recombinant expression carrier pET32a-3AB1B2 of fusion protein rtagP3AB1B2, imports Escherichia coli for pET32a-3AB1B2 The rtagP3AB1B2 of BL21 (DE3) acquisition solubility expression.It is diagnosed as a purpose with the rtagP3AB1B2 of solubility expression anti- Non-structural protein antibody in former and/or detection antigen detection serum can be used to distinguish vaccine immunity animal and wild virus infection is dynamic Object；Secondly non-structural protein is regardless of serotype, with rtagP3AB1B2 diagnostic antigen and/or the inspection as a purpose of solubility expression The ELISA and time-resolved fluoroimmunoassay for surveying the non-structural protein antibody in the detection serum that antigen is established can be with counterparts Seven kinds of serotypes of fever aphthous are detected.

Not only solubility expression ratio is high by rtagP3AB1B2 of the invention, but also compared with 3ABC holoantigen, detection it is quick Perceptual suitable, specificity is more preferable, and accuracy is higher:

1, the present invention accounts for the 62% of bacterial protein using the rtagP3AB1B2 of Bacillus coli expression, and 95% is solvable.This The rtag3AB1B2 for the soluble-expression that invention is obtained using Escherichia coli identifies aftosa wild virus infection and epidemic disease for further exploitation Seedling immune antiboidy detection kit has established extraordinary basis.

2, the indirect ELISA that the rtag3AB1B2 of soluble-expression is established as envelope antigen is detected into antibodies against foot-and-mouth disease virus The sensitivity for the indirect ELISA detection foot and mouth disease virus antibody method that the sensibility and rtag3ABC of method are established as envelope antigen Property it is suitable, but be apparently higher than the indirect ELISA inspection established using rtag3B1B2, rtagP3A and rtag3A as envelope antigen The sensibility for surveying antibodies against foot-and-mouth disease virus method, is also apparently higher than the quick of Switzerland PRIONICS aftosa NS antibody assay kit Perception.

3, the indirect ELISA that the rtag3AB1B2 of soluble-expression is established as envelope antigen is detected into antibodies against foot-and-mouth disease virus Method and aftosa Switzerland PRIONICS aftosa NS antibody assay kit total coincidence rate be significantly higher than respectively with Rtag3ABC, rtag3B1B2, rtagP3A and rtag3A are anti-as the indirect ELISA detection foot and mouth disease virus that envelope antigen is established The method of body.

The present invention is tried by the antibody test of diagnostic antigen and/or detection antigen preparation of the rtagP3AB1B2 of soluble-expression Agent box sensibility is high, and accuracy is high, easy to operate, quick, is suitable for base's Veterinary office at different levels and Entry-Exit Inspection and Quarantine Bureau To the rapid, high volume selective mechanisms of aftosa wild virus infection serum antibody, important technology hand is provided for the prevention and control and purification of aftosa Section.

Detailed description of the invention

Fig. 1 is the SDS-PAGE electrophoresis spectrum that each bacterial strain expresses albumen.

In figure, swimming lane Marker be protein molecular weight standard, from top to bottom respectively 90kDa, 65kDa, 50kDa, 38kDa, 26kDa, 15kDa, 10kDa, 1 is BL21 (DE3)/pET30a whole bacterial protein liquid of inducing expression, and 2 be inducing expression BL21 (DE3)/pET32a-3AB1B2 whole bacterial protein liquid, 3 be inducing expression BL21 (DE3)/pET32a-3AB1B2 Contain albumen precipitation containing albumen supernatant, 4 for BL21 (DE3)/pET32a-3AB1B2 of inducing expression, 5 be the BL21 of inducing expression (DE3)/pET32a-3ABC whole bacterial protein liquid, 6 be BL21 (DE3)/pET32a-3ABC supernatant containing albumen of inducing expression Liquid, 7 for inducing expression BL21 (DE3)/pET32a-3ABC contain albumen precipitation, 8 for inducing expression BL21 (DE3)/ The whole bacterial protein liquid of pET32a-3B1B2,9 for inducing expression BL21 (DE3)/pET32a-3B1B2 contain albumen supernatant, 10 Contain albumen precipitation for BL21 (DE3)/pET32a-3B1B2 of inducing expression, 11 be BL21 (DE3)/pET32a- of inducing expression The whole bacterial protein liquid of P3A, 12 contain albumen supernatant for BL21 (DE3)/pET32a-P3A of inducing expression, and 13 be inducing expression BL21 (DE3)/pET32a-P3A contain albumen precipitation, 14 be inducing expression BL21 (DE3)/pET32a-3A whole bacterial protein Liquid, 15 for inducing expression BL21 (DE3)/pET32a-3A contain albumen supernatant, 16 for inducing expression BL21 (DE3)/ PET32a-3A contains albumen precipitation, and 17 be the rtagP3AB1B2 albumen of ni-sepharose purification, and 18 be the rtagP3AB1B2 of molecular sieve purification Albumen.

The molecular sieve purification identification and Structural Identification that Fig. 2 is recombinant protein rtagP3AB1B2.Arrow meaning is purifying Destination protein peak, the entitled retention volume (liter) for retaining purification column of abscissa.

Specific embodiment

The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining The bright present invention, the range being not intended to be limiting of the invention.Examples provided below can be used as the art ordinary skill The guide that personnel are further improved, is not construed as limiting the invention in any way.

Experimental method in following embodiments is unless otherwise specified conventional method.Material as used in the following examples Material, reagent etc., are commercially available unless otherwise specified.

PET32a (+) in following embodiments is Novagen Products.Europium labelled element (Eu3+) is Guangzhou Da Ruisheng Object technical concern Co., Ltd product.Rabbit-anti goat secondary antibody is Sigma Products.Auto-DELFIA1235 time resolution is glimmering Optical detector is purchased from PerkinElmer limited liability company.ELISA Plate is purchased from U.S. Costar company.In following embodiments Switzerland PRIONICS aftosa NS antibody assay kit isFMDV NS Antibody ELISA test Kit(FMDV NS FMDV Antibody test Kit, ELISA), lot number is F161101L, and article No. is 7610770。

Sheep FMDV non-structural protein Positive Sera in following embodiments is clinical sheep blood serum through Switzerland PRIONICS The serum of aftosa NS antibody assay kit test positive, sheep FMDV non-structural protein negative antibody serum are clinical sheep blood Negative serum is detected as through Switzerland PRIONICS aftosa NS antibody assay kit clearly.

The solubility expression of embodiment 1, foot and mouth disease virus recombinant protein r3AB1B2 and rtag3AB1B2

1, the building of recombinant expression carrier

The building of 1.1 foot and mouth disease virus recombinant protein gene recombinant vectors

1.1.1 the design of foot and mouth disease virus recombinant protein gene

The application devises 5 kinds of foot and mouth disease virus recombinant protein genes, is shown in SEQ ID No.1 respectively Rtag3ABC gene (crt gene 1) shown in rtagP3AB1B2 gene, SEQ ID No.3, shown in SEQ ID No.5 RtagP3A gene (crt gene 3) shown in rtag3B1B2 gene (crt gene 2), SEQ ID No.7 and SEQ ID No.9 Shown in rtag3A gene (crt gene 4).

1.1.1.1 rtagP3AB1B2 gene (gene of the present invention)

SEQ ID No.1 is made of 873 nucleotide, wherein the 1-873 CDS for rtagP3AB1B2 gene, the The 496-846 nucleotide sequences for rP3AB1B2 gene, 1-495 and the 847-873 sequences for pET32a (+), The gene order (1-120 of GenBank:AY614501.1) of the 496-615 3A protein fragments for foot and mouth disease virus, The gene order (430-498 of GenBank:AY614501.1) of the 661-729 3B1 albumen for foot and mouth disease virus, The gene order (499-570 of GenBank:AY614501.1) of the 775-846 3B2 albumen for foot and mouth disease virus.

1.1.1.2 rtag3ABC gene (foot and mouth disease virus overall length 3ABC genetic contrast)

SEQ ID No.3 is made of 1803 nucleotide, wherein the 1-1803 CDS for rtag3ABC gene, the The 496-1776 nucleotide sequences (sequence with GenBank:AY614501.1 1-1281) for 3ABC gene, 1- The 495 and 1777-1803 sequences for pET32a (+), the gene sequence of the 496-924 3A albumen for foot and mouth disease virus It arranges (1-429 of GenBank:AY614501.1), the gene order of the 925-993 3B1 albumen for foot and mouth disease virus (430-498 of GenBank:AY614501.1), the gene order of the 994-1065 3B2 albumen for foot and mouth disease virus (499-570 of GenBank:AY614501.1), the gene sequence of the 1066-1137 3B3 albumen for foot and mouth disease virus It arranges (571-642 of GenBank:AY614501.1), the gene sequence of the 1138-1776 3C albumen for foot and mouth disease virus It arranges (643-1281 of GenBank:AY614501.1).

1.1.1.3 rtag3B1B2 gene (foot and mouth disease virus 3B13B2 genetic contrast)

SEQ ID No.5 is made of 708 nucleotide, wherein the 1-708 CDS for rtag3B1B2 gene, the The 496-681 nucleotide sequences for 3B1B2 gene, 1-495 and the 682-708 sequences for pET32a (+), the The gene order (430-498 of GenBank:AY614501.1) of the 496-564 3B1 albumen for foot and mouth disease virus, the The gene order (499-570 of GenBank:AY614501.1) of the 610-681 3B2 albumen for foot and mouth disease virus.

1.1.1.4 rtagP3A gene (control of foot and mouth disease virus 3A fragment gene)

SEQ ID No.7 is made of 642 nucleotide, wherein the 1-642 CDS, 496- for rtagP3A gene The gene order (1-120 of GenBank:AY614501.1) of 615 3A protein fragments for foot and mouth disease virus, 1- The 495 and 616-642 sequences for pET32a (+).

1.1.1.5 rtag3A gene (control of foot and mouth disease virus 3A full-length gene)

SEQ ID No.9 is made of 951 nucleotide, wherein the 1-951 CDS, 496- for rtag3A gene The gene order (1-429 of GenBank:AY614501.1) of the 924 3A albumen for foot and mouth disease virus, 1-495 It is the sequence of pET32a (+) with 925-951.

1.1.2 the building of recombinant expression carrier

1.1.2.1 the building of rtagP3AB1B2 gene recombinant vectors (expression vector of the invention)

With nucleotide sequence be SEQ ID No.1 490-852 DNA replacement pET32a (+) BamH I and Segment (small fragment including BamH I recognition site and XhoI recognition site) between XhoI recognition site keeps pET32a Other sequences of (+) are constant, obtain rtagP3AB1B2 gene recombinant vectors, are named as pET32a-3AB1B2.pET32a- 3AB1B2 contains the DNA molecular that nucleotide sequence is SEQ ID No.1.PET32a-3AB1B2 contains rtagP3AB1B2 gene, The nucleotide sequence of rtagP3AB1B2 gene is SEQ ID No.1, and coded sequence is the 1-873 nucleosides of SEQ ID No.1 Acid, rtagP3AB1B2 gene encode protein rtagP3AB1B2 shown in SEQ ID No.2.

1.1.2.2 rtag3ABC gene recombinant vectors (control of foot and mouth disease virus overall length 3ABC expression vector)

With nucleotide sequence be SEQ ID No.3 490-1782 DNA replacement pET32a (+) BamH I and Segment (small fragment including BamH I recognition site and XhoI recognition site) between XhoI recognition site keeps pET32a Other sequences of (+) are constant, obtain rtag3ABC gene recombinant vectors, are named as pET32a-3ABC.pET32a-3ABC It is the DNA molecular of SEQ ID No.3 containing nucleotide sequence.PET32a-3ABC contains rtag3ABC gene, rtag3ABC gene Nucleotide sequence be SEQ ID No.3, coded sequence is the 1-1803 nucleotide of SEQ ID No.3, rtag3ABC base Because of protein rtag3ABC shown in coding SEQ ID No.4.

1.1.2.3 rtag3B1B2 gene recombinant vectors (control of foot and mouth disease virus 3B13B2 expression vector)

With nucleotide sequence be SEQ ID No.5 490-687 DNA replacement pET32a (+) BamH I and Segment (small fragment including BamH I recognition site and XhoI recognition site) between XhoI recognition site keeps pET32a Other sequences of (+) are constant, obtain rtag3B1B2 gene recombinant vectors, are named as pET32a-3B1B2.pET32a- 3B1B2 contains the DNA molecular that nucleotide sequence is SEQ ID No.5.PET32a-3B1B2 contains rtag3B1B2 gene, The nucleotide sequence of rtag3B1B2 gene is SEQ ID No.5, and coded sequence is the 1-708 nucleosides of SEQ ID No.5 Acid, rtag3B1B2 gene encode protein rtag3B1B2 shown in SEQ ID No.6.

1.1.2.4 rtagP3A gene recombinant vectors (control of foot and mouth disease virus 3A fragment gene expression vector)

With nucleotide sequence be SEQ ID No.7 490-621 DNA replacement pET32a (+) BamH I and Segment (small fragment including BamH I recognition site and XhoI recognition site) between XhoI recognition site keeps pET32a Other sequences of (+) are constant, obtain rtagP3A gene recombinant vectors, are named as pET32a-P3A.PET32a-P3A contains Nucleotide sequence is the DNA molecular of SEQ ID No.7.PET32a-P3A contains rtagP3A gene, the nucleosides of rtagP3A gene Acid sequence is SEQ ID No.7, and coded sequence is the 1-642 nucleotide of SEQ ID No.7, and rtagP3A gene encodes SEQ Protein rtagP3A shown in ID No.8.

1.1.2.5 rtag3A gene recombinant vectors (control of foot and mouth disease virus 3A full-length gene expression vector)

With nucleotide sequence be SEQ ID No.9 490-930 DNA replacement pET32a (+) BamH I and Segment (small fragment including BamH I recognition site and XhoI recognition site) between XhoI recognition site keeps pET32a Other sequences of (+) are constant, obtain rtag3A gene recombinant vectors, are named as pET32a-3A.PET32a-3A contains core Nucleotide sequence is the DNA molecular of SEQ ID No.9.PET32a-3A contains rtag3A gene, the nucleotide sequence of rtag3A gene It is SEQ ID No.9, coded sequence is the 1-951 nucleotide of SEQ ID No.9, and rtag3A gene encodes SEQ ID Protein rtag3A shown in No.10.

2, the building of recombinant bacterium

PET32a-3AB1B2, pET32a-3ABC that step 1 is constructed, pET32a-3B1B2, pET32a-P3A, This 5 kinds of expression vectors of pET32a-3A individually convert e. coli bl21 (DE3) competent cell.It is spread evenly across On LB plate containing ampicillin (50 μ g/mL), 37 DEG C are cultivated 16 hours.Single colonie shaken cultivation stay overnight, extract plasmid into Row sequencing, shows that the recombination bacillus coli containing pET32a-3AB1B2 is named as BL21 (DE3)/pET32a- for sequencing result Sequencing result is shown that the recombination bacillus coli containing pET32a-3ABC is named as BL21 (DE3)/pET32a- by 3AB1B2 Sequencing result is shown that the recombination bacillus coli containing pET32a-3B1B2 is named as BL21 (DE3)/pET32a- by 3ABC Sequencing result is shown that the recombination bacillus coli containing pET32a-P3A is named as BL21 (DE3)/pET32a-P3A by 3B1B2, will Sequencing result shows that the recombination bacillus coli containing pET32a-3A is named as BL21 (DE3)/pET32a-3A.Simultaneously by pET32a (+) import e. coli bl21 (DE3) obtain being named as containing the recombination bacillus coli of pET32a (+) BL21 (DE3)/ PET32a, as empty vector control.

3, the analysis and identification of protein expression form

By BL21 (DE3)/pET32a-3AB1B2, BL21 (DE3)/pET32a-3ABC, BL21 (DE3)/pET32a- This 6 bacterial strain difference of 3B1B2, BL21 (DE3)/pET32a-P3A, BL21 (DE3)/pET32a-3A and BL21 (DE3)/pET32a It is individually inoculated in the LB liquid medium containing 50 μ g/ml ampicillins and (ampicillin is added extremely in LB liquid medium The concentration of ampicillin is the obtained culture medium of 50 μ g/ml) in, 37 DEG C, using Thermo MaxQ6000 type warm oscillator entirely 200rpm shaken cultivation is to 0D₆₀₀Value (using the LB liquid medium containing 50 μ g/ml ampicillins as blank control) reaches 0.6 When, isopropylthio-β-D-galactoside (IPTG) is added and carries out inducing expression.The inducing expression of above-mentioned 6 plants of bacterium is to use The IPTG of 0.75mM 16 DEG C induce 13 hours (the inducing expression condition be by the conditions such as temperature, time, IPTG concentration into The highly-soluble inducing expression condition that row optimization obtains).

Take above-mentioned inducing expression fermentation liquid for protein expression form analysis.The specific steps are take 1m L fermentation liquid to be placed in It in 1.5mL centrifuge tube, marks, 8500rpm/min is centrifuged 45min under the conditions of 4 DEG C, discards supernatant, collects bacterial sediment. 1m L PBS is added, precipitating is resuspended, 8000rpm/min is centrifuged 5min, discards supernatant.It is added into the bacterial sediment washed 200 μ L PBS, high pressure are crushed thallus, and cracking is no longer sticky to bacterium solution, obtains whole bacterial protein liquid.By whole bacterial protein liquid in 4 18000rpm/min is centrifuged 45min in DEG C centrifuge, collects supernatant (being named as containing albumen supernatant) respectively and precipitating (is named For containing albumen precipitation), 50 μ L PBS resuspension washing precipitating is added in albumen precipitation to containing.To whole bacterial protein liquid, containing on albumen Clear liquid and containing 10 μ L 5 × SDS-PAGE loading Buffer are added in albumen precipitation, after mixing well, sets in boiling water bath and boils Boil 5min, after sample is cooling, with hand held centrifuge wink from.Take 6 μ L for SDS-PAGE electrophoretic analysis, and binding protein is grey Degree analysis software preliminary analysis protein content.Gel after electrophoresis is needed on NC film, the sheep anti-mouse antibody with anti-His label is Binding antibody DAB colour developing, carries out Western-blot identification.By above-mentioned whole bacterial protein liquid and containing albumen supernatant with 0.22 μm After membrane filtration loading in advance with solution 1 (solute and its concentration are as follows: 20mM Tris, 150mM NaCl, and solvent is water, The solution of pH8.0) nickel column that has balanced.Nickel column is accessed on AKTA machine, respectively with solution 1 and 10 column of 10 column volumes (solute and its concentration are as follows: 20mM Tris, 150mM NaCl, 50mM imidazoles, and solvent is water, and pH8.0's is molten for the solution 2 of volume Liquid) impurity protein in cleaning nickel column, and protein peak is monitored on AKTA machine.With solution 3 (solute and its concentration are as follows: 20mM Tris, 150mM NaCl, 300mM imidazoles, solvent are water, the solution of pH8.0) destination protein hung in nickel column is rinsed, And the elution samples for destination protein peak occur are collected using AKTA, which is known as ni-sepharose purification destination protein sample.

The Superdex200 gel column that the destination protein sample of ni-sepharose purification GE company is produced passes through molecular sieve into one Step purifying.Mobile phase uses solution 1.By can remove a large amount of imidazoles contained in sample after molecular sieve purification, collection is washed De- peak, obtains the destination protein sample of molecular sieve purification, right using NanoDrop2000 ultramicrospectrophotometer (ND2000) The content of albumen (i.e. soluble destination protein) carries out quantitative analysis in the destination protein sample of obtained molecular sieve purification.It is used in combination NanoDrop2000 ultramicrospectrophotometer (ND2000) measures the protein content in whole bacterial protein liquid, and it is total to obtain thallus Protein content.It will contain after albumen precipitation dissolves with urea, be measured with NanoDrop2000 ultramicrospectrophotometer (ND2000) Content containing the protein in albumen precipitation.

3.1BL21 (DE3)/pET32a-3AB1B2 expression

The result shows that BL21 (DE3)/pET32a-3AB1B2 whole bacterial protein liquid of inducing expression, contain albumen supernatant With containing in albumen precipitation containing size be 31kDa destination protein rtagP3AB1B2；The BL21 (DE3) of inducing expression/ Destination protein rtagP3AB1B2 in the whole bacterial protein liquid of pET32a-3AB1B2 accounts for bacterial protein (full bacterium total protein) 62%, BL21 (DE3)/pET32a-3AB1B2 of inducing expression is accounted for containing the destination protein rtagP3AB1B2 in albumen supernatant The 95% of destination protein rtagP3AB1B2 in BL21 (DE3)/pET32a-3AB1B2 whole bacterial protein liquid of inducing expression, should 95% destination protein rtagP3AB1B2 is soluble protein；BL21 (DE3)/pET32a-3AB1B2 of inducing expression contains egg Destination protein rtagP3AB1B2 in white precipitating accounts for BL21 (DE3)/pET32a-3AB1B2 whole bacterial protein liquid of inducing expression The 5% of destination protein rtagP3AB1B2 in body, the 5% destination protein rtagP3AB1B2 are insoluble inclusion body albumen；It says BL21 (DE3)/pET32a-3AB1B2 destination protein rtagP3AB1B2 of bright inducing expression accounts for the 62% of bacterial protein, 95% is soluble protein in BL21 (DE3)/pET32a-3AB1B2 expression destination protein rtagP3AB1B2, and 5% is insoluble Property inclusion body protein (Fig. 1).

3.2 BL21 (DE3)/pET32a-3ABC expression

The result shows that BL21 (DE3)/pET32a-3ABC whole bacterial protein liquid of inducing expression and containing in albumen precipitation The destination protein rtag3ABC for being 66kDa containing size, and BL21 (DE3)/pET32a-3ABC of inducing expression is containing on albumen The destination protein rtag3ABC for being 66kDa without containing size in clear liquid.BL21 (DE3)/pET32a-3ABC of inducing expression is complete Destination protein rtag3ABC in mycoprotein liquid accounts for the 17% of bacterial protein (full bacterium total protein).Illustrate BL21 (DE3)/ The destination protein rtag3ABC of pET32a-3ABC expression is insoluble inclusion body albumen, and BL21 (DE3)/pET32a-3ABC cannot Express soluble destination protein (Fig. 1).

The expression of 3.3 pET32a-3B1B2

The result shows that BL21 (DE3)/pET32a-3B1B2 whole bacterial protein liquid of inducing expression and contain albumen supernatant In be 25kDa containing size destination protein rtag3B1B2, and BL21 (DE3)/pET32a-3B1B2 of inducing expression contains The destination protein rtag3B1B2 for being 25kDa without containing size in albumen precipitation.BL21 (DE3)/pET32a- of inducing expression Destination protein rtag3B1B2 in the whole bacterial protein liquid of 3B1B2 accounts for the 26% of bacterial protein (full bacterium total protein).Explanation BL21 (DE3)/pET32a-3B1B2 expression destination protein rtag3B1B2 is soluble protein, BL21 (DE3)/pET32a- 3B1B2 cannot express inclusion body destination protein (Fig. 1).

3.4BL21 (DE3)/pET32a-P3A expression

The result shows that BL21 (DE3)/pET32a-P3A whole bacterial protein liquid of inducing expression and containing in albumen supernatant The destination protein rtagP3A for being 23kDa containing size, and BL21 (DE3)/pET32a-P3A of inducing expression is heavy containing albumen The destination protein rtagP3A for being 23kDa without containing size in shallow lake.The full bacterium egg of BL21 (DE3)/pET32a-P3A of inducing expression Destination protein rtagP3A in white liquor body accounts for the 14% of bacterial protein (full bacterium total protein).Illustrate BL21 (DE3)/pET32a- The destination protein rtagP3A of P3A expression is soluble protein, and BL21 (DE3)/pET32a-P3A cannot express inclusion body purpose egg White (Fig. 1).

3.5BL21 (DE3)/pET32a-3A expression

The result shows that BL21 (DE3)/pET32a-3A whole bacterial protein liquid of inducing expression, containing albumen supernatant and containing The destination protein rtag3A for being 35kDa containing size in albumen precipitation；BL21 (DE3)/pET32a-3A of inducing expression is complete Destination protein rtag3A in mycoprotein liquid accounts for the 8% of bacterial protein (full bacterium total protein), the BL21 of inducing expression (DE3)/pET32a-3A accounts for BL21 (DE3)/pET32a- of inducing expression containing the destination protein rtag3A in albumen supernatant The 62% of destination protein rtag3A in the whole bacterial protein liquid of 3A, the 62% destination protein rtag3A are soluble protein；It lures BL21 (the DE3)/pET32a-3A for leading expression accounts for the BL21 of inducing expression containing the destination protein rtag3A in albumen precipitation (DE3) the 38% of destination protein rtag3A in/pET32a-3A whole bacterial protein liquid, the 38% destination protein rtag3A are Insoluble inclusion body albumen；Illustrate that BL21 (DE3)/pET32a-3A destination protein rtag3A of inducing expression accounts for the total egg of thallus 62% is soluble protein in white 8%, BL21 (DE3)/pET32a-3A expression destination protein rtag3A, and 38% is insoluble Property inclusion body protein (Fig. 1).

3.6 expression BL21 (DE3)/pET32a

The result shows that BL21 (DE3)/pET32a whole bacterial protein liquid of inducing expression does not have the expression of destination protein.

Although 3.1 and 3.2 result illustrates using same expression vector pET32a (+) and same host strain large intestine bar The expression of bacterium BL21 (DE3), different external source target gene differ greatly, and rtagP3AB1B2 gene is passed through pET32a (+), which imports e. coli bl21 (DE3), can get the solution expression with high efficiency of rtagP3AB1B2 gene, by rtag3ABC gene It is imported in e. coli bl21 (DE3) by pET32a (+), rtag3ABC gene cannot but obtain solubility expression.

In addition, the present inventor research and develop the present invention during, by identical rP3AB1B2 encoding gene (SEQ DNA molecular shown in 496-846 of ID No.1) insertion pET32a (+) EcoR I and XhoI recognition site between obtain Recombinant prokaryotic expression vector does not express fusion protein but in e. coli bl21 (DE3).As it can be seen that by same foreign gene It is inserted into the different location of same prokaryotic expression carrier, also will affect the expression of results of the foreign gene.

In addition, the present inventor research and develop the present invention during, by identical rP3AB1B2 encoding gene (SEQ DNA molecular shown in 496-846 of ID No.1) it is inserted into the recombination obtained between the site Nde I and XhoI of pET30a (+) Prokaryotic expression carrier, in e. coli bl21 (DE3) although in can obtain the destination protein of solubility expression, destination protein Amount of soluble expression is very low, is mostly destination protein for inclusion body, the destination protein of solubility expression accounts for the destination protein of expression The 27% of total amount.As it can be seen that same foreign gene to be inserted into different prokaryotic expression carriers, the expression of the foreign gene also will affect As a result.

4, the solubility expression of rtagP3AB1B2 and purifying

BL21 (DE3)/pET32a-3AB1B2 is inoculated in the LB liquid medium containing 50 μ g/ml ampicillins (in LB The concentration that ampicillin is added to ampicillin in fluid nutrient medium is the culture medium that 50 μ g/ml are obtained) in, it 37 DEG C, adopts With Thermo MaxQ6000 type entirely warm oscillator 200rpm shaken cultivation to 0D₆₀₀Value is (to contain the LB of 50 μ g/ml ampicillins Fluid nutrient medium is blank control) when reaching 0.6, IPTG is added and carries out inducing expression.The IPTG of inducing expression 0.75mM In 16 DEG C of induction 13h.Fermentation liquid after taking IPTG inducing expression 13h collects bacterial sediment.PBS is added, precipitating is resuspended, 8000rpm/min is centrifuged 5min, discards supernatant.PBS is added into the bacterial sediment washed, high pressure is crushed thallus, cracking It is no longer sticky to bacterium solution, in 4 DEG C of centrifuges 16000rpm/min be centrifuged 30min, collect supernatant (be named as BL21 (DE3)/ PET32a-3AB1B2 contains albumen supernatant), abandon precipitating.BL21 (DE3)/pET32a-3AB1B2 is contained into albumen supernatant with 0.22 After μm membrane filtration loading in advance with solution 1 (solute and its concentration are as follows: 20mM Tris, 150mM NaCl, and solvent is water, The solution of pH8.0) nickel column that has balanced.Nickel column is accessed on AKTA machine, respectively with solution 1 and 10 column of 10 column volumes (solute and its concentration are as follows: 20mM Tris, 150mM NaCl, 50mM imidazoles, and solvent is water, and pH8.0's is molten for the solution 2 of volume Liquid) impurity protein in cleaning nickel column, and protein peak is monitored on AKTA machine.With solution 3 (solute and its concentration are as follows: 20mM Tris, 150mM NaCl, 300mM imidazoles, solvent are water, the solution of pH8.0) rinse nickel column hang over the purpose in nickel column Albumen, and the elution samples for destination protein peak occur are collected using AKTA, which is known as to the RtagP3AB1B2 of ni-sepharose purification Albumen (ni-sepharose purification destination protein sample, Fig. 1 in swimming lane 17).

The Superdex200 gel column that the rtagP3AB1B2 albumen of ni-sepharose purification is produced with GE company is passed through into molecular sieve It is further purified, obtains the rtagP3AB1B2 albumen (swimming lane 18 in Fig. 1) of molecular sieve purification.Flowing in the molecular sieve purification Mutually use above-mentioned solution 1.By can remove a large amount of imidazoles contained in sample, rtagP3AB1B2 egg after molecular sieve purification White structure is monomer structure (Fig. 2).The eluting peak for collecting monomer structure, obtains the rtagP3AB1B2 albumen of molecular sieve purification (molecular sieve purification destination protein sample), using NanoDrop2000 ultramicrospectrophotometer (ND2000) to obtained albumen Purity carry out quantitative analysis.

The rtagP3AB1B2 albumen of molecular sieve purification is analyzed by mass spectrometry its amino acid sequence, the results showed that The amino acid sequence of rtagP3AB1B2 is as shown in SEQ ID No.2.

5, the expression and purifying of reference protein matter

The solubility expression of 5.1 rtag3B1B2, rtagP3A and rtag3A and purifying

Referring to the method for step 4, BL21 (DE3)/pET32a-3B1B2, BL21 (DE3)/pET32a-P3A, BL21 is utilized (DE3)/pET32a-3A carries out inducing expression, respectively obtains the following reference protein matter (soluble protein) of molecular sieve purification: Rtag3B1B2, rtagP3A and rtag3A.

The expression of 5.2 rtag3ABC and inclusion body are denaturalized renaturation

BL21 (DE3)/pET32a-3ABC that step 3 obtains is inoculated in the training of the LB liquid containing 50 μ g/ml ampicillins Support in base, 37 DEG C, using Thermo MaxQ6000 type entirely warm oscillator 200rpm shaken cultivation to 0D₆₀₀Value is (to contain 50 μ g/ml The LB liquid medium of ampicillin is blank control) when reaching 0.6, IPTG is added and carries out inducing expression.The inducing expression With the IPTG of 0.75mM in 16 DEG C of induction 13h.Fermentation liquid after taking IPTG inducing expression 13h collects bacterial sediment.PBS weight is added Outstanding precipitating, 8000rpm/min are centrifuged 5min, discard supernatant.PBS is added into the bacterial sediment washed, high pressure is crushed bacterium Body, cracking is no longer sticky to bacterium solution, and 16000rpm/min is centrifuged 30min in 4 DEG C of centrifuges, collects precipitating and (is named as BL21 (DE3)/pET32a-3ABC contains albumen precipitation), abandon supernatant.By BL21 (DE3)/pET32a-3ABC containing albumen precipitation with containing The buffer (20mM Tris, 150mM NaCl, 8M urea, solvent are water, the solution of pH8.0) of the urea of 8M dissolves.Lysate With loading after 0.22 μm of membrane filtration to solution 1, (solute and its concentration are as follows: 20mM Tris, 150mM NaCl, 8M in advance Urea, solvent are water, the solution of pH8.0) nickel column that has balanced.Nickel column is accessed on AKTA machine, respectively with 10 column volumes The solution 2 of solution 1 and 10 column volume (solute and its concentration are as follows: 20mM Tris, 150mM NaCl, 8M urea, 50mM Imidazoles, solvent are water, the solution of pH8.0) impurity protein in cleaning nickel column, and protein peak is monitored on AKTA machine.With molten (solute and its concentration are as follows: 20mM Tris, 150mM NaCl, 300mM imidazoles, 8M urea, and solvent is water, and pH8.0's is molten for liquid 3 Liquid) flushing nickel column hangs over the destination protein in nickel column, and the elution samples for destination protein peak occur are collected using AKTA, by the sample Product are known as the rtag3ABC inclusion body protein of ni-sepharose purification.

Embodiment 2 as the enzyme linked immunological kit of the diagnosis aftosa of antigen or detects hoof-and-mouth disease using rtag3AB1B2 The enzyme linked immunological kit of malicious antibody

Using rtag3AB1B2 as the enzyme linked immunological kit of the diagnosis aftosa of antigen or detection provided by the present embodiment The enzyme linked immunological kit of antibodies against foot-and-mouth disease virus includes envelope antigen, and envelope antigen is pure for the molecular sieve prepared in embodiment 1 The rtag3AB1B2 albumen (hereinafter referred to as rtag3AB1B2 albumen) of change.

The enzyme linked immunological kit further includes ELIAS secondary antibody, coating buffer, cleaning solution, secondary antibody diluent.

In the enzyme linked immunological kit, ELIAS secondary antibody is the rabbit-anti goat IgG of HRP label.

Be coated with buffer: the sodium carbonate-bicarbonate buffer (pH9.6) of 0.05mol/L, solvent is water, solute and its Concentration is as follows: Na₂CO₃1.59g/L and NaHCO₃ 2.93g/L。

Cleaning solution is 0.5% tween cleaning solution (PBST cleaning solution).0.5% tween cleaning solution is prepared as follows: It is 0.01M in concentration, it is 5mL/L that the content of polysorbas20 to polysorbas20, which is added, in the PBS buffer solution that pH value is 7.4, obtains 0.5% Tween cleaning solution.

Confining liquid is 1%BSA confining liquid.1%BSA confining liquid is prepared as follows: being 0.01M, pH value in concentration 1%BSA confining liquid is obtained until the volumn concentration of BSA is 1% for the BSA solution for adding 10% in 7.4 PBS buffer solution.

Secondary antibody diluent: being 0.01M in concentration, the content that BSA to BSA is added in the PBS buffer solution that pH value is 7.4 is 1% (volumn concentration), obtains secondary antibody diluent.

Wherein, concentration 0.01M, the preparation for the PBS buffer solution that pH value is 7.4: 8.5g NaCl, 0.2g KCl, 2.9g Na₂HPO₄·12H₂O、0.59g NaH₂PO₄·2H₂O, 1L deionized water.

1, it is established by optimization experiment and detects aftosa by envelope antigen indirect ELISA method of rtag3AB1B2 albumen The optimization experimental method (hereinafter referred to as rtag3AB1B2 optimizes indirect ELISA method) of antiviral antibody is as follows:

1.1 coatings: the rtag3AB1B2 albumen of molecular sieve purification in embodiment 1 is diluted (hereinafter referred to as with coating buffer Rtag3AB1B2 albumen) to rtag3AB1B2 albumen concentration be 1.0 μ g/ml, obtain coating original solution, with the coating original solution It is coated with experimental port, 100 holes μ L/ add to ELISA Plate, 4 DEG C of incubation 16h.

1.2 washings: incline hole endoperidium original solution, is washed 5 times with PBST cleaning solution, each 3min；It pats dry.

1.3 closings: 1%BSA confining liquid, 250 holes μ L/, 37 DEG C of incubation 2h are added.

1.4 sample-addings:

1.4.1 sample well

Sheep FMDV non-structural protein Positive Sera is diluted 50 times with coating buffer, obtains test serum.By 100 μ L test serum is added on ELISA Plate, 37 DEG C of reaction 1h, then liquid in hole of inclining is washed 5 times with cleaning solution.

1.4.2 blank control wells

Difference with 1.4.1, which is only that, replaces with test serum isometric high purity water, and other steps are constant.

1.5 plus ELIAS secondary antibody: addition carries out the diluted HRP label rabbit-anti goat IgG of 1:50000 with secondary antibody diluent, and 100 The hole μ L/, 37 DEG C of 30min.

1.6 colour developings: being added TMB, and 10min is reacted in 100 holes μ L/.

1.7 terminate: 0.2mol/L H is added₂SO₄Solution terminates reaction, 100 holes μ L/.

1.8 measurement: reading each hole OD with microplate reader_450nmNumerical value.

2, the determination of ELISA yin and yang attribute critical value

400 parts of sheep FMDV non-structural protein negative antibody serum are optimized into indirect ELISA using the rtag3AB1B2 of step 1 Sheep FMDV non-structural protein Positive Sera in 1.4.1 (is replaced with 400 parts of sheep FMDV non-structural proteins by method respectively Negative antibody serum, other operations are identical) indirect ELISA detection is carried out, calculate 400 parts of sheep FMDV non-structural protein antibody yin The average value (X) and standard deviation (SD) of property serum.It is judged to the positive；It is judged to feminine gender.

The result shows that 400 parts of sheep FMDV non-structural protein negative antibody serum average valuesIt is for 0.178, SD 0.057, therefore yin and yang attribute critical valueIt is 0.349.

3, specific test

Sheep perituberculosis positive serum, sheep cloth using the rtag3AB1B2 optimization indirect ELISA method of step 1 to each 10 parts Sick positive serum, peste des petits ruminants positive serum and clostridium perfringens disease positive serum are detected, observation and Other diseases There is no cross reaction.Wherein, the sheep FMDV non-structural protein Positive Sera in 1.4.1 is replaced with into above-mentioned serum respectively, Other operations are identical.The result shows that the rtag3AB1B2 optimizes indirect ELISA method to sheep perituberculosis positive serum, sheep cloth disease sun Property serum, peste des petits ruminants positive serum and clostridium perfringens disease positive serum are detected, OD₄₅₀Value is respectively as follows: 0.188,0.231,0.196,0.284, respectively less than critical value 0.349 show rtag3AB1B2 and sheep perituberculosis positive serum, sheep Cloth disease positive serum, peste des petits ruminants positive serum, the equal no cross reaction of clostridium perfringens disease positive serum, step 1 Rtag3AB1B2, which optimizes indirect ELISA method, has good specificity.

4, sensitivity tests

Rtag3AB1B2 in the rtag3AB1B2 optimization indirect ELISA method of step 1 is replaced with into rtag3ABC, it is other Operate it is constant, establish rtag3ABC optimization indirect ELISA method.

Rtag3AB1B2 in the rtag3AB1B2 optimization indirect ELISA method of step 1 is replaced with into rtag3B1B2, Its operation is constant, establishes rtag3B1B2 optimization indirect ELISA method.

Rtag3AB1B2 in the rtag3AB1B2 optimization indirect ELISA method of step 1 is replaced with into rtagP3A, it is other Operate it is constant, establish rtagP3A optimization indirect ELISA method.

Rtag3AB1B2 in the rtag3AB1B2 optimization indirect ELISA method of step 1 is replaced with into rtag3A, Qi Tacao Make it is constant, establish rtag3A optimization indirect ELISA method.

Sheep FMDV non-structural protein Positive Sera is subjected to doubling dilution, the rtag3AB1B2 of step 1 is respectively adopted Optimization indirect ELISA method, rtag3ABC optimize indirect ELISA method, rtag3B1B2 optimizes indirect ELISA method, RtagP3A optimization indirect ELISA method and rtag3A optimization indirect ELISA method are detected, when obtaining positive critical value Greatest dilution.

The result shows that sheep FMDV non-structural protein Positive Sera is carried out doubling dilution since 1:4, using step 1 Rtag3AB1B2 optimization indirect ELISA method carry out detection sheep FMDV non-structural protein Positive Sera dilution be 1: It is still positive when 1024；The sheep FMDV non-structural protein detected using Switzerland PRIONICS aftosa NS antibody assay kit The greatest dilution of white Positive Sera is 1: 512；Using rtag3ABC optimization indirect ELISA method, rtag3B1B2 optimization Indirect ELISA method, rtagP3A optimization indirect ELISA method and rtag3A optimization indirect ELISA method examined It surveys, the greatest dilution of sheep FMDV non-structural protein Positive Sera is respectively 1: 1024,1: 512,1: 512 and 1: 256.Table The sensibility of the bright indirect ELISA detection foot and mouth disease virus antibody method established using rtag3AB1B2 as envelope antigen with Rtag3ABC is suitable as the sensibility for the indirect ELISA detection foot and mouth disease virus antibody method that envelope antigen is established, but bright It is aobvious to be higher than the indirect ELISA detection foot and mouth disease virus established using rtag3B1B2, rtagP3A and rtag3A as envelope antigen The sensibility of antibody method is also apparently higher than the sensibility of Switzerland PRIONICS aftosa NS antibody assay kit.

5, repetitive test

Using the rtag3AB1B2 optimization indirect ELISA method of step 1 to 6 parts of sheep FMDV non-structural protein antibody positive blood It is detected clearly, is measured in parallel 5 times respectively on same batch plate and different batches plate, calculate batch interior, interassay coefficient of variation (CV). The results show that batch interior coefficient of variation that repeats repeats the coefficient of variation less than 9% (table 1) between 2%~8%, between batch.As a result table Bright, the rtag3AB1B2 optimization indirect ELISA method of step 1 has good repeatability.

The rtag3AB1B2 of 1. step 1 of table optimizes indirect ELISA method repetitive test

6, accordance is tested

It is (agriculture from China Animal Disease Control And Prevention Center using Switzerland PRIONICS aftosa NS antibody assay kit Rural area portion Disease Diagnosis of Veterinary center) save sheep blood serum picked out 90 parts of sheep FMDV non-structural protein Positive Seras and 90 parts Sheep FMDV non-structural protein negative antibody serum.The rtag3AB1B2 optimization that step 1 is respectively adopted to this 180 parts of sheep blood serums is indirect ELISA method, the rtag3ABC optimization indirect ELISA method of step 4, rtag3B1B2 optimization indirect ELISA method, rtagP3A Optimization indirect ELISA method and rtag3A optimization indirect ELISA method are detected, and are calculated and Switzerland PRIONICS aftosa NS The coincidence rate of antibody assay kit.

The result shows that 180 parts of sheep blood serums, the rtag3AB1B2 optimization indirect ELISA method of step 1 and Switzerland Total coincidence rate of PRIONICS aftosa NS antibody assay kit is 94.44% (positive coincidence rate 94.44%, feminine gender symbol Conjunction rate be 94.44%), the rtag3ABC of step 4 optimization indirect ELISA method, rtag3B1B2 optimization indirect ELISA method, RtagP3A optimization indirect ELISA method and rtag3A optimization indirect ELISA method carry out detection and aftosa Switzerland PRIONICS Total coincidence rate of aftosa NS antibody assay kit method is respectively 87.78% (positive coincidence rate 97.78%, feminine gender symbol Conjunction rate be 77.78%), 88.89% (positive coincidence rate 80%, negative match-rate 97.78%), 79.44% (positive meets Rate is 61.11%, negative match-rate 97.78%), 82.22% (positive coincidence rate 70%, negative match-rate are 94.44%) (table 2- table 6).

Table 2.rtag3AB1B2 optimizes indirect ELISA method blood serum sample testing result

By 2 result of table, it can be concluded that, the rtag3AB1B2 of step 1 optimizes indirect ELISA method, detected 85 parts of sun Property, 85 parts of feminine genders.The sample that testing result is not consistent is 10 parts.The positive coincidence rate of two methods is 94.44%, and feminine gender meets Rate is 94.44%, and total coincidence rate is 94.44% (being shown in Table 3).

Table 3.rtag3ABC optimizes indirect ELISA method blood serum sample testing result

By 3 result of table, it can be concluded that, the rtag3ABC of step 4 optimizes indirect ELISA method, detected 108 parts of sun Property, 72 parts of feminine genders.The sample that testing result is not consistent is 22 parts.The positive coincidence rate of two methods is 97.78%, and feminine gender meets Rate is 77.78%, and total coincidence rate is 87.78% (being shown in Table 3).

Table 4.rtag3B1B2 optimizes indirect ELISA method blood serum sample testing result

By 4 result of table, it can be concluded that, the rtag3B1B2 of step 4 optimizes indirect ELISA method, detected 74 parts of sun Property, 106 parts of feminine genders.The sample that testing result is not consistent is 20 parts.The positive coincidence rate of two methods is 80%, negative match-rate It is 97.78%, total coincidence rate is 88.89% (being shown in Table 4).

Table 5.rtagP3A optimizes indirect ELISA method blood serum sample testing result

By 5 result of table, it can be concluded that, the rtagP3A of step 4 optimizes indirect ELISA method, detected 57 parts of positives, 143 parts of feminine genders.The sample that testing result is not consistent is 37 parts.The positive coincidence rate of two methods is 61.11%, negative match-rate It is 97.78%, total coincidence rate is 79.44% (being shown in Table 5).

Table 6.rtag3A optimizes indirect ELISA method blood serum sample testing result

By 6 result of table, it can be concluded that, the rtag3A of step 4 optimizes indirect ELISA method, detected 68 parts of positives, 112 parts of feminine genders.The sample that testing result is not consistent is 32 parts.The positive coincidence rate of two methods is 70%, and negative match-rate is 94.44%, total coincidence rate is 82.22% (being shown in Table 6).

Illustrate the method for the indirect ELISA established using rtag3AB1B2 as envelope antigen detection antibodies against foot-and-mouth disease virus with Total coincidence rate of aftosa Switzerland PRIONICS aftosa NS antibody assay kit be significantly higher than respectively with rtag3ABC, The method for the indirect ELISA detection antibodies against foot-and-mouth disease virus that rtag3B1B2, rtagP3A and rtag3A are established as envelope antigen.

The present invention is had been described in detail above.To those skilled in the art, do not depart from spirit of the invention and Range, and without carrying out under unnecessary experimental conditions, can synchronization parameters, concentration and under the conditions of, it is real in a wider range Apply the present invention.Although The present invention gives particular embodiments, it is understood that, the present invention can be improved further. In short, pressing the principle of the present invention, the application is intended to include any change, purposes or improvement of the present invention, including departing from this Shen Please in the open scope, and the change carried out with routine techniques known in the art.By the range of following attached claims, It can carry out the application of some essential characteristics.

<110>China Animal Disease Control And Prevention Center (technique center is butchered by the Ministry of Agriculture)

<120>enzyme linked immunological kit and its application of mouth disease virus infection antibody are detected

<130> GNCFH182286

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 873

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

atgagcgata aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg 60

gacggggcga tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc 120

ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac 180

atcgatcaaa accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg 240

ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg 300

aaagagttcc tcgacgctaa cctggccggt tctggttctg gccatatgca ccatcatcat 360

catcattctt ctggtctggt gccacgcggt tctggtatga aagaaaccgc tgctgctaaa 420

ttcgaacgcc agcacatgga cagcccagat ctgggtaccg acgacgacga caaggccatg 480

gctgatatcg gatccatttc aatcccttcc cagaaggctg tgttgtactt tctcattgag 540

aagggccagc acgaagcagc aattgagttc ttcgagggta tggtccacga ctccatcaag 600

gaggagctcc ggcctggtgg cggtggaatc ggaggtggtg gaagcggagg aggtggaagc 660

ggaccctacg ctgggccact cgagcgtcag aaacctctta aagtgaaagc caggctgcca 720

caacaggagg gtggcggtgg aatcggaggt ggtggaagcg gaggaggtgg aagcggaccc 780

tacgccggcc caatggagag acagaaacca ctaaaggtga aagcaaaagt ccccgtcgtg 840

aaggaactcg agcaccacca ccaccaccac tga 873

<210> 2

<211> 290

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<213>artificial sequence (Artificial Sequence)

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Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp

1 5 10 15

Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp

20 25 30

Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp

35 40 45

Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn

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Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu

65 70 75 80

Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser

85 90 95

Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly

100 105 110

Ser Gly His Met His His His His His His Ser Ser Gly Leu Val Pro

115 120 125

Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln

130 135 140

His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met

145 150 155 160

Ala Asp Ile Gly Ser Ile Ser Ile Pro Ser Gln Lys Ala Val Leu Tyr

165 170 175

Phe Leu Ile Glu Lys Gly Gln His Glu Ala Ala Ile Glu Phe Phe Glu

180 185 190

Gly Met Val His Asp Ser Ile Lys Glu Glu Leu Arg Pro Gly Gly Gly

195 200 205

Gly Ile Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Pro Tyr Ala

210 215 220

Gly Pro Leu Glu Arg Gln Lys Pro Leu Lys Val Lys Ala Arg Leu Pro

225 230 235 240

Gln Gln Glu Gly Gly Gly Gly Ile Gly Gly Gly Gly Ser Gly Gly Gly

245 250 255

Gly Ser Gly Pro Tyr Ala Gly Pro Met Glu Arg Gln Lys Pro Leu Lys

260 265 270

Val Lys Ala Lys Val Pro Val Val Lys Glu Leu Glu His His His His

275 280 285

His His

290

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atgagcgata aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg 60

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ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac 180

atcgatcaaa accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg 240

ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg 300

aaagagttcc tcgacgctaa cctggccggt tctggttctg gccatatgca ccatcatcat 360

catcattctt ctggtctggt gccacgcggt tctggtatga aagaaaccgc tgctgctaaa 420

ttcgaacgcc agcacatgga cagcccagat ctgggtaccg acgacgacga caaggccatg 480

gctgatatcg gatccatttc aatcccttcc cagaaggctg tgttgtactt tctcattgag 540

aagggccagc acgaagcagc aattgagttc ttcgagggta tggtccacga ctccatcaag 600

gaggagctcc ggcctctcat ccagcagacc tcgtttgtac aacgcgcctt caagcgcctg 660

aaggagaact ttgagattgt agctctgtgt ttaaccctct tggcaaacat agtgattatg 720

ctccgccaag cgcgcaagag acgccagtcg gtggatgacc cactggacgg cgacataact 780

cttggcgacg cggaaaagga ccctctggag gcgcgtggcg ctagcgctgt cggtttcaga 840

gagagaccac ccaccgagca agagacgtgc gaagacgcga acgctgagcc tgtcgtgctc 900

gggagggaac aaccgcgagc tgaaggaccc tacgctgggc cactcgagcg tcagaaacct 960

cttaaagtga aagccaggct gccacaacag gagggaccct acgccggccc aatggagaga 1020

cagaaaccac taaaggtgaa agcaaaagtc cccgtcgtga aggaaggacc ttacgaggga 1080

ccggtgaaga aacctgtcgc tttgaaagtg aaagcaaaga acttgatagt cactgagagt 1140

ggtgcgccac cgaccgactt gcaaaagatg gtcatgggca acactaagcc ggtcgagctt 1200

atcctcgacg gcaagacggt ggccatctgt tgtgccaccg gagtgtttgg cactgcctac 1260

ctcgtgcctc gtcacctctt cgcggagaag tatgacaaga tcatgttgga cggtagagcc 1320

ttaacagaca gcgactacag agtgttcgag tttgagatta aagtaaaagg acaggacatg 1380

ctctcagacg ccgctctcat ggtgttgcac cgtgggaacc gcgtgcgcga tatcacgaaa 1440

cactttcgtg atgtggcgag aatgaagaag ggaacccccg tcgtcggtgt gatcaacaat 1500

gctgacgtag ggagactcat attctctggt gaagccctta cttacaagga catcgtcgtg 1560

tgtatggacg gagacaccat gcctgggctc ttcgcctaca gagcgtccac caaggcaggc 1620

tactgtggag gagccgtcct cgcaaaggac ggtgccgaga cgttcatcgt tggcactcac 1680

tccgcaggtg gtaacggtat aggatactgc tcctgcgtct cccgatcgat gctcatgaag 1740

atgaaggcac acatcgaccc cgaaccacac cacgagctcg agcaccacca ccaccaccac 1800

tga 1803

<210> 4

<211> 600

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 4

Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp

1 5 10 15

Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp

20 25 30

Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp

35 40 45

Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn

50 55 60

Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu

65 70 75 80

Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser

85 90 95

Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly

100 105 110

Ser Gly His Met His His His His His His Ser Ser Gly Leu Val Pro

115 120 125

Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln

130 135 140

His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met

145 150 155 160

Ala Asp Ile Gly Ser Ile Ser Ile Pro Ser Gln Lys Ala Val Leu Tyr

165 170 175

Phe Leu Ile Glu Lys Gly Gln His Glu Ala Ala Ile Glu Phe Phe Glu

180 185 190

Gly Met Val His Asp Ser Ile Lys Glu Glu Leu Arg Pro Leu Ile Gln

195 200 205

Gln Thr Ser Phe Val Gln Arg Ala Phe Lys Arg Leu Lys Glu Asn Phe

210 215 220

Glu Ile Val Ala Leu Cys Leu Thr Leu Leu Ala Asn Ile Val Ile Met

225 230 235 240

Leu Arg Gln Ala Arg Lys Arg Arg Gln Ser Val Asp Asp Pro Leu Asp

245 250 255

Gly Asp Ile Thr Leu Gly Asp Ala Glu Lys Asp Pro Leu Glu Ala Arg

260 265 270

Gly Ala Ser Ala Val Gly Phe Arg Glu Arg Pro Pro Thr Glu Gln Glu

275 280 285

Thr Cys Glu Asp Ala Asn Ala Glu Pro Val Val Leu Gly Arg Glu Gln

290 295 300

Pro Arg Ala Glu Gly Pro Tyr Ala Gly Pro Leu Glu Arg Gln Lys Pro

305 310 315 320

Leu Lys Val Lys Ala Arg Leu Pro Gln Gln Glu Gly Pro Tyr Ala Gly

325 330 335

Pro Met Glu Arg Gln Lys Pro Leu Lys Val Lys Ala Lys Val Pro Val

340 345 350

Val Lys Glu Gly Pro Tyr Glu Gly Pro Val Lys Lys Pro Val Ala Leu

355 360 365

Lys Val Lys Ala Lys Asn Leu Ile Val Thr Glu Ser Gly Ala Pro Pro

370 375 380

Thr Asp Leu Gln Lys Met Val Met Gly Asn Thr Lys Pro Val Glu Leu

385 390 395 400

Ile Leu Asp Gly Lys Thr Val Ala Ile Cys Cys Ala Thr Gly Val Phe

405 410 415

Gly Thr Ala Tyr Leu Val Pro Arg His Leu Phe Ala Glu Lys Tyr Asp

420 425 430

Lys Ile Met Leu Asp Gly Arg Ala Leu Thr Asp Ser Asp Tyr Arg Val

435 440 445

Phe Glu Phe Glu Ile Lys Val Lys Gly Gln Asp Met Leu Ser Asp Ala

450 455 460

Ala Leu Met Val Leu His Arg Gly Asn Arg Val Arg Asp Ile Thr Lys

465 470 475 480

His Phe Arg Asp Val Ala Arg Met Lys Lys Gly Thr Pro Val Val Gly

485 490 495

Val Ile Asn Asn Ala Asp Val Gly Arg Leu Ile Phe Ser Gly Glu Ala

500 505 510

Leu Thr Tyr Lys Asp Ile Val Val Cys Met Asp Gly Asp Thr Met Pro

515 520 525

Gly Leu Phe Ala Tyr Arg Ala Ser Thr Lys Ala Gly Tyr Cys Gly Gly

530 535 540

Ala Val Leu Ala Lys Asp Gly Ala Glu Thr Phe Ile Val Gly Thr His

545 550 555 560

Ser Ala Gly Gly Asn Gly Ile Gly Tyr Cys Ser Cys Val Ser Arg Ser

565 570 575

Met Leu Met Lys Met Lys Ala His Ile Asp Pro Glu Pro His His Glu

580 585 590

Leu Glu His His His His His His

595 600

<210> 5

<211> 708

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

atgagcgata aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg 60

gacggggcga tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc 120

ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac 180

atcgatcaaa accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg 240

ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg 300

aaagagttcc tcgacgctaa cctggccggt tctggttctg gccatatgca ccatcatcat 360

catcattctt ctggtctggt gccacgcggt tctggtatga aagaaaccgc tgctgctaaa 420

ttcgaacgcc agcacatgga cagcccagat ctgggtaccg acgacgacga caaggccatg 480

gctgatatcg gatccggacc ctacgctggg ccactcgagc gtcagaaacc tcttaaagtg 540

aaagccaggc tgccacaaca ggagggtggc ggtggaatcg gaggtggtgg aagcggagga 600

ggtggaagcg gaccctacgc cggcccaatg gagagacaga aaccactaaa ggtgaaagca 660

aaagtccccg tcgtgaagga actcgagcac caccaccacc accactga 708

<210> 6

<211> 235

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 6

Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp

1 5 10 15

Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp

20 25 30

Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp

35 40 45

Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn

50 55 60

Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu

65 70 75 80

Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser

85 90 95

Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly

100 105 110

Ser Gly His Met His His His His His His Ser Ser Gly Leu Val Pro

115 120 125

Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln

130 135 140

His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met

145 150 155 160

Ala Asp Ile Gly Ser Gly Pro Tyr Ala Gly Pro Leu Glu Arg Gln Lys

165 170 175

Pro Leu Lys Val Lys Ala Arg Leu Pro Gln Gln Glu Gly Gly Gly Gly

180 185 190

Ile Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Pro Tyr Ala Gly

195 200 205

Pro Met Glu Arg Gln Lys Pro Leu Lys Val Lys Ala Lys Val Pro Val

210 215 220

Val Lys Glu Leu Glu His His His His His His

225 230 235

<210> 7

<211> 642

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

atgagcgata aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg 60

gacggggcga tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc 120

ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac 180

atcgatcaaa accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg 240

ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg 300

aaagagttcc tcgacgctaa cctggccggt tctggttctg gccatatgca ccatcatcat 360

catcattctt ctggtctggt gccacgcggt tctggtatga aagaaaccgc tgctgctaaa 420

ttcgaacgcc agcacatgga cagcccagat ctgggtaccg acgacgacga caaggccatg 480

gctgatatcg gatccatttc aatcccttcc cagaaggctg tgttgtactt tctcattgag 540

aagggccagc acgaagcagc aattgagttc ttcgagggta tggtccacga ctccatcaag 600

gaggagctcc ggcctctcga gcaccaccac caccaccact ga 642

<210> 8

<211> 213

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 8

Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp

1 5 10 15

Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp

20 25 30

Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp

35 40 45

Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn

50 55 60

Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu

65 70 75 80

Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser

85 90 95

Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly

100 105 110

Ser Gly His Met His His His His His His Ser Ser Gly Leu Val Pro

115 120 125

Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln

130 135 140

His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met

145 150 155 160

Ala Asp Ile Gly Ser Ile Ser Ile Pro Ser Gln Lys Ala Val Leu Tyr

165 170 175

Phe Leu Ile Glu Lys Gly Gln His Glu Ala Ala Ile Glu Phe Phe Glu

180 185 190

Gly Met Val His Asp Ser Ile Lys Glu Glu Leu Arg Pro Leu Glu His

195 200 205

His His His His His

210

<210> 9

<211> 951

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

atgagcgata aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg 60

gacggggcga tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc 120

ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac 180

atcgatcaaa accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg 240

ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg 300

aaagagttcc tcgacgctaa cctggccggt tctggttctg gccatatgca ccatcatcat 360

catcattctt ctggtctggt gccacgcggt tctggtatga aagaaaccgc tgctgctaaa 420

ttcgaacgcc agcacatgga cagcccagat ctgggtaccg acgacgacga caaggccatg 480

gctgatatcg gatccatttc aatcccttcc cagaaggctg tgttgtactt tctcattgag 540

aagggccagc acgaagcagc aattgagttc ttcgagggta tggtccacga ctccatcaag 600

gaggagctcc ggcctctcat ccagcagacc tcgtttgtac aacgcgcctt caagcgcctg 660

aaggagaact ttgagattgt agctctgtgt ttaaccctct tggcaaacat agtgattatg 720

ctccgccaag cgcgcaagag acgccagtcg gtggatgacc cactggacgg cgacataact 780

cttggcgacg cggaaaagga ccctctggag gcgcgtggcg ctagcgctgt cggtttcaga 840

gagagaccac ccaccgagca agagacgtgc gaagacgcga acgctgagcc tgtcgtgctc 900

gggagggaac aaccgcgagc tgaactcgag caccaccacc accaccactg a 951

<210> 10

<211> 316

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 10

Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp

1 5 10 15

Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp

20 25 30

Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp

35 40 45

Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn

50 55 60

Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu

65 70 75 80

Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser

85 90 95

Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly

100 105 110

Ser Gly His Met His His His His His His Ser Ser Gly Leu Val Pro

115 120 125

Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln

130 135 140

His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met

145 150 155 160

Ala Asp Ile Gly Ser Ile Ser Ile Pro Ser Gln Lys Ala Val Leu Tyr

165 170 175

Phe Leu Ile Glu Lys Gly Gln His Glu Ala Ala Ile Glu Phe Phe Glu

180 185 190

Gly Met Val His Asp Ser Ile Lys Glu Glu Leu Arg Pro Leu Ile Gln

195 200 205

Gln Thr Ser Phe Val Gln Arg Ala Phe Lys Arg Leu Lys Glu Asn Phe

210 215 220

Glu Ile Val Ala Leu Cys Leu Thr Leu Leu Ala Asn Ile Val Ile Met

225 230 235 240

Leu Arg Gln Ala Arg Lys Arg Arg Gln Ser Val Asp Asp Pro Leu Asp

245 250 255

Gly Asp Ile Thr Leu Gly Asp Ala Glu Lys Asp Pro Leu Glu Ala Arg

260 265 270

Gly Ala Ser Ala Val Gly Phe Arg Glu Arg Pro Pro Thr Glu Gln Glu

275 280 285

Thr Cys Glu Asp Ala Asn Ala Glu Pro Val Val Leu Gly Arg Glu Gln

290 295 300

Pro Arg Ala Glu Leu Glu His His His His His His

305 310 315

Claims (10)

1. application of the protein in reagent preparation box, it is characterised in that: the kit is aftosa diagnostic kit or inspection Survey the kit of antibodies against foot-and-mouth disease virus, the protein is R1), R2) or protein R3):

R1) amino acid sequence is the protein of SEQ ID No.2,

R2) amino acid sequence is the 166-282 protein of SEQ ID No.2,

R3) in R1) or R2) shown in protein c-terminus or/and having of obtaining of aminoterminal fusion tag albumen and R1) or R2) identical active soluble fusion protein.

2. application according to claim 1, it is characterised in that: the protein is according to the method system included the following steps It is standby: to make the encoding gene of the protein be expressed to obtain the protein in biology, the biology is microorganism, plant Or non-human animal.

3. application according to claim 2, it is characterised in that: make the encoding gene of protein carry out table in biology Up to including that the encoding gene of the protein is imported recipient microorganism, the recombinant microorganism for expressing the protein is obtained, is trained The recombinant microorganism is supported, expression obtains the protein.

Any one of 4. application according to claim 3, it is characterised in that: the recipient microorganism is C1)-C4):

C1) prokaryotic micro-organisms,

C2) gramnegative bacterium,

C3) Escherichia bacteria,

C4) e. coli bl21 (DE3).

5. according to application described in any claim in claim 2-4, it is characterised in that: the encoding gene of the protein Be following 1) -4) in any DNA molecular:

1) coded sequence is the DNA molecular of SEQ ID No.1,

2) nucleotide sequence is the DNA molecular of SEQ ID No.1,

3) coded sequence is the DNA molecular of the 496-846 nucleotide of SEQ ID No.1,

4) nucleotide sequence is the DNA molecular of the 496-846 nucleotide of SEQ ID No.1.

6. according to application described in any claim in claim 3-5, it is characterised in that: the recombinant microorganism is will PET32a-3AB1B2 imports the protein that the express amino acid sequence that e. coli bl21 (DE3) is obtained is SEQ ID No.2 Recombinant microorganism, the recombinant microorganism are named as BL21 (DE3)/pET32a-3AB1B2, and the pET32a-3AB1B2 is to use Nucleotide sequence be SEQ ID No.1 490-852 DNA replacement pET32a (+) BamHI and XhoI recognition site between Segment, keep pET32a (+) other sequences it is constant, obtained recombinant expression carrier.

7. according to application described in any claim in claim 2-6, it is characterised in that: described to be expressed as inducing expression.

8. application according to claim 7, it is characterised in that: the inducing expression is to be lured with the IPTG of 0.75mM at 16 DEG C It leads 13-16 hours or 13-24 hours or 13 hours or 16 hours.

9. kit, it is characterised in that: the kit is the enzyme linked immunological kit or detection hoof-and-mouth disease for diagnosing aftosa The enzyme linked immunological kit of malicious antibody, the kit include envelope antigen, and the envelope antigen is any in claim 1-6 The protein.

10. application of the kit as claimed in claim 9 in preparation aftosa monitoring reagent box.