CN109293747A - Foot and mouth disease virus non-structural protein 3B epitope peptide and its application - Google Patents

Foot and mouth disease virus non-structural protein 3B epitope peptide and its application Download PDF

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CN109293747A
CN109293747A CN201811126936.3A CN201811126936A CN109293747A CN 109293747 A CN109293747 A CN 109293747A CN 201811126936 A CN201811126936 A CN 201811126936A CN 109293747 A CN109293747 A CN 109293747A
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foot
mouth disease
epitope
disease virus
monoclonal antibody
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CN109293747B (en
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周国辉
于力
杨德成
王海伟
孙超
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Harbin Veterinary Research Institute of CAAS
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    • G01N2333/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
    • G01N2333/09—Foot-and-mouth disease virus

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Abstract

The invention discloses foot and mouth disease virus non-structural protein 3B epitope peptide and its applications.The present invention is screened by cell-fusion techniques obtains a strain of hybridoma system 2H1, and the monoclonal antibody broad spectrum activity of secretion is good, conservative is high, affinity is strong, and has strong competitiveness with detection serum.The epitope that precise Identification of the present invention monoclonal antibody 2H1 is identified, sequence are shown in SEQ ID No.2；The present invention obtains series mutants to the epitope polypeptide mutant.The FMDV Positive Sera presentation sound response ability of epitope peptide and its mutant provided by the invention and infection of foot-and-mouth disease pig, ox, sheep, the antibody ELISA kit for using it to establish as envelope antigen can accurately distinguish FMDV infection serum and antigen immune serum, and the serum of any animal species can be detected with a kind of detection kit, species barrier is spanned, has many advantages, such as that high reliablity, application are wide, broad spectrum activity is good, detection is more efficient and convenient.

Description

Foot and mouth disease virus non-structural protein 3B epitope peptide and its application
Technical field
The present invention relates to foot and mouth disease virus antigen epitopes, and the invention particularly relates to foot and mouth disease virus non-structural protein 3B antigens Epitope and its application, the invention further relates to them to distinguish aftosa natural infected animal and answering in immunity inoculation animal With belonging to foot and mouth disease virus non-structural protein epitope and its application field.
Background technique
Aftosa (foot-and-mouth disease, FMD) is the main infringement caused by foot and mouth disease virus (FMDV) The domestic animals such as ox, pig, sheep and a variety of wild highly contagious diseases artiodactylous, people can also infect.The disease causes cub Dead, output of milk decline, carnivorous reduction, meat declines, the production performance of animal reduces, and has extremely strong infectiousness, can A wide range of prevalence is formed, huge economic loss is caused.Foot and mouth disease virus belong to microRNA Viraceae Hostis at Member is single-stranded positive RNA, geneome RNA overall length about 8.5kb.According to immunogenicity, foot and mouth disease virus have O, A, C, SAT1, The hypotype of SAT2, SAT3 and Asial totally 7 serotypes and 65 or more, without Cross immunogenicity between each serotype.Currently, Chinese Major Epidemic is O-shaped and A type FMD.
Prevention and control aftosa key be efficient vaccine application and accurate diagnostic method.Traditional inactivated vaccine Immunity inoculation be prevention and control aftosa most effective means, since existing detection method cannot accurately distinguish aftosa Natural infected animal and immunity inoculation animal, cause aftosa that cannot be controlled effectively.
Different degrees of the lacking there are following several respects of monoclonal antibody of the prior art for detecting foot and mouth disease virus It falls into:
(1) the targeted epitope of monoclonal antibody is not identified, not can determine that the degree of monoclonal antibody broad spectrum activity: existing Situations such as targeted epitope sequences of monoclonal antibody and its key amino acid or important amino acid, is unclear, if key amino acid and Important amino acid has variation between strain, and the degree of broad spectrum activity will change, and cannot ensure its wide spectrum to aftosa strain Property, lack real application value accordingly；
(2) monoclonal antibody broad spectrum activity is poor: the epitope conservative that monoclonal antibody is directed to is poor, leads to its one with aftosa A little strains do not react；
(3) competitiveness of monoclonal antibody is poor: the competitiveness of some monoclonal antibodies and infection of foot-and-mouth disease serum is bad, causes not It can establish and be effectively blocked ELISA detection kit.It is only suitable for using without competitive or competitive poor monoclonal antibody FMDV 3ABC antigen is captured in coating elisa plate, then can only add one of pig, ox or sheep blood serum to be checked, finally use HRP marks the IgG antibody of anti-pig, ox perhaps sheep to be established with this without competitive or competitive poor monoclonal antibody Aftosa differential diagnostic method not only wizard is cumbersome, more seriously, cannot be at one not across animal species barrier The serum of any animal species is detected in reaction system.
Due to it is existing for detect foot and mouth disease virus monoclonal antibody it is different degrees of there are drawbacks described above, lead to this A little monoclonal antibodies, which cannot be established, is effectively blocked ELISA art formula.The ELISA detection kit established with these existing monoclonal antibodies, It is more seriously, every to detect a kind of move in addition to there are detection sensitivities low, broad spectrum activity (being directed to the conservative of epitope) difference is outer Object just needs to establish a kind of detection kit, lacks actual application value.Therefore, develop a kind of broad spectrum activity is good, conservative is high, Affinity is strong, and has strong emulative foot and mouth disease virus non-structural protein monoclonal antibody with detection serum, and identify to it Epitope carry out accurate identification, this accurately distinguishes the blocking of aftosa natural infected animal Yu immunity inoculation animal for foundation ELISA detection kit etc. is of great significance.
Summary of the invention
An object of the present invention is to provide plant height effect, stably excreting foot and mouth disease virus non-structural protein monoclonal antibody Hybridoma cell line；
The second object of the present invention is to provide the foot-and-mouth disease virus resistant non-structural protein secreted by the hybridoma cell line Monoclonal antibody, broad spectrum activity is good, conservative is high, affinity is strong, and has strong competitiveness with detection serum；
The third object of the present invention is the antigen that accurate identification foot and mouth disease virus non-structural protein monoclonal antibody is identified Epitope；
The fourth object of the present invention is to construct a kind of foot-and-mouth disease antibody as envelope antigen using identified epitope ELISA kit is detected, aftosa natural infected animal is accurately distinguished using the ELISA detection kit and immunity inoculation is dynamic Object.
Above-mentioned purpose of the invention is achieved through the following technical solutions:
BALB/c mouse is immunized with the aftosa non-structural protein that eukaryotic system is expressed in the present invention, and extracting spleen cell is thin with SP2/0 Born of the same parents' fusion obtains the FMDV NSP antibody in the hybridoma indirect ELISA of acquisition and IFA method detection culture supernatant Antibody positive hybridoma, antibody positive hybridoma are subcloned through 3 limiting dilution assays, are finally screened and are obtained 5 plants of energy The hybridoma of stably excreting monoclonal antibody, is respectively designated as 4B7,7G9,3A3,3A10 and 2H1.The present invention further will The hybridoma culture supernatant of hybridoma 4B7,7G9,3A3,3A10,2H1 are respectively with infection A, O and Asia1 type FMDV's BHK-21 cell carries out indirect immunofluorescene assay；Testing result shows, only list secreted by hybridoma 2H1 and 3A10 It is anti-to be positive with A, O, Asia1 type strain, monoclonal antibody secreted by hybridoma 4B7,7G9 and 3A3 only with portion Divide the infection cell of FMDV strain that specific reaction occurs；Immunofluorescence test the result shows that, hybridoma 2H1 and 3A10 The monoclonal antibody of secretion has broad spectrum activity, and monoclonal antibody secreted by hybridoma 4B7,7G9 and 3A3 does not have broad spectrum activity.
The present invention further determines monoclonal antibody secreted by positive hybridoma cell 4B7,7G9,3A3,3A10 and 2H1 Affinity；Affinity measurement result shows that the relative affinity constant of monoclonal antibody 4B7,7G9,3A3,3A10 and 2H1 are respectively 2.0mol/L, 2.5mol/L, 1.0mol/L, 3.5mol/L and 3.5mol/L, according to measurement result as it can be seen that monoclonal antibody 2H1 and The affinity of 3A10 is significantly higher than the affinity of monoclonal antibody 4B7,7G9 and 3A3.
It is according to indirect immunofluorescene assay and affinity determination test result as it can be seen that miscellaneous in all positives screened It hands in oncocyte, only monoclonal antibody secreted by hybridoma cell line 2H1 and 3A10 has broad spectrum activity, remaining positive hybridoma is thin Monoclonal antibody secreted by born of the same parents does not have broad spectrum activity；In addition, monoclonal antibody 2H1 and 3A10 is also significantly greater than in this performance of affinity The affinity of monoclonal antibody 4B7,7G9,3A3；To sum up, monoclonal antibody secreted by hybridoma cell line 2H1 and 3A10 exists Far superior to other hybridoma cell lines in the performances such as broad spectrum activity, conservative, affinity；It can be as the blocking side ELISA Detection antibody in method is for distinguishing or identifying aftosa natural infected animal and immunity inoculation animal.
The hybridoma cell line 2H1 of stably excreting foot and mouth disease virus non-structural protein monoclonal antibody is submitted to by the present invention The mechanism that patent is approved carries out preservation, and deposit number is: CGMCC No.16212；The preservation time is: on August 1st, 2018；Point Class name is: the hybridoma cell line of secretion foot and mouth disease virus non-structural protein monoclonal antibody 2H1；Depositary institution is: China General Microbiological Culture preservation administrative center；Preservation address is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences Institute of microbiology.
The present invention submits the hybridoma cell line 3A10 of stably excreting foot and mouth disease virus non-structural protein monoclonal antibody The mechanism approved to patent carries out preservation, and deposit number is: CGMCC No.16213；The preservation time is: on August 1st, 2018； Classification naming is: the hybridoma cell line of secretion foot and mouth disease virus non-structural protein monoclonal antibody 3A10；Depositary institution is: in State's General Microbiological Culture preservation administrative center；Preservation address is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese science Institute of microbiology of institute.
In order to examine monoclonal antibody secreted by hybridoma cell line 2H1 in foot and mouth disease virus non-structural protein antibody detection method In application value, the present invention use monoclonal antibody 3A10 as coated antibody, HRP mark monoclonal antibody 2H1 as detection antibody establish one Kind blocks ELISA detection method, examines application effect of the 2H1 in FMD immune antiboidy and natural infection antibody differential diagnostic method Fruit；The blocking ELISA detection method that testing result shows that monoclonal antibody 2H1 is established as detection antibody can accurately distinguish aftosa Natural infected animal and immunity inoculation animal.Therefore, the present invention provides hybridoma cell line 2H1 and secreted monoclonal antibody 2H1 It can be used for detecting the detection antibody in the blocking ELISA detection kit of FMDV non-structural protein antibody.
The present invention provides a kind of for distinguishing the blocking of aftosa natural infected animal Yu immunity inoculation animal as a result, ELISA detection kit, comprising: ELISA detection plate, foot-and-mouth disease virus antigen, foot and mouth disease virus non-structural protein packet coating are anti- Body marks the foot and mouth disease virus non-structural protein of substance markers to detect antibody, foot and mouth disease virus positive control serum, negative control blood Cleer and peaceful terminate liquid；Wherein, the detection antibody is monoclonal antibody secreted by hybridoma cell line 2H1；The antigen It can be the non-structural protein of FMDV, for example FMDV 3AB albumen.The marker can be enzyme, fluorescent marker, phosphorescence Any one of marker, luminescent marker object or radioactively labelled substance；Preferably, the marker is enzyme, preferential, institute The enzyme stated can be any one in horseradish peroxidase (HRP), catalase, alkaline phosphatase or beta galactosidase Kind；The foot and mouth disease virus non-structural protein coated antibody is preferably monoclonal antibody 3A10.
Further, also may include in the blocking ELISA detection kit: serum dilution, washing lotion and substrate are molten Liquid；The serum dilution can be the PBS containing 0.05% tween (V/V) and 0.5%BSA (bovine serum albumin(BSA)) (mg/ml) Solution, the pH of the PBS solution are 7.4；The washing lotion is PBST solution；The substrate solution can be 3,3', 5,5'- tetramethyl Base benzidine (TMB) substrate solution；The terminate liquid is 2M H2SO4。
Invention further provides the foot and mouth disease virus non-structural proteins of application FMDV monoclonal antibody 2H1 identification Linear epitope:
The present invention shows that monoclonal antibody 2H1 identifies foot and mouth disease virus non-structural protein 3B-4 peptide using expression technique study is truncated Section,8KPLKVK13(it is in FMDV 3B specific location34KPLKVK39) it is the minimal reaction activity unit that monoclonal antibody 2H1 identifies epitope, The motif of the 3B epitope of as monoclonal antibody 2H1 identification.
For further appreciate that monoclonal antibody 2H1 identify epitope in each amino acid residue with work played in monoclonal antibody cohesive process With the present invention is with 2H1 epitope motifs34KPLKVK39Based on, amino acid one by one, which is carried out, from N-terminal to C-terminal with alanine (A) replaces Change, through SDS-PAGE and Western blot analysis shows that, in the amino acid sequence of the identified epitope of 2H1, Lys34、Lys37 And Val38For key amino acid, the replacement of remaining residue is on epitope activity without influence.
For the conservative for verifying the identified epitope of 2H1, the present invention chooses 274 FMDV3B albumen that GenBank is included Amino acid sequence is compared, and the amino acid sequence of wherein 12 FMDV 3B is compared；Comparative analysis result is aobvious Show, the amino acid sequence for the epitope that 2H1 is identified is highly conserved between tetra- kinds of serotype FMDV strains of A, O, Asia1, C 's.From sequence alignment as it can be seen that there is amino acid mutation to occur in Leu36And Lys39, Pro is sported respectively36And Arg39, but it is to be somebody's turn to do The nonessential amino acid of epitope does not have an impact epitope activity after mutation；And Lys34、Lys37And Val38It reflects as this test The key amino acid of fixed 2H1 epitope is highly conserved in the 3B amino acid sequence for having delivered strain；These result tables Bright, the epitope that monoclonal antibody 2H1 is identified is a highly conserved linear epitope for tetra- kinds of serotype strains of FMDV.
In the 3B epitope motifs for identifying monoclonal antibody 2H1 and being identified34KPLKVK39Afterwards, the present invention is further by epitope34KPLKVK39Each amino acid carry out direct mutagenesis and obtain multiple mutant, determine the epitope and prominent by testing Can variant be used as the significant diagnostic antigen of virus nonstructural protein antibody in detection mouth disease virus infection serum:
The FMDV 3B epitope that the present invention respectively identifies 2H134KPLKVK39Each amino acid carry out direct mutagenesis, point Do not sport Ala (A), by GST fusion in a manner of be expressed as mutation epitope peptide, be respectively designated as 34A, 35A, 36A, 37A, 38A and 39A.Then, the epitope peptide that these GST are merged is coated with elisa plate as antigen respectively, while setting up GST empty carrier Control detects infection of foot-and-mouth disease serum and negative serum containing foot and mouth disease virus non-structural protein antibody, to detect list Anti- 2H1 identifies epitope sequences34KPLKVK39And its mutant and foot and mouth disease virus non-structural protein antibody (infection of foot-and-mouth disease blood Respond clearly) thus evaluates the FMDV 3B epitope peptide of 2H1 identification34KPLKVK39Valence in aftosa antidiastole Value；Test result shows: the FMDV 3B epitope peptide and its mutant 35A, 36A and 39A of monoclonal antibody 2H1 identification and foot and mouth disease virus Good respond, epitope mutation peptide 34A, 37A and 38A and mouth hoof is presented in non-structural protein antibody (infection of foot-and-mouth disease serum) Epidemic disease virus nonstructural protein antibody (infection of foot-and-mouth disease serum) and FMDV negative serum do not react.These testing result tables It is bright, with the amino acid residue Lys found during 2H1 identification Epitope Identification34、Lys37And Val38It is the key that the epitope activity Amino acid and the replacement of remaining residue is consistent on the active result without influence of the epitope, thereby determined that monoclonal antibody 2H1 identification FMDV 3B epitope34KPLKVK39And its a series of epitope mutation peptides identify for establishing foot and mouth disease virus non-structural protein antibody The value and application potential of diagnostic method.
Therefore, invention further provides by anti-the identified foot and mouth disease virus non-structural protein of FMDV monoclonal antibody 2H1 White linear epitope peptide, amino acid sequence are as follows: KX1X2KVX3(SQE ID NO.1), wherein X1、X2Or X3For any ammonia Base acid residue, it is preferred that the X1For proline (P) or alanine (A)；The X2For leucine (L) or alanine (A)；It is described X3For lysine (K) or alanine (A)；Most preferably, the non-knot of the identified foot and mouth disease virus of the anti-FMDV monoclonal antibody 2H1 The linear epitope peptide of structure albumen are as follows: KPLKVK (SQE ID NO.2).
The gene for encoding the linear epitope peptide also belongs within protection category of the invention.
Linear epitope peptide provided by the present invention and its mutant can be used as diagnosis marker for detecting aftosa nature Infection animal and immunity inoculation animal can for example be used the linear epitope peptide as envelope antigen building ELISA detection kit In detection aftosa natural infected animal and immunity inoculation animal.Correspondingly, the present invention also provides a kind of inspections of foot-and-mouth disease antibody Survey ELISA kit, comprising: ELISA detection plate, foot and mouth disease virus envelope antigen, the antibody for marking substance markers, foot and mouth disease virus Infect serum, immune serum, foot and mouth disease virus negative serum, dilution, cleaning solution, substrate solution and developing solution；Wherein, described Foot and mouth disease virus envelope antigen is the linear epitope for the foot and mouth disease virus non-structural protein that anti-FMDV monoclonal antibody 2H1 is identified Peptide；The marker can be appointing in enzyme, fluorescent marker, phosphorescent labels, luminescent marker object or radioactively labelled substance What is a kind of；Preferably, the marker is enzyme, the enzyme can be horseradish peroxidase (HRP), catalase, Any one of alkaline phosphatase or beta galactosidase；The antibody can be goat-anti ox IgG antibody；The substrate Liquid can be OPD.
The present invention still further provides the foot and mouth disease virus non-structural protein identified using FMDV monoclonal antibody 3A10 Linear epitope:
The present invention is shown using expression technique study is truncated7ERTLP11(it is in FMDV 3A specific location34KPLKVK39) The minimal reaction activity unit of epitope, the motif of the 3A epitope of as monoclonal antibody 3A10 identification are identified for monoclonal antibody 3A10.
For further appreciate that monoclonal antibody 3A10 identify epitope in each amino acid residue with played in monoclonal antibody cohesive process Effect, the present invention is with 3A10 epitope motifs126ERTLP130Based on, amino acid one by one is carried out from N-terminal to C-terminal with alanine (A) Replacement,126ERTLP130As positive control, GST empty carrier as negative control.It is analyzed through SDS-PAGE and Western blot It shows, in the amino acid sequence of the identified epitope of 3A10, Arg127And Leu129For key amino acid, the replacement of remaining residue is to table Position activity is without influence.
For the conservative for verifying 3A10 epitope, the present invention chooses the 3A amino acid sequence for 274 FMDV that GenBank is included It is compared, using the 3A amino acid sequence of wherein 21 FMDV as representative, compares and analyze the amino acid the results show that 3A10 epitope Sequence has higher conservative between 7 kinds of serotype FMDV strains.It is found in sequence alignment, in the epitope amino acid sequence Mutation is Glu126、Thr128And Pro130, but it is the nonessential amino acid of the epitope, it will not to epitope activity after mutation It has an impact；And Arg127And Leu129As the key amino acid for the 3A10 epitope that the present invention identifies, the two amino acid residues There is higher conservative in the epitope amino acid sequence for having delivered strain；It is above-mentioned these results indicate that 3A10 epitope is needle To a conservative linear epitope of 7 serotype strains of FMDV.
In the epitope motifs for identifying monoclonal antibody 3A10 and being identified126ERTLP130Afterwards, the present invention is further by epitope126ERTLP130Each amino acid carry out direct mutagenesis and obtain multiple mutant, determine the epitope and prominent by testing Can variant be used as the significant diagnostic antigen of virus nonstructural protein antibody in detection mouth disease virus infection serum:
To determine the epitope126ERTLP130And its can mutant non-structural to detect foot and mouth disease virus as diagnostic antigen Protein antibodies (infection of foot-and-mouth disease serum), the present invention is by the amino acid sequence of the 3A10 FMDV 3A epitope identified126ERTLP130Into Row direct mutagenesis, it may be assumed that E126Sport Ala (A)；R127Sport Ala (A)；T128Sport Ala (A)；L129Sport Ala (A)；P130Sport Ala (A).It is expressed in a manner of GST fusion, is respectively designated as 126A, 127A, 128A, 129A and 130A. Then, the epitope peptide that these GST are merged is coated with elisa plate as antigen respectively, meanwhile, it sets up GST empty carrier and is used as control, Foot and mouth disease virus non-structural protein antibody (infection of foot-and-mouth disease serum) and negative serum are detected, to detect monoclonal antibody 3A10 identification The amino acid sequence of FMDV epitope126ERTLP130And its mutant and foot and mouth disease virus non-structural protein antibody (infection of foot-and-mouth disease Serum) respond, thus evaluate 3A10 identification FMDV epitope amino acid sequence126ERTLP130In aftosa diagnosis Value；Testing result shows: the FMDV epitope peptide and its epitope mutation peptide 126A, 128A and 130A and mouth of monoclonal antibody 3A10 identification Good respond is presented in aphtovirus non-structural protein antibody (infection of foot-and-mouth disease serum)；Epitope mutation peptide 127A and 129A It is not reacted with foot and mouth disease virus non-structural protein antibody (infection of foot-and-mouth disease serum) and FMDV negative serum；These detections As a result illustrate, with the amino acid residue Arg found during 3A10 identification Epitope Identification127And Leu129It is the pass of the epitope activity Key amino acid, and the replacement of remaining residue is consistent on the active result without influence of the epitope；As a result, present invention determine that monoclonal antibody The amino acid sequence of the FMDV epitope of 3A10 identification126ERTLP130And its series epitope mutant peptide is non-for establishing foot and mouth disease virus The value and application potential of structural proteins antibody diagnosis method.
The present invention is also further provided by the non-knot of the identified foot and mouth disease virus of anti-FMDV monoclonal antibody 3A10 as a result, The linear epitope peptide of structure albumen, amino acid sequence are as follows: X1RX2LX3(SQE ID NO.3), wherein X1、X2Or X3It is any one Kind amino acid residue, it is preferred that the X1For glutamic acid (E) or alanine (A)；The X2For threonine (T) or alanine (A)； The X3For proline (P) or alanine (A)；Preferably, the identified foot and mouth disease virus of the anti-FMDV monoclonal antibody 3A10 The linear epitope peptide of non-structural protein is ERTLP (SQE ID NO.4).
The gene for encoding the linear epitope peptide also belongs within protection category of the invention.
Therefore, the present invention still further provides a kind of foot-and-mouth disease antibody detection ELISA kit, comprising: ELISA detection Plate, foot and mouth disease virus envelope antigen, antibody, mouth disease virus infection serum, the immune serum, foot and mouth disease virus for marking substance markers Negative serum, dilution, cleaning solution, substrate solution and developing solution；Wherein, the foot and mouth disease virus envelope antigen is above-mentioned resists The linear epitope peptide for the foot and mouth disease virus non-structural protein that FMDV monoclonal antibody 3A10 is identified；The marker can be Any one of enzyme, fluorescent marker, phosphorescent labels, luminescent marker object or radioactively labelled substance；Preferably, the mark Remember that object is enzyme, the enzyme can be horseradish peroxidase (HRP), catalase, alkaline phosphatase or beta galactose glycosides Any one of enzyme；The antibody can be goat-anti ox IgG antibody；The substrate solution can be OPD.
Linear epitope peptide of the present invention by artificial synthesized (such as solid phase synthesis process of polypeptide) or can pass through It is prepared by the method for genetic engineering；Wherein the gene engineering method can be the encoding gene and table of linear epitope peptide It is ligated and transformed into inducing expression in host cell up to carrier is operable, collecting expressed albumen and carrying out purifying can obtain To the epitope peptide.
The expression vector can be prokaryotic expression carrier or carrier for expression of eukaryon；The host cell can be original Nucleus or eukaryocyte, such as the prokaryotic cell can be Escherichia coli；The eukaryocyte can be yeast cells, Zooblast or insect cell etc., preferably Escherichia coli or Pichia pastoris (Pichia pastoris) cell.
The present invention further can also carry out modification or optimization appropriate to the gene for encoding the linear epitope peptide, to improve Its expression quantity or expression efficiency in host cell, these modifications or optimization method are that those skilled in the art institute is skilled It grasps.
The targeted foot-and-mouth disease epitope of existing aftosa monoclonal antibody does not identify that some important performances include competing Striving property, broad spectrum activity, epitope Property Identification etc. are uncertain, and the epitope that monoclonal antibody is directed to is not identified, epitope sequences and its crucial ammonia Base acid and important amino acid are unclear, if key amino acid and important amino acid have variation, the journey of broad spectrum activity between strain Degree will change or cannot ensure it with broad spectrum activity, and the art formula for the detection method established with such monoclonal antibody is not competition ELISA, not only poor reliability, but also detect different animal species and just need to establish corresponding different kits, in practice substantially There is no application value.
The present invention screens monoclonal antibody 2H1 and 3A10 secreted by the 2 strain of hybridoma systems obtained, with infection of foot-and-mouth disease blood Clear competitiveness is high, and broad spectrum activity is good, conservative is high, affinity is strong；In addition, monoclonal antibody 2H1 and 3A10 targeted antigen table By accurate identification, the targeted epitope of the two is positioned in the different position 3ABC for position；Wherein, the epitope of monoclonal antibody 2H1 identification It is positioned at 3B albumen, sequence is a highly conserved linear epitope for tetra- kinds of serotype strains of FMDV；Monoclonal antibody 3A10 For the epitope mapping of identification in 3A albumen, sequence is a highly conserved linear list for seven kinds of serotype strains of FMDV Position, the two space is apart from each other will not to generate space steric effect, can be used for simultaneously respectively as detection antibody and capture antibody In one blocking ELISA detection kit, aftosa natural infected animal and immunity inoculation animal can not only be accurately distinguished, by This blocking ELISA detection kit established can also cross over species barrier, can detect any animal with a kind of detection kit The serum of kind has many advantages, such as that high reliablity, application are wide, detection is more efficient and convenient.
Detailed description of the invention
Fig. 1 IFA detects monoclonal antibody 2H1,4B7,7G9,3A3,3A10 and A type, O-shaped, Asia1 type FMDV strain reacts knot Fruit.
The measurement result of the relative affinity constant of Fig. 2 monoclonal antibody 2H1,4B7,7G9 and 3A3.
The measurement result of the relative affinity constant of Fig. 3 monoclonal antibody 3A10,4B7,7G9 and 3A3.
The ELISA detection method detection infection of foot-and-mouth disease and be immunized that Fig. 4 is established using aftosa non-structural protein antibody 2H1 Pig, ox, sheep blood serum and the aftosa feminine gender pig of inoculation, ox, sheep blood serum testing result.
Fig. 5 prokaryotic expression 3A and 3B albumen result of indirect ELISA.
Fig. 6 3B synthetic peptide result of indirect ELISA.
SDS-PAGE (a) and the Western blot of Fig. 7 truncated peptide gst fusion protein analyze (b).
SDS-PAGE (a) and the Western blot of Fig. 8 Alanine scanning mutagenesis synthetic peptide analyze (b).
The conservative Analysis of Fig. 9 2H1 epitope peptide amino acid sequence.
The serial epitope mutant peptide and mouth disease virus infection and the pig of vaccine immunity, ox, sheep blood of Figure 10 GST amalgamation and expression Clear and aftosa negative serum immunoreactivity result.
Figure 11 monoclonal antibody 3A10 identifies the albumen positioning of epitope.
SDS-PAGE (a) and the Western blot of Figure 12 truncated peptide gst fusion protein analyze (b).
SDS-PAGE (a) and the Western blot of Figure 13 Alanine scanning mutagenesis synthetic peptide analyze (b).
The conservative Analysis of Figure 14 3A10 epitope peptide amino acid sequence.
The serial epitope mutant peptide of Figure 15 GST amalgamation and expression and infection of foot-and-mouth disease and the pig being immunized, ox, sheep blood serum and The immunoreactivity result of aftosa negative serum.
Specific embodiment
Further describe the present invention below in conjunction with specific embodiment, the advantages and features of the present invention will be with description and It is apparent.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art Member it should be understood that can modify without departing from the spirit and scope of the invention to details and form of the invention or Replacement, but these modifications and replacement are fallen within the protection scope of the present invention.
The screening and identification of 1 foot and mouth disease virus non-structural protein monoclonal antibody 2H1 of embodiment
1 test material
1.1 viruses, cell, bacterial strain and experimental animal
Carrying out specificity analysis institute using strain to monoclonal antibody with IFA includes: A FMDV A/KT/58 (GenBank accession number:AJ131665),A/XJBC/CHA/2010,Asia1FMDV Asia1/YS/CHA/05 (GU931682),O FMDV O/Tibet/CHA/99(AJ539138),O/GD/86(AJ131468)(Wang et al., 2011)；Newborn hamster kidney cell BHK-21, SP2/0 myeloma cell is the preservation of this laboratory；Cleaning grade female BAl BIc/c mouse purchase In Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center.A type, O and Asia1 type FMDV infection and immune serum By Xinjiang, Tian Kang biology Co., Ltd is given.Baculovirus expression FMDV 3ABC albumen, prokaryotic system express FMDV 3AB albumen It is saved by this laboratory.The monoclonal antibody 3A10 of foot and mouth disease virus nonstructural protein 3A is saved by this laboratory.
2.1 main agents
The sheep anti mouse that fusion agent PEG/DMSO (Mw, 1450), HAT salt (50x), HT salt (50x), HRP or FITC are marked IgG, Freund's complete adjuvant and incomplete Freund's adjuvant are purchased from Sigma company；Monoclonal antibody subgroup identification kit is purchased from Southern Biotech company；L-Glutamine (glutamine), glycine are purchased from Amresco company；Dimethyl sulfoxide (DMSO), o-phenylenediamine (OPD), PEG6000 are purchased from Solarbio company；DMEM in high glucose dry powder is purchased from GIBCO company；Import is excellent Grade fetal calf serum (PAA) is purchased from Spain Nalgene company；96 porocyte culture plates are purchased from Canada JET Biochemical Company.
2 test methods
The immune preparation with monoclonal antibody of 2.1 mouse
(1) it is only mixed with French 206 adjuvants of equivalent with 00 μ g/ of the FMDV 3ABC antigen 1 of bar expressing viral, subcutaneous route It is inoculated with 6 week old female BAl BIcs/c mouse, is immunized for second of intramuscular routes after two weeks, then is immune through two weeks abdominal channels third times. Docking blood sampling after two weeks detects antibody titer with indirect ELISA, the mouse of 1:6400 or more is reached to potency, is reinforced through abdominal cavity The immune antigen without adjuvant doubling dosage, takes mouse boosting cell to be merged after 3 days.
(2) cell fusion
A) preparation of feeder cells, eye scissors, ophthalmic tweezers the preparation of feeder cells: are carried out on the day before cell fusion The test toolss such as son, plate do steriling test with preceding high temperature hot air sterilization, HAT complete culture solution.2 BALB/c mouses are taken to extract Eyeball bloodletting, separates negative serum according to a conventional method.It draws neck dislocation to put to death mouse, is soaked in 75% alcohol, after 10min Move into superclean bench.Mouse web portion is fixed on upwards on mouse frame, lifts the positive middle part skin of abdomen with tweezers, eye scissors are lateral An osculum is cut, is sure not to break peritonaeum, skin is torn up and down with scissors and tweezers, sufficiently exposes peritonaeum.Gently lift abdomen with tweezers The 8-10mL HAT complete culture solution drawn in 10mL syringe is injected abdominal cavity, is sure not to puncture internal organs and enteron aisle, syringe by film It not extract, aspirate 5-10 times, then massaged two sides abdomen 30 seconds or so with tweezers, then aspirated, weight back and forth intraperitoneal Multiple the above operation 2-3 times.With the intraperitoneal liquid of syringe resorption.Pay attention to avoiding mesenterium and adipose tissue, in order to avoid blocking syringe needle. It will be added in 55mL HAT culture solution from the abdominal cavity cell taken out in 2 mouse, dispel mixing cell, be sub-packed in 6 with 100 holes μ L/ 96 well culture plate of block, is placed in 37 DEG C, 5%CO2It is cultivated in incubator.
B) preparation of myeloma cell: myeloma cell is expanded and is cultivated by 36-48h before merging, and so that cell is in logarithm raw For a long time.On the fusion same day, cell is blown down from bottle wall with 15mL DMEM basic culture solution, is collected in 50mL centrifuge tube, 1000rpm is centrifuged 10min.Cell precipitation resuspension is set in 20mL DMEM basic culture solution, is mixed.Take a small amount of myeloma cell Suspension, platform expects blue dyeing counting, spare.
C) preparation of immune spleen cell: eyeball of mouse blood sampling is extractd before fusion, prepares positive serum.Mouse is drawn into neck dislocation It is lethal, it is soaked in 75% alcohol, super-clean bench is put in after 10min.Sterile opening abdominal cavity separates connective tissue and takes out spleen, Spleen is put into equipped with sterilizing nylon wire and is filled in the plate of 15mLDMEM basic culture solution, with sterilizing glass syringe inner core Spleen is ground, so that splenocyte is all passed through mesh and enters in plate.Spleen cell solutions are transferred in 50mL centrifuge tube, DMEM base is added Plinth culture solution about to 30mL, mixes, and 1000rpm is centrifuged 8min, abandons supernatant.Cell precipitation is suspended from the training of the basis 10mL DMEM In nutrient solution, mix.Cell suspension is taken, platform expects blue dyeing counting, spare.
D) splenocyte is merged with myeloma cell: taking 65mLHAT culture solution, the basis 15mL DMEM culture solution and 1mL50% PEG is preheated in 37 DEG C of water-baths, and another standby 200mL beaker fills 37 DEG C of water.Add 1 part of myeloma cell's number by 5 parts of spleens cell numbers Corresponding cell suspension amount is taken, is added in 50mL glass centrifuge tube, DMEM basic culture solution is added to 30mL, mixes.1 000rpm It is centrifuged 10min, abandons supernatant, is fallen as far as possible dry.Centrifugation bottom of the tube is tapped with palm, keeps sedimentation cell loose uniformly in the pasty state.It will Centrifuge tube is put into the 200mL beaker for filling 37 DEG C of water, proficiency uniform rotation centrifuge tube, and the other hand is drawn with 1mL pasteur pipet It is added in 37 DEG C of 50%PEG solution 1mL, 1min, stands 2min.Be added fastly after then first slow DMEM basic culture solution terminate it is anti- It answers, 37 DEG C of water-baths stand 10min.1000rpm is centrifuged 10min, abandons supernatant, and 65mLHAT culture medium is added, and lightly dispels thin Born of the same parents, every hole 0.1mL, which is inoculated in, has cultivated have feeder cells 6 piece of 96 well culture plate, sets the CO of 37 DEG C, 5%2It is trained in incubator It supports.The later half amount of 5d changes liquid, changes liquid entirely after 8d, wait clone it is long to hole floor space 1/4-1/3 when take supernatant to be detected and change HT training Nutrient solution.
2.2 antibody test ELISA
With 96 orifice plate of FMDV 3AB antigen coat of prokaryotic expression, 100uL Hybridoma Cell Culture supernatant, 37 DEG C of temperature are added It is washed after educating 1h, the sheep anti mouse secondary antibody of the diluted HRP label of 1:5000 is added, substrate TMB is added after washing and is protected from light colour developing 10min measures light absorption value in wavelength 450nm.
2.3 indirect immunofluorescence
By the hybridoma culture supernatant of monoclonal antibody 2H1,4B7,7G9,3A3 respectively with infection A, O and Asia1 type FMDV BHK- 21 cells carry out indirect immunofluorescene assay, and the specific method is as follows:
96 orifice-plate microporosity plate culture BHK-21 cells are inoculated with foot and mouth disease virus 4 × 10 to single layer3TCID50, there is CPE in/hole It is fixed before with cold dehydrated alcohol, 50uL Hybridoma Cell Culture supernatant is added, PBS washing three after 37 DEG C of incubation 40min It is secondary, the diluted FITC of 1:200 is added and marks sheep anti-mouse igg antibody, 37 DEG C of incubation 40min, in inverted fluorescence microscope after washing Lower observation fluorescence intensity.
The measurement test of 2.4 monoclonal antibody affinity constants
Monoclonal antibody 4B7,7G9,3A3 and 2H1 respectively in conjunction with envelope antigen 3ABC after, with the thiocyanate salt solution of various concentration It is eluted, measures affinity constant.
3. test result
The screening and identification of 3.1FMDV non-structural protein monoclonal antibody
Mouse, extracting spleen cell and SP2/0 cell fusion is immunized in the NSP of eukaryotic expression, and the hybridoma of acquisition is with indirectly ELISA and IFA method detects the FMDV NSP antibody in culture supernatant, Positive judgement standards are as follows: in Hybridoma Cell Culture Clearly to the OD of 3AB antigen450Absorbance value and normal BALB/c mouse serum, SP2/0 cells and supernatant are to 3AB antigen OD450 The ratio between value is all larger than 2.1, and hybridoma supernatant and normal BHK-21 cell do not generate fluorescence reaction, are judged to the positive.Antibody Positive hybridoma cell is subcloned through 3 limiting dilution assays, obtains the hybridoma of 5 plants of energy stably excreting monoclonal antibodies, It is respectively designated as 4B7,7G9,3A3,3A10 and 2H1.Immunoglobulin subclass identification display, monoclonal antibody 4B7 heavy chain type are IgG2, Light chain is κ type.Monoclonal antibody 7G9 heavy chain type is IgG1, and light chain is κ type.Monoclonal antibody 3A3 heavy chain type is IgG1, and light chain is κ class Type.Monoclonal antibody 3A10 heavy chain type is IgG1, and light chain is κ type.Monoclonal antibody 2H1 heavy chain type is IgG1, and light chain is κ type.
The broad spectrum activity measurement result of 3.2 monoclonal antibodies
By the hybridoma culture supernatant of monoclonal antibody 2H1,4B7,7G9,3A3 and 3A10 respectively with infection A, O and Asia1 type The BHK-21 cell of FMDV carries out indirect immunofluorescene assay (Fig. 1), the results show that 2H1 and 3A10 and A, O, Asia1 type poison Strain is positive, and shows that monoclonal antibody 2H1 and 3A10 have broad spectrum activity, and monoclonal antibody 4B7,7G9 and 3A3 are only malicious with part FMDV Specific reaction occurs for the infection cell of strain.
The affinity measurement result of 3.3 monoclonal antibodies
Affinity as the result is shown (Fig. 2, Fig. 3), distinguish by the relative affinity constant of monoclonal antibody 4B7,7G9,3A3,3A10 and 2H1 For 2.0mol/L, 2.5mol/L, 1.0mol/L, 3.5mol/L and 3.5mol/L, the parent of affinity 2H1 and 3A10 as the result is shown It is significantly higher than the affinity of 4B7,7G9 and 3A3 monoclonal antibody with power.
The assembling of 2 antibodies against foot-and-mouth disease virus of embodiment blocking ELISA detection kit
Antibodies against foot-and-mouth disease virus detection blocks ELISA kit, comprising: the monoclonal of horseradish peroxidase-labeled detects Antibody, elisa plate, coated antibody, the foot-and-mouth disease virus antigen, foot and mouth disease virus for being coated with foot and mouth disease virus non-structural protein antibody Positive control serum, foot and mouth disease virus negative control sera, serum dilution, washing lotion, substrate solution and terminate liquid.
The foot and mouth disease virus of the horseradish peroxidase-labeled detects monoclonal antibody: being CCTCC by deposit number The cell strain of NO:16212 is secreted, then purified to be made；
The coated antibody is secreted by the cell strain that deposit number is CCTCC NO:16213, then purified to be made；
Positive control serum: pig and cow's serum after foot and mouth disease virus inactivated vaccine is immune；
Negative control sera: SPF chicken serum；
Serum dilution: contain PBS (pH7.4) solution of 0.05% (V/V) tween and 0.5%BSA (mg/ml)；
Washing lotion: PBST solution；
Substrate solution: tmb substrate solution；
Terminate liquid: 2M H2SO4。
The assembling of 3 foot-and-mouth disease antibody of embodiment detection ELISA kit
Foot-and-mouth disease antibody detects ELISA kit, comprising: ELISA detection plate, foot and mouth disease virus envelope antigen, enzyme label Antibody, mouth disease virus infection serum, immune serum, foot and mouth disease virus negative serum, dilution, cleaning solution, substrate solution and Developing solution；
Wherein, the foot and mouth disease virus envelope antigen is linear epitope peptide (KPLKVK)；
The antibody of the enzyme label is the goat-anti ox IgG antibody of HRP label；
It is coated with the elisa plate of foot and mouth disease virus inactivation antigen: being 1ng/ μ by the concentration dilution of foot and mouth disease virus envelope antigen L, 100 holes μ L/ are coated with elisa plate, and 37 DEG C are incubated for 2h or 4 DEG C overnight；
Dilution: contain PBS (pH7.4) solution of 0.05% (V/V) tween and 0.5%BSA (mg/ml)；
The substrate solution is OPD；
Cleaning solution: PBST solution；
Substrate solution: tmb substrate solution.
The foundation of 1 FMD NSP blocking ELISA antibody detection method of test example
1 test method
The purifying of 1.1 monoclonal antibody ascites
In view of the broad spectrum activity and affinity of monoclonal antibody 2H1 and 3A10, further research monoclonal antibody 2H1 and 3A10 exists for this test The immune value with antidiastole in natural infection serum of FMD.
Monoclonal antibody 2H1 or 3A10 ascites prepared by embodiment 1, using octanoic acid-saturated ammonium sulfate method to the ascites of preparation into Row purifying, operating procedure are summarized as follows:
(1) ascites for taking 3ml pretreated adds 6ml acetate buffer solution (0.06mol, pH4.8)；In ascites plus 99ul is sad It is stirred at room temperature 30 minutes, 4 DEG C stand 2 hours or more；It takes out 11000rpm to be centrifuged 30 minutes, abandons precipitating；Supernatant 2mol/L NaOH tune pH to 7.4；
(2) saturated ammonium sulfate is added under the conditions of 4 DEG C to 50% saturation degree, ice bath stirring acts on 30 minutes, and 4 DEG C stand 2 Hour or more；11000rpm is centrifuged 30 minutes, abandons supernatant；Precipitating is dissolved in 5ml PBS (0.1M, pH7.4)；
(3) add appropriate saturated ammonium sulfate solution while stirring into precipitating suspended matter, make concentration in ammonium sulfate 30%-40%, ice bath stirring act on 30 minutes, and 4 DEG C 2 hours or more static；11000rpm is centrifuged 30 minutes, is taken precipitating, will be sunk It forms sediment and incorporates in 2ml PBS；
(4) precipitation suspension is fitted into dialysis band, is dialysed, is changed the liquid once with PBS at 4 DEG C within 2 hours, dialysed 2 days.Dialysis After the completion, the purifying ascites in bag filter is recycled, ultraviolet specrophotometer measures protein concentration, then with DEAE-Sephadex A- Monoclonal antibody, the monoclonal antibody after recovery purifying is further purified in 50 (GE company) columns chromatography, and ultraviolet specrophotometer is surveyed Determine protein concentration, carry out purity detecting with SDS-PAGE, marks HRP with improvement Over-voltage protection.
1.2 foot and mouth disease virus non-structural proteins (FMD NSP) blocking ELISA antibody detection method
To examine monoclonal antibody 2H1 and its hybridoma to tie up to and establish answering in aftosa non-structural protein antibody detection method With value, it is coated with elisa plate with FMDV non-structural protein monoclonal antibody 3A10, FMDV 3AB albumen is as antigen, the monoclonal antibody of HRP label 2H1 is tentatively established a kind of for detecting the ELISA method of aftosa non-structural protein antibody, detection mouth hoof as detection antibody Epidemic disease infection and immune pig, ox, sheep blood serum and aftosa negative serum.
1) it is pressed using concentration dilution coating monoclonal antibody 3A10 with the carbonate buffer solution of pH9.6 in elisa plate, 100 holes μ L/, 4 DEG C overnight.Next day takes out elisa plate, discards liquid, with pH7.4PBST board-washing 5 times, drying；
2) 3AB antigen is diluted to using concentration, 100 holes μ L/, 37 DEG C of incubation 1h, ibid board-washing 5 times with PBS；
3) negative serum, positive serum are diluted with 10 times of PBS, 50 holes μ L/, 37 DEG C of incubation 1h, ibid board-washing 5 times；
4) it being added in elisa plate and uses the diluted HRP-2H1 of PBS, 50 holes μ L/, abundant shake mixes, 37 DEG C of incubation 1h, Ibid board-washing 5 times；
5) substrate solution TMB, 50 holes μ L/ is added, room temperature is protected from light 15min.After 15min, 2M H is added2SO4Terminate liquid, 50 μ The hole L/ terminates reaction, OD is read in microplate reader450Nm value.
Result judgement: it by detection known negative and positive serum, calculates and inhibits percentage (Percentage inhibition,PI)。
2 test results
The purification result of 2.1 monoclonal antibody ascites
The concentration of the monoclonal antibody 2H1 of ultraviolet specrophotometer measurement after purification is 5.23mg/mL, ultraviolet specrophotometer measurement The concentration of monoclonal antibody 3A10 after purification is 6.17mg/mL；Antibody 2H1 again through DEAE-sephadex a-50 ion-exchange chromatography into The purifying of one step marks HRP with improvement Over-voltage protection.
2.2 coating monoclonal antibodies and detection monoclonal antibody use the determination of concentration
To 3AB antigen, coating monoclonal antibody 3A10 and the upright titration of use concentration progress for detecting monoclonal antibody 2H1, as a result such as 1 institute of table Show, when capture monoclonal antibody 1:2000 dilute (3.1 μ g/ml), 3AB antigen 1: 1000 dilution, HRP label detection monoclonal antibody be 1:4000 When diluting (1.31 μ g/ml), OD450It is optimal combination that nm value, which is 1.82,.
The best peridium concentration of 1 monoclonal antibody of table and the square matrix titration results for detecting monoclonal antibody optimal use concentration
2.3 aftosa non-structural protein antibody tests
Aftosa non-structural protein antibody test result is as shown in figure 4, testing result shows to apply the aftosa non-structural Blocking ELISA detection method constructed by protein antibodies 3A10 and 2H1 is capable of infection serum, the immune blood of specificity differentiation FMDV Clear and negative serum.And it can also be seen that the blocking ELISA detection method of the invention established due to strong competitive single from Fig. 4 The advantage of anti-2H1 infects the detected value of serum (80% or more PI value) and immune serum and negative serum (20% or less PI value) Significant difference.Above-mentioned test result explanation, monoclonal antibody 3A10 and 2H1 have weight in aftosa non-structural protein antibody detection method The application value wanted.
The screening and identification of the foot and mouth disease virus non-structural protein 3B epitope of 2 monoclonal antibody 2H1 of test example identification
1, test material and test method
1.1 main agents
Elisa plate is purchased from Corning company, and DAB developing solution is purchased from Bioisystech Co., Ltd of Beijing Zhong Shan Golden Bridge, BL21 competence is purchased from Tiangeng company, and pGEX-6p-1 is saved by this laboratory.Prokaryotic expression FMDV 3A and 3B albumen is by this Laboratory saves.A type, O and Asia1 type FMDV infection and immune serum are given by Xinjiang Tian Kang biology Co., Ltd.3B synthesis Peptide fragment is purchased from BeiJing ZhongKe Yaguang Biology Science Co., Ltd.
1.2 prokaryotic expression 3A and 3B proteantigen indirect ELISAs
1) it is pressed with the carbonate buffer solution of pH9.6 and dilutes coating aftosa recombinant protein 3A and 3B albumen respectively using concentration In elisa plate, 100 holes μ L/, 4 DEG C overnight.Next day takes out elisa plate, discards liquid, with pH7.4PBST board-washing 5 times, drying； 2) monoclonal antibody 2H1 ascites is diluted to using concentration, 100 holes μ L/, 37 DEG C of incubation 1h, ibid board-washing 5 times with PBST；3) dilute with PBST Sheep anti mouse HRP is released to using concentration, 100 holes μ L/, 37 DEG C of incubation 1h；4) it is same as above board-washing 5 times, substrate solution TMB, 50 μ L/ is added Hole, room temperature are protected from light 15min.After 15min, 2M H is added2SO4Terminate liquid, 50 holes μ L/ terminate reaction, read in microplate reader OD450Nm value.
1.3 3B synthetic peptide indirect ELISAs
Foot and mouth disease virus non-structural protein 3B albumen is divided into 6 amino acid of overlapping between 7 sections, every two sections, is named as 3B1,3B2,3B3,3B4,3B5,3B6,3B7 carry out sequent synthesis, distribution such as table 2 respectively.
2 3B of table synthesizes peptide sequence
It is pressed using concentration dilution coating synthetic peptide 3B1 to 3B7 with the carbonate buffer solution of pH9.6 in elisa plate, 0.5 μ g/ Hole, 4 DEG C overnight.Next day takes out elisa plate, discards liquid, with pH7.4PBST board-washing 5 times, drying；2) monoclonal antibody is diluted with PBST 2H1 ascites is to using concentration, 100 holes μ L/, 37 DEG C of incubation 1h, ibid board-washing 5 times；3) with PBST dilution sheep anti mouse HRP to use Concentration, 100 holes μ L/, 37 DEG C of incubation 1h；5) it is same as above board-washing 5 times, substrate solution TMB, 50 holes μ L/ is added, room temperature is protected from light 15min Colour developing.2M H is added2SO450 holes μ L/ terminate reaction, OD are read in microplate reader450Nm value.
The GST amalgamation and expression of 1.4 epitope peptides
Artificial synthesized 3B epitope and its monamino acid truncate two complementary oligonucleotide chains of the coding DNA of epitope one by one, Upstream introduces BamHI viscosity extending end, and downstream introduces terminator codon and SalI viscosity extending end, primer sequence are shown in Table 3。
Table 3 encodes the complementary oligonucleotide chain of the DNA of truncated peptide and its truncated mutant
By two complementary strands directly anneal formed the double-stranded DNA with cohesive end, be inserted into pGEX-6p-1 BamHI and Between SalI, BL21 competent cell is converted.Individually positive bacterium colony is incubated overnight simultaneously sequence verification to picking, and then 1:100 dilutes It is inoculated in fresh LB liquid medium, 37 DEG C of shake cultures to OD600nm=0.6, inducer IPTG is added to final concentration of 1mmol/L, 37 DEG C are continued to cultivate 4h, are collected thallus and are resuspended with suitable PBS.The fusion protein of expression is respectively designated as 2h1- 1-5 to 2h1-1-14 and 2h1-2-15 to 2h1-10-15.
The GST amalgamation and expression of 1.5 serial epitope mutant peptides
Two complementary oligonucleotide chains of artificial synthesized 3B epitope coding DNA, upstream introduce BamHI viscosity extending end, Downstream introduces terminator codon and SalI viscosity extending end, primer sequence are shown in Table 5.
Table 4 is used for the synthetic peptide and its amino acid sequence of this test
aThe amino acid sequence (8~13aa) of ia1/YS/CHA/05 plants of 3B epitope peptides of foot and mouth disease virus As
bAmino acid equal overstriking in mutational site is simultaneously marked with underscore, successively has a residue alanine by left and right sequence (A) it replaces
The complementary oligonucleotide chain of the coding of table 5 synthetic peptide DNA
By two complementary strands directly anneal formed the double-stranded DNA with cohesive end, be inserted into pGEX-6p-1 BamHI and Between SalI, BL21 competent cell is converted, individually positive bacterium colony is incubated overnight simultaneously sequence verification to picking, and then 1:100 dilutes It is inoculated in fresh LB liquid medium, 37 DEG C of shake cultures to OD600nm=0.6, inducer IPTG is added to final concentration of 1mmol/L, 37 DEG C of expression for continuing to cultivate 4h inducible protein are collected thallus and are resuspended with suitable PBS, the GST fusion of expression Albumen is respectively designated as 8A, 9A, 10A, 11A, 12A and 13A.
1.6SDS-PAGE electrophoresis and Western blot detection
SDS-PAGE electrophoresis (12%Tris-glycine) separates each fusion protein and is transferred on nitrocellulose filter (90V, 60min), overnight with 4 DEG C of 5% skimmed milk-PBS confining liquid closings, with monoclonal antibody 2H1 in 37 DEG C of temperature 1h, with HRP label Sheep anti-mouse igg room temperature acts on 1h, DAB (6mg/10mL) colour developing.
1.7 antibody test ELISA
It uses the epitope peptide of the GST amalgamation and expression in above-mentioned material method 1.5 as 96 orifice plate of antigen coat, 100uL is added 100 times of diluted infection of foot-and-mouth disease and immune pig, ox, sheep blood serum and aftosa negative serum wash after 37 DEG C of incubation 1h, The goat-anti ox IgG antibody of the diluted HRP label of 1:5000 is added, substrate OPD is added after washing and is protected from light colour developing 10min, in wavelength 492nm measures light absorption value.
2 test results
2.1 monoclonal antibody 2H1 identify the albumen positioning of epitope
Prokaryotic expression 3A and 3B albumen result of indirect ELISA such as Fig. 5, monoclonal antibody 2H1 identifies that foot and mouth disease virus is non-as the result is shown Structural proteins 3B.3B synthetic peptide result of indirect ELISA such as Fig. 6, monoclonal antibody 2H1 identifies foot and mouth disease virus non-structural protein as the result is shown 3B-4 peptide fragment.
The accurate positioning of 2.2 monoclonal antibody 2H1 identification epitope
For the epitope of further qualitative monoclonal antibody 2H1 identification, annealed for the purpose of forming target fragment by upstream and downstream primer in turn 20 pairs of primers (table 3) are devised, is gradually successively decreased from the N-terminal of 3B-4 small peptide and C-terminal with 1 amino acid residue design peptide fragment respectively, Until C-terminal or N-terminal are left the small peptide of 5 amino acid composition, above-mentioned peptide fragment is subjected to GST amalgamation and expression and SDS- PAGE electrophoresis (Fig. 7-a) and Western blot (Fig. 7-b) analysis.
Analysis is the results show that MAb 2H1 can be with the end 3B-4N1AGPMERQKPLKVKA13Extremely1AGPMERQKPLKVKAK15 And the end 3B-4C2GPMERQKPLKVKAK15Extremely8KPLKVKAK15Specifically bind, and with other fusogenic peptides and GST Control is not reacted.The above results show8KPLKVK13(it is in FMDV 3B specific location34KPLKVK39) it is that monoclonal antibody 2H1 is identified The motif of the 3B epitope of the minimal reaction activity unit of epitope, as monoclonal antibody 2H1 identification.
The key amino acid of 2.3 2H1 identification epitope
For further appreciate that monoclonal antibody 2H1 identify epitope in each amino acid residue with work played in monoclonal antibody cohesive process With with 2H1 epitope motifs34KPLKVK39Based on, amino acid substitution (4 He of table one by one is carried out from N-terminal to C-terminal with alanine (A) Table 5),34KPLKVK39As positive control, GST empty carrier as negative control.Through SDS-PAGE (Fig. 8-a) and Western Blot analyzes (Fig. 8-b) display, in the amino acid sequence of the identified epitope of 2H1, Lys34、Lys37And Val38For crucial amino Acid, the replacement of remaining residue is on epitope activity without influence.
2.4 2H1 epitopes are the epitopes conserved amongsts of each genotype strain of seven serotype FMDV
For the conservative for verifying 2H1 epitope, choose the amino acid sequence of 274 FMDV3B albumen that GenBank is included into Row compares, and the amino acid sequence of wherein 12 FMDV 3B is compared.Comparative analysis result is as shown in figure 9,2H1 table The amino acid sequence of position is highly conserved between tetra- kinds of serotype FMDV strains of A, O, Asia1, C.From sequence alignment as it can be seen that There is amino acid mutation to occur in Leu36And Lys39, Pro is sported respectively36And Arg39, but it is the non-essential amino of the epitope Acid will not have an impact epitope activity after mutation；And Lys34、Lys37And Val38The pass of 2H1 epitope as this test for identification Key amino acid is highly conserved in the 3B amino acid sequence for having delivered strain.These results indicate that 2H1 epitope is to be directed to One highly conserved linear epitope of tetra- kinds of serotype strains of FMDV.
2.5 monoclonal antibody 2H1 identify that the specificity of epitope and its mutant and foot and mouth disease virus non-structural protein antibody is anti- It answers
In the 3B epitope motifs for identifying monoclonal antibody 2H1 and being identified34KPLKVK39Afterwards, this test further determines that the antigen table The significant diagnosis that can position and its mutant be used as virus nonstructural protein antibody in detection mouth disease virus infection serum is anti- It is former.
The FMDV 3B epitope that 2H1 is identified respectively34KPLKVK39Each amino acid carry out direct mutagenesis, be mutated respectively For Ala (A), the epitope peptide of mutation is expressed as in a manner of GST fusion, be respectively designated as 34A, 35A, 36A, 37A, 38A and 39A.Then, the epitope peptide that these GST are merged is coated with elisa plate as antigen respectively, while setting up GST empty vector control, Infection of foot-and-mouth disease serum and negative serum containing foot and mouth disease virus non-structural protein antibody are detected, to detect monoclonal antibody 2H1 Identify epitope sequences34KPLKVK39And its mutant and foot and mouth disease virus non-structural protein antibody (infection of foot-and-mouth disease serum) is anti- Should be able to power, thus evaluate 2H1 identification FMDV3B epitope peptide34KPLKVK39Value in aftosa antidiastole.
Test result (Figure 10) shows: monoclonal antibody 2H1 identification FMDV 3B epitope peptide and its mutant 35A, 36A and 39A with Good respond, epitope mutation peptide 34A, 37A is presented in foot and mouth disease virus non-structural protein antibody (infection of foot-and-mouth disease serum) It is not reacted with 38A with foot and mouth disease virus non-structural protein antibody (infection of foot-and-mouth disease serum) and FMDV negative serum.This A little testing results show to identify the amino acid residue Lys found during Epitope Identification with 2H134、Lys37And Val38It is the table The active key amino acid in position and the replacement of remaining residue is consistent on the active result without influence of the epitope, thereby determine that The FMDV 3B epitope of monoclonal antibody 2H1 identification34KPLKVK39And its a series of epitope mutation peptides are non-structural for establishing foot and mouth disease virus The value and application potential of protein antibodies differential diagnostic method.
The screening and identification of the foot and mouth disease virus nonstructural protein 3A epitope of 3 monoclonal antibody 3A10 of test example identification
1, test material and test method
1.1 main agents
Elisa plate is purchased from Corning company, and DAB developing solution is purchased from Bioisystech Co., Ltd of Beijing Zhong Shan Golden Bridge, BL21 competence is purchased from Tiangeng company, and pGEX-6p-1 is saved by this laboratory.Prokaryotic expression FMDV 3A and 3B albumen is by this Laboratory saves.A type, O and Asia1 type FMDV infection and immune serum are given by Xinjiang Tian Kang biology Co., Ltd.3B synthesis Peptide fragment is purchased from BeiJing ZhongKe Yaguang Biology Science Co., Ltd.
1.2 prokaryotic expression 3A and 3B proteantigen indirect ELISAs
1) it is pressed with the carbonate buffer solution of pH9.6 and dilutes coating aftosa recombinant protein 3A and 3B albumen respectively using concentration In elisa plate, 100 holes μ L/, 4 DEG C overnight.Next day takes out elisa plate, discards liquid, with pH7.4PBST board-washing 5 times, drying； 2) monoclonal antibody 2H1 ascites is diluted to using concentration, 100 holes μ L/, 37 DEG C of incubation 1h, ibid board-washing 5 times with PBST；3) dilute with PBST Sheep anti mouse HRP is released to using concentration, 100 holes μ L/, 37 DEG C of incubation 1h；4) it is same as above board-washing 5 times, substrate solution TMB, 50 μ L/ is added Hole, room temperature are protected from light 15min.After 15min, 2M H is added2SO4Terminate liquid, 50 holes μ L/ terminate reaction, read in microplate reader OD450Nm value.
The truncation of 1.3 3A albumen is expressed
According to FMDV Asia1/YS/CHA/05 (GenBank accession number:GU931682) strain 3A albumen sequence Column, design 7 pairs of specific primers, albumen are divided into 7 truncated segments and is expressed.All upstream and downstream primers introduce respectively BamHI, SalI digestion enzyme site, primer sequence are shown in Table 6, are synthesized by Invitrogen company.
The coding of table 6 truncates the complementary oligonucleotide chain of 3A peptide
7 pairs of primers are respectively used for amplifying 7 truncated gene segments, and distribution is as shown in table 6.Genetic fragment after digestion is inserted Enter between the BamHI of pGEX-6p-1 and SalI, converts BL21 competent cell, individually positive bacterium colony is incubated overnight and surveys picking Sequence verifying, then 1:100 dilution are inoculated in fresh LB liquid medium, 37 DEG C of shake cultures to OD600Nm=0.6 is added Inducer IPTG to final concentration of 0.1mmol/L, 37 DEG C are continued to cultivate 4h, are collected thallus and are resuspended with appropriate PBS, fusion protein It is respectively designated as 3A1,3A2,3A3,3A4,3A5,3A6 and 3A7.
The GST amalgamation and expression of 1.4 epitope peptides
Epitope synthesis and its monamino acid truncate two complementary oligonucleotide chains of the coding DNA of epitope one by one, on Trip introduces BamHI viscosity extending end, and downstream introduces terminator codon and SalI viscosity extending end, primer sequence are shown in Table 7.
Table 7 encodes truncated peptide and its complementary oligonucleotide chain of truncated mutant DNA
By two complementary strands directly anneal formed the double-stranded DNA with cohesive end, be inserted into pGEX-6p-1 BamHI and Between SalI, BL21 competent cell is converted.Individually positive bacterium colony is incubated overnight simultaneously sequence verification to picking, and then 1:100 dilutes It is inoculated in fresh LB liquid medium, 37 DEG C of shake cultures to OD600nm=0.6, inducer IPTG is added to final concentration of 1mmol/L, 37 DEG C are continued to cultivate 4h, are collected thallus and are resuspended with suitable PBS.The fusion protein of expression is respectively designated as, 3A1-15,3A10-25 and 3A20-34,3A10-1-5 to 3A10-1-14 and 3A10-2-15 to 3A10-10-15.
The GST amalgamation and expression of 1.5 serial epitope mutant peptides
Two complementary oligonucleotide chains of epitope synthesis coding DNA, upstream introduce BamHI viscosity extending end, under Trip introduces terminator codon and SalI viscosity extending end, primer sequence are shown in Table 8 and table 9.
Table 8 is used for the synthetic peptide and its amino acid sequence of this research
a7~11aa peptide of aphthovirus Asial/YS/CHA/05 plants of 3A
bAmino acid equal overstriking in mutational site is simultaneously marked with underscore, successively has a residue alanine by left and right sequence (A) it replaces
The complementary oligonucleotide chain of the coding of table 9 synthetic peptide DNA
By two complementary strands directly anneal formed the double-stranded DNA with cohesive end, be inserted into pGEX-6p-1 BamHI and Between SalI, BL21 competent cell is converted, individually positive bacterium colony is incubated overnight simultaneously sequence verification to picking, and then 1:100 dilutes It is inoculated in fresh LB liquid medium, 37 DEG C of shake cultures to OD600nm=0.6, inducer IPTG is added to final concentration of 1mmol/L, 37 DEG C of expression for continuing to cultivate 4h inducible protein are collected thallus and are resuspended with suitable PBS, the GST fusion of expression Albumen is respectively designated as 126A, 127A, 128A, 129A and 130A.
1.6SDS-PAGE electrophoresis and Western blot detection
SDS-PAGE electrophoresis (12%Tris-glycine) separates each fusion protein, is transferred on nitrocellulose filter (90V, 60min), overnight with 4 DEG C of 5% skimmed milk-PBS confining liquid closings, with monoclonal antibody 3A10 in 37 DEG C of temperature 1h, with HRP label Sheep anti-mouse igg room temperature acts on 1h, DAB (6mg/10mL) colour developing.
1.7 antibody test ELISA
It uses the epitope peptide of the GST amalgamation and expression in above-mentioned material method 1.5 as 96 orifice plate of antigen coat, 100uL is added 100 times of diluted infection of foot-and-mouth disease and immune pig, ox, sheep blood serum and aftosa negative serum wash after 37 DEG C of incubation 1h, Goat-anti pig, the ox, sheep IgG antibody of the diluted HRP label of 1:5000 is added, substrate OPD is added after washing and is protected from light colour developing 10min, Light absorption value is measured in wavelength 492nm.
2 test results
2.1 monoclonal antibody 3A10 identify the albumen positioning of epitope
Prokaryotic expression 3A and 3B albumen result of indirect ELISA (Figure 11-a) shows that monoclonal antibody 3A10 identifies the non-knot of foot and mouth disease virus Structure albumen 3A, and there is no specific reaction with 3B.3A1,3A2,3A3,3A4,3A5,3A6,3A7 of GST amalgamation and expression are carried out SDS-PAGE (Figure 11-b) separation, and using the diluted monoclonal antibody 3A10 of 1:1000 as the sheep of primary antibody, the diluted HRP label of 1:5000 Anti-mouse antibody carries out Western blot analysis (Figure 11-c) as secondary antibody；The results show that the epitope that 3A10 is identified is located at 3A7 On glycoprotein segments.It is 3 sections (3A1-15,3A10-25 and 3A20-35) by 3A7 points to further position the epitope of 3A10 identification Further truncate expression, SDS-PAGE (Figure 11-d) and Western blot analysis (Figure 11-e) display, the table that 3A10 is identified Position is located at the segment 3A1-15.
2. the accurate positioning of monoclonal antibody 3A10 identification epitope
For the epitope of further qualitative monoclonal antibody 3A10 identification, this test anneals to form target fragment as mesh using upstream and downstream primer So devise 20 pairs of primers, gradually successively decrease designed peptide from the N-terminal of 3A1-15 small peptide and C-terminal with 1 amino acid residue respectively Section, until C-terminal or N-terminal are left the small peptide of 5 amino acid composition, by above-mentioned peptide fragment carry out GST amalgamation and expression and SDS-PAGE electrophoresis (Figure 12-a) and Western blot (Figure 12-b) analysis.The results show that MAb 3A10 can be with the end 3A7N1STVGFRERTLP11Extremely1STVGFRERTLPGRKT15And the end 3A7C2TVGFRERTLPGRKT15Extremely7ERTLPGRKT15Occur Specific binding, and do not reacted with other fusogenic peptides and GST control.The above results show7ERTLP11It is (specific in FMDV 3A Position is34KPLKVK39) identified for the minimal reaction activity unit of the MAb 3A10 true epitope identified, as monoclonal antibody 3A10 The motif of 3A epitope.
3. the key amino acid of 3A10 identification epitope
For further appreciate that monoclonal antibody 3A10 identify epitope in each amino acid residue with played in monoclonal antibody cohesive process Effect, with 3A10 epitope motifs126ERTLP130Based on, amino acid substitution one by one is carried out from N-terminal to C-terminal with alanine (A),126ERTLP130As positive control, GST empty carrier as negative control.Through SDS-PAGE (Figure 13-a) and Western blot (Figure 13-b) is analyzed to show, in the amino acid sequence of the identified epitope of 3A10, Arg127And Leu129For key amino acid, remaining is residual The replacement of base is on epitope activity without influence.
4. the epitopes conserved amongst that 3A10 epitope is each genotype strain of seven serotype FMDV
For the conservative for verifying 3A10 epitope, the 3A amino acid sequence for choosing 274 FMDV that GenBank is included is compared It is right, using the 3A amino acid sequence of wherein 21 FMDV as representative, compare analysis result (Figure 14) display, the amino acid of 3A10 epitope Sequence has higher conservative between 7 kinds of serotype FMDV strains.It is found in sequence alignment, in the epitope amino acid sequence Mutation is Glu126、Thr128And Pro130, but it is the nonessential amino acid of the epitope, it will not to epitope activity after mutation It has an impact；And Arg127And Leu129As the key amino acid for the 3A10 epitope that the present invention identifies, the two amino acid residues There is higher conservative in the epitope amino acid sequence for having delivered strain, these results indicate that the epitope of 3A10 identification is For a conservative linear epitope of 7 serotype strains of FMDV.
5. monoclonal antibody 3A10 identifies that the specificity of epitope and its mutant and foot and mouth disease virus non-structural protein antibody is anti- It answers
In the epitope motifs for identifying monoclonal antibody 3A10 and being identified126ERTLP130It afterwards, can conduct for the determining epitope Diagnostic antigen detects foot and mouth disease virus non-structural protein antibody (infection of foot-and-mouth disease serum), and the present invention is first by 3A10 identification The amino acid sequence of FMDV 3A epitope126ERTLP130Carry out direct mutagenesis: E126Sport Ala (A)；R127Sport Ala (A)； T128Sport Ala (A)；L129Sport Ala (A)；P130Sport Ala (A).It is expressed in a manner of GST fusion, is named respectively For 126A, 127A, 128A, 129A and 130A.Then, the epitope peptide that these GST are merged is coated with ELISA as antigen respectively Plate, meanwhile, set up GST empty carrier as control, detect foot and mouth disease virus non-structural protein antibody (infection of foot-and-mouth disease serum) and Negative serum, to detect the amino acid sequence of the FMDV epitope of monoclonal antibody 3A10 identification126ERTLP130And its mutant and aftosa The respond of virus nonstructural protein antibody (infection of foot-and-mouth disease serum) thus evaluates the amino of the FMDV epitope of 3A10 identification Acid sequence126ERTLP130Value in aftosa diagnosis.
Testing result (Figure 15) shows: monoclonal antibody 3A10 identification FMDV epitope peptide and its epitope mutation peptide 126A, 128A and Good respond is presented in 130A and foot and mouth disease virus non-structural protein antibody (infection of foot-and-mouth disease serum)；Epitope mutation peptide 127A and 129A and foot and mouth disease virus non-structural protein antibody (infection of foot-and-mouth disease serum) and FMDV negative serum are without anti- It answers.The amino acid residue Arg found during these testing results, with 3A10 identification Epitope Identification127And Leu129It is the epitope Active key amino acid, and the replacement of remaining residue is consistent on the active result without influence of the epitope, has thereby determined that list The amino acid sequence of the FMDV epitope of anti-3A10 identification126ERTLP130And its serial epitope mutant peptide is for establishing foot and mouth disease virus The value and application potential of non-structural protein antibody diagnosis method.
4 present invention of test example blocks the coincidence rate of ELISA detection kit to detect test
Test object: antibodies against foot-and-mouth disease virus prepared by embodiment 2 blocks ELISA detection kit；
Commercial reagent box: Ceditest kit (Dutch sadie diagnostic companies, aftosa NSP antibody assay kit).
According to the coincidence rate testing result of table 10 as it can be seen that total coincidence rate of present invention blocking ELISA detection kit is 98.68%, this also shows that monoclonal antibody 3A10 and 2H1 have good broad spectrum activity indirectly.
There are 39 parts of Swine serums and 37 parts of cow's serums in 76 parts of serum detected, in conjunction with strong competitive monoclonal antibody 2H1 of the invention Advantage, the present invention blocks ELISA detection kit that can detect pig and cow's serum simultaneously in a reaction system.
10 present invention of table blocks the coincidence rate testing result of ELISA detection kit
SEQUENCE LISTING
<110>Harbin Veterinary Medicine Inst., China Academy of Agriculture
<120>foot and mouth disease virus non-structural protein 3B epitope peptide and its application
<130> HLJ-2001-180711A
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 6
<212> PRT
<213> Foot and Mouth Disease Virus
<400> 1
Lys X1 X2 Lys Val X3
1 5
<210> 2
<211> 6
<212> PRT
<213> Foot and Mouth Disease Virus
<400> 2
Lys Pro Leu Lys Val Lys
1 5
<210> 3
<211> 5
<212> PRT
<213> Foot and Mouth Disease Virus
<400> 3
X1 Arg X2 Leu X3
1 5
<210> 4
<211> 6
<212> PRT
<213> Foot and Mouth Disease Virus
<400> 4
Glu Arg Thr Leu Pro
1 5

Claims (10)

1. foot and mouth disease virus non-structural protein 3B epitope peptide, which is characterized in that its amino acid sequence is SEQ ID NO.1 institute Show；Wherein, X1、X2Or X3For any amino acid residue.
2. foot and mouth disease virus non-structural protein 3B epitope peptide described in accordance with the claim 1, which is characterized in that the X1For Proline or alanine；The X2For leucine or alanine；The X3For lysine or alanine.
3. foot and mouth disease virus non-structural protein 3B epitope peptide according to claim 2, which is characterized in that the X1For Proline；The X2For leucine；The X3For lysine.
4. encoding the gene of any one of the claim 1-3 foot and mouth disease virus non-structural protein 3B epitope peptide.
5. the expression vector containing gene described in claim 4.
6. any one of the claim 1-3 foot and mouth disease virus non-structural protein 3B epitope peptide distinguishes aftosa in preparation Purposes in natural infected animal and immunity inoculation animal reagent.
7. use of the gene described in claim 4 in preparation detection aftosa natural infected animal and immunity inoculation animal reagent On the way.
8. a kind of foot-and-mouth disease antibody detects ELISA kit, comprising: ELISA detection plate, foot and mouth disease virus envelope antigen, label Antibody, mouth disease virus infection serum, immune serum, foot and mouth disease virus negative serum and the substrate solution of substance markers；Its feature Be: the foot and mouth disease virus envelope antigen is the epitope peptide of any one of claim 1-3.
9. foot-and-mouth disease antibody detects ELISA kit according to claim 8, which is characterized in that further include dilution, wash Wash liquid or developing solution.
10. foot-and-mouth disease antibody detects ELISA kit according to claim 8, which is characterized in that the marker packet Include enzyme, fluorescent marker, phosphorescent labels, luminescent marker object or radioactively labelled substance；The antibody is goat-anti ox IgG anti- Body；The substrate solution is OPD.
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