CN111208302B

CN111208302B - Chemiluminescence detection kit for detecting O-type antibody of pig foot-and-mouth disease by using multi-epitope tandem protein

Info

Publication number: CN111208302B
Application number: CN202010037597.2A
Authority: CN
Inventors: 常惠芸; 刘伟; 邵军军; 常艳燕
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2020-01-14
Filing date: 2020-01-14
Publication date: 2021-04-13
Anticipated expiration: 2040-01-14
Also published as: CN111208302A

Abstract

The invention relates to a chemiluminescence detection kit for detecting a swine foot-and-mouth disease O-type antibody by using multi-epitope tandem protein, which belongs to the technical field of biology and comprises a chemiluminescence plate coated with an M2 antigen, an enzyme-labeled secondary antibody, a serum diluent, a 20 xPBST washing solution, a positive control serum, a negative control serum, a chemiluminescence substrate A solution and a chemiluminescence substrate B solution. The M2 antigen is a multi-epitope tandem protein capable of being distinguished, and the multi-epitope tandem protein is obtained by connecting differential epitopes of different isolates in 3 topological types of O-type foot-and-mouth disease in series; the identification type epitope is 140-154 amino acids of the G-H ring. The kit has the advantages of high sensitivity, good specificity, good broad spectrum, no cross reaction with immune A-type and Asia 1-type monovalent vaccine serum, simple operation, short reaction time and good application prospect in the detection of foot-and-mouth disease O-type antibodies.

Description

Chemiluminescence detection kit for detecting O-type antibody of pig foot-and-mouth disease by using multi-epitope tandem protein

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a chemiluminescence detection kit for detecting an O-type antibody of swine foot-and-mouth disease by using multi-epitope tandem protein.

Background

Foot-and-mouth disease is an acute, febrile and highly infectious epidemic disease mainly infecting artiodactyls caused by foot-and-mouth disease virus (FMDV). At present, the prevention and control of foot-and-mouth disease in China mainly takes comprehensive prevention and control measures of immunization prevention as main and killing as auxiliary, and simultaneously, the titer detection of immune antibodies is combined to evaluate the immune effect. FMDV has seven serotypes, A, O, C, SAT1, SAT2, SAT3 and Asia 1. Foot-and-mouth disease strains epidemic in China have 5 epidemic strains of two serotypes, wherein O type 4 strains and A type 1 strains are O type Mya-98 strains, O type CATHAY strains, O type PanAia strains, O type Ind-2001 strains and A type Sea-97 strains respectively. The 4 circulating O-type strains are classified into 3 topologies, namely SEA, CATHAY and ME-SA topologies. At present, the domestic immune A, O bivalent vaccine is abundant, if the detection method can not distinguish the type well, if only one strain has good antigenicity (the integrity of 146S) after the immune, the antibody generated by the animal can be detected as the positive of the foot-and-mouth disease antibody by the diagnosis method which can not distinguish the type, because the cross immune protection is not available between different serotypes, and when the other strain is infected, the immune protection can be generated, so the diagnosis method which can distinguish the different serotypes is particularly important. In recent years, the prevalence of the type O foot-and-mouth disease is on the rise, and a simple and effective antibody detection method is an index for evaluating the immune effect and is also an important basis for formulating the immune program of the foot-and-mouth disease. The world animal health Organization (OIE) recommends methods for evaluating the immune effect of FMD, such as neutralization assay (VNT), liquid phase blocking ELISA (LPB-ELISA), and solid phase competition ELISA (SPC-ELISA). VNT is the best diagnostic method to evaluate the immune efficacy, but it requires the use of live virus and therefore cannot be used widely. And then gradually replaced by LPB-ELISA, which is a method established by using inactivated virus and polyclonal antibody, so that the method has cross reaction with different serotypes of immune animals, and is complex in operation, long in used time (about 4h) and high in cost. In recent years, various national scholars establish detection methods aiming at various serotypes by utilizing type-specific monoclonal antibodies, but the method has high requirements on the performance of the monoclonal antibodies, and the screening of the type-specific monoclonal antibodies is a key and difficult point.

The traditional ELISA diagnosis method is long in time consumption and low in sensitivity. Chemiluminescence immunoassay (CLIA) is a new detection technology developed on the basis of ELISA. Compared to ELISA, CLIA derives all its energy from chemical reactions, thus eliminating background fluorescence signals, has extremely high sensitivity, and can detect extremely low concentrations of analytes over a wide linear range.

Disclosure of Invention

In order to solve the defects in the prior art, the invention aims to provide a chemiluminescence detection kit for detecting the pig foot-and-mouth disease O-type antibody by utilizing multi-epitope tandem protein, the kit has high sensitivity and good specificity, does not have cross reaction with A-type and Asia I-type monovalent vaccine immune serum, and can really realize the distinction between types; the kit is short in time, and the whole experiment only needs 25 min.

In order to achieve the purpose, the invention adopts the specific scheme that:

a chemiluminescence detection kit for detecting a swine foot-and-mouth disease O-type antibody by using multi-epitope tandem protein comprises a chemiluminescence plate coated with an M2 antigen, an enzyme-labeled secondary antibody, a serum diluent, a 20 xPBST washing solution, a positive control serum, a negative control serum, a chemiluminescence substrate A solution and a chemiluminescence substrate B solution;

the M2 antigen is a multi-epitope tandem protein capable of being distinguished, and the multi-epitope tandem protein is obtained by connecting differential epitopes of different isolates in 3 topological types of O-type foot-and-mouth disease in series; the identification type epitope is 140-154 amino acids of the G-H ring.

Further, the preparation method of the multi-epitope tandem protein comprises the following steps:

(1) and screening of the differential epitope: comparing amino acid sequences of FMDV isolates on GenBank, selecting sites with differences among different serotypes, designing primers, annealing to form double-stranded DNA molecules respectively encoding epitope proteins, and connecting the double-stranded DNA molecules with linearized plasmids which are cut by the same enzyme to obtain a connection product; then the ligation product is transformed and expressed; after IPTG induced expression, WB is verified; performing ELISA verification after further purifying the protein available for verification;

(2) tandem of discriminatory epitopes: finally determining the amino acids at position 140-154 of the G-H ring as the dominant antigen epitope of the O-type foot-and-mouth disease; and then serially connecting the dominant antigen epitopes of 6 isolates of 3 topological type foot-and-mouth disease O type, wherein the 6 isolates are respectively: O/MYA98/BY/2010, OS 99, O/SCGH/CHA/2016, Akesu/58, O/CHA/7/2011, O/XJBC/CHA/2017; then constructing a recombinant expression plasmid;

(3) and expressing and purifying the multi-epitope tandem protein: and D, transforming the recombinant expression plasmid obtained in the step two, carrying out IPTG induced expression, centrifuging and ultrasonically crushing the bacterial liquid, and taking the supernatant for purification to obtain a multi-epitope tandem protein product.

Further: the amino acid sequence of the multi-epitope tandem protein is SEQ ID NO: 2, respectively. Further, the base sequence of the multi-epitope tandem protein is SEQ ID NO: 1 is shown.

Further, the chemiluminescence plate is prepared by diluting purified M2 antigen protein to 500ng/ml with carbonate buffer solution, adding 100 μ l/well into the chemiluminescence plate, and coating overnight at 4 ℃; after PBST washing 3-4 times, 200. mu.l of blocking solution was added to each well and blocked at 37 ℃ for 2 h. Drying at room temperature for 2h after throwing off and patting, bagging, vacuum sealing, and storing at 4 deg.C.

Further: the blocking solution is PBS buffer solution containing 0.05 percent of Tween-20 by volume fraction, 5 percent of sucrose by mass fraction, 1 percent of BSA by mass fraction and 0.1 percent of proclin-300 by mass fraction.

Further, the enzyme-labeled secondary antibody is a rabbit anti-pig IgG antibody labeled by horseradish peroxidase; the serum diluent is a phosphate buffer solution containing 1% of casein by mass, 0.05% of Tween-20 by volume, 0.1% of proclin-300 by mass, 2% of sucrose by mass and 2% of escherichia coli lysate by volume; the 20 XPBST is phosphate buffer solution with pH 7.2-7.4 and concentration of 0.2mol/L, and contains 1% of Tween-20 by volume fraction; the chemiluminescence solution A contains 0.1mmol/L luminol and 0.07mol/L luminescence enhancer IPP, and the chemiluminescence solution B is 3mmol/L H₂O₂And (3) solution.

Furthermore, the preparation method of the escherichia coli lysate comprises the following steps: transferring the pET-32a plasmid into escherichia coli, culturing for 6h at 37 ℃, and centrifugally collecting thalli; then dissolving the thalli in PBS, and carrying out ultrasonic treatment for 20min under the ice bath condition; then centrifuging, and taking the supernatant to obtain the Escherichia coli lysate.

Further, the positive control serum is serum collected BY immunizing a pig with the foot-and-mouth disease O/Mya98/BY/2010 monovalent inactivated vaccine for 3 times; the negative control serum was healthy pig serum that was not immunized with any vaccine.

Has the advantages that:

1. the M2 antigen used by the chemiluminescence method for detecting the swine foot-and-mouth disease O-type virus antibody is epitope tandem protein which can be distinguished, and the epitope tandem protein comprises 3 topological epitopes of the domestic epidemic O-type foot-and-mouth disease. Foot-and-mouth disease virus is RNA virus, the mutation speed is high, the difficulty of screening specific monoclonal antibody is high, and the affinity between individual mutant strain and specific antibody is low, which leads to the omission of detection; or the monoclonal antibody has poor specificity and can react with viruses of other serotypes, so that the serotypes cannot be distinguished. The epitope tandem protein can greatly reduce the difficulty, and if the affinity to a new mutant strain or different strains transmitted from abroad is low, the epitope of the strain can be tandem-connected to the original sequence, so that the renewal can be realized in a short time.

2. In the preparation process of the M2 antigen, different linear epitopes are connected to a pGEX-4T-1 vector, WB and ELISA are used for verification, so that differential epitopes capable of being distinguished are screened, dominant epitopes capable of distinguishing O-type from A-type and Asia 1-type blood serum are determined, the epitopes of 3 topological 6 isolates of foot-and-mouth disease O-type are connected in series, and the obtained M2 antigen is used as an envelope protein, so that the distinction between types can be realized.

3. The chemiluminescence method for detecting the swine foot-and-mouth disease O-type virus antibody has the advantages of high sensitivity, good specificity, good broad spectrum, no cross reaction with immune A-type and Asia 1-type monovalent vaccine serum, simple operation, short reaction time, capability of quickly and effectively detecting whether the serum contains the foot-and-mouth disease O-type antibody or not only within 25min, good application prospect in the detection of the foot-and-mouth disease O-type antibody, and technical support for comprehensive prevention and treatment of the foot-and-mouth disease.

Drawings

FIG. 1 is a discriminatory epitope differentiating serotypes by WB screening;

FIG. 2 is a discriminatory epitope differentiating serotypes by WB screening;

FIG. 3 is a SDS-PAGE pattern of M2 protein purification, wherein M is marker, 1 is pre-loading, 2 is flow-through, and 3 is purified M2;

FIG. 4 is a graph comparing the optimization of antigen coating concentration and serum dilution by checkerboard titration;

FIG. 5 is a ROC plot of M2-CLIA antibody detection kit detecting known background porcine serum;

FIG. 6 is a background cross-point diagram of M2-CLIA antibody detection kit for detecting known background pig serum, wherein 0 represents foot-and-mouth disease negative serum, and 1 represents foot-and-mouth disease O type positive serum;

FIG. 7 shows M2-CLIA, domestic reagent kit O-LPBE, Italy O-SPCE, French ID

Comparison of analytical sensitivity for FMD Type O composition;

FIG. 8 is a graph of the stability of the chemiluminescent plate at 37 ℃.

Detailed Description

A chemiluminescence method for detecting swine foot-and-mouth disease virus O-type antibody comprises a chemiluminescence plate coated with antigen, an enzyme-labeled secondary antibody, a serum diluent, a 20 xPBST washing solution, a positive control serum, a negative control serum, a chemiluminescence substrate A solution and a chemiluminescence substrate B solution;

the chemiluminescent plate coated with the antigen is a white detachable polystyrene 96-well plate coated with different O-type FMDV strain tandem epitopes, and the preparation method comprises the following steps: different O type FMDV strains are connected in series, expression and purification are carried out to obtain the envelope antigen, then the fusion protein is diluted to be 500ng/ml by carbonate buffer solution with pH 9.6, the solution is added into a 96-hole white board according to the amount of 100 mu l/hole, and the solution is enveloped overnight at 4 ℃ (12 h); PBST washing solution is washed for 3-4 times; adding sealing liquid 200 μ l/hole, sealing at 37 deg.C for 2 hr, drying at room temperature for 2 hr, packaging, sealing, and storing at 4 deg.C.

Further, the preparation method of the tandem epitope of different O-type FMDV strains comprises the following steps:

(1) and screening of the differential epitope: by comparing amino acid sequences of FMDV isolates on GenBank, sites with differences among different serotypes are selected, primers are designed, double-stranded DNA molecules which respectively code epitope proteins are formed by annealing, the double-stranded DNA molecules are respectively connected with pGEX-4T-1 linearized plasmids which are cut by the same enzyme, and then the connection products are transformed into escherichia coli BL21(DE3) competent cells. After IPTG induced expression, WB was verified. The validated proteins were further purified for ELISA validation.

(2) Tandem of discriminatory epitopes: finally, the G-H loop (amino acid 154-140) is determined to be the dominant epitope of the O-type foot-and-mouth disease, and the O-type can be distinguished from the A-type and Asia 1-type serum. Then, the epitopes of 3 topological 6 isolates of the foot-and-mouth disease O type are connected in series (O/MYA98/BY/2010, OS 99, O/SCGH/CHA/2016, Akesu/58, O/CHA/7/2011, O/XJBC/CHA/2017), and a recombinant expression plasmid pET-32a-M2 is constructed.

(3) Expression and purification of tandem epitope M2: the recombinant plasmid pET-32a-M2 is transformed into BL21(DE3) competent cells, after IPTG induction expression, the bacterial liquid is centrifuged and ultrasonically crushed, and the supernatant is taken and purified by a His prepacked column.

Further, the blocking solution is phosphate buffer solution containing 0.05% Tween-20, 5% sucrose, 1% BSA, 0.1% proclin-300.

The enzyme-labeled secondary antibody is a rabbit anti-pig IgG antibody labeled by horseradish peroxidase.

The serum diluent is a phosphate buffer solution containing 1% of casein, 0.05% of Tween-20, 0.1% of proclin-300, 2% of sucrose and 2% of Escherichia coli lysate.

Further, the Escherichia coli lysate is obtained by transferring pET-32a plasmid into Escherichia coli, culturing at 37 ℃ for 6h, and centrifuging to collect thalli. Then, the cells were dissolved in PBS (20-fold concentration, i.e., cells collected from 500ml of the cell suspension were dissolved in 25ml of PBS), and sonicated for 20min under ice bath conditions (sonication for 4s, interval 4 s). Then, the mixture is centrifuged, and the supernatant is taken, so that the Escherichia coli lysate is obtained.

The 20 XPBST was 0.2M phosphate buffer (pH 7.2-7.4) containing 1% Tween-20, sterilized with a 0.22 μ M filter.

The positive negative control serum: taking the serum collected from 3 times of immunized pigs with univalent inactivated vaccine against foot-and-mouth disease O/Mya98/BY/2010 as standard positive control serum, wherein the antibody titer detected BY a domestic commercial reagent kit foot-and-mouth disease O type liquid phase blocking ELISA antibody detection kit (O-LPBE) is more than 1:512, the blocking rate detected BY Italy Solid Phase Competition ELISA (SPCE) detection kit (O-SPCE) is 93%, and French ID is used

The percent Competition (S/N%) for the FMD Type O Competition test was 4%; serum from healthy pigs not immunized with any vaccine, with an antibody titer < 1:8 as determined by O-LPBE and a blocking rate of 2% as determined by O-SPCE in Italy, was used as a standard negative control serum, with ID in France

Percentage of Competition measured by FMD Type O Competition (S/N%) was 102% using

FMDV NS ELISA the blocking rate was-7%.

The chemiluminescence solution A contains 0.1mM luminol and 0.07M luminescence enhancer IPP, and the chemiluminescence solution B is 3mM H₂O₂And (3) solution.

The chemiluminescence method for detecting the swine foot-and-mouth disease O-type virus antibody comprises the following operation steps:

1. and (3) balancing at room temperature: the kit taken out at 4 ℃ was returned to room temperature for further use.

2. Preparing a washing solution: diluting 20 times of PBST in the kit by deionized water or distilled water.

3. Serum dilution: in a U-shaped dilution plate, the serum to be detected and the positive negative control serum are diluted by 1:20 times by using serum diluent, and each plate is provided with 3 positive control sera and 3 negative control sera. Shaking and mixing evenly.

4. Sample adding and incubation: serum from the dilution plate of step 3 was transferred to an antigen-coated chemiluminescence plate in order and reacted at 37 ℃ for 10 min.

5. Washing the plate: discarding the reaction solution, adding 300 μ l of diluted washing solution into each well, washing for 4-6 times, shaking and washing for 30-60s in the middle, and finally drying.

6. Adding enzyme for incubation: mu.l of rabbit anti-pig IgG labeled with horseradish peroxidase was added to each well and reacted at 37 ℃ for 10 min.

7. Washing the plate: the same as step 5.

8. And (3) detection: mixing chemiluminescence A solution and B solution 1:1, adding 100 μ l into each hole, reacting at room temperature for 5min, and detecting chemiluminescence value with chemiluminescence detector.

And (3) judging a detection result:

1. the chemiluminescence value of the positive control should be greater than or equal to 7,000,000.

2. The chemiluminescence of the negative control should be < 50,000.

3. The test results were evaluated as Percent Positive (PP) × 100% (CLIA value of test sample-average CLIA value of negative control)/(average CLIA value of positive control-average CLIA value of negative control). When the PP is more than or equal to 3.5 percent, judging that the foot-and-mouth disease O-type antibody is positive; PP is more than or equal to 2.2 and less than 3.5, and is judged to be suspicious; and the PP is less than 2.2 percent, and the foot-and-mouth disease is judged to be negative O type.

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

Example 1 foot-and-mouth disease O-type differential epitope screening and construction of epitope tandem proteins

1. Screening of the identifying epitope: current studies indicate that there are 5 neutralizing epitopes for type O foot-and-mouth disease, of which only the G-H loop and the C-terminus of VP1 are linear epitopes. In order to be able to screen out epitopes that can distinguish between type O and type A and type Asia 1, the present study compared the amino acid sequences of FMDV isolates on GenBank, selected sites that differ in the C-terminus of the G-H loop and VP1 of different serotypes, as well as in the topology of type O (representative strains in different topologies), and then designed primers (see Table 1) and annealed to form double-stranded DNA molecules that encode the proteins of each epitope, respectively. The specific operation is as follows: mu.l of each of the 100. mu.M forward primer and 100. mu.M reverse primer was added to the PCR tube, heated in a 95 ℃ water bath for 5min, and then slowly returned to room temperature. Mu.l of the above target product recovered to room temperature, 2. mu.l of pGEX-4T-1 linearized plasmid which had been digested with the same enzymes, 1. mu. l T4 ligase and 1. mu.l of 10 XT 4 ligase buffer were ligated at 37 ℃ overnight. And then transforming the ligation product into escherichia coli DH5 alpha for sequencing verification, extracting plasmids from the escherichia coli with correct sequencing, transferring the plasmids into BL21(DE3) competent cells, coating an LB plate, selecting a single clone to transfer into LB with ampicillin resistance, culturing for 2.5 hours (OD is 0.6-0.8), adding 1mM IPTG (isopropyl-beta-thiogalactoside) for induction for 6 hours, collecting thalli, resuspending the thalli with PBS, sampling, quantifying proteins by a Braford method, and verifying WB. The primary antibody is hyperimmune serum collected after 3 times immunization of pigs with 1: 200-fold diluted foot-and-mouth disease O (P1507), A (P1510) and Asia 1(P1522) monovalent vaccine pig serum (the antibody titer detected BY a domestic commercial reagent kit foot-and-mouth disease O, A, Asia type liquid phase blocking ELISA antibody detection kit is more than 1:512), the hyperimmune serum is incubated for 1h at room temperature, and 1: 5000-fold diluted horse radish peroxidase-labeled rabbit anti-pig IgG is added after PBST washing.

And purifying the protein which is available for verification and then performing ELISA verification. The purification method comprises the following specific steps: the cells collected after induction were washed 1 time with PBS, resuspended in PBS and sonicated, centrifuged at 10,000 rpm for 20min, the supernatant was removed and filtered through a 0.22 μm filter. 2ml of GST resin packing was loaded into a column, 5 column volumes were washed with PBS, and the filtered protein was added to the column and incubated at room temperature for 30 min. After washing 5 column volumes with PBS, the protein of interest was eluted with 3 column volumes of Elution buffer. The purity of the purification was characterized by SDS-PAGE and further verified by ELISA after quantification by Braford.

2. Tandem of the discriminatory epitopes: WB results show that the G-H ring epitope of the hyperimmune serum prepared BY immunizing the O/Mya98/BY/2010 monovalent vaccine has strong reactivity with SEA and ME-SA topological strains, and the reactivity with CATHAY topological strains is weak; the C-terminus of VP1 was poorly reactive with hyperimmune serum, indicating that VP 1C-terminus is not a dominant epitope in type O foot-and-mouth disease (FIG. 1, FIG. 2). Through WB and ELISA verification, the amino acid position 140-154 of the G-H loop is finally determined to be the dominant epitope of O-type foot-and-mouth disease, and O-type can be distinguished from A-type and Asia 1-type blood serum, so the epitope of 3 topological 6 isolates of the foot-and-mouth disease O-type is connected in series in the research (O/MYA98/BY/2010, OS 99, O/SCGH/CHA/2016, Akesu/58, O/CHA/7/2011, O/XJBC/CHA/2017), and the base sequence of a recombinant expression plasmid pET-32a-M2, M2 is shown as SEQ ID NO: 1 and an amino acid sequence shown as SEQ ID NO: 2, respectively.

3. Expression and purification of tandem epitope M2: the recombinant plasmid pET-32a-M2 is transformed into BL21(DE3) competent cells, after IPTG induction expression, the bacterial liquid is centrifuged and crushed by ultrasound, and the supernatant is filtered by a 0.22 μ M filter. The recombinant protein was purified by connecting to 1ml His excel pre-packed column using AKTA pure protein purifier to obtain recombinant protein of foot-and-mouth disease virus O2 (FIG. 3).

Table 1 nucleotide sequence of each epitope:

example 2 optimization of chemiluminescence method for detecting O-type antibodies of swine foot-and-mouth disease by using multi-epitope tandem proteins

The serum collected from 3 times of pigs immunized with univalent inactivated vaccine against foot-and-mouth disease O/Mya98/BY/2010 was used as standard positive control serum with antibody titer > 1:512 detected BY O-LPBE and blocking rate 93% detected BY Italy's Solid Phase Competitive ELISA (SPCE) detection kit (O-SPCE), and French ID

FMDV NS ELISA the blocking rate was-7%. Antigen coating concentration (4, 2, 1, 0.5, 0.25, 0.125. mu.g/ml) and serum dilution (1:10, 1:20, 1:40, 1:80) were optimized by checkerboard titration, and the optimal coating concentration of M2 antigen was determined to be 0.5. mu.g/M by considering signal-to-noise ratio and economic factors, as shown in FIG. 4The optimal dilution of serum was 1: 20.

Example 3 determination of Cut-off value, diagnostic sensitivity and diagnostic specificity of the method by known background sera

1. The method comprises the following operation steps

Diluting the serum to be detected and the positive negative control serum by 1:20 times by using serum diluent in a U-shaped dilution plate, namely adding 114 mu l of serum diluent in the U-shaped dilution plate, then adding 6 mu l of the serum to be detected and the positive negative control serum, setting 3 positive control sera and 3 negative control sera in each plate, and shaking and uniformly mixing. The diluted sera from the above dilution plates were then transferred in order to a chemiluminescence plate coated with M2, 100. mu.l/well, and reacted at 37 ℃ for 10 min. Then, the reaction solution was discarded, washed with PBST for 5 times, and then 100-fold diluted enzyme-labeled secondary antibody was added thereto, and reacted at 37 ℃ for 10 min. Washing with PBST for 5 times after discarding, adding 100 μ l chemiluminescent solution A and B at a ratio of 1:1, and detecting chemiluminescence value with chemiluminescence detector after 5 min. The test results were evaluated as Percent Positive (PP) × 100% (CLIA value of test sample-average CLIA value of negative control)/(average CLIA value of positive control-average CLIA value of negative control).

2. Establishment of serum tray

a. The total 208 sera come from clinically healthy pigs which are not immunized with the foot-and-mouth disease vaccine, and the O-LPBE and the A-LPBE are used for detecting the sera, so that the sera are proved to have no O-type and A-type antibodies of the foot-and-mouth disease; by French ID

The percent Competition (S/N%) measured by FMD Type O Competition is<50 percent. These sera were used to evaluate the diagnostic specificity of the competitive chemiluminescence method.

b. The diagnostic specificity of the method is evaluated by collecting 50 parts of pig serum immunized by the A type monovalent vaccine (AF72), namely 5 pigs immunized by the self-made AF72 monovalent vaccine, 0d is recorded as primary immunization, 21d is used for secondary immunization, 42d is used for tertiary immunization, 3d, 7d, 14d, 21d, 28d, 35d, 42d, 49d, 56d and 63d after immunization.

c. A total of 49 porcine sera immunized with Asia 1 type monovalent vaccine (JS05), i.e. 5 pigs immunized with homemade JS05 monovalent vaccine, 0d as first immunization, 21d for secondary immunization, 42d for tertiary immunization, 3d, 7d, 14d, 21d, 28d, 35d, 42d, 49d, 56d, 63d after immunization, and sera were collected to evaluate the diagnostic specificity of the method.

d. A total of 40 parts of pig serum immunized BY O type monovalent vaccine (O/Mya98/BY/2010), namely 5-head pigs are immunized BY the self-made O/Mya98/BY/2010 monovalent vaccine, 0d is recorded as primary immunization, 21d is subjected to secondary immunization, 42d is subjected to tertiary immunization, 3d, 7d, 14d, 21d, 28d, 35d, 42d, 49d, 56d and 63d after immunization are used for collecting serum, and the diagnostic sensitivity of the method is evaluated BY using the serum 14d-63d after immunization.

e. 52 sera of pigs immunized with monovalent vaccine type O (O/Mya98/XJ/2010+ O/GX/09-7) were collected 35d, 56d, 84d after immunization and evaluated for diagnostic sensitivity.

f. 26 sera were collected from pigs challenged with O/MYA98/BY/2010 strain in the laboratory 10-20 days after challenge and were used to evaluate the diagnostic sensitivity of the method.

g. 30 parts of O, A swine serum immunized with bivalent vaccine (O/MYA98/BY/2010 strain + Re-A/WH/09 strain) and used for evaluating M2-CLIA and ID

The match rate of FMD Type O completion.

h. 165 parts of O, A swine serum immunized with a bivalent vaccine (OHM/02 strain + AKT-III strain), wherein 30 parts of the serum are swine immunized 5 to 15 times with the vaccine; in addition, 21 out of 135 sera were collected at immunization 28d, 40 at immunization 30d, 38 at immunization 42d, and 36 at immunization 56 d. The sera were used for evaluation of M2-CLIA and ID

The match rate of FMD Type O completion.

i. 68 parts of swine serum immunized with monovalent vaccine type O (O/Mya98/XJ/2010+ O/GX/09-7) collected 3 weeks after immunization, and the sera were usedEvaluation of M2-CLIA and ID

The match rate of FMD Type O completion.

j. 46 sera were collected from pigs immunized with O, A bivalent vaccine in the field and used to evaluate M2-CLIA and ID

The match rate of FMD Type O completion.

k. 22 sera were collected from pigs immunized with the polypeptide vaccine and used to evaluate M2-CLIA for ID

The match rate of FMD Type O completion.

3. Cut-off value, diagnostic sensitivity and diagnostic specificity of the method

The Cut-off value of this method was determined using serum of known clear background and evaluated for diagnostic sensitivity and specificity. Cut-off values were analyzed using the ROC curve (FIG. 5) and a background interaction plot (FIG. 6). The test results showed that in the detection of pig serum, the diagnostic sensitivity was 100% and the diagnostic specificity was 99.37% at a PP value of 2.18% (FIG. 5). In order to better distinguish the types and improve the specificity, the Cut-off value is determined to be 3.5, namely when the PP is more than or equal to 3.5 percent, the foot-and-mouth disease O-type antibody is judged to be positive; PP is more than or equal to 2.2 and less than 3.5, and is judged to be suspicious; and the PP is less than 2.2 percent, and the foot-and-mouth disease is judged to be negative O type.

4. M2-CLIA, O-LPBE, O-SPCE in Italy, ID in France

FMD Type O Competition test for the rate of coincidence of known background sera

M2-CLIA、O-LPBE、O-SPCE、ID

The FMD Type O Competition examined 425 clear background porcine sera. Results of the experimentAs shown in Table 2, M2-CLIA, O-LPBE, ID were detected in 208 non-immunized negative sera

The detection coincidence rate of FMD Type O Competition is 100%, which shows that the three methods are good in the specificity of detecting the serum sample of the non-immune animal. However, when 50 parts of serum collected by a self-made AF72 monovalent vaccine immunized pig is detected by using O-LPBE, false positive often appears after the third immunization, and the coincidence rate is 86%; when the self-made Asia 1/JS05 monovalent vaccine is used for immunizing pig serum, the cross reaction is serious, and the serum is detected to be positive 7 days after secondary immunization, so the coincidence rate is very low and is only 36.73 percent. When the AF72 monovalent vaccine immune pig serum is detected by O-SPCE, false positive begins to appear 21d after the second immunization, and when Asia 1/JS05 monovalent vaccine immune pig serum is detected, the serum is detected to be positive 7d after the second immunization. By ID

When the FMD Type O Competition detects the AF72 monovalent vaccine immunized pig serum and the Asia 1/JS05 monovalent vaccine immunized pig serum, doubtful or positive results are often generated after three-immunity, and the coincidence rate is 78% and 71.43% respectively. And M2-CLIA can well distinguish O type from A type and Asia 1 type, only one suspicious part is detected in each pig serum immune-collected by AF72 univalent vaccine and Asia 1/JS05 univalent vaccine, which shows that the method has good specificity and can realize type distinguishing.

When M2-CLIA is used for detecting pig serum immunized BY O/Mya98/BY/2010 monovalent vaccine, only one pig is detected to be suspicious at 14d and 21d after immunization, and the coincidence rate is 95%; by ID

In FMD Type O Competition detection, 5 sera which are total are detected as suspicious only at 14d and 21d after immunization, and the coincidence rate is 87.5%; when the O-SPCE is used for detection, all pigs are detected to be positive at 28d after immunization, so that the sensitivity is low, and the coincidence rate is only 77.5%; O-LPBE was found to be positive at 21d after immunization, and the coincidence rate was 85%.

In addition to the above-mentioned evaluation kitIn addition to sensitivity and specificity, broad spectrum is also important. Therefore, pig serum immunized with O/Mya98/XJ/2010+ O/GX/09-7 monovalent vaccine and pig serum challenged with O/MYA98/BY/2010 were used to evaluate the sensitivity and broad spectrum of the diagnostic method, and the results showed M2-CLIA and ID

The sensitivity and broad spectrum of FMD Type O composition are good.

Table 2: four methods for detecting coincidence rate of known background serum

Example 4 evaluation of M2-CLIA, O-LPBE, O-SPCE in Italy, ID in France

Analytical sensitivity of FMD Type O Competition

The standard positive sera were diluted in multiples (1:5-1:1280) and then treated with M2-CLIA, O-SPCE in Italy, ID in France

FMD Type O composition was performed. The results are shown in FIG. 7, O-SPCE and ID

The highest dilution of FMD Type O Competition detection standard positive serum is slightly more than 1:160, the antibody titer of O-LPBE detection standard positive serum is slightly more than 1:512, and the highest dilution of M2-CLIA detection standard positive serum is slightly more than 1:640, so that M2-CLIA has higher analysis sensitivity.

Example 5M2-CLIA and French ID

FMD Type O Competition detection O, A porcine serum immunized by bivalent vaccine and porcine blood immunized by O Type univalent vaccine (XJ/2010+ GX/09-7) for 3 weeksAnd cleaning and polypeptide vaccine, and comparing the diagnostic ability.

With M2-CLIA and ID

FMD Type O Competition detects O, A bivalent vaccine (BY/2010+ WH/09 and OHM/02+ AKT-III) immune pig serum with good coincidence rate, which is 100%, 100% and 94.07% respectively. The two methods were used to detect the low coincidence rate (72.06%) in 3-week immunized pigs, and positive sera with M2-CLIA compared to ID

The FMD Type O Competition is more probably due to the high sensitivity of M2-CLIA analysis. ID when 2-8 times of sera from O, A bivalent seedlings in field and 135 parts of sera from O, A bivalent seedlings (OHM/02+ AKT-III) were tested

FMD Type O Competition was more positive than M2-CLIA, probably due to ID

False positive exists between FMD Type O Competition and A Type vaccine immune serum, and after A Type vaccine containing immune serum, especially after multiple immunizations, individual false positive appears, so that the detection positive rate of the kit is higher than that of M2-CLIA. The results of two methods for detecting the polypeptide vaccine show that the coincidence rate of the two methods is good and reaches 95.45 percent (Table 3). The result further shows that M2-CLIA has good sensitivity and broad spectrum, and has good application prospect in the detection of O type foot-and-mouth disease antibodies.

Table 3: comparison of M2-CLIA with ID

Diagnostic capabilities of FMD Type O Competition

Example 6 stability of M2-CLIA plaques

The chemiluminescent plate coated with M2 was placed at 37 ℃ for 7 days for accelerated storage experiments. The results show that: after the CLIA plate was left for 7 days, 4 positive sera and 2 negative sera were still very reactive (fig. 8). The stability of the coated plate is good, and the coated plate can be placed at 4 ℃ for 1 year.

It should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that certain insubstantial modifications and adaptations of the present invention can be made without departing from the spirit and scope of the invention.

SEQUENCE LISTING

<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences

<120> chemiluminescence detection kit for detecting O-type antibodies of foot-and-mouth disease by using multi-epitope tandem proteins

<130> 1

<160> 28

<170> PatentIn version 3.3

<210> 1

<211> 339

<212> DNA

<213> Artificial Synthesis

<400> 1

ggtggttcat caggtggttc actgcccaac gtgagaggcg atctccaagt gctggctcag 60

aagccaggcc caggcagtgc caataacgtc agaggcgacc tacacgtgtt ggctaagaac 120

ggcggccgcg tgagcaacgt gaggggtgac cttcaagtgt tggctcagaa gggctcaggc 180

tcaggcccgg tgaccaaagt aagaggtgac cttcaagtgt tggctcagaa gggcggcgct 240

gtgaccaacg tgaggggtga cttgcaagtg ttggctcaga agccaggccc aggccccgtg 300

accaacgtga ggggtgacct acaagtgttg gcccagaag 339

<210> 2

<211> 113

<212> PRT

<213> Escherichia coli BL21(DE3)

<400> 2

Gly Gly Ser Ser Gly Gly Ser Leu Pro Asn Val Arg Gly Asp Leu Gln

1 5 10 15

Val Leu Ala Gln Lys Pro Gly Pro Gly Ser Ala Asn Asn Val Arg Gly

20 25 30

Asp Leu His Val Leu Ala Lys Asn Gly Gly Arg Val Ser Asn Val Arg

35 40 45

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

50 55 60

Thr Lys Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Gly Gly Ala

65 70 75 80

Val Thr Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Pro Gly

85 90 95

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

100 105 110

Lys

<210> 3

<211> 54

<212> DNA

<213> Artificial Synthesis

<400> 3

gatcctacgg tgacaccagc gctaacaacg tgagaggtga ccttcaagtg taac 54

<210> 4

<211> 54

<212> DNA

<213> Artificial Synthesis

<400> 4

tcgagttaca cttgaaggtc acctctcacg ttgttagcgc tggtgtcacc gtag 54

<210> 5

<211> 54

<212> DNA

<213> Artificial Synthesis

<400> 5

gatcctacgc cgggggttca ctgaccaacg tgagaggcga tctccaagtg taac 54

<210> 6

<211> 54

<212> DNA

<213> Artificial Synthesis

<400> 6

tcgagttaca cttggagatc gcctctcacg ttggtcagtg aacccccggc gtag 54

<210> 7

<211> 69

<212> DNA

<213> Artificial Synthesis

<400> 7

gatccgggtc agggtcaggg tacgccgggg gttcactgac caacgtgaga ggcgatctcc 60

aagtgtaac 69

<210> 8

<211> 69

<212> DNA

<213> Artificial Synthesis

<400> 8

tcgagttaca cttggagatc gcctctcacg ttggtcagtg aacccccggc gtaccctgac 60

cctgacccg 69

<210> 9

<211> 84

<212> DNA

<213> Artificial Synthesis

<400> 9

gatccaagaa ttgcaaatac gccgggggtt cactgaccaa cgtgagaggc gatctccaag 60

tgctggctca gaaggcggcg aggc 84

<210> 10

<211> 84

<212> DNA

<213> Artificial Synthesis

<400> 10

gttcttaacg tttatgcggc ccccaagtga ctggttgcac tctccgctag aggttcacga 60

ccgagtcttc cgccgctccg agct 84

<210> 11

<211> 66

<212> DNA

<213> Artificial Synthesis

<400> 11

gatccaacgt gagaggcgat ctccaagtgc tggctcagaa ggcggcgagg ccgctgccta 60

cttctc 66

<210> 12

<211> 66

<212> DNA

<213> Artificial Synthesis

<400> 12

gttgcactct ccgctagagg ttcacgaccg agtcttccgc cgctccggcg acggatgaag 60

agagct 66

<210> 13

<211> 36

<212> DNA

<213> Artificial Synthesis

<400> 13

gatccagagg cgatctccaa gtgctgactc cgaagc 36

<210> 14

<211> 36

<212> DNA

<213> Artificial Synthesis

<400> 14

tcgagcttcg gagtcagcac ttggagatcg cctctg 36

<210> 15

<211> 36

<212> DNA

<213> Artificial Synthesis

<400> 15

gatccagagg cgatctccaa gtgctggctc agaagc 36

<210> 16

<211> 36

<212> DNA

<213> Artificial Synthesis

<400> 16

tcgagcttct gagccagcac ttggagatcg cctctg 36

<210> 17

<211> 66

<212> DNA

<213> Artificial Synthesis

<400> 17

gatccaagca cccgagtgcg gctagacaca aacagaaaat agtggcacct gtaaagcagt 60

ccttgc 66

<210> 18

<211> 66

<212> DNA

<213> Artificial Synthesis

<400> 18

tcgagcaagg actgctttac aggtgccact attttctgtt tgtgtctagc cgcactcggg 60

tgcttg 66

<210> 19

<211> 75

<212> DNA

<213> Artificial Synthesis

<400> 19

gatcctacgc cgggggctca ctgcccaacg tgagaggcga tctccaagtg ctggctcaga 60

aggcagcgag gccgc 75

<210> 20

<211> 75

<212> DNA

<213> Artificial Synthesis

<400> 20

tcgagcggcc tcgctgcctt ctgagccagc acttggagat cgcctctcac gttgggcagt 60

gagcccccgg cgtag 75

<210> 21

<211> 51

<212> DNA

<213> Artificial Synthesis

<400> 21

gatcctcact gcccaacgtg agaggcgatc tccaagtgct ggctcagaag c 51

<210> 22

<211> 51

<212> DNA

<213> Artificial Synthesis

<400> 22

tcgagcttct gagccagcac ttggagatcg cctctcacgt tgggcagtga g 51

<210> 23

<211> 51

<212> DNA

<213> Artificial Synthesis

<400> 23

gatcccccgt gaccaacgtg aggggtgacc tacaagtgtt ggcccagaag c 51

<210> 24

<211> 51

<212> DNA

<213> Artificial Synthesis

<400> 24

tcgagcttct gggccaacac ttgtaggtca cccctcacgt tggtcacggg g 51

<210> 25

<211> 75

<212> DNA

<213> Artificial Synthesis

<400> 25

gatcctacgg tgacgccagt gccaacaacg tcagaggcga ccttcacgtg ttagctaaga 60

acgcggaaag aactc 75

<210> 26

<211> 75

<212> DNA

<213> Artificial Synthesis

<400> 26

tcgagagttc tttccgcgtt cttagctaac acgtgaaggt cgcctctgac gttgttggca 60

ctggcgtcac cgtag 75

<210> 27

<211> 51

<212> DNA

<213> Artificial Synthesis

<400> 27

gatccagtgc caacaacgtc agaggcgacc ttcacgtgtt agctaagaac c 51

<210> 28

<211> 51

<212> DNA

<213> Artificial Synthesis

<400> 28

tcgaggttct tagctaacac gtgaaggtcg cctctgacgt tgttggcact g 51

Claims

1. A chemiluminescence detection kit for detecting the O-type antibody of the pig foot-and-mouth disease by utilizing multi-epitope tandem protein is characterized in that: comprises a chemiluminescence plate coated with an M2 antigen, an enzyme-labeled secondary antibody, a serum diluent, 20 xPBST cleaning solution, positive control serum, negative control serum, a chemiluminescence substrate A solution and a chemiluminescence substrate B solution;

the M2 antigen is a multi-epitope tandem protein capable of being distinguished, and the multi-epitope tandem protein is obtained by connecting differential epitopes of different isolates in 3 topological types of O-type foot-and-mouth disease in series; the identification epitope is 140-154 amino acids of the G-H ring;

the preparation method of the multi-epitope tandem protein comprises the following steps:

(2) tandem of discriminatory epitopes: finally determining the amino acids at position 140-154 of the G-H ring as the dominant antigen epitope of the O-type foot-and-mouth disease; and then serially connecting the dominant antigen epitopes of 6 isolates in 3 topotypes of foot-and-mouth disease O type, wherein the 6 isolates are respectively: O/MYA98/BY/2010, OS 99, O/SCGH/CHA/2016, Akesu/58, O/CHA/7/2011, O/XJBC/CHA/2017; then constructing a recombinant expression plasmid;

(3) and expressing and purifying the multi-epitope tandem protein: and (3) transforming the recombinant expression plasmid obtained in the step (2), carrying out IPTG induced expression, centrifuging and ultrasonically crushing the bacterial liquid, and taking the supernatant for purification to obtain a multi-epitope tandem protein product.

2. The chemiluminescence detection kit for detecting the O-type antibody of the foot-and-mouth disease of the pigs by utilizing the multi-epitope tandem protein as claimed in claim 1, is characterized in that: the amino acid sequence of the multi-epitope tandem protein is SEQ ID NO: 2, respectively.

3. The chemiluminescence detection kit for detecting the O-type antibody of the foot-and-mouth disease by using the multi-epitope tandem protein according to claim 2, is characterized in that: the base sequence of the multi-epitope tandem protein is SEQ ID NO: 1 is shown.

4. The chemiluminescence detection kit for detecting the O-type antibody of the foot-and-mouth disease of the pigs by utilizing the multi-epitope tandem protein according to any one of claims 1 to 3, is characterized in that: the chemiluminescence plate is prepared by diluting purified M2 antigen protein to 500ng/ml by carbonate buffer solution, adding 100 mul/hole into the chemiluminescence plate, and coating overnight at 4 ℃; after PBST is washed for 3-4 times, 200 mul of blocking solution is added into each hole, and the holes are blocked for 2h at 37 ℃; drying at room temperature for 2h after throwing off and patting, bagging, vacuum sealing, and storing at 4 deg.C.

5. The chemiluminescent detection kit for detecting the O-type antibody of foot-and-mouth disease of pigs by using the multi-epitope tandem protein as claimed in claim 4, characterized in that: the blocking solution is PBS buffer solution containing 0.05 percent of Tween-20 by volume fraction, 5 percent of sucrose by mass fraction, 1 percent of BSA by mass fraction and 0.1 percent of proclin-300 by mass fraction.

6. The chemiluminescence detection kit for detecting the O-type antibody of the foot-and-mouth disease of the pigs by utilizing the multi-epitope tandem protein as claimed in claim 1, is characterized in that: the enzyme-labeled secondary antibody is horseradishPeroxidase-labeled rabbit anti-pig IgG antibody; the serum diluent is a PBS buffer solution containing casein with the mass fraction of 1%, Tween-20 with the volume fraction of 0.05%, proclin-300 with the mass fraction of 0.1%, cane sugar with the mass fraction of 2% and escherichia coli lysate with the volume fraction of 2%; the 20 XPBST is PBS buffer solution with the pH value of 7.2-7.4 and the concentration of 0.2mol/L, and contains 1% of Tween-20 by volume fraction; the chemiluminescence solution A contains 0.1mmol/L luminol and 0.07mol/L luminescence enhancer IPP, and the chemiluminescence solution B is 3mmol/L H₂O₂And (3) solution.

7. The chemiluminescent detection kit for detecting the O-type antibodies to porcine foot and mouth disease according to claim 6 utilizing the multi-epitope tandem protein is characterized in that: the preparation method of the escherichia coli lysate comprises the following steps: transferring the pET-32a plasmid into escherichia coli, culturing for 6h at 37 ℃, and centrifugally collecting thalli; then dissolving the thalli in PBS buffer solution, and carrying out ultrasonic treatment for 20min under the ice bath condition; then centrifuging, and taking the supernatant to obtain the Escherichia coli lysate.

8. The chemiluminescence detection kit for detecting the O-type antibody of the foot-and-mouth disease of the pigs by utilizing the multi-epitope tandem protein as claimed in claim 1, is characterized in that: the positive control serum is collected BY immunizing a pig with the foot-and-mouth disease O/Mya98/BY/2010 monovalent inactivated vaccine for 3 times; the negative control serum was healthy pig serum that was not immunized with any vaccine.