CN111961120B - African swine fever virus MGFs and CD2v ELISA antibody detection kit - Google Patents

African swine fever virus MGFs and CD2v ELISA antibody detection kit Download PDF

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CN111961120B
CN111961120B CN202010904919.9A CN202010904919A CN111961120B CN 111961120 B CN111961120 B CN 111961120B CN 202010904919 A CN202010904919 A CN 202010904919A CN 111961120 B CN111961120 B CN 111961120B
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张蕾
董春娜
李静
毕建敏
肖进
齐鹏
刘新月
李玲
李鹏宇
吴冬荀
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China Animal Husbandry Industry Co Ltd
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Abstract

The invention discloses an African swine fever virus MGFs and CD2v ELISA antibody detection kit. The kit comprises an enzyme-linked reaction plate and an enzyme-labeled secondary antibody, wherein the enzyme-labeled plate is coated with an African swine fever virus antigen epitope polypeptide composition. The antigen epitope polypeptide composition is one or any combination of more than two of a polypeptide shown in a sequence 1 in a sequence table, a polypeptide shown in a sequence 2 in the sequence table and a polypeptide shown in a sequence 3 in the sequence table. The kit uses chemically synthesized antigen peptide to coat the ELISA plate, has small antigen dosage and high sensitivity and specificity, and can efficiently detect whether MGF360-505R and CD2v antibodies generated by African swine fever virus are present, thereby realizing differential diagnosis. The kit has the advantages of high sensitivity, good specificity, convenient operation and good market prospect.

Description

African swine fever virus MGFs and CD2v ELISA antibody detection kit
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to an African swine fever virus ELISA antibody detection kit, in particular to an African swine fever virus MGF360-505R and CD2v synthetic peptide ELISA antibody detection kit.
Background
African swine fever is an acute, febrile and highly contagious animal infectious disease of pigs caused by African Swine Fever Virus (ASFV), and is clinically characterized by high fever, reticuloendothelial system bleeding and high mortality, and the mortality of susceptible swine herds reaches up to 100%.
ASFV belongs to the African swine fever virus family (Asfarviridae) genus African swine fever virus (Asfivirus). ASFV is an enveloped virus, the viral genome is a double-stranded DNA molecule, is 170-193kb in length, contains 151-167 Open Reading Frames (ORFs), encodes more than 150 polypeptides, and about 30% of the ORFs have multiple copies, i.e., multigene families (MGFs). The CD2v protein is a transmembrane protein, is an ASFV late expression protein, is coded by EP402R gene, and is involved in mediating the binding of erythrocytes to infected cells or virus particles to cause the phenomenon of erythrocyte adsorption.
The vaccine is the most effective and economical method for preventing and controlling the epidemic situation of the African swine fever. Although, at present, no African swine fever vaccine approved for sale at home and abroad exists; however, the current research shows that the African swine fever gene deletion vaccine is the most probable and most promising African swine fever vaccine, and particularly, the gene deletion strain or natural low virulent strain of MGF360-505R multigenome family and CD2v has good safety and immune protection effect, and can provide the virus attack protection against the homologous genotype and the heterologous genotype after immunization. "attenuated African swine fever virus with gene deletion and application thereof as vaccine" (application No: CN201910348878.7) disclosed in Kazakhstan veterinary research institute of Chinese academy of agricultural sciences, 8/6.2019, Pig/CN/HLJ/2018 is used as parent strain, and gene deletion virus with MGF360-505R deletion and combined deletion of CD2V and MGF360-505R is obtained by gene engineering technology. Experiments show that the two strains can provide 100 percent immune protection for the Chinese epidemic virulent strain of the African swine fever, can be used as a safe and effective vaccine for preventing and controlling the epidemic situation of the Chinese African swine fever, and has great social value. An African swine fever virus vaccine strain and a vaccine containing the vaccine strain, which are disclosed in 11/1/2019 and military veterinary research institute of military medical academy of sciences (application number: CN201910700685.3), are constructed by taking an ASFV SY18 strain as a parent strain through a genetic engineering technology, so that the vaccine strain lacking CD2V and MGF360-505R is reduced in virulence, can provide virus attack against the SY18 strain, has an immune protection effect, and can be used as a candidate vaccine strain. Therefore, the deletion of MGF360-505R and CD2V is the direction for developing future African swine fever vaccines. Therefore, the method for effectively identifying the ASFV gene deletion vaccine strain and the wild strain is an important means for establishing the effective prevention and control of ASFV, and a differential diagnosis method aiming at MGF360-505R and CD2v needs to be developed urgently to provide technical support for the application of the subsequent ASFV gene deletion inactivated vaccine, which has profound significance for establishing an African swine fever prevention and control system.
Disclosure of Invention
The invention aims to provide an indirect ELISA detection kit for detecting African swine fever virus MGF360-505R and CD2v protein antibodies, the kit utilizes African swine fever virus MGF360-505R and CD2v epitope polypeptide as coating antigen, establishes an indirect ELISA method with good specificity, sensitivity and repeatability, and is used for detecting whether the swine serum contains the antibodies of the African swine fever virus specific MGF360-505R and CD2 v.
In order to achieve the purpose, the African swine fever virus epitope polypeptide composition with excellent performance is obtained through screening, and the African swine fever virus epitope polypeptide composition provided by the invention is one or any combination of more than two of a polypeptide shown as a sequence 1 in a sequence table, a polypeptide shown as a sequence 2 in the sequence table and a polypeptide shown as a sequence 3 in the sequence table. When the polypeptide composition is two of the polypeptide shown in the sequence 1, the polypeptide shown in the sequence 2 and the polypeptide shown in the sequence 3, the mass ratio of the two polypeptides is (0.5-1.5): (0.5 to 1.5); preferably, the mass ratio of the components is 1: 1; when the polypeptide composition is three of the polypeptide shown in the sequence 1, the polypeptide shown in the sequence 2 and the polypeptide shown in the sequence 3, the mass ratio of any three polypeptides is (0.5-1.5): (0.5-1.5): (0.5 to 1.5); preferably, the mass ratio of the components is 1:1: 1;
the invention also claims an African swine fever virus antigen epitope polypeptide which is a polypeptide shown as a sequence 1 in a sequence table, a polypeptide shown as a sequence 2 in the sequence table or a polypeptide shown as a sequence 3 in the sequence table.
The African swine fever virus MGF360-505R and CD2v synthetic peptide ELISA antibody detection kit comprises an enzyme-linked reaction plate, positive control serum, negative control serum, enzyme-labeled secondary antibody, sample diluent, 20-time concentrated washing liquid, substrate liquid A, substrate liquid B and stop solution, wherein the enzyme-linked reaction plate is coated with the African swine fever virus epitope polypeptide composition.
The enzyme-linked reaction plate is a detachable 96-hole enzyme label plate; the polypeptides in the African swine fever virus epitope polypeptide composition are all obtained by chemical artificial synthesis.
The optimal preparation method and conditions of the ELISA reaction plate are that the African swine fever virus epitope polypeptide composition is dissolved in a carbonate solution with the pH value of 9.6, then the African swine fever virus epitope polypeptide composition is added into a 96-hole polystyrene ELISA reaction plate, 150ng of polypeptide or the polypeptide composition is placed at the temperature of 2-8 ℃ for 8-12 hours, the polypeptide antigen is fully combined with the ELISA reaction plate, then PBS buffer solution containing 5mg/ml Bovine Serum Albumin (BSA) and having the pH value of 7.4 is added into each hole according to 300 mu l, sealing treatment is carried out at the temperature of 37 ℃ for 2-3 hours, and after being dried, the ELISA reaction plate is sealed and stored at the temperature of 4 ℃.
The positive control serum is hyperimmune serum prepared by joint immunization of the polypeptide shown in the African swine fever virus antigen sequence 1, the polypeptide shown in the sequence 2 in the sequence table and the polypeptide shown in the sequence 3 in the sequence table; the positive control serum is obtained by the following steps: 1) and (2) respectively taking the polypeptide shown in the sequence 1, the polypeptide shown in the sequence 2 in the sequence table and the polypeptide shown in the sequence 3 in the sequence table for immunization, preparing hyperimmune serum as positive stock serum of the kit, and properly diluting the hyperimmune serum by using a sample diluent to obtain the positive control serum of the kit.
The negative control serum is swine serum free of Specific Pathogens (SPF).
The enzyme-labeled secondary antibody is a horse radish peroxidase-labeled rabbit anti-pig IgG antibody.
The substrate solution A is a citrate phosphate buffer solution containing 0.6mg/ml of urea hydrogen peroxide, and the substrate solution B is a tetramethylbenzidine solution containing 0.2mg/ml, and the substrate solution A and the substrate solution B are mixed in a ratio of 1:1 when in use. The stop solution is a 2mol/L sulfuric acid solution.
The kit also comprises a sample diluent and a 20-time concentrated washing solution; the sample diluent was phosphate buffer containing 5mg/ml casein with a value of 0.01M, pH of 7.4; the concentrated washing solution is 0.01M phosphate buffer solution with pH value of 7.4 and contains Tween-20 with concentration of 0.8-1.2% (ml/ml).
The detection program of the kit of the invention is as follows:
1. balancing: taking out the kit from the refrigeration environment, and standing at room temperature for balancing for 30min for later use; the liquid reagents were mixed well before use.
2. Preparing a liquid: diluting the concentrated washing solution by 20 times of distilled water or deionized water to obtain a washing buffer solution;
3. setting: 2 negative control holes and 2 positive control holes, and the rest are sample holes to be detected.
4. Pre-diluting a sample to be detected: and (3) using the sample diluent to mix the serum of the sample to be detected, the negative control serum and the positive control serum according to the ratio of 1: 20 in proportion.
5. Sample adding: each well was pre-diluted with 100. mu.l of the test sample. The time span of the sample application process should be as short as possible.
6. And (3) incubation: shaking and mixing evenly, placing in an incubator at 37 ℃ and reacting for 30 min.
7. Washing the plate: discarding the reaction solution, adding 300. mu.l of diluted washing buffer solution into each hole, soaking for 15s, throwing off the washing solution, continuously washing the plate for 4 times, and then patting dry.
8. Adding an enzyme: mu.l of horseradish peroxidase labeled rabbit anti-pig IgG antibody was added to each well.
9. And (3) incubation: the reaction mixture was placed in an incubator at 37 ℃ and reacted for 30 min.
10. Washing the plate: discarding the reaction solution, adding 300 mu l of diluted washing buffer solution into each hole, soaking for 15s, throwing away the washing solution, continuously washing the plate for 4 times, and then drying by beating.
11. Adding 100 μ l of substrate working solution (substrate working solution is obtained by mixing substrate solution A and substrate solution B in equal amount, and is prepared at present), shaking, mixing, placing in 37 deg.C incubator, and reacting for 15min in dark.
12. 50. mu.l of chromogenic stop solution was added to each well, and the reaction was stopped by shaking and mixing.
13. Determination of OD per well450nmValue (reaction plate with stop solution should read OD within 15min450nmValue).
And (3) judging a detection result:
1. negative control OD450nmThe average value should be less than or equal to 0.15, otherwise it is not effective.
2. Each test value of the positive control should be between 1.0 and 2.5, otherwise, the positive control is invalid.
3. Calculation of the critical value: critical value 0.17 × positive control OD450nmValue average.
Determination of OD in serum to be examined450nmIf the value is larger than or equal to the critical value, judging the value as positive; determination of OD in serum to be examined450nmValue of<The critical value is judged to be negative.
The kit can be used for detecting the African swine fever virus antibody so as to judge whether the African swine fever virus antibody generated after infection exists in the detected animal.
The application of the kit in the preparation of the kit for detecting whether the African swine fever virus is infected belongs to the protection scope of the invention;
the invention has the positive effects that: the invention adopts a bioinformatics method to accurately analyze the epitope of the African swine fever virus, and selects the peptide fragment suitable for ELISA detection from the main epitope on the proteins of MGF360, MGF505 and CD2 v. The peptide fragment concentrates the epitope and has the advantages of high sensitivity and strong specificity.
Meanwhile, the advanced solid-phase peptide synthesis technology is adopted to synthesize the polypeptide antigen for preparing the coated enzyme-labeled reaction plate.
In addition, the coating antigen used in the kit is a chemically synthesized polypeptide, does not contain hybrid protein, has high purity, and further improves the efficiency of detecting the African swine fever virus antibody so as to judge whether the tested animal is infected by the African swine fever virus.
In a word, the kit adopts the antigen peptide of the main antigen sites of the chemically synthesized structural proteins MGF360, MGF505 and CD2v to coat the enzyme-linked reaction plate, has the advantages of small antigen dosage, high sensitivity and strong specificity, and can effectively detect the antibody generated after the African swine fever virus wild strain is infected. The experimental result shows that the kit has good repeatability, strong specificity and high sensitivity. Can meet the requirements of personnel at different levels, and has wide market prospect and good economic and social benefits.
The African swine fever virus enzyme-linked immunosorbent assay diagnostic kit is used for detecting whether animals are infected with African swine fever virus wild strains, and is favorable for building an African swine fever virus prevention and control system in China.
Detailed Description
The methods in the following examples are all conventional methods unless otherwise specified.
Example 1 preparation of envelope antigen of ELISA antibody detection kit for synthetic peptides of African swine fever virus MGF360-505R and CD2v
The test adopts a bioinformatics method to accurately analyze the main antigen epitopes of MGF360, MGF505 and CD2v proteins of a Pig/HLJ/2018 strain sequence (MK333180.1) published by GenBank, screens out proper peptide fragments, respectively synthesizes three peptides by using a full-automatic polypeptide synthesizer, wherein the sequences are respectively shown as a sequence 1, a sequence 2 and a sequence 3 in a sequence table, and prepares a coating antigen with the purity of about 80 percent, so that the coating antigen can cover the main antigen epitopes of African swine fever virus MGF360, MGF505 and CD2v proteins, and the positive detection rate of the antibody is improved. The polypeptide synthesis method can be a conventional method, and the three polypeptides of the invention are synthesized by the following method as a coating antigen of the kit of the invention.
The coated antigen of the present invention can be prepared using an Applied Biosystem full-automatic polypeptide synthesizer (model 433A). The method applies a Merrifield solid phase synthesis method, adopts Fmoc (9-fluoromethylenecarbonyl, 9-fluorenylmethyloxycarbonyl) modified amino acid, and uses Rink Amide MBHA resin as a solid phase carrier. The production process comprises five parts of polypeptide antigen solid phase synthesis, polypeptide cracking and identification, antigen purification, freeze drying and storage. The following are described separately:
first, solid phase synthesis of envelope antigen
1. Preparation of synthetic reagents
The amino acid sequences of the synthetic envelope antigen are shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3.
Appropriate Fmoc-modified amino acids (purchased from NOVA) were prepared according to the coating antigen sequence and synthesis scale and added to the corresponding cartidge. Similarly, 5g of the resin was weighed on the desired synthesis scale, placed in the reaction chamber, the upper and lower caps were tightened, labeled, and the name of the synthesized peptide, the batch number, TARE in the reaction chamber and the weight of the resin weighed were recorded. The reaction chamber was loaded into the synthesizer. Appropriate amounts of synthesis reagents were prepared including 100% NMP, 3% AIM (hexanoyl imidazole), 35% PIP (piperidine), 100% MeOH (methanol), etc. and placed in corresponding reagent bottles.
2. Detection of synthesizer state
Checking 433A polypeptide synthesis instrument to determine whether it is normal, starting the instrument, running Run Self Test program, and Self-checking whether each index is normal. Further check if nitrogen is sufficient and system gauge pressure is normal (433A normal gauge pressure 10.2 psi). The flow rate of each synthesis reagent is measured because the performance of the instrument is known prior to synthesis. 433A synthesizer: sending Flow Rate1-18 to synthesizer, selecting Main Menu-Module Test-finding Module A, Module D, Module I, Module A according to Prer or next-measuring or observing according to Start-measuring according to more, if the Flow is not proper, regulating lower valve pressure until reaching the requirement (the specific detection requirement is shown in Table 1).
TABLE 1 flow rate test standard table for polypeptide synthesizer
Reagent Bottle number Module Standard range of
35％Piperidine 1 A 1.0～1.2ml
3％AIM 4 D 1.0～1.2ml
100％MeOH 9 I 3.5～4.0ml
DIC 8 I 0.45～0.55g
100％NMP 10 A 2.6～2.8ml
3. Initiation of coating antigen Synthesis
The amino acid sequence to be synthesized was sent to the Std Fmoc 1.0 Sol DIC90 on the synthesizer in the 433A synthesizer program. File-New-Sequence-editing the Sequence of the synthetic peptide, and storing. File-New-Run, check whether Chemistry is Std Fmoc 1.0 Sol DIC 90; whether Sequence is the stored name; setting Cycles; and (5) storing. And finally sending the data to a synthesizer.
Main Menu-Cycle Monitor-begin, run.
4. Coating antigen synthesis
The removal of Fmoc group and the electron-withdrawing action of fluorene ring system of Fmoc group make 9-H have acidity and be easily removed by weak base, during the reaction, piperidine (PIP) is used to attack 9-H, beta is eliminated to form dibenzofluorene alkene, and the dibenzofluorene alkene is easily attacked by secondary cyclic amine to form stable addition compound. Removal of the Fmov group exposes the "-NH 2" group for the synthesis reaction. The activated amino acid protected with the next Fmoc group and 1-Hydroxybenzotriazole (HOBT) were then added to the reactor.
The above polypeptide sequence is synthesized by repeating the synthesis steps (the synthesizer is automatically completed according to the program, and the specific cycle steps are as shown in the following table 2) from the C-terminal to the N-terminal in sequence according to a specific sequence. During which the recorded reagent dosage and the running condition were observed.
TABLE 2 coating antigen Synthesis cycle procedure
Figure GDA0003561374180000071
5. End of coating antigen Synthesis
The synthesizer will automatically stop after the synthesis of the coated antigen is complete and the peptide resin (peptide now also attached to the resin) is essentially washed clean. Then taking off the reactor from the polypeptide synthesizer, washing the peptide resin with 100% methanol for 3 times, drying in a fume hood, transferring all the polypeptide resin into a brown polyethylene bottle, placing into a refrigerator at-20 deg.C, and sealing with a sealing film for use.
Secondly, the cleavage and identification of the envelope antigen
1. Cleavage of polypeptide antigens
The polypeptide obtained by the above reaction is chemically bonded to the solid support, and the polypeptide must be separated from the solid support by acid hydrolysis using a specific strong organic acid. The acid hydrolysis also removes the protecting group on each amino acid function. The method comprises the following steps:
the synthesized polypeptide resin (meaning the peptide is also attached to the resin) was removed from the refrigerator, placed in a 2L round bottom flask, 90ml of Trifluoroacetic acid (TFA), 10ml of Tripropylsilane (TIS) and a magnetic stirrer were added to the flask in a fume hood, and the flask was then placed on a magnetic stirrer steadily and stirred at room temperature for 1h until the reaction was complete. After the reaction is finished, continuously evaporating for 30-120 min by using a rotary evaporator with a cold trap to remove TFA in the crude product. Then using dimethyl formamide (DMF) to wash the crude product of the polypeptide antigen for multiple times, and finally filtering the mixed resin by using a sand core funnel to obtain the coating antigen.
2. Identification of coating antigens
After the polypeptide antigen is synthesized, performing qualitative and quantitative analysis by matrix-assisted laser desorption time of flight mass spectrometry (MODAL-TOF) and reversed-phase high-pressure liquid chromatography (RP-HPLC), and identifying the synthesized peptide by common amino acid analysis.
3. Purification of coating antigen
The cyclized polypeptide antigen is ultrafiltered by using a circulating Tangential filtration membrane (a Tangential Flow Device circulating Tangential filtration membrane manufactured by PALL company and a peristaltic pump matched with the circulating Tangential filtration membrane), the polypeptide antigen as a macromolecule can not pass through a filtration membrane with a certain pore diameter, and small molecular impurities formed or introduced in the early synthesis process and the later cyclization reaction can pass through the filtration membrane. Then sterilizing through a filter with the pore diameter of 0.2 mu m, subpackaging the finally obtained solution into sterile plastic bottles, and labeling. The name, serial number, production lot number, concentration, production date, storage life and storage condition of the polypeptide are marked on the label, and the label is subpackaged and stored at-20 ℃ or-40 ℃ for later use.
4. Freeze drying of coated antigens
For long-term storage and transportation, the coated antigen needs to be freeze-dried to obtain the polypeptide in a solid state. And (3) placing the pre-frozen coated antigen on a freeze dryer of Labconco for drying to obtain the coated antigen in a solid state. And (4) labeling the packaged polypeptide, wherein the name, the number, the production batch number, the concentration, the production date, the storage period and the storage condition of the polypeptide are marked on the label.
Example 2 preparation of Positive control serum for ELISA antibody detection kit for African Swine fever Virus MGF360-505R and CD2v synthetic peptide
1. The immune animals are isolated and observed for 7 days by healthy pigs with the weight of about 40kg (which are negative through the detection of commercial African swine fever virus antigen and antibody detection kit).
2. The preparation of immunogen uses sterile deionized water to dilute epitope polypeptide (mass ratio of each antigen peptide is 1:1:1) containing sequence 1, sequence 2 and sequence 3 into 2mg/ml solution, and the solution is mixed with equal volume of Freund's complete adjuvant, fully emulsified for the first immunization, mixed with equal volume of Freund's incomplete adjuvant, fully emulsified for the second immunization.
3. The immunization program is divided into 2 immunizations, which are respectively carried out at 0 week and 3 weeks, the immunization dose is respectively 2ml and 4ml, and the injection is carried out in the muscle after the auricular root.
4. The preparation of positive control serum uses indirect ELISA to detect the serum antibody level, when the serum dilution potency is above 1:1280, the anterior vena cava aseptically collects blood, centrifugally separates serum, inactivates at 60 ℃ for 30 minutes, and the serum is used as positive stock serum (stored at minus 80 ℃) after passing the test. And (3) diluting the positive stock serum by using a sample diluent in a multiple ratio to determine the titer, and taking the positive stock serum according to the determined titer, and properly diluting the positive stock serum by using the sample diluent to ensure that the OD value is between 1.5 and 2.5, thereby obtaining the positive control serum of the kit.
Example 3 preparation of ELISA antibody detection kit for African swine fever Virus MGF360-505R and CD2v synthetic peptide
The method comprises the following steps:
(1) a 96-hole detachable polystyrene enzyme-linked reaction plate coated with African swine fever virus MGF360-505R and CD2v epitope polypeptide antigen; 2X 96 wells.
(2) Positive control serum: the hyperimmune serum prepared by immunizing epitope polypeptides shown in the sequence 1, the sequence 2 and the sequence 3 of the African swine fever virus is diluted by a sample diluent and used as positive control serum (1 tube, 1.5 ml/tube) of the kit.
(3) Negative control serum: is Specific Pathogen Free (SPF) pig serum as negative control serum for the kit (1 tube, 1.5 ml/tube).
(4) Enzyme-labeled secondary antibody: the sample was prepared by diluting horseradish peroxidase-labeled rabbit anti-porcine IgG (purchased from sigma, cat. Inc., cat. No. A5670) at a ratio of 1:30000, and then prepared into 2 bottles (12 ml/bottle).
(5) Sample diluent: 1 vial (24 ml/vial) of 0.01M, pH value 7.4 phosphate buffer containing 5mg/ml casein.
(6) Substrate solution A: is citrate phosphate buffer solution (1 bottle, 12 ml/bottle) containing 0.6mg/ml urea hydrogen peroxide
(7) Substrate solution B: a 0.2mg/ml solution of Tetramethylbenzidine (TMB) (1 vial, 12 ml/vial).
(8) Stopping liquid: 2mol/L sulfuric acid solution (1 bottle, 12 ml/bottle).
(9)20 times of concentrated washing solution: is 0.01M phosphate buffer solution (50 ml/bottle, 2 bottles) with pH value of 7.4 and containing Tween-20 with concentration of 0.8-1.2% (ml/ml).
Serum dilution plates (2, 96 wells/block) may also be present in the kit for dilution of serum samples, as desired.
The preparation method of the 96-hole detachable polystyrene enzyme-linked reaction plate coated with the African swine fever virus MGF360-505R and CD2v epitope polypeptide antigen comprises the following steps: 1. dissolving the polypeptide antigen prepared in example 1 in a carbonate solution with pH of 9.6, adding the solution to a 96-well polystyrene enzyme-linked reaction plate, wherein 150ng of polypeptide is added to each well (seven groups of treatments are arranged, QS2019001 is 150ng of polypeptide shown in sequence 1, QS2019002 is 150ng of polypeptide shown in sequence 2, QS2019003 is 150ng of polypeptide shown in sequence 3, QS2019004 is 75ng of polypeptide shown in sequence 1 and 75ng of polypeptide shown in sequence 2, QS2019005 is 75ng of polypeptide shown in sequence 2 and 75ng of polypeptide shown in sequence 3, QS2019006 is 75ng of polypeptide shown in sequence 1 and 75ng of polypeptide shown in sequence 3, QS 9007 is added to each well with 50ng of polypeptide shown in sequence 1, 50ng of polypeptide shown in sequence 2 and 50ng of polypeptide shown in sequence 3, placing the mixture at a pH of 2-8 ℃ for 8-12 hours to fully combine the polypeptide antigen with the enzyme-linked reaction plate, and then adding BSA buffer (5 mg of albumin) containing albumin (4 mg/ml) to 300. mu.l/well Sealing at 37 ℃ for 2-3 hours, drying, and sealing and storing at 4 ℃ after the enzyme-linked reaction plate is dried.
Example 4 sensitivity test
Use method of ELISA antibody detection kit for synthetic peptides of African swine fever virus MGF360-505R and CD2v
1. Balancing: taking out the kit from the refrigeration environment, and standing at room temperature for balancing for 30min for later use; the liquid reagents were mixed well before use.
2. Preparing liquid: diluting the concentrated washing solution by 20 times of distilled water or deionized water to obtain a washing buffer solution;
3. setting: 2 negative control holes and 2 positive control holes, and the rest are sample holes to be detected.
4. Pre-diluting a sample to be detected: and (3) using the sample diluent to mix the serum of the sample to be detected, the negative control serum and the positive control serum according to the ratio of 1: 20 in proportion.
5. Sample adding: each well was pre-diluted with 100. mu.l of the test sample. The time span of the sample application process should be as short as possible.
6. And (3) incubation: shaking and mixing evenly, placing in an incubator at 37 ℃ and reacting for 30 min.
7. Washing the plate: discarding the reaction solution, adding 300 μ l of diluted washing buffer solution into each well, soaking for 15s, throwing away the washing solution, continuously washing the plate for 4 times, and then drying by beating.
8. Adding an enzyme: mu.l of horseradish peroxidase labeled rabbit anti-pig IgG antibody was added to each well.
9. And (3) incubation: the mixture was placed in an incubator at 37 ℃ and reacted for 30 min.
10. Washing the plate: discarding the reaction solution, adding 300 mu l of diluted washing buffer solution into each hole, soaking for 15s, throwing away the washing solution, continuously washing the plate for 4 times, and then drying by beating.
11. Adding 100 μ l of substrate working solution (substrate working solution is obtained by mixing substrate solution A and substrate solution B in equal amount, and is prepared at present), shaking, mixing, placing in 37 deg.C incubator, and reacting for 15min in dark.
12. 50. mu.l of chromogenic stop solution was added to each well, and the reaction was stopped by shaking and mixing.
13. Determination of OD per well450nmValue (reaction plate with stop solution should read OD within 15min450nmValue).
And (3) judging a detection result:
1. negative control OD450nmThe average value should be less than or equal to 0.15, otherwise it is not effective.
2. Each test value of the positive control should be between 1.0 and 2.5, otherwise, the positive control is invalid.
3. Calculation of the critical value: critical value 0.17 × positive control OD450nmValue average.
Determination of OD in serum to be examined450nmIf the value is larger than or equal to the critical value, judging the value as positive; determination of OD in serum to be examined450nmValue of<The critical value is judged to be negative.
Second, sensitivity test
Using seven batches of african swine fever virus MGF360-505R and CD2v synthetic peptide ELISA antibody detection kits (batches QS 2019001-QS 2019007) prepared according to the method of example 3, 25 sera of pigs to be detected (all of which are wild virus infected sera provided by southern hua agriculture university and all of which are inactivated for 30min at 60 ℃ according to the method of using the african swine fever virus MGF360-505R and CD2v synthetic peptide ELISA antibody detection kits, and are detected as positive by the spanish girna african swine fever blocking ELISA antibody detection kit developed by OIE reference laboratory) collected from a pig farm, the experimental results are shown in table 3, 8 parts of the kit of QS2019001 of the invention are detected in total, and the sensitivity of the kit to 25 sera to be detected is 32.0%; the QS2019002 batch number kit disclosed by the invention detects 7 parts of the serum, and the sensitivity of the kit to 25 parts of serum to be detected is 28.0%; 16 parts of QS2019003 batch number kit are detected together, and the sensitivity of the kit to 25 parts of serum to be detected is 64.0%; 14 parts of QS2019004 batch number kit are detected together, and the sensitivity of the kit to 25 parts of serum to be detected is 56.0%; 21 parts of QS2019005 batch number kit are detected together, and the sensitivity of the kit to 25 parts of serum to be detected is 84.0%; in the QS2019006 batch number, 19 parts are detected, and the sensitivity of the kit to 25 parts of serum to be detected is 76.0%; 23 parts of QS2019007 batch number kit are detected together, and the sensitivity of the kit to 25 parts of serum to be detected is 92.0%.
TABLE 3 results of sensitivity test
Kit batch number Detection rate Sensitivity of the composition
QS2019001 (sequence 1) 8/25 32.0％
QS2019002 (sequence 2) 7/25 28.0％
QS2019003 (SEQ ID NO: 3) 16/25 64.0％
QS2019004 (SEQ ID NO: 1+ SEQ ID NO: 2) 14/25 56.0％
QS2019005 (SEQ ID NO: 2+ SEQ ID NO: 3) 21/25 84.0％
QS2019006 (SEQ ID NO: 1+ SEQ ID NO: 3) 19/25 76.0％
QS2019007 (SEQ ID NO: 1+ SEQ ID NO: 2+ SEQ ID NO: 3) 23/25 92.0％
Example 5 specificity test
Using the seven kits of example 2, 50 healthy swine serum (supplied from midrange stock company, ltd.), 2 swine fever positive serum (CSF) (purchased from the institute of chinese veterinary medicine), 2 swine foot and mouth disease virus O-type (FMD-O) positive serum (supplied from midrange stock company, ltd.), and 2 swine foot and mouth disease virus a-type (FMD-a) positive serum (supplied from midrange stock company, ltd) were each detected using the ELISA kits of synthetic peptides of african swine fever virus MGF360-505R and CD2v described in example 3.
The results of the specific detection of the kit are shown in the following table (table 4), and the results of the detection of 50 healthy pig sera show that the specificity of all batches of the kit is 100.0%. The detection results of 2 parts of swine fever positive serum CSF), 2 parts of swine foot-and-mouth disease virus type O (FMD-O) positive serum and 2 parts of swine foot-and-mouth disease virus type A (FMD-A) positive serum are negative, so that the detection specificity of the seven batches of the kit on the 6 parts of the relevant pathogen positive serum is 100%.
TABLE 4 specific detection results of ELISA antibody detection kit for synthetic peptides of African swine fever virus MGF360-505R and CD2v
Figure GDA0003561374180000131
Figure GDA0003561374180000141
Sequence listing
<110> Zhongmu industries GmbH
<120> African swine fever virus MGFs and CD2v ELISA antibody detection kit
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<210> 1
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<213> Artificial Sequence (Artificial Sequence)
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Tyr Asn Leu Lys Glu Ala Ile Gln Tyr Phe Tyr Gln Lys Tyr Thr His
1 5 10 15
Leu Asn Thr Trp Arg Leu Thr Cys Ala Leu Cys Phe Asn Asn Val Phe
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<213> Artificial Sequence (Artificial Sequence)
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Glu Lys His Ile Ile Ser Leu Phe Thr Met Lys Val Met Thr Glu Glu
20 25 30
Glu Lys Asn Leu Cys Leu Glu Ile Leu Tyr Lys
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<210> 3
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Asn Tyr Ser Thr Ser Ile Tyr Asn Ile Thr Asn Asn Cys Ser Leu Thr
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Ile Phe Pro His Asn Asp Val Phe Asp Thr Thr Tyr Gln Val Val Trp
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Asn Gln Ile Ile Asn Tyr Thr Ile
35 40

Claims (9)

1. The African swine fever virus antigen epitope polypeptide composition consists of a polypeptide shown as a sequence 1 in a sequence table, a polypeptide shown as a sequence 2 in the sequence table and a polypeptide shown as a sequence 3 in the sequence table.
2. The African swine fever virus epitope polypeptide composition of claim 1, wherein: the mass ratio of the polypeptide shown in the sequence 1 to the polypeptide shown in the sequence 2 to the polypeptide shown in the sequence 3 is (0.5-1.5): (0.5-1.5): (0.5 to 1.5).
3. The African swine fever virus epitope polypeptide composition of claim 1, wherein: the mass ratio of the polypeptide shown in the sequence 1 to the polypeptide shown in the sequence 2 to the polypeptide shown in the sequence 3 is 1:1: 1.
4. an ELISA antibody detection kit for synthetic peptides of African swine fever virus MGF360-505R and CD2v comprises an enzyme-linked reaction plate and an enzyme-labeled secondary antibody; wherein the enzyme-linked reaction plate is coated with the African swine fever virus epitope polypeptide composition of any one of claims 1-3.
5. The ELISA kit of claim 4, wherein: the enzyme-linked reaction plate is a detachable 96-hole enzyme label plate; the African swine fever virus epitope polypeptide is obtained by chemical artificial synthesis; the method for obtaining the ELISA reaction plate comprises the steps of dissolving the African swine fever virus epitope polypeptide composition of any one of claims 1-3 in 100ul of a carbonate solution with pH of 9.6, adding the solution into a 96-hole polystyrene ELISA reaction plate, placing 150ng of the African swine fever virus epitope polypeptide composition of any one of claims 1-3 in each hole at 2-8 ℃ for 8-12 hours to fully combine the African swine fever virus epitope polypeptide composition with the ELISA reaction plate, adding a PBS buffer solution containing 5mg/ml of bovine serum albumin with pH of 7.4 according to 300 ul/hole, sealing at 37 ℃ for 2-3 hours, drying, and sealing and storing at 4 ℃ after the ELISA reaction plate is dried.
6. The ELISA detection kit of claim 4, characterized in that: the kit further comprises negative control serum; the negative control serum is swine serum without specific pathogens; the enzyme-labeled secondary antibody is a horse radish peroxidase-labeled rabbit anti-pig IgG antibody.
7. The ELISA kit of claim 4, wherein: the kit also comprises a substrate solution A, a substrate solution B and a stop solution; the substrate solution A is a citrate phosphate buffer solution containing 0.6mg/ml urea hydrogen peroxide, the substrate solution B is a tetramethylbenzidine solution containing 0.2mg/ml, and the substrate solution A and the substrate solution B are mixed according to the proportion of 1:1 when in use; the stop solution is a 2mol/L sulfuric acid solution.
8. The ELISA detection kit of claim 4, characterized in that: the kit also comprises a sample diluent and a 20-time concentrated washing solution; the sample diluent is a phosphate buffer solution with a value of 7.4 and 0.01mol/L, pH containing 5mg/ml casein; the concentrated washing solution is 0.01mol/L phosphate buffer solution containing 0.8-1.2% of Tween-20 by volume percentage and has a pH value of 7.4.
9. Use of the African swine fever virus epitope polypeptide composition of any one of claims 1-3 for the preparation of a kit for detecting African swine fever virus.
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