Portable immunochromatography device for detecting African swine fever virus neutralizing antibody and application of portable immunochromatography device

Abstract

The invention discloses a portable immunochromatography device for detecting an African swine fever virus neutralizing antibody and application thereof.The device comprises a reading module, a sample preparation module and a detection module, the sample preparation module and the detection module are integrally connected up and down, and the sample preparation module is sequentially provided with a sample adding hole, a filtering chromatography cavity and a communicating pipe which are communicated from top to bottom; a filtering chromatography part is arranged in the filtering chromatography cavity, the detection module is provided with a fluorescence immunochromatography test strip and a shell, the fluorescence immunochromatography test strip is located in the shell, a sample pad, a fluorescence marker pad, an NC membrane and a water absorption pad of the fluorescence immunochromatography test strip are sequentially overlapped and adhered to a PVC bottom plate, and the two ends of a conduction pipe are communicated with the filtering chromatography cavity and the shell respectively. One end of the fluorescence immunochromatography test strip is located below the filtration chromatography component, the other end of the fluorescence immunochromatography test strip is located above the sample pad, the water absorption pad of the fluorescence immunochromatography test strip and the NC membrane are sequentially inserted into the reading module, and the reading module is connected with the shell through a buckle and reads a quality control line and a detection line of the inserted NC membrane.

Description

Technical Field

[0001] The present invention relates to the field of detection technology. More specifically, the present invention relates to a portable immunochromatographic device for detecting African swine fever virus neutralizing antibodies and its application. Background Art

[0002] African swine fever (ASF) is an acute, highly contagious and fatal infectious disease caused by African swine fever virus (ASFV), which has had a huge impact on the pig farming industry and is a class of animal diseases that China focuses on preventing. Currently, the main diagnostic methods for ASF are etiological examination and serological examination. The PCR molecular detection method dominates in etiological examination, and serological examination mainly uses methods such as ELISA, IFA, and colloidal gold test strips for analysis. Due to the existence of recovered pigs and attenuated strains, during the non-virus excretion period, the PCR detection method has a risk of missed detection, and ASFV antibodies usually can be maintained in serum for a long time. Therefore, the detection of ASFV-specific antibodies is still one of the main tools for diagnosing and controlling this disease. Therefore, carrying out antibody detection, especially the detection of neutralizing antibodies, is of great significance for the prevention and control of ASFV.

[0003] Currently, the most commonly used antibody detection method in veterinary diagnosis is the ELISA detection method, which takes about 2 hours for detection, has a complicated operation, requires high operation requirements for the detection personnel, is prone to false positive and false negative results due to improper operation, and is affected by equipment, with relatively low detection linear range and detection sensitivity. The level of immune antibodies is a reference index for evaluating the immune effect, and the antibody that best represents protection in the immune effect is the neutralizing antibody. Therefore, through the screening of antibody recognition epitopes of key encoded proteins of ASFV, recombinant expression is carried out after different combinations, and according to the identification of the characteristics of the recombinant protein, a recombinant antigen that can recognize ASFV neutralizing antibodies is obtained. Furthermore, a field rapid detection device suitable for the detection of ASFV neutralizing antibodies is developed using this recombinant antigen and applied to areas with simple conditions such as farms, providing important support for the detection device for the prevention and control of African swine fever virus. Summary of the Invention

[0004] The purpose of the present invention is to provide a portable immunochromatographic device for detecting African swine fever virus neutralizing antibodies, which can be applied to the detection of African swine fever virus neutralizing antibodies in pig blood samples.

[0005] According to one aspect of the present invention, a portable immunochromatographic device for detecting African swine fever virus neutralizing antibodies is provided, which includes a reading module, a sample preparation module and a detection module that are integrally connected up and down. The sample preparation module is successively provided with a sample addition hole, a filtration chromatography chamber and a conduction tube from top to bottom, which are connected in communication. A filtration chromatography component is arranged in the filtration chromatography chamber, and the filtration chromatography component is a sheep anti-pig fibrinogen antibody-magnetic nanocellulose chromatography pad. The detection module is provided with a fluorescence immunochromatographic test strip and a housing. The fluorescence immunochromatographic test strip is located inside the housing. The fluorescence immunochromatographic test strip includes a sample pad, a fluorescence marker pad, an NC membrane, a water absorption pad and a PVC bottom plate. The sample pad, the fluorescence marker pad, the NC membrane and the water absorption pad are successively lapped and adhered to the PVC bottom plate. A detection line and a quality control line are successively arranged on the NC membrane along the sample flow direction. The two ends of the conduction tube are respectively connected in communication with the filtration chromatography chamber and the housing. One end is located below the sheep anti-pig fibrinogen antibody-magnetic nanocellulose chromatography pad, and the other end is located above the sample pad. The water absorption pad and the NC membrane of the fluorescence immunochromatographic test strip are successively inserted into the reading module. The reading module is connected to the housing by a buckle and reads the quality control line and the detection line of the inserted NC membrane.

[0006] In some embodiments, the preparation method of the filtration chromatography component includes the following steps: S1, preparing a uniform nanocellulose suspension with a concentration of 1-2 wt% by acid hydrolysis method, wherein the molecular weight of the nanocellulose is 12-14 kDa; S2, adding FeCl3 and FeCl2 with a molar ratio of 2:1 to the uniformly dispersed nanocellulose suspension for iron salt loading, and then preparing magnetic nanocellulose by in-situ precipitation synthesis method; S3, performing amination modification and DEAE group grafting on the magnetic nanocellulose prepared in S2 to obtain DEAE-magnetic nanocellulose; S4, adding the DEAE-magnetic nanocellulose prepared in S3 and sheep anti-pig fibrinogen antibody to a PBS solution in a mass ratio of 1:5, gently stirring to make the sheep anti-pig fibrinogen antibody supersaturatedly bind to the DEAE-magnetic nanocellulose, then adding Tween-20 to a final concentration of 0.1%, continuing to stir, placing the filtration chromatography component in the above mixed solution, soaking overnight at 4°C, and performing low-temperature drying the next day to obtain a sheep anti-pig fibrinogen antibody-magnetic nanocellulose chromatography pad, that is, the filtration chromatography component, cutting it into a suitable size and storing it sealed for later use.

[0007] In some embodiments, the housing of the detection module covers the sample pad and the fluorescence marker pad. The detection line, the quality control line of the NC membrane and the water absorption pad are all located outside the housing. The housing is also provided with a clamping device, and the clamping device clamps and fixes one end of the sample pad of the fluorescence immunochromatographic test strip. The buckle is located at the open end of the housing.

[0008] In some embodiments, the reading module is provided with a test strip insertion hole, a display screen and a card slot. The absorbent pad and the NC membrane of the fluorescence immunochromatographic test strip are sequentially inserted into the test strip insertion hole. The reading module performs fluorescence reading on the detection results displayed on the detection line and the quality control line of the NC membrane, and displays the detection value on the display screen. The card slot is located outside the test strip insertion hole, and the buckle is correspondingly clamped with the card slot.

[0009] In some embodiments, the fluorescence marker pad contains a fluorescently labeled p54-F3 recombinant fusion protein. The detection line is coated with rabbit anti-pig antibody, and the quality control line is coated with a polyclonal antibody against the p54-F3 recombinant fusion protein.

[0010] In some embodiments, the p54-F3 recombinant fusion protein is obtained by fusing the p54-3 recombinant protein and the SC-F protein in a mass ratio of 1:1. The amino acid sequence of the p54-3 recombinant protein is as shown in SEQ ID NO: 18, and the amino acid sequence of the SC-F protein is as shown in SEQ ID NO: 20, that is, the amino acid sequence shown in SEQ ID NO: 15 is fused with the amino acid sequence shown in SEQ ID NO: 14 through a flexible linker.

[0011] In some embodiments, the method for preparing the p54-3 recombinant protein includes the following steps: (1) The amino acid sequence shown in SEQ ID NO: 12 and the amino acid sequence shown in SEQ ID NO: 9 are sequentially concatenated six times in series, and then sequentially concatenated six times with the amino acid sequence shown in SEQ ID NO: 10 through a flexible linker to obtain the amino acid sequence shown in SEQ ID NO: 18; (2) The amino acid sequence shown in SEQ ID NO: 18 is constructed onto the pcDNA3.1 vector, the plasmid is extracted, and after being identified by sequencing, the p54 recombinant plasmid is obtained. The p54 recombinant plasmid is transfected into 293F cells, cultured, centrifuged, the supernatant of the transfected cells is collected to remove impurities, and purified to obtain the p54-3 recombinant protein with the amino acid sequence shown in SEQ ID NO: 18.

[0012] In some embodiments, the method for preparing the SC-F protein is: The amino acid sequence shown in SEQ ID NO: 15 was fused with the amino acid sequence shown in SEQ ID NO: 14 through a flexible linker to obtain the amino acid sequence shown in SEQ ID NO: 20. It was constructed onto the pET28a prokaryotic expression vector and induced for expression using the Escherichia coli expression system. The ferritin fused with Spycatcher was harvested through nickel column purification and molecular sieve. After determining the protein concentration by the BCA method, it was adjusted to 1 mg / mL with PBS for standby. The purified protein was denoted as SC-F protein.

[0013] In some embodiments, the polyclonal antibody is prepared by immunizing animals with the p54-F3 recombinant fusion protein.

[0014] According to another aspect of the present invention, there is provided an application of a portable immunochromatographic device for detecting African swine fever virus neutralizing antibodies in the immunological detection for non-disease diagnosis purposes of African swine fever virus neutralizing antibodies.

[0015] Advantages of the present invention: 1. The portable immunochromatographic device of the present invention can be directly used for the detection of blood without separating serum. The detection time is short, the operation is simple, and the detection result is judged by an instrument, avoiding the error caused by human judgment of the result. 2. The portable immunochromatographic device of the present invention is provided with a filtration chromatography component, which is DEAE-magnetic nanocellulose saturated with sheep anti-pig fibrinogen antibody. The sheep anti-pig fibrinogen antibody binds to fibrinogen in pig blood, can effectively adsorb fibrinogen in pig blood, and at the same time forms a fibrin network structure on the filtration chromatography component, which can effectively block red blood cells and other impurities in the blood from passing through the filtration chromatography component. Only the serum liquid containing ASFV neutralizing antibody can pass through the filtration chromatography component and smoothly enter the detection module, thus omitting the cumbersome steps of sample pretreatment, removing impurities, and improving the detection sensitivity. 3. The recombinant protein obtained by expressing the neutralizing antibody epitope of African swine fever virus p54 protein in multiple tandem has good antigenicity and reactivity. Indirect ELISA after coupling with ferritin through the SpyCatcher / Spytag system has good specificity and sensitivity, can effectively recognize the neutralizing antibody in serum, and exposes multiple antigen epitopes on the surface of ferritin through the SpyCatcher / Spytag system, which is equivalent to increasing the binding sites of the neutralizing antibody, thus greatly improving the detection sensitivity of the African swine fever virus neutralizing antibody in serum. Moreover, the recombinant protein has good specificity and can be applied to the detection of African swine fever virus neutralizing antibody. Brief Description of the Drawings

[0016] Figure 1Schematic diagram of the structure of the portable immunochromatographic device in Example 1 of the present invention.

[0017] Figure 2 Schematic diagram of the structure of the sample preparation module and the detection module of the portable immunochromatographic device in Example 1 of the present invention.

[0018] Figure 3 Schematic diagram of the structure of the reading module of the portable immunochromatographic device in Example 1 of the present invention.

[0019] Figure 4 Scanning electron microscopy result diagram of the magnetic nanocellulose prepared in Example 2 of the present invention.

[0020] Figure 5 Hydrophilic exposed region diagram of the p54 protein predicted by NetSurfP-2.0 in Example 3 of the present invention.

[0021] Figure 6 Secondary structure result diagram of the transmembrane region of the p54 protein predicted by PSIPRED in Example 3 of the present invention.

[0022] Figure 7 Indirect immunofluorescence detection result diagram of Example 3 of the present invention, where a is the indirect immunofluorescence detection result diagram of the polyclonal antibody of polypeptide 1, b is the indirect immunofluorescence detection result diagram of the polyclonal antibody of polypeptide 2, and c is the indirect immunofluorescence detection result diagram of the polyclonal antibody of polypeptide 3.

[0023] Figure 8 SDS-PAGE detection result diagram of the four kinds of p54 recombinant proteins prepared in Example 4 of the present invention, where Line1 is p54-1; Line2 is p54-2; Line3 is p54-3; Line4 is p54-4.

[0024] Figure 9 SDS-PAGE detection result diagram of the four kinds of recombinant fusion proteins p54-F1, p54-F2, p54-F3, p54-F4 prepared in Example 4 of the present invention, where Line1 is p54-F1; Line2 is p54-F2; Line3 is p54-F3; Line4 is p54-F4.

[0025] Figure 10 Specificity detection result diagram of the four kinds of recombinant fusion proteins p54-F1, p54-F2, p54-F3, p54-F4 in Example 4 of the present invention.

[0026] Figure 11 Sensitivity detection result diagram of the three kinds of recombinant fusion proteins p54-F1, p54-F2, p54-F3 in Example 4 of the present invention.

[0027] Figure 12 This is the ROC curve graph of the p54-F3 fluorescence immunochromatographic test strip in Example 5 of the present invention. Detailed implementation manners

[0028] The present invention will be further described in detail through specific implementation cases. It should be understood that these implementation cases are only used to illustrate the present invention and not to limit the protection scope of the present invention. After reading the present invention, various equivalent modifications made by those skilled in the art to the present invention fall within the scope defined by the appended claims of this application. Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents that can be purchased in the conventional market.

[0029] According to the technical solution of the present disclosure, certain amino acids in the amino acid sequence can be conservatively substituted without changing the activity or function of the protein. See Table 1.

[0030] Table 1 Amino acid table for conservative substitution

[0031] In addition, due to the degeneracy of bases, the bases of the polynucleotide sequence can be substituted without changing the activity or function of the polynucleotide sequence. The recombinant protein for detecting African swine fever virus neutralizing antibody provided by the present invention is soluble and can form a correctly folded functional conformation during the expression in eukaryotic cells, which is closer to the structure and function of the real protein and avoids the biosafety risks brought by using real viruses.

[0032] Embodiments and drawings are provided below to help understand the present disclosure. However, it should be understood that these embodiments and drawings are only used to illustrate the present disclosure but do not constitute any limitation. The actual protection scope of the present disclosure is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present disclosure.

[0033] Embodiment 1 A portable immunochromatographic device for detecting African swine fever virus neutralizing antibody Reference Figures 1 to 3 , a portable immunochromatographic device for detecting African swine fever virus neutralizing antibody, includes a reading module 3, and a sample preparation module 1 and a detection module 2 which are integrally connected up and down. The sample preparation module 1 is successively provided with a sample adding hole 11, a filtration chromatography chamber 12 and a conduction tube 13 which are communicated from top to bottom. A filtration chromatography component is arranged in the filtration chromatography chamber 12, and the filtration chromatography component is a sheep anti-pig fibrinogen antibody-magnetic nanocellulose chromatography pad 121. The sheep anti-pig fibrinogen antibody-magnetic nanocellulose chromatography pad 121 is located between the sample adding hole 11 and the conduction tube 13.

[0034] The sheep anti - porcine fibrinogen antibody - magnetic nanocellulose chromatography pad 121 adsorbs DEAE - magnetic nanocellulose. The surface of the DEAE - magnetic nanocellulose is supersaturated with the sheep anti - porcine fibrinogen antibody, and the sheep anti - porcine fibrinogen antibody can effectively adsorb fibrinogen in porcine blood. At the same time, a fibrin network structure is formed on the sheep anti - porcine fibrinogen antibody - magnetic nanocellulose chromatography pad 121, which can effectively block blood cells and impurities in the blood. In this way, the serum liquid containing African swine fever virus neutralizing antibody can smoothly pass through the sheep anti - porcine fibrinogen antibody - magnetic nanocellulose chromatography pad 121 and flow into the conduction tube 13.

[0035] The detection module 2 is provided with a fluorescence immunochromatographic test strip 21 and a housing 22. The fluorescence immunochromatographic test strip 21 is located inside the housing 22. The fluorescence immunochromatographic test strip includes a sample pad 211, a fluorescence marker pad 212, an NC membrane 213, a water absorption pad 214, and a PVC bottom plate 215. The sample pad 211, the fluorescence marker pad 212, the NC membrane 213, and the water absorption pad 214 are sequentially overlapped and adhered to the PVC bottom plate 215. Along the sample flow direction on the NC membrane 213, a detection line 216 and a quality control line 217 are sequentially arranged. The fluorescence marker pad 212 contains a fluorescence - labeled p54 - F3 recombinant fusion protein, the detection line 216 is coated with rabbit anti - porcine antibody, and the quality control line 217 is coated with a polyclonal antibody against the p54 - F3 recombinant fusion protein.

[0036] Both ends of the conduction tube 13 extend into the sample preparation module 1 and the housing 22 respectively. One end is located below the sheep anti - porcine fibrinogen antibody - magnetic nanocellulose chromatography pad 121, and the other end is located above the sample pad 211. The sample serum liquid passing through the sheep anti - porcine fibrinogen antibody - magnetic nanocellulose chromatography pad 121 enters the detection module 2 through the conduction tube 13 and drips onto the sample pad 211.

[0037] The housing 22 is cylindrical and covers the sample pad 211 and the fluorescence marker pad 212. The detection line 216, the quality control line 217 of the NC membrane 213, and the water absorption pad 214 are all located outside the housing 22. A clamping device 221 and a buckle 222 are arranged inside the housing 22. The clamping device 221 clamps and fixes one end of the sample pad 211 of the fluorescence immunochromatographic test strip 21. The buckle 222 is located at the open end of the housing 22.

[0038] The reading module 3 is provided with a test strip insertion hole 31, a display screen 32 and a card slot 33. The absorbent pad 214 of the fluorescent immunochromatographic test strip 21 and the NC membrane 213 are sequentially inserted into the test strip insertion hole 31. The reading module 3 performs fluorescent readings on the test results displayed by the detection line 216 and the quality control line 217 of the NC membrane 213, and displays the test values ​​on the display screen 32. The card slot 33 is located on the outside of the test strip insertion hole 31, and the buckle 222 is correspondingly mounted with the card slot 33. The existence of the buckle 222 allows the reading module to be reused, saving production costs. The reading module 3 is provided by Shenzhen Sanfangyuan Biotechnology Co., Ltd.

[0039] When this embodiment is used, the collected pig blood sample is added into the filtration chromatography chamber 12 from the sample addition hole 11, and the pig blood sample drips on the sheep anti-porcine fibrinogen antibody-magnetic nanocellulose chromatography pad 121. In the process of flowing through the sheep anti-porcine fibrinogen antibody-magnetic nanocellulose chromatography pad 121, the fibrinogen in the pig blood sample is adsorbed by the sheep anti-porcine fibrinogen antibody, and a fibrin network structure is formed on the surface of the filtration chromatography component, so that blood cells and other impurities in the pig blood sample are blocked above the sheep anti-porcine fibrinogen antibody-magnetic nanocellulose chromatography pad 121, and the filtration chromatography treatment of the pig blood sample is realized. The serum solution containing the neutralizing antibody of the African swine fever virus flows directly to the sample pad 211 of the detection module 2 through the conducting pipe 13 under the action of gravity, passes through the fluorescent marker pad 212 through chromatography, and finally chromatographs to the NC membrane 213 to excite fluorescence. After the pig blood sample is added, the timing is set for 10 minutes, and one end of the absorbent pad 214 of the immunochromatographic test strip 21 is inserted into the test strip insertion hole 31 of the reading module 3, so that the absorbent pad 214 and the NC membrane 213 are fully inserted. The reading module 3 performs fluorescence readings on the detection line 216 and the quality control line 217 on the NC membrane 213, and the detection value and the detection result are displayed on the display screen 32 of the reading module 3 after the reading.

[0040] When this embodiment is used in certain circumstances, a syringe whose outer diameter of the injection port matches the inner diameter of the sample addition hole is used to add the pig blood sample to be tested into the filtration chromatography cavity 12 through the sample addition hole, and then air is injected into the filtration chromatography cavity 12 through the syringe for pressurization, so that the pig blood sample in the filtration chromatography cavity 12 passes through the filtration chromatography component faster, which can shorten the filtration chromatography processing time and improve the detection efficiency. Because the outer diameter of the injection port matches the inner diameter of the sample addition hole, the syringe blocks the sample addition hole, which can prevent the injected air from escaping from the sample addition hole, thereby achieving the purpose of pressurized filtration of the pig blood sample in the filtration chromatography cavity 12.

[0041] The portable immunochromatographic device for detecting African swine fever virus neutralizing antibodies of the present invention can be used for detecting complex blood samples such as blood and plasma, without the need for serum separation, with a short detection time, convenient and fast. The detection results are interpreted by an instrument, which is scientific and accurate.

[0042] Example 2 Preparation of the filtration chromatography component 2.1 Preparation of nanocellulose by acid hydrolysis 2.1.1 Pretreatment: Disperse 10 g of microcrystalline cellulose with a particle size of 50 μm in 200 mL of deionized water, stir for 30 minutes, and filter to remove impurities; 2.1.2 Acid hydrolysis: Add the pretreated cellulose to 500 mL of 64 wt% sulfuric acid, keep the temperature at 45 °C, and react with magnetic stirring at 500 rpm for 2 hours; 2.1.3 Terminate the reaction: Add a large amount of ice water (volume ratio 1:10) to terminate the reaction, centrifuge at 10,000×g for 15 minutes, and collect the precipitate; 2.1.4 Washing and purification: Centrifuge and wash the precipitate with deionized water until neutral (pH≈7), and then wash with ethanol 3 times to remove residual acid; 2.1.5 Dialysis treatment: Load the suspension into a dialysis bag with a molecular weight cut-off of 12-14 kDa, and dialyze in deionized water for 3 days (change water 3 times a day) to remove small molecule impurities; 2.1.6 Ultrasonic dispersion: The dialyzed suspension is ultrasonically treated (power 300 W, 30 minutes) to obtain a uniform nanocellulose suspension with a concentration of 1-2 wt%.

[0043] 2.2 Preparation of magnetic nanocellulose 2.2.1 Disperse nanocellulose: Take 100 mL of a 1wt% nanocellulose suspension, and ultrasonically treat (200W, 20 minutes) to ensure uniform dispersion; 2.2.2 Iron salt loading: Add 0.1 M FeCl3 and 0.05 M FeCl2, and stir for 30 minutes to adsorb Fe³⁺ / Fe²⁺ on the surface of nanocellulose; 2.2.3 In-situ precipitation: Raise the temperature to 75 °C, protect with N2, slowly add ammonia water to pH 10, and react for 2 hours; 2.2.4 Post-treatment: Magnetically separate and collect the composite material, wash it with deionized water and ethanol in turn, and freeze-dry at -50 °C for 24 hours to obtain magnetic nanocellulose.

[0044] 2.3 Surface functionalization 2.3.1 Amination modification: Magnetic nanocellulose was dispersed in anhydrous toluene, and 5% 3-aminopropyltriethoxysilane (APTES) was added. The mixture was refluxed at 80 °C for 6 hours, and then centrifuged and washed to obtain aminated magnetic nanocellulose; 2.3.2 Grafting of DEAE groups: Aminated magnetic nanocellulose was reacted with 10% (mass / volume) diethylaminoethyl chloride-borate buffer solution (pH 8.5) at 60 °C for 12 hours, and then washed to obtain DEAE-magnetic nanocellulose.

[0045] 2.4 Characterization and performance testing 2.4.1 SEM analysis of morphology: The acceleration voltage was 5 kV, and the sample was sputter-coated with gold. The morphology of DEAE-magnetic nanocellulose was observed. The results are shown in Figure 4 . It can be seen from Figure 4 that the diameter of DEAE-magnetic nanocellulose is 20 - 50 nm, and the surface of the nanocellulose is covered with grayish-white magnetic particles.

[0046] 2.4.2 Magnetic property testing: VSM: The magnetic field range was ±20 kOe, and the saturation magnetization intensity was measured. It was determined that the saturation magnetization intensity of DEAE-magnetic nanocellulose > 40 emu / g, indicating that the prepared DEAE-magnetic nanocellulose has strong magnetism.

[0047] 2.4.3 Adsorption property testing: Plasma experiment: 1 mL of plasma was mixed with 10 mg of DEAE-magnetic nanocellulose, and the mixture was shaken at 37 °C for 30 minutes. After magnetic separation, the change in antibody concentration in the plasma was detected by ELISA. At the same time, a control of pure plasma sample was set. After detection, it was found that the antibody concentration in the plasma decreased significantly after magnetic separation.

[0048] 2.5 Preparation of filtration chromatography component 10 mg of the DEAE-magnetic nanocellulose prepared in 2.3 and sheep anti-pig fibrinogen antibody were added to 10 mL of PBS at a mass ratio of 1:5. The mixture was gently stirred at 4 °C for 6 h, and then Tween-20 was added to a final concentration of 0.1%. Stirring was continued at 4 °C for 1 h. The filtration chromatography component was placed in the above mixed solution and soaked at 4 °C overnight. The next day, it was dried at low temperature to obtain a sheep anti-pig fibrinogen antibody-magnetic nanocellulose chromatography pad 121, that is, the filtration chromatography component, which was cut into appropriate sizes and stored in a sealed manner for later use.

[0049] The sheep anti-pig fibrinogen antibody added in the above preparation process is supersaturated with respect to DEAE-magnetic nanocellulose and can bind to DEAE-magnetic nanocellulose in a supersaturated manner.

[0050] The sheep anti - porcine fibrinogen antibody - magnetic nanocellulose chromatography pad 121 can effectively adsorb fibrinogen in blood. At the same time, after the adsorbed fibrinogen is converted into fibrin, the formed network structure can also effectively prevent the passage of blood cells, thereby realizing the filtration of blood cells in the blood sample and enabling the serum in the blood to flow downward smoothly through the sheep anti - porcine fibrinogen antibody - magnetic nanocellulose chromatography pad 121. Microscopic examination of the outflowing liquid can show a significant reduction in blood cells, indicating that the sheep anti - porcine fibrinogen antibody - magnetic nanocellulose chromatography pad 121 in this example has an obvious filtering and blocking effect on blood cells.

[0051] Example 3 Prediction and Screening of Epitopes Recognized by Neutralizing Antibodies Against African Swine Fever Virus p54 Protein Materials, Reagents and Instruments Used in the Example: Materials 1. Bacterial Strains E. coli competent cell DH5α was purchased from Beijing TransGen Biotech Co., Ltd. The pCAGGS vector, pcNDA3.1 vector, and 293F cells were stored in our laboratory. 2. Sera The ASFV - positive serum inactivated with 0.3% TNBP and 1% triton X - 100 was kindly provided by China Animal Husbandry Research Institute. The PRRSV - vaccine - virus - positive serum, PPV - vaccine - virus - positive serum, PRV - vaccine - virus - positive serum, and PCV3 - positive serum inactivated with 0.3% TNBP and 1% triton X - 100 were stored in our laboratory. 50 inactivated African swine fever - positive sera and 150 ASFV - negative sera were kindly provided by the National Veterinary Research Institute of Poland and the African Swine Fever National Reference Laboratory of the Institute of Animal Health of Sardinia, Italy. Porcine negative control serum was purchased from Gibco. Reagents ​SMM 293-TIS Expression Medium serum-free medium and PEI were purchased from Beijing Sino Biological Inc., Ni-NTA agarose gel resin and plasmid extraction kit were purchased from QIAGEN, BCA protein concentration assay kit was purchased from Beijing Solarbio Science & Technology Co., Ltd., fetal bovine serum was purchased from Gibico, BamH I / Xho I restriction enzymes, T4 ligase, and TaKaRa LA PCR™ Kit were purchased from TaKaRa, DNA extraction kit DNeasy Blood and Tissue Kit was purchased from Qiagen, agarose gel DNA purification kit, IPTG, X-gal, and ampicillin were purchased from Beijing TransGen Biotech Co., Ltd., rabbit anti-pig IgG HRP was purchased from Bioworld Technology; FITC-labeled goat anti-mouse antibody was purchased from Abcam; Instrument Ordinary PCR instrument of ABI company (AB Applied Biosystems), tabletop centrifuge of sigma company (3-18k), constant temperature oscillating incubator (HZQ-F100), fluorescence microscope (ZEISS, AXIO), Liuyi electrophoresis instrument, dual infrared laser scanning imaging system (Proteinsimple).

[0052] 3.1 Screening of neutralizing antibody epitopes of African swine fever virus p54 protein According to the full-length amino acid sequence of the p54 protein of African swine fever virus Heilongjiang strain (MK333180.1) published in GenBank (SEQ ID NO: 1), the secondary structure of the p54 protein was preliminarily analyzed and predicted using online sequence analysis software https: / / services.healthtech.dtu.dk / services / NetSurfP-2.0 / , http: / / cho-fas.sourceforge.net / , http: / / bioinf.cs.ucl.ac.uk / psipred / , and https: / / www.iedb.org / home_v3.php. According to the prediction of NetSurfP-2.0, the hydrophilic exposed region of the p54 protein was obtained, as shown in Figure 5 . According to the prediction of PSIPRED, the secondary structure results of the transmembrane region of the p54 protein were obtained, as shown in Figure 6 . Combining the antigen recognition epitopes of the p54 protein published on the IEDB website, the possible neutralizing antibody recognition epitopes of the p54 protein were preliminarily screened, as shown in Table 2. Three different combinations were designed for artificial synthesis, as shown in Table 3, and Nanjing Peptide Valley Biotechnology Co., Ltd. was commissioned for polypeptide synthesis.

[0053] Summary Table of p54 Protein Epitopes Screened

[0054] Table 3 Three Different Combinations of p54 Synthetic Peptides

[0055] 3.2 Preparation of Polyclonal Antibodies and Comparison of Their Neutralization Effects Dilute the synthetic polypeptides 1, 2, and 3 in 3.1 to 1 mg / mL with PBS, mix them with Freund's complete adjuvant, and then immunize mice by subcutaneous injection. Each polypeptide is used to immunize mice at three doses (50 μg, 100 μg, 150 μg). Two weeks after the primary immunization, inject again using the same method, but use Freund's incomplete adjuvant. 7 - 10 days later, collect blood from the mice and separate the serum. Use this polyclonal antiserum for neutralization tests to compare the neutralization titers of the polyclonal antisera prepared from the above three polypeptides.

[0056] Dilute the ASFV virus solution to 100 TCID 50 / 0.1 mL. Dilute the prepared polyclonal antibody by 2-fold serial dilution, with a total of 2 dilution factors, and 4 replicates for each dilution factor. Also set up a positive control (containing only the virus solution without antibody) and a negative control (empty cells). Mix the diluted virus solution with polyclonal antibodies at different dilution factors and incubate at 4°C for 2 h, then add it to a 96-well cell plate pre-coated with PAM cells and continue culturing for 24 h. Discard the culture medium, fix the cells, and perform an indirect immunofluorescence assay with FITC-labeled goat anti-mouse antibody. After the reaction, take pictures with a fluorescence microscope to record. Observe the results of the indirect immunofluorescence assay. If the fluorescence decreases as the antibody concentration increases, it indicates that the virus has been neutralized by the antibody. If the number of fluorescence does not change, it means the virus has not been neutralized by the antibody.

[0057] The indirect immunofluorescence results of various polyclonal antibodies are shown in Figure 7 a~ Figure 7 c. After comparison, the polyclonal antibodies produced by mice immunized with polypeptides 1 and 2 at 10 μg have a higher neutralization effect. The fluorescence in each well of cells decreases as the concentration of the polyclonal antibody increases. However, compared with the virus control, the fluorescence in the cell wells incubated with the polyclonal antibody of polypeptide 3 does not change much. Thus, it can be seen that the epitopes composed of polypeptides 1 and 2 can well recognize neutralizing antibodies. Therefore, polypeptides 1 and 2 are selected for subsequent research.

[0058] Example 4 Expression and Purification of Recombinant Protein of p54 Neutralizing Antibody Recognition Epitope 4.1 Design and Expression of Recombinant Protein of p54 Neutralizing Antibody Recognition Epitope SpyCatcher / SpyTag is a commonly used protein conjugation system at present. Its operation is simple and fast, and it can achieve in vitro self-assembly of proteins. Therefore, in this example, the SpyCatcher / SpyTag (SC-ST) system is used to design recombinant proteins for identifying epitopes of 4 p54 neutralizing antibodies. The sequence design method is as follows, and the sequences are shown in SEQ ID NO: 16-19 respectively: p54-1: His tag + polypeptide 1 + polypeptide 2 + spy tag, (SEQ ID NO: 16); p54-2: His tag ++ polypeptide 1 conjugated 3 times + GGGGGGGG (flexible linker) + polypeptide 2 conjugated 3 times + spy tag, (SEQ ID NO: 17); p54-3: His tag + polypeptide 1 conjugated 6 times + GGGGGGGG (flexible linker) + polypeptide 2 conjugated 6 times + spy tag, (SEQ ID NO: 18); p54-4: His tag + polypeptide 1 conjugated 9 times + GGGGGGGG (flexible linker) + polypeptide 2 conjugated 9 times + spy tag, (SEQ ID NO: 19).

[0059] The sequences of His tag, spy tag, SpyCatcher and Ferritin are shown in SEQ ID NO: 12-15, as shown in Table 4 specifically.

[0060] Table 4 Amino acid sequence table of His tag, spy tag, SpyCatcher

[0061] The sequences of SEQ ID NO: 16-19 above are respectively constructed into the pcDNA3.1 vector, and the plasmids are extracted. After being identified by sequencing, they are transfected into 293F cells. In 1 L of cell culture medium, a mixed solution containing 2 mg of p54 recombinant plasmid and 12 mg of PEI is prepared. When the cell density is 2×10 6 / mL, the mixed solution is transfected into 293F cells. At 48 hours after transfection, the cells are centrifuged at 1000×g for 20 min, and the transfected cell supernatant is collected. The impurities are removed with a 0.22 μm filter membrane. The supernatant is added to the pre-treated Ni-NTA beads, and purified according to the operation steps of nickel column purification. The effluent and protein eluate of the target protein are collected, concentrated with a 10 ku ultrafiltration tube, and 10 μL of each tube is taken for sample preparation for SDS-PAGE detection. The results are shown in Figure 8, the protein concentration was determined by the BCA method and adjusted to 1 mg / mL with PBS for standby. The purified proteins were respectively denoted as recombinant proteins p54-1, p54-2, p54-3, and p54-4.

[0062] Meanwhile, the Ferritin protein with the sequence shown in SEQ ID NO: 15 was fused with SpyCatcher with the sequence shown in SEQ ID NO: 14 using a flexible linker (GGGGGGGG) and constructed onto the pET28a prokaryotic expression vector. It was induced and expressed using the Escherichia coli expression system, and the ferritin fused with Spycatcher was harvested through nickel column purification and molecular sieve. After determining the protein concentration by the BCA method, it was adjusted to 1 mg / mL with PBS for standby. The purified protein was denoted as SC-F protein, and the amino acid sequence of the SC-F protein was shown in SEQ ID NO: 20.

[0063] 4.2 Preparation of p54 protein and ferritin complex The 4 purified p54 recombinant proteins in 4.1 were respectively mixed with SC-F ferritin at a mass ratio of 1:1 and left overnight at 4°C. After the reaction was completed, for the proteins that did not react completely, size exclusion chromatography molecular sieve chromatography columns were used for separation. After filtering the protein solution through a 0.22 μm filter membrane, the column was equilibrated with 2 column volumes of equilibration buffer (0.02 mol / L sodium phosphate, 0.5 mol / L sodium chloride, pH = 7.4) and then loaded. It was rinsed with another 2 column volumes. Proteins with different molecular weights eluted and peaked at different times. The elution peaks of the target proteins were collected for SDS-PAGE electrophoresis verification. The electrophoresis results are shown in Figure 9 .

[0064] As can be seen from Figure 9 , after the reaction products were separated by SEC, fusion proteins of p54 protein and ferritin with high purity were obtained, which were respectively labeled as recombinant fusion proteins p54-F1, p54-F2, p54-F3, and p54-F4. The concentrations of the above proteins were determined and adjusted to 1 mg / mL with PBS for standby.

[0065] 4.3 Specificity comparison of recombinant fusion proteins p54-F1, p54-F2, p54-F3, and p54-F4 The indirect ELISA was performed by coating ELISA plates with recombinant fusion proteins p54-F1, p54-F2, p54-F3, and p54-F4 respectively to identify the specificity of recombinant fusion proteins p54-F1, p54-F2, p54-F3, and p54-F4. The primary antibodies were inactivated ASFV positive serum, PRRSV vaccine virus positive serum, PPV vaccine virus positive serum, PRV vaccine virus positive serum, PCV3 positive serum, and porcine negative serum respectively, and the secondary antibody was HRP-labeled rabbit anti-pig IgG. At the same time, positive and negative controls were set. After the reaction, the OD 450nm value was read, the S / N value was calculated, and a S / N value ≥ 2.1 was judged as positive, while a S / N value < 2.1 was judged as negative. The specific test results are shown in Figure 10 .

[0066] As can be seen from Figure 10 , recombinant fusion proteins p54-F1, p54-F2, and p54-F3 only showed obvious positive reactions with ASFV positive serum, and did not cross-react with PRRSV vaccine virus positive serum, PPV vaccine virus positive serum, PRV vaccine virus positive serum, PCV3 positive serum, and porcine negative serum. Among them, the distinction between positive and negative of p54-F3 was more obvious, while p54-F4 cross-reacted with PRV vaccine virus positive serum and had poor specificity.

[0067] 4.4 Comparison of the sensitivity of recombinant fusion proteins p54-F1, p54-F2, and p54-F3 The ASFV standard positive serum was serially diluted (1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560, 1:5120), and three fusion proteins p54-F1, p54-F2, and p54-F3 were coated respectively. The serially diluted ASFV standard positive serum was detected by indirect ELISA, and a negative control was set at the same time to determine the detection sensitivity of the three fusion proteins to the standard positive serum. The test results are shown in Figure 11 .

[0068] As can be seen from Figure 11 , recombinant fusion protein p54-F3 could detect ASFV positive serum diluted 1:2560, with the highest detection sensitivity, while p54-F1 could only detect positive serum diluted 1:160, and p54-F2 could only detect positive serum diluted 1:640. The above results indicate that the best coupling method in the present invention is to repeat the coupling of the main neutralizing antibody epitope of p54 six times, which can not only provide detection sensitivity but also avoid non-specific binding. Therefore, recombinant fusion protein p54-F3 was used for the subsequent development of test strips. At the same time, p54-F3 mouse polyclonal antibody was prepared according to the method for preparing polyclonal antibody in 3.2 of Example 3 with recombinant fusion protein p54-F3.

[0069] Example 5 Preparation of a Fluorescent Immunochromatographic Test Strip for Detecting African Swine Fever Virus Neutralizing Antibodies 5.1 Preparation of the Recombinant Fusion Protein p54-F3-Fluorescent Label Take 100 μL of the fluorescent microsphere solution and label 100 μg of the recombinant fusion protein p54-F3 according to the instructions. Finally, resuspend the p54-F3-fluorescent label conjugate with 300 μL of a dilution solution (pH 8.0) containing 5 g·L -1 BSA, 5 g·L -1 PVP, 50 mmol / L Tris, and 0.2% sodium azide, and store it at 4°C for later use.

[0070] 5.2 Preparation of the p54-F3 Fluorescent Immunochromatographic Test Strip Use a film spraying instrument to evenly spray the p54-F3-fluorescent label conjugate onto the fluorescent label pad at a rate of 5 μL / cm, and dry it at 37°C for 2 hours. The NC membrane is provided with a test line (T line) and a quality control line (C line). The test line (T line) is coated with rabbit anti-pig antibody at a coating concentration of 0.5 - 1.2 mg / mL, and the quality control line (C line) is coated with a polyclonal antibody against the recombinant fusion protein p54-F3 at a coating concentration of 0.25 - 0.6 mg / mL. The coating concentrations of the test line (T line) and the quality control line (C line) are in a ratio of 2:1. Then, sequentially lap and paste the sample pad, the fluorescent label pad, the NC membrane, and the absorbent pad onto the PVC bottom plate. After pasting, cut the prepared test strip into strips 4 mm wide, store the prepared test strip in an aluminum foil bag and seal it, and place it in a dry place for later use.

[0071] 5.3 Determination of the Detection Threshold of the p54-F3 Fluorescent Immunochromatographic Test Strip Use the p54-F3 fluorescent test strip to detect 200 known background sera (50 positive sera and 150 negative sera), and use the ROC curve (receiver operating characteristic curve) to determine the negative and positive judgment thresholds of the test strip.

[0072] Use the true positive rate (sensitivity) as the ordinate and the false positive rate (100 - specificity) as the abscissa, and draw an ROC curve based on the detection results of 200 sera by the p54-F3 fluorescent microsphere detection method, as shown in Figure 12 .

[0073] From Figure 12It can be seen that the area under the curve value is 0.982, indicating that this method has high accuracy. For the Youden index (the sum of sensitivity and specificity minus 1) corresponding to T / C of each sample on the curve, the T / C = 0.47 corresponding to the maximum Youden index (0.921) was selected as the critical value of this method. At this time, the corresponding sensitivity was 94.8% and the specificity was 97.3%. Therefore, the determination criteria of this method were determined: when T / C ≥ 0.47, it was determined as positive for ASFV neutralizing antibody; when T / C < 0.47, it was determined as negative for ASFV neutralizing antibody.

[0074] Example 6 Comparison of the Measured Effects of a Portable Immunochromatographic Device for Detecting African Swine Fever Virus Neutralizing Antibodies 6.1 Assembly of the portable immunochromatographic device: The fluorescence immunochromatographic test strip prepared in Example 5 was installed in the detection module of Example 1, and one end of the sample pad of the fluorescence immunochromatographic test strip was clamped and fixed by the clamping device to prepare a portable immunochromatographic detection device 1. An adsorption filtration chromatography component of sheep anti-pig fibrinogen antibody-magnetic nanocellulose, namely a sheep anti-pig fibrinogen antibody-magnetic nanocellulose chromatography pad, was provided in the device 1.

[0075] At the same time, a portable immunochromatographic detection device 2 and a portable immunochromatographic detection device 3 were prepared with reference to Example 1. Among them, a filtration chromatography component that only adsorbed magnetic nanocellulose, namely a magnetic nanocellulose chromatography pad, was provided in the device 2. A filtration chromatography component that did not adsorb sheep anti-pig fibrinogen antibody and did not adsorb magnetic nanocellulose, namely a chromatography pad, was provided in the device 3.

[0076] 6.3 Sensitivity detection: The portable immunochromatographic detection devices 1-3 prepared in 6.2 were used to detect the fresh blood of pigs immunized with recombinant protein p54-F3 diluted in a series, and the dilution ratios were: 1:10, 1:100, 1:200, 1:400, 1:800, 1:1600. 500 μL of the above-mentioned serially diluted blood samples to be tested were respectively dropped into the sample addition holes of the portable immunochromatographic detection devices 1-3 prepared in 6.2. After 10 minutes, the fluorescence readings of the detection line and the quality control line of the fluorescence test strips of the above-mentioned devices 1-3 were taken using the reading module. The specific results are shown in Table 5.

[0077] Table 5 Results of sensitivity detection

[0078] As shown in Table 5, in terms of sensitivity detection, the portable immunochromatographic detection device 1 (equipped with an adsorption filtration chromatography component of sheep anti-pig fibrinogen antibody-magnetic nanocellulose) could detect the pig blood immunized with recombinant protein p54-F3 diluted 1:800 times, and the detection sensitivity was relatively high.

[0079] The portable immunochromatographic detection device 2 (equipped with a filtration chromatography component that only adsorbs magnetic nanocellulose), when recombinant protein p54-F3-immunized porcine blood is added to the device 2, since magnetic nanocellulose can adsorb ASFV antibodies in the blood, the ASFV antibodies passing through the filtration chromatography component are greatly reduced. And substances such as blood cells and some fibrinogens in the blood will flow to the sample pad of the test strip under the action of gravity along with the liquid. Fibrinogen condenses on the sample pad to form fibrin, affecting the chromatography of the sample to the NC membrane. Under the dual effects of antibody adsorption and affected chromatography, the detection sensitivity of the device 2 is very low.

[0080] The filtration chromatography component provided in the portable immunochromatographic detection device 3 is just a simple chromatography pad, which neither adsorbs magnetic nanocellulose nor is supersaturated with sheep anti-pig fibrinogen antibody bound. Although the chromatography pad can filter and block some impurities in the blood, the effect is minimal. And various proteins and blood cells in the blood cannot be filtered by the chromatography pad, but fall onto the sample pad of the test strip along with the serum through the conduction tube, forming solid substances such as coagulated fibrin on the sample pad, seriously affecting the chromatography of the sample on the test strip, resulting in a very low detection sensitivity.

[0081] Therefore, in terms of sensitivity detection, for the portable immunochromatographic detection device 1 (equipped with a filtration chromatography component that adsorbs sheep anti-pig fibrinogen antibody-magnetic nanocellulose), when the blood flows down, fibrinogen in the blood binds to the sheep anti-pig fibrinogen antibody, and the sheep anti-pig fibrinogen antibody is adsorbed on the magnetic nanocellulose, thus forming a fibrin network structure that blocks the passage of blood cells and other blood impurities. Only the blood containing ASFV antibodies can pass through smoothly and flow to the fluorescence immunochromatographic test strip below, so that the sensitivity of the test strip can be as high as 1:800 times.

[0082] 6.4 Specificity detection The portable immunochromatographic detection devices 1-3 prepared in 6.2 were used to detect ASFV antibody-positive serum and control sera (PRRSV vaccine virus-positive serum, PPV vaccine virus-positive serum, PRV vaccine virus-positive serum, PCV3-positive serum, porcine negative serum) respectively. The detection results are shown in Table 6.

[0083] Table 6 Specificity detection result table

[0084] The specificities of the above detection devices 1-3 are all relatively good. This has a great deal to do with the fact that the present invention uses the recombinant fusion protein p54-F3, which is a tandem expression of neutralizing antibody recognition epitopes, as an antigen. Moreover, the recombinant antigen capable of recognizing neutralizing antibody epitopes expressed in the present invention uses the SpyCatcher / SpyTag system, incorporates ferritin, improves the antigenicity of the recombinant antigen complex, and displays multiple neutralizing antibody epitopes on the surface of ferritin through the SpyTag system, thereby ensuring the high efficiency and specificity of the detection results.

[0085] The above are only some embodiments of the present invention. For those of ordinary skill in the art, without departing from the creative concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention.

Claims

1. A portable immunochromatographic device for detecting African swine fever virus neutralizing antibodies, characterized in that, It includes a reading module, a sample preparation module and a detection module which are integrally connected up and down. The sample preparation module is successively provided with a sample adding hole, a filtration chromatography chamber and a conduction tube communicating with each other from top to bottom. A filtration chromatography component is arranged in the filtration chromatography chamber. The filtration chromatography component is a sheep anti-pig fibrinogen antibody-magnetic nanocellulose chromatography pad. The detection module is provided with a fluorescence immunoassay chromatography strip and a housing. The fluorescence immunoassay chromatography strip is located inside the housing. The fluorescence immunoassay chromatography strip includes a sample pad, a fluorescence marker pad, an NC membrane, a water absorption pad and a PVC bottom plate. The sample pad, the fluorescence marker pad, the NC membrane and the water absorption pad are successively lapped and adhered to the PVC bottom plate. A detection line and a quality control line are successively arranged on the NC membrane along the sample flow direction. Two ends of the conduction tube are respectively communicated with the filtration chromatography chamber and the housing. One end of the conduction tube is located below the sheep anti-pig fibrinogen antibody-magnetic nanocellulose chromatography pad, and the other end is located above the sample pad. The water absorption pad and the NC membrane of the fluorescence immunoassay chromatography strip are successively inserted into the reading module. The reading module is connected to the housing through a buckle and reads the quality control line and the detection line of the inserted NC membrane.

2. The portable immunochromatographic device for detecting African swine fever virus neutralizing antibody according to claim 1, characterized in that, The preparation method of the filtration chromatography component includes the following steps: S1. A uniform nanocellulose suspension with a concentration of 1-2 wt% is prepared by an acid hydrolysis method, where the molecular weight of the nanocellulose is 12-14 kDa; S2. FeCl3 and FeCl2 with a molar ratio of 2:1 are added to the uniformly dispersed nanocellulose suspension for iron salt loading, and then magnetic nanocellulose is prepared by an in-situ precipitation synthesis method; S3. The magnetic nanocellulose prepared in S2 is subjected to amination modification and DEAE group grafting to obtain DEAE-magnetic nanocellulose; S4. The DEAE-magnetic nanocellulose prepared in S3 and the sheep anti-pig fibrinogen antibody are added to a PBS solution at a mass ratio of 1:5, and gently stirred to make the sheep anti-pig fibrinogen antibody and the DEAE-magnetic nanocellulose be supersaturatedly combined. Then Tween-20 is added to a final concentration of 0.1%, and stirring is continued. The chromatography pad is placed in the above mixed solution and soaked overnight at 4°C. The next day, it is dried at low temperature to obtain a sheep anti-pig fibrinogen antibody-magnetic nanocellulose chromatography pad, that is, the filtration chromatography component, which is cut into a suitable size, sealed and stored for later use.

3. The portable immunochromatographic device for detecting African swine fever virus neutralizing antibody according to claim 1, characterized in that, The housing of the detection module covers the sample pad and the fluorescence marker pad. The detection line, the quality control line of the NC membrane and the water absorption pad are all located outside the housing. The housing is also provided with a clamping device, and the clamping device clamps and fixes one end of the sample pad of the fluorescence immunoassay chromatography strip. The buckle is located at the opening end of the housing.

4. The portable immunochromatographic device for detecting African swine fever virus neutralizing antibody according to claim 1, characterized in that, The reading module is provided with a test strip insertion hole, a display screen and a card slot. The absorbent pad and the NC membrane of the fluorescence immunochromatographic test strip are sequentially inserted into the test strip insertion hole. The reading module performs fluorescence reading on the detection results displayed on the detection line and the quality control line of the NC membrane, and displays the detection value on the display screen. The card slot is located outside the test strip insertion hole, and the buckle is correspondingly clamped with the card slot.

5. The portable immunochromatographic device for detecting African swine fever virus neutralizing antibody according to claim 1, characterized in that, The fluorescence marker pad contains a fluorescently labeled p54-F3 recombinant fusion protein. The detection line is coated with rabbit anti-pig antibody, and the quality control line is coated with a polyclonal antibody against the p54-F3 recombinant fusion protein.

6. The portable immunochromatographic device for detecting African swine fever virus neutralizing antibody according to claim 5, wherein, The p54-F3 recombinant fusion protein is obtained by fusing the p54-3 recombinant protein and the SC-F protein in a mass ratio of 1:

1. The amino acid sequence of the p54-3 recombinant protein is as shown in SEQ ID NO: 18, and the amino acid sequence of the SC-F protein is as shown in SEQ ID NO: 20, that is, the amino acid sequence shown in SEQ ID NO: 15 is fused with the amino acid sequence shown in SEQ ID NO: 14 through a flexible linker.

7. The portable immunochromatographic device for detecting African swine fever virus neutralizing antibody according to claim 6, wherein The method for preparing the p54-3 recombinant protein according to claim 6 comprises the following steps: (1) The amino acid sequence shown in SEQ ID NO: 12 and the amino acid sequence shown in SEQ ID NO: 9 are sequentially concatenated six times, and then sequentially concatenated six times with the amino acid sequence shown in SEQ ID NO: 10 through a flexible linker to obtain the amino acid sequence shown in SEQ ID NO: 18; (2) The amino acid sequence shown in SEQ ID NO: 18 is constructed onto the pcDNA3.1 vector, the plasmid is extracted, and after being identified by sequencing, the p54 recombinant plasmid is obtained. The p54 recombinant plasmid is transfected into 293F cells, cultured, centrifuged, the supernatant of the transfected cells is collected to remove impurities, purified, and the p54-3 recombinant protein with the amino acid sequence shown in SEQ ID NO: 18 is obtained after separation and purification.

8. The portable immunochromatographic device for detecting African swine fever virus neutralizing antibody according to claim 6, characterized in that, The method for preparing the SC-F protein according to claim 6 is: The amino acid sequence shown in SEQ ID NO: 15 is fused with the amino acid sequence shown in SEQ ID NO: 14 through a flexible linker to obtain the amino acid sequence shown in SEQ ID NO:

20. It is constructed onto the pET28a prokaryotic expression vector, induced and expressed using the Escherichia coli expression system, and the ferritin fused with Spycatcher is harvested by nickel column purification and molecular sieve. After measuring the protein concentration by the BCA method, it is adjusted to 1 mg / mL with PBS for standby. The purified protein is denoted as the SC-F protein.

9. The portable immunochromatographic device for detecting African swine fever virus neutralizing antibody according to claim 6, wherein The polyclonal antibody is prepared by immunizing an animal with the p54-F3 recombinant fusion protein according to claim 6.

10. Use of a portable immunochromatographic device for detecting African swine fever virus neutralizing antibodies according to claims 1-9 in the immunological detection of African swine fever virus neutralizing antibodies for non-disease diagnosis purposes.

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