Portable immunochromatographic device for detecting antibodies against african swine fever virus and use thereof

Abstract

The application discloses a portable immunochromatography device for detecting African swine fever virus neutralizing antibodies and application thereof, which comprises a reading module, a sample preparation module and a detection module which are integrally connected in sequence from top to bottom, the sample preparation module is sequentially provided with a sample adding hole, a filter chromatography cavity and a through pipe which are in communication from top to bottom, the filter chromatography cavity is provided with a filter chromatography component, the detection module is provided with a fluorescent immunochromatography test strip and a shell, the fluorescent immunochromatography test strip is located in the shell, a sample pad, a fluorescent marker pad, an NC membrane and a water absorption pad of the fluorescent immunochromatography test strip are sequentially overlapped and adhered on a PVC bottom plate, two ends of the through pipe are respectively in communication with the filter chromatography cavity and the shell, one end of the through pipe is located below the filter chromatography component, and the other end of the through pipe is located above the sample pad, the water absorption pad and the NC membrane of the fluorescent immunochromatography test strip are sequentially inserted into the reading module, the reading module is connected with the shell through buckling, and the quality control line and the detection line of the inserted NC membrane are read.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection, and more particularly, to a portable immunochromatographic device for detecting neutralizing antibodies of African swine fever virus and application thereof. BACKGROUND

[0002] African swine fever (ASF) is an acute, highly infectious and fatal infectious disease caused by African swine fever virus (ASFV), which has caused great impact on the pig breeding industry and is a key animal disease to be prevented in China. At present, the diagnosis methods of ASF mainly include pathogen examination and serological examination, and the PCR molecular detection method dominates the pathogen examination, and the serological examination mainly uses ELISA, IFA and colloidal gold test strip methods for analysis. Due to the existence of resistant pigs and weak strains, there is a risk of missed detection by PCR detection method during the non-detoxification period, and ASFV antibodies can usually maintain in serum for a long time, so ASFV specific antibody detection is still one of the main tools for diagnosis and control of the disease. Therefore, it is of great significance to carry out antibody detection, especially neutralizing antibody detection, for the prevention and control of ASFV.

[0003] The most commonly used antibody detection method for veterinary diagnosis at present is the ELISA detection method, which needs about 2 hours for detection, and the operation is complicated, the operation requirements for the detection personnel are high, and false positive and false negative results are easy to occur due to improper operation, and the detection linear range and detection sensitivity are relatively low due to the influence of equipment. The immune antibody level is a reference index for evaluating the immune effect, and the antibody that can best represent protection in the immune effect is the neutralizing antibody. Therefore, through the screening of antibody recognition epitopes of key coding proteins of ASFV, the present application carries out recombination expression after different combinations, and according to the identification of the characteristics of the recombinant protein, the recombinant antigen capable of recognizing the neutralizing antibody of ASFV is obtained, and then a field rapid detection device suitable for the detection of ASFV neutralizing antibody is developed by using the recombinant antigen, which is applied to breeding farms and other areas with poor conditions, and provides an important detection device support for the prevention and control of African swine fever virus. SUMMARY

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

[0005] According to one aspect of the present application, a portable immunochromatographic device for detecting African swine fever virus neutralizing antibodies is provided, comprising a reading module, a sample preparation module and a detection module connected integrally from top to bottom, the sample preparation module is sequentially provided with a sample addition hole, a filter chromatography cavity and a through pipe in communication from top to bottom, the filter chromatography cavity is provided with a filter chromatography component, the filter chromatography component is a sheep anti-porcine fibrinogen antibody-magnetic nanocellulose chromatography pad, the detection module is provided with a fluorescent immunochromatographic test strip and a shell, the fluorescent immunochromatographic test strip is located in the shell, and the fluorescent immunochromatographic test strip comprises a sample pad, a fluorescent marker pad, an NC membrane, a water absorption pad and a PVC bottom plate, the sample pad, the fluorescent marker pad, the NC membrane and the water absorption pad are sequentially overlapped and adhered on the PVC bottom plate, the NC membrane is sequentially provided with a detection line and a quality control line in the sample flow direction, and the two ends of the through pipe are in communication with the filter chromatography cavity and the shell respectively, one end of the through pipe is located below the sheep anti-porcine fibrinogen antibody-magnetic nanocellulose chromatography pad, and the other end of the through pipe is located above the sample pad, the water absorption pad and the NC membrane of the fluorescent immunochromatographic test strip are sequentially inserted into the reading module, the reading module is connected with the shell through buckling, and the quality control line and the detection line of the inserted NC membrane are read.

[0006] In some embodiments, the preparation method of the filter chromatography component comprises the following steps:

[0007] S1, a uniform nanocellulose suspension with a concentration of 1-2 wt% is prepared by acid hydrolysis, wherein the molecular weight of the nanocellulose is 12-14 kDa;

[0008] S2, FeCl3 and FeCl2 with a molar ratio of 2:1 are added to the uniformly dispersed nanocellulose suspension, iron salt loading is performed, and then magnetic nanocellulose is prepared by in-situ precipitation synthesis;

[0009] S3, the magnetic nanocellulose prepared in S2 is subjected to amination modification and DEAE group grafting to obtain DEAE-magnetic nanocellulose;

[0010] S4, the DEAE-magnetic nanocellulose prepared in S3 and sheep anti-porcine fibrinogen antibody are added to a PBS solution in a mass ratio of 1:5, the sheep anti-porcine fibrinogen antibody is combined with the DEAE-magnetic nanocellulose by over-saturation through gentle stirring, Tween-20 is then added to a final concentration of 0.1%, and the filter chromatography component is immersed in the above mixed solution at 4℃ overnight, followed by low-temperature drying the next day to obtain a sheep anti-porcine fibrinogen antibody-magnetic nanocellulose chromatography pad, i.e., the filter chromatography component, which is cut to an appropriate size, sealed and stored for use.

[0011] In some embodiments, the detection module is provided with a shell cover on the sample pad and the fluorescent marker pad, the detection line, the quality control line and the water absorption pad of the NC membrane are located outside the shell, the shell is further provided with a clamping device for clamping and fixing one end of the sample pad of the fluorescent immunochromatography test strip, and the buckle is located at the opening end of the shell.

[0012] In some embodiments, the reading module is provided with a test strip insertion hole, a display screen and a card slot, the water absorption pad and the NC membrane of the fluorescent immunochromatography test strip are sequentially inserted into the test strip insertion hole, the reading module performs fluorescent reading on the detection results displayed by 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.

[0013] In some embodiments, the fluorescent marker pad contains a fluorescently labeled p54-F3 recombinant fusion protein, the detection line is coated with rabbit anti-swine antibody, and the quality control line is coated with polyclonal antibody against the p54-F3 recombinant fusion protein.

[0014] In some embodiments, the p54-F3 recombinant fusion protein is obtained by fusing p54-3 recombinant protein and SC-F protein at a mass ratio of 1:1, the amino acid sequence of the p54-3 recombinant protein is shown in SEQ ID NO: 18, and the amino acid sequence of the SC-F protein is shown in SEQ ID NO: 20, i.e. the amino acid sequence shown in SEQ ID NO: 15 is fused to the amino acid sequence shown in SEQ ID NO: 14 through a flexible linker.

[0015] In some embodiments, the method for preparing the p54-3 recombinant protein comprises the following steps:

[0016] (1) sequentially connecting the amino acid sequence shown in SEQ ID NO: 12 and the amino acid sequence shown in SEQ ID NO: 9 six times, and then connecting the amino acid sequence shown in SEQ ID NO: 10 six times through a flexible linker to obtain the amino acid sequence shown in SEQ ID NO: 18;

[0017] (2) constructing the amino acid sequence shown in SEQ ID NO: 18 to a pcDNA3.1 vector, extracting a plasmid, and obtaining a p54 recombinant plasmid after sequencing identification, transfecting the p54 recombinant plasmid into 293F cells, culturing, centrifuging, collecting the supernatant of the transfected cells to remove impurities, purifying, and obtaining the p54-3 recombinant protein with the amino acid sequence shown in SEQ ID NO: 18 after separation and purification.

[0018] In some embodiments, the method for preparing the SC-F protein is:

[0019] The amino acid sequence shown as SEQ ID NO: 15 is fused to the amino acid sequence shown as SEQ ID NO: 14 through a flexible linker to obtain an amino acid sequence shown as SEQ ID NO: 20, which is constructed on a pET28a prokaryotic expression vector, induced for expression by using an E. coli expression system, and the ferritin fused with Spycatcher is harvested by nickel column purification and molecular sieve, and after the protein concentration is determined by a BCA method, it is adjusted to 1 mg / mL with PBS for standby, and the purified protein is recorded as SC-F protein.

[0020] In some embodiments, the polyclonal antibody is prepared by immunizing animals with the p54-F3 recombinant fusion protein.

[0021] According to another aspect of the application, there is provided a use of a portable immunochromatographic device for detecting African swine fever virus neutralizing antibodies in immunological detection of African swine fever virus neutralizing antibodies for non-disease diagnosis purposes.

[0022] The application has the following beneficial effects: 1. The portable immunochromatographic device can be directly used for blood detection without the need for separating serum, and the detection time is short, the operation is simple, the detection result is instrumentally determined, and errors caused by human determination are avoided.

[0023] 2. The portable immunochromatographic device comprises a filter chromatography component, which is DEAE-magnetic nanocellulose saturated with sheep anti-porcine fibrinogen antibodies, wherein the sheep anti-porcine fibrinogen antibodies are combined with fibrinogen in pig blood, can effectively adsorb fibrinogen in pig blood, and simultaneously solidify to form a fibrin network structure on the filter chromatography component, which can effectively block red blood cells and other impurities in the blood from passing through the filter chromatography component, so that only serum liquid containing ASFV neutralizing antibodies can pass through the filter chromatography component and smoothly enter the detection module, thereby omitting the cumbersome steps of sample pretreatment, removing impurities, and improving detection sensitivity.

[0024] 3. The recombinant protein obtained by expressing the p54 protein neutralizing antibody epitope of African swine fever virus in series has good antigenicity and reactivity, indirect ELISA is performed after the ferritin is coupled by the SpyCatcher / Spytag system, and good specificity and sensitivity are obtained, which can effectively recognize neutralizing antibodies in serum, the SpyCatcher / Spytag system exposes multiple antigen epitopes on the surface of ferritin, which is equivalent to increasing the binding sites of neutralizing antibodies, so that the detection sensitivity of the recombinant protein to African swine fever virus neutralizing antibodies in serum is greatly improved, and the recombinant protein has good specificity and can be applied to the detection of African swine fever virus neutralizing antibodies. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Structure diagram of the portable immunochromatographic device in Example 1 of the present application.

[0026] Figure 2 Structure diagram of the sample preparation module and detection module of the portable immunochromatographic device in Example 1 of the present application.

[0027] Figure 3 Structure diagram of the reading module of the portable immunochromatographic device in Example 1 of the present application.

[0028] Figure 4 Scanning electron microscope result diagram of the magnetic nanocellulose prepared in Example 2 of the present application.

[0029] Figure 5 p54 protein hydrophilic exposure region diagram predicted by NetSurfP-2.0 in Example 3 of the present application.

[0030] Figure 6 p54 protein membrane inner region secondary structure result diagram predicted by PSIPRED in Example 3 of the present application.

[0031] Figure 7 Indirect immunofluorescence detection result diagram of Example 3 of the present application, wherein a is the indirect immunofluorescence detection result diagram of polypeptide 1 polyclonal antibody, b is the indirect immunofluorescence detection result diagram of polypeptide 2 polyclonal antibody, and c is the indirect immunofluorescence detection result diagram of polypeptide 3 polyclonal antibody.

[0032] Figure 8 SDS-PAGE detection result diagram of four kinds of p54 recombinant proteins prepared in Example 4 of the present application, wherein Line 1 is p54-1; Line 2 is p54-2; Line 3 is p54-3; and Line 4 is p54-4.

[0033] Figure 9 SDS-PAGE detection result diagram of four kinds of recombinant fusion proteins p54-F1, p54-F2, p54-F3, and p54-F4 prepared in Example 4 of the present application, wherein Line 1 is p54-F1; Line 2 is p54-F2; Line 3 is p54-F3; and Line 4 is p54-F4.

[0034] Figure 10 Specificity detection result diagram of four kinds of recombinant fusion proteins p54-F1, p54-F2, p54-F3, and p54-F4 in Example 4 of the present application.

[0035] Figure 11 Sensitivity detection result diagram of three kinds of recombinant fusion proteins p54-F1, p54-F2, and p54-F3 in Example 4 of the present application.

[0036] Figure 12 1 is a ROC curve diagram of the p54-F3 fluorescent immunochromatographic test strip in Example 5 of the present invention. DETAILED DESCRIPTION

[0037] The present invention will be further described in detail through specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, modifications of various equivalent forms of the present invention by those skilled in the art are all within the scope of the appended claims. Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents available on the conventional market.

[0038] 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.

[0039] Table 1 Conservatively substituted amino acids

[0040]

[0041] In addition, due to the degeneracy of bases, bases in the polynucleotide sequence can be substituted without changing the activity or function of the polynucleotide sequence. The recombinant protein for detecting neutralizing antibodies to African swine fever virus provided by the present invention is soluble and can form a correctly folded functional conformation during eukaryotic cell expression, which is closer to the structure and function of the real protein and avoids the biosafety risks brought about by the use of real viruses.

[0042] The following examples and figures are provided to facilitate understanding of the present disclosure. However, it should be understood that these examples and figures are intended to illustrate the present disclosure only and are not intended to limit the present disclosure in any way. The actual scope of protection of the present disclosure is set forth in the claims. It should be understood that any modifications and variations may be made without departing from the spirit of the present disclosure.

[0043] Example 1 A portable immunochromatographic device for detecting neutralizing antibodies to African swine fever virus

[0044] refer to Figures 1-3 A portable immunochromatographic device for detecting neutralizing antibodies to African swine fever virus comprises a reading module 3, a sample preparation module 1, and a detection module 2 integrally connected from top to bottom. The sample preparation module 1 is provided with a sample loading hole 11, a filtration chromatography cavity 12, and a conducting tube 13, which are interconnected from top to bottom. The filtration chromatography cavity 12 is provided with a filtration chromatography component, which is a sheep anti-porcine fibrinogen antibody-magnetic nanocellulose chromatography pad 121. The sheep anti-porcine fibrinogen antibody-magnetic nanocellulose chromatography pad 121 is located between the sample loading hole 11 and the conducting tube 13.

[0045] The sheep anti-porcine fibrinogen antibody-magnetic nanocellulose chromatography pad 121 has DEAE-magnetic nanocellulose adsorbed therein, and the DEAE-magnetic nanocellulose has sheep anti-porcine fibrinogen antibodies over-saturatedly bound to the surface of the DEAE-magnetic nanocellulose. The sheep anti-porcine fibrinogen antibodies can effectively adsorb fibrinogen in the blood of the pig. 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. Therefore, the serum liquid containing the neutralizing antibodies of the African swine fever virus can smoothly pass through the sheep anti-porcine fibrinogen antibody-magnetic nanocellulose chromatography pad 121 and flow into the through pipe 13.

[0046] The detection module 2 is provided with a fluorescent immunochromatography test strip 21 and a shell 22. The fluorescent immunochromatography test strip 21 is located in the shell 22. The fluorescent immunochromatography test strip includes a sample pad 211, a fluorescent marker pad 212, an NC membrane 213, a water absorption pad 214, and a PVC bottom plate 215. The sample pad 211, the fluorescent marker pad 212, the NC membrane 213, and the water absorption pad 214 are sequentially and adhesively connected to the PVC bottom plate 215. The NC membrane 213 contains a detection line 216 and a quality control line 217 arranged in sequence along the sample flow direction. The fluorescent marker pad 212 contains fluorescently labeled p54-F3 recombinant fusion protein. The detection line 216 is coated with rabbit anti-porcine antibodies, and the quality control line 217 is coated with polyclonal antibodies against the p54-F3 recombinant fusion protein.

[0047] The through pipe 13 has two ends respectively extending into the sample preparation module 1 and the shell 22, and 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 passes through the sheep anti-porcine fibrinogen antibody-magnetic nanocellulose chromatography pad 121 and enters the detection module 2 through the through pipe 13, and then drops on the sample pad 211.

[0048] The shell 22 is in a cylindrical shape, covers the sample pad 211 and the fluorescent marker pad 212, and the detection line 216 and the quality control line 217 of the NC membrane 213 and the water absorption pad 214 are located outside the shell 22. The shell 22 is provided with a clamping device 221 and a buckle 222. The clamping device 221 clamps and fixes one end of the sample pad 211 of the fluorescent immunochromatography test strip 21. The buckle 222 is located at the opening end of the shell 22.

[0049] The reading module 3 is provided with a test strip insertion hole 31, a display screen 32 and a clamping groove 33. The water absorption pad 214 and the NC membrane 213 of the fluorescent immunochromatographic test strip 21 are sequentially inserted into the test strip insertion hole 31. The reading module 3 performs fluorescent reading on the detection results displayed by the detection line 216 and the quality control line 217 of the NC membrane 213, and displays the detection values on the display screen 32. The clamping groove 33 is located outside the test strip insertion hole 31, and the buckle 222 is clamped in correspondence with the clamping groove 33. The presence of the buckle 222 makes the reading module reusable, saving production costs. The reading module 3 is provided by Shenzhen Sanfangyuan Biotechnology Co., Ltd.

[0050] In use, the collected pig blood sample is added from the sample addition hole 11 into the filter chromatography cavity 12, and the pig blood sample drops on the sheep anti-pig fibrinogen antibody-magnetic nanocellulose chromatographic pad 121. In the process of flowing through the sheep anti-pig fibrinogen antibody-magnetic nanocellulose chromatographic pad 121, the fibrinogen in the pig blood sample is adsorbed by the sheep anti-pig fibrinogen antibody, and a fibrin network structure is formed on the surface of the filter chromatography component, so that other impurities such as blood cells in the pig blood sample are blocked above the sheep anti-pig fibrinogen antibody-magnetic nanocellulose chromatographic pad 121, realizing the filter chromatography treatment of the pig blood sample. The serum solution containing the neutralizing antibody of the African swine fever virus directly flows to the sample pad 211 of the detection module 2 through the through 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 immunochromatographic test strip 21 is inserted into the test strip insertion hole 31 of the reading module 3 after 10 minutes, so that the water absorption pad 214 and the NC membrane 213 are fully inserted. The reading module 3 performs fluorescent reading on the detection line 216 and the quality control line 217 on the NC membrane 213, and the detection values and detection results are displayed on the display screen 32 of the reading module 3 after reading.

[0051] In some cases, the injection port outer diameter of the syringe matches the inner diameter of the sample addition hole, and the pig blood sample to be tested is added into the filter chromatography cavity 12 through the sample addition hole, and then air is injected into the filter chromatography cavity 12 through the syringe to pressurize, so that the pig blood sample in the filter chromatography cavity 12 can pass through the filter chromatography component more quickly, which can shorten the filter chromatography processing time and improve the detection efficiency. Because the injection port outer diameter of the syringe matches the inner diameter of the sample addition hole, the sample addition hole is blocked by the syringe, which can avoid the escape of the injected air, so as to achieve the purpose of pressurized filtration of the pig blood sample in the filter chromatography cavity 12.

[0052] The portable immunochromatographic device for detecting African swine fever virus neutralizing antibodies can be used for the detection of complex blood samples such as blood and plasma, without the need for serum separation, short detection time, and convenient and fast. The detection result is instrumentally interpreted, scientific and accurate.

[0053] Example 2 Preparation of the filtration chromatography part

[0054] 2.1 Preparation of nanocellulose by acid hydrolysis

[0055] 2.1.1 Pretreatment: 10 g of microcrystalline cellulose with a particle size of 50 μm was dispersed in 200 mL of deionized water, stirred for 30 minutes, and filtered to remove impurities;

[0056] 2.1.2 Acid hydrolysis: The pretreated cellulose was added to 500 mL of 64 wt% sulfuric acid, the temperature was kept at 45℃, and the reaction was carried out under 500 rpm magnetic stirring for 2 hours;

[0057] 2.1.3 Reaction termination: The reaction was terminated by adding a large amount of ice water (volume ratio 1:10), centrifuged at 10,000xg for 15 minutes, and the precipitate was collected;

[0058] 2.1.4 Washing and purification: The precipitate was washed with deionized water by centrifugation until neutral (pH ≈ 7), and then washed with ethanol for 3 times to remove residual acid;

[0059] 2.1.5 Dialysis treatment: The suspension was placed in a dialysis bag with a molecular weight cutoff of 12-14 kDa, and dialyzed in deionized water for 3 days (water was changed 3 times a day), to remove small molecular impurities;

[0060] 2.1.6 Ultrasonic dispersion: After dialysis, the suspension was treated by ultrasonic (power 300 W, 30 minutes) to obtain a uniform nanocellulose suspension with a concentration of 1-2 wt%.

[0061] 2.2 Preparation of magnetic nanocellulose

[0062] 2.2.1 Disperse nanocellulose: Take 100 mL of nanocellulose suspension with a concentration of 1 wt%, and ultrasonic treatment (200 W, 20 minutes) to ensure uniform dispersion;

[0063] 2.2.2 Iron salt loading: Add 0.1 M FeCl3 and 0.05 M FeCl2, stir for 30 minutes to make Fe³⁺ / Fe²⁺ adsorbed on the surface of nanocellulose;

[0064] 2.2.3 In-situ precipitation: Increase the temperature to 75℃, protect with N2, slowly add ammonia water to pH 10, and react for 2 hours;

[0065] 2.2.4 Post-treatment: The composite material was collected by magnetic separation, washed with deionized water and ethanol successively, and freeze-dried at -50℃ for 24 hours to obtain magnetic nanocellulose.

[0066] 2.3 Surface functionalization

[0067] 2.3.1 Amino-modification: The magnetic nanocellulose was dispersed in anhydrous toluene, 5% 3-aminopropyltriethoxysilane (APTES) was added, and refluxed at 80℃ for 6 hours. The amino-modified magnetic nanocellulose was obtained by centrifugal washing.

[0068] 2.3.2 DEAE group grafting: The amino-modified magnetic nanocellulose was reacted with 10% diethylaminoethyl chloride-borate buffer (pH 8.5) by mass fraction at 60℃ for 12 hours. The DEAE-magnetic nanocellulose was obtained by washing.

[0069] 2.4 Characterization and performance test

[0070] 2.4.1 SEM analysis of morphology: The morphology of the DEAE-magnetic nanocellulose was observed by SEM at an acceleration voltage of 5 kV and with gold spraying treatment. The results are shown in Figure 4 From the results, it can be seen that the diameter of the DEAE-magnetic nanocellulose is 20-50 nm, and the surface of the nanocellulose is covered with gray magnetic particles. Figure 4

[0071] 2.4.2 Magnetic performance test:

[0072] VSM: The magnetic field range was ±20 kOe, and the saturation magnetization was measured. The measured saturation magnetization of the DEAE-magnetic nanocellulose was >40 emu / g, indicating that the prepared DEAE-magnetic nanocellulose has strong magnetism.

[0073] 2.4.3 Adsorption performance test:

[0074] Plasma experiment: 1 mL of plasma was mixed with 10 mg of DEAE-magnetic nanocellulose, and oscillated at 37℃ for 30 minutes. After magnetic separation, the change in antibody concentration in the plasma was detected by ELISA. A simple plasma sample was set as a control. It was found that the antibody concentration in the plasma after magnetic separation decreased significantly.

[0075] 2.5 Preparation of filter chromatography components

[0076] ​The DEAE-magnetic nanocellulose prepared in 2.3 and sheep anti-porcine fibrinogen antibody were added into 10 mL PBS at a mass ratio of 1:5, 10 mg in total, and combined at 4°C with gentle stirring for 6 h, then Tween-20 was added to a final concentration of 0.1%, and the stirring was continued at 4°C for 1 h. The filter chromatography component was placed in the above mixed solution, soaked at 4°C overnight, and dried at low temperature the next day to obtain a sheep anti-porcine fibrinogen antibody-magnetic nanocellulose chromatography pad 121, i.e. a filter chromatography component, which was cut to an appropriate size, sealed and stored for use.

[0077] The sheep anti-porcine fibrinogen antibody added in the above preparation process is supersaturated with respect to the DEAE-magnetic nanocellulose, and can be combined with the DEAE-magnetic nanocellulose in a supersaturated manner.

[0078] The sheep anti-porcine fibrinogen antibody-magnetic nanocellulose chromatography pad 121 can effectively adsorb fibrinogen in blood, and after the adsorbed fibrinogen is converted into fibrin, the fibrin forms a network structure that can effectively prevent the passage of blood cells, thereby achieving filtration of blood cells in the blood sample and allowing the serum in the blood to flow smoothly through the sheep anti-porcine fibrinogen antibody-magnetic nanocellulose chromatography pad 121. Microscopic examination of the effluent liquid shows a significant reduction in blood cells, indicating that the sheep anti-porcine fibrinogen antibody-magnetic nanocellulose chromatography pad 121 of this embodiment has a significant filtering and blocking effect on blood cells.

[0079] Example 3 Prediction and screening of African swine fever virus p54 protein neutralizing antibody recognition epitopes

[0080] Materials, reagents and instruments used in the examples:

[0081] Materials

[0082] 1. Bacterial strain

[0083] The E. coli competent cells DH5α were purchased from Beijing Quanshi Gold Biotechnology Co., Ltd., the pCAGGS vector and pcNDA3.1 vector, and 293F cells were preserved in the laboratory;

[0084] 2. Serum

[0085] ASFV positive serum inactivated with 0.3% TNBP and 1% triton X-100 was a gift from the Institute of Animal Health and Technology of the Middle East, PRRSV vaccine virus positive serum, PPV vaccine virus positive serum, PRV vaccine virus positive serum, PCV3 positive serum inactivated with 0.3% TNBP and 1% triton X-100 were preserved in our laboratory; 50 inactivated African swine fever positive serum and 150 ASFV negative serum were donated by the National Veterinary Institute of Poland and the National Reference Laboratory for African Swine Fever of the Institute of Animal Health of Sardinia, Italy; Pig negative control serum was purchased from Gibco Company;

[0086] Reagents

[0087] SMM 293-TIS Expression Medium, PEI were purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., Ni-NTA agarose gel resin, plasmid extraction kit were purchased from QIAGEN company, BCA protein concentration determination kit was purchased from Beijing Solabio Technology Co., Ltd., fetal bovine serum was purchased from Gibico company, BamH I / Xho I restriction endonuclease, T4 ligase, TaKaRa LA PCR™ Kit were purchased from TaKaRa company, DNA extraction kit DNeasy Blood and Tissue Kit was purchased from Qiagen company, Agarose Gel DNA Purification Kit, IPTG, X-gal, ampicillin were purchased from Beijing Quanshi Gold Biotechnology Co., Ltd., rabbit anti-pig IgG HRP was purchased from Bioawell; FITC-labeled goat anti-mouse antibody was purchased from Abeam company;

[0088] Instruments

[0089] ABI's general PCR instrument (AB Applied Biosystems), sigma's table centrifuge (3-18k), constant temperature oscillation incubator (HZQ-F100), fluorescence microscope (ZEISS, AXIO), six one electrophoresis instrument, double infrared laser scanning imaging system (Proteinsimple).

[0090] 3.1 Screening of neutralizing antibody epitopes in African swine fever virus p54 protein

[0091] According to the full-length amino acid sequence (SEQ ID NO: 1) of the p54 protein of the African swine fever virus Heilongjiang strain (MK333180.1) published in GenBank, the secondary structure of the p54 protein was preliminarily analyzed and predicted by 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 intramembrane region of the p54 protein were obtained, as shown in Figure 6 Combined with 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 entrusted to synthesize polypeptides.

[0092] Table 2 Summary of screened p54 protein antigen epitopes

[0093]

[0094] Table 3 Three different combinations of p54 synthetic peptides

[0095]

[0096] 3.2 Preparation of polyclonal antibodies and comparison of neutralizing effects of polyclonal antibodies

[0097] The polypeptide 1, polypeptide 2 and polypeptide 3 synthesized in 3.1 were diluted to 1 mg / mL with PBS, mixed with Freund's complete adjuvant, and then subcutaneously injected into mice for immunization. Each polypeptide was used to immunize mice at three doses (50 μg, 100 μg, 150 μg). Two weeks after the initial immunization, the same method was used for injection again, but Freund's incomplete adjuvant was used. 7-10 days later, blood was collected to separate mouse serum, and the neutralization test was performed using the polyclonal antibody serum to compare the neutralization titers of the polyclonal antibody sera prepared from the above three polypeptides.

[0098] The ASF virus liquid was diluted to 100 TCID 50 / 0.1 mL. The prepared polyclonal antibodies were diluted 2-fold by 2 dilutions, and each dilution was repeated 4 times. A positive control (containing only virus liquid without antibody) and a negative control (empty cells) were also set. After mixing the diluted virus liquid with different dilutions of polyclonal antibodies, the mixture was incubated at 4°C for 2 hours, and then PAM cells were added to the 96-well cell plate for further culture for 24 hours. The culture solution was discarded, and the cells were fixed for indirect immunofluorescence test with FITC-labeled goat anti-mouse antibody. After the reaction, the results were recorded by fluorescence microscopy. If the fluorescence decreases with the increase of antibody concentration, it indicates that the virus is neutralized by the antibody. If the number of fluorescence does not change, it indicates that the virus is not neutralized by the antibody.

[0099] The results of indirect immunofluorescence of various polyclonal antibodies are shown in Figure 7 a~ Figure 7 c, After comparison, the polyclonal antibodies produced by mice immunized with 10 μg of polypeptide 1 and polypeptide 2 have higher neutralizing effect, and the fluorescence in each cell decreases with the increase of polyclonal antibody concentration. However, the fluorescence in the cell hole incubated with polypeptide 3 polyclonal antibody does not change much compared with the virus control. Therefore, it can be seen that the antigen epitopes of polypeptide 1 and polypeptide 2 can be well recognized and neutralized by antibodies, and therefore polypeptide 1 and polypeptide 2 are selected for further study.

[0100] Example 4 Expression and purification of p54 neutralizing antibody recognition epitope recombinant protein

[0101] 4.1 Design and expression of p54 neutralizing antibody recognition epitope recombinant protein

[0102] SpyCatcher / SpyTag is a commonly used protein coupling system, which is simple, fast and can realize in vitro self-assembly of proteins. Therefore, in this embodiment, SpyCatcher / SpyTag (SC-ST) system is used to design four kinds of p54 neutralizing antibody recognition epitope recombinant proteins. The sequence design method is as follows, and the sequences are shown in SEQ ID NO: 16-19:

[0103] p54-1: His tag + polypeptide 1 + polypeptide 2 + spy tag, (SEQ ID NO: 16);

[0104] p54-2: His tag + polypeptide 1 coupled 3 times + GGGGGGGG (flexible linker) + polypeptide 2 coupled 3 times + spy tag, (SEQ ID NO: 17);

[0105] p54-3: His tag + polypeptide 1 coupled 6 times + GGGGGGGG (flexible linker) + polypeptide 2 coupled 6 times + spy tag, (SEQ ID NO: 18);

[0106] p54-4: His tag + polypeptide 1 repeated 9 times coupling + GGGGGGGG (flexible linker) + polypeptide 2 repeated 9 times coupling + spy tag, (SEQ ID NO: 19).

[0107] The sequences of His tag, spy tag, SpyCatcher and Ferritin are shown in SEQ ID NO: 12-15, and are specifically shown in Table 4.

[0108] Table 4 His tag, spy tag, SpyCatcher amino acid sequence table

[0109]

[0110] The above SEQ ID NO: 16-19 sequences are respectively constructed into pcDNA3.1 vector, and after extraction of plasmid and identification by sequencing, 293F cells are transfected, a mixture containing 2 mg of p54 recombinant plasmid and 12 mg of PEI is prepared in 1 L of cell culture medium, and the mixture is transfected into 293F cells when the cell density is 2 x 10 6 / mL, centrifuged at 1 000 x g for 20 min at 48 h after transfection, and the transfected cell supernatant is collected, impurities are removed with a 0.22 μm filter membrane, and the supernatant is added to the pretreated Ni-NTA beads. Purification is performed according to the nickel column purification operation steps, the effluent of the target protein and the protein eluent are collected, concentrated with a 10 ku ultrafiltration tube, and 10 μL per tube is taken for SDS-PAGE detection, the results are shown in Figure 8 , the protein concentration is determined by BCA method, and adjusted to 1 mg / mL with PBS for standby, and the purified proteins are respectively recorded as recombinant proteins p54-1, p54-2, p54-3, and p54-4.

[0111] Meanwhile, the Ferritin protein with the sequence shown in SEQ ID NO: 15 is fused with the SpyCatcher with the sequence shown in SEQ ID NO: 14 using a flexible linker (GGGGGGGG), and is constructed into a pET28a prokaryotic expression vector. The ferritin fused with Spycatcher is harvested by nickel column purification and molecular sieve using an Escherichia coli expression system for induction expression. After determining the protein concentration by BCA method, the protein is adjusted to 1 mg / mL with PBS for standby, and the purified protein is recorded as SC-F protein. The amino acid sequence of SC-F protein is shown in SEQ ID NO: 20.

[0112] 4.2 Preparation of p54 protein and ferritin complex

[0113] The four p54 recombinant proteins purified in 4.1 were mixed with SC-F ferritin at a mass ratio of 1:1 and incubated at 4°C overnight. After the reaction was complete, the unreacted proteins were separated using a size exclusion chromatography molecular sieve column. The protein solution was filtered through a 0.22 μm filter membrane and equilibrated with 2 column volumes of equilibration buffer (0.02 mol / L sodium phosphate, 0.5 mol / L sodium chloride, pH = 7.4) before loading the column. The column was then rinsed for another 2 column volumes. Proteins of different molecular weights eluted at different times. The target protein elution peak was collected for SDS-PAGE electrophoresis verification. The electrophoresis results are shown in Figure 2. Figure 9 .

[0114] Depend on Figure 9 It can be seen that after the reaction products were separated by SEC, fusion proteins of p54 protein and ferritin with high purity were obtained, which were labeled as recombinant fusion proteins p54-F1, p54-F2, p54-F3, and p54-F4, respectively. The concentrations of the above proteins were measured and adjusted to 1 mg / mL with PBS for later use.

[0115] 4.3 Comparison of specificity of recombinant fusion proteins p54-F1, p54-F2, p54-F3, and p54-F4

[0116] Recombinant fusion proteins p54-F1, p54-F2, p54-F3 and p54-F4 were coated on ELISA plates for indirect ELISA 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 pig negative serum, and the secondary antibody was HRP-labeled rabbit anti-pig IgG. At the same time, positive and negative controls were set. After the reaction was completed, the OD was read. 450nm The S / N value was calculated and a S / N value ≥ 2.1 was considered positive and a S / N value < 2.1 was considered negative. Figure 10 .

[0117] from Figure 10 It can be seen that the 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 pig negative serum. Among them, p54-F3 had a clearer positive and negative distinction, while p54-F4 cross-reacted with PRV vaccine virus positive serum and had poor specificity.

[0118] 4.4 Comparison of the sensitivity of recombinant fusion proteins p54-F1, p54-F2, and p54-F3

[0119] The ASFV standard positive serum was diluted in multiples (1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560, 1:5120) and coated with the three fusion proteins p54-F1, p54-F2, and p54-F3, respectively. The diluted ASFV standard positive serum was detected by indirect ELISA. At the same time, a negative control was set up to determine the detection sensitivity of the three fusion proteins to the standard positive serum. The test results are shown in Figure 11 .

[0120] Depend on Figure 11 It can be seen that the recombinant fusion protein p54-F3 can detect ASFV-positive serum diluted 1:2560, with the highest detection sensitivity, while p54-F1 can only detect positive serum diluted 1:160, and p54-F2 can only detect positive serum diluted 1:640. The above results show that the present invention's repeated coupling of the p54 main neutralizing antibody epitope 6 times is the best coupling method, which can not only improve detection sensitivity but also avoid non-specific binding. Therefore, the recombinant fusion protein p54-F3 was used for the subsequent development of test strips. At the same time, the recombinant fusion protein p54-F3 was used to prepare the p54-F3 mouse polyclonal antibody according to the polyclonal antibody preparation method in 3.2 of Example 3.

[0121] Example 5 Preparation of a Fluorescent Immunochromatographic Test Strip for Detecting Neutralizing Antibodies to African Swine Fever Virus

[0122] 5.1 Preparation of recombinant fusion protein p54-F3-fluorescent marker

[0123] Take 100 μL of fluorescent microsphere solution and label 100 μg of recombinant fusion protein p54-F3 according to the instructions. Finally, the p54-F3-fluorescent marker conjugate is washed with 300 μL of 5 g·L -1 BSA, 5 g L -1 Resuspend in a diluent containing PVP, 50 mmol / L Tris and 0.2% sodium azide (pH 8.0) and store at 4°C until use.

[0124] 5.2 Preparation of p54-F3 Fluorescent Immunochromatographic Test Strips

[0125] The p54-F3-fluorescent label conjugate was uniformly sprayed on the fluorescent label pad by a film sprayer at an amount of 5 μL / cm, and dried at 37°C for 2 hours. The NC membrane was provided with a test line (T line) and a quality control line (C line). The test line (T line) was coated with rabbit anti-swine antibody at a coating concentration of 0.5-1.2 mg / mL, and the quality control line (C line) was coated with polyclonal antibody against recombinant fusion protein p54-F3 at a coating concentration of 0.25-0.6 mg / mL. The coating concentration of the test line (T line) and the quality control line (C line) was 2:1. The sample pad, the fluorescent label pad, the NC membrane, and the water absorption pad were sequentially laminated and pasted on the PVC bottom plate, and then cut into 4 mm wide test strips. The prepared test strips were stored in an aluminum foil bag, sealed, and placed in a dry place for storage.

[0126] 5.3 Determination of the detection threshold of the p54-F3 fluorescent immunochromatographic test strip

[0127] The detection results of 200 known background sera (50 positive sera and 150 negative sera) by the p54-F3 fluorescent test strip were determined by ROC curve (receiver operating characteristic curve).

[0128] The ROC curve was plotted according to the detection results of 200 sera by the p54-F3 fluorescent microsphere detection method, as shown in Figure 12 .

[0129] As can be seen from Figure 12 , the area under the curve is 0.982, indicating that the method has high accuracy. For each sample on the curve, the Youden index (the sum of sensitivity and specificity minus 1) corresponding to T / C, the maximum Youden index (0.921) corresponding to T / C=0.47 was selected as the critical value of the method, and the corresponding sensitivity was 94.8% and the specificity was 97.3%. Therefore, the determination criteria of the method are as follows: T / C≥0.47, determined as ASFV neutralizing antibody positive; and T / C<0.47, determined as ASFV neutralizing antibody negative.

[0130] Example 6 Comparison of the actual measurement effect of the portable immunochromatographic device for detecting African swine fever virus neutralizing antibodies

[0131] 6.1 Assembly of the portable immunochromatographic device:

[0132] The fluorescent immunochromatographic test strip card prepared in Example 5 was loaded into the detection module of Example 1, so that one end of the sample pad of the fluorescent immunochromatographic test strip was clamped and fixed by the clamping device, and a portable immunochromatographic detection device 1 was prepared. The device 1 is provided with a filter chromatography component that adsorbs sheep anti-porcine fibrinogen antibody-magnetic nanocellulose, i.e., a sheep anti-porcine fibrinogen antibody-magnetic nanocellulose chromatography pad.

[0133] Meanwhile, the portable immunochromatographic detection device 2 and the portable immunochromatographic detection device 3 of Example 1 were prepared. The device 2 is provided with a filter chromatography component that only adsorbs magnetic nanocellulose, i.e., a magnetic nanocellulose chromatography pad. The device 3 is provided with a filter chromatography component that does not adsorb sheep anti-porcine fibrinogen antibody and does not adsorb magnetic nanocellulose, i.e., a chromatography pad.

[0134] 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 at different dilution ratios, i.e., 1:10, 1:100, 1:200, 1:400, 1:800, and 1:1600. 500 μL of the blood sample at each dilution ratio was added dropwise to the sample addition hole of the portable immunochromatographic detection devices 1-3 prepared in 6.2, and after 10 min, the fluorescence reading module was used to read the fluorescence of the test line and the quality control line of the fluorescent test strip of the devices 1-3. The specific results are shown in Table 5.

[0135] Table 5 Sensitivity detection results

[0136]

[0137] As shown in Table 5, in terms of sensitivity detection, the portable immunochromatographic detection device 1 (provided with a filter chromatography component that adsorbs sheep anti-porcine fibrinogen antibody-magnetic nanocellulose) can detect the blood of pigs immunized with recombinant protein p54-F3 at a dilution ratio of 1:800, and has high detection sensitivity.

[0138] The portable immunochromatographic detection device 2 (provided with a filter chromatography component that only adsorbs magnetic nanocellulose) has low detection sensitivity. When the blood of pigs immunized with recombinant protein p54-F3 is added to the device 2, the magnetic nanocellulose can adsorb the ASFV antibodies in the blood, which greatly reduces the ASFV antibodies passing through the filter chromatography component. However, the blood cells and part of the fibrinogen in the blood will flow to the test strip sample pad under the action of gravity, and the fibrinogen will coagulate to form fibrin on the sample pad, which affects the chromatography of the sample to the NC membrane. Under the dual effects of antibody adsorption and chromatography influence, the detection sensitivity of the device 2 is very low.

[0139] The filter chromatography component provided in the portable immunochromatographic detection device 3 is only a simple chromatography pad, which neither adsorbs the magnetic nanocellulose nor saturates the sheep anti-porcine fibrinogen antibody, although the chromatography pad can filter and block some impurities in the blood, the effect is small, and various proteins and blood cells and other substances in the blood cannot be filtered by the chromatography pad, but fall onto the sample pad of the test strip through the through pipe with the serum, forming solid substances such as coagulated fibrin on the sample pad, which seriously affects the chromatography of the sample on the test strip, thereby resulting in very low detection sensitivity.

[0140] Therefore, in terms of sensitivity detection, the portable immunochromatographic detection device 1 (provided with a filter chromatography component adsorbing sheep anti-porcine fibrinogen antibody-magnetic nanocellulose), when the blood flows downward, the fibrinogen in the blood is combined with the sheep anti-porcine fibrinogen antibody, and the sheep anti-porcine fibrinogen antibody is adsorbed on the magnetic nanocellulose, thereby forming a fibrin network structure, blocking the passage of blood cells and other blood impurities, and only the blood containing the ASFV antibody can smoothly flow to the fluorescent immunochromatographic test strip below, thereby making the sensitivity of the test strip as high as 1:800 times.

[0141] 6.4 Specificity detection

[0142] The portable immunochromatographic detection devices 1-3 prepared in 6.2 were used to detect ASFV antibody positive serum and control serum (PRRSV vaccine virus positive serum, PPV vaccine virus positive serum, PRV vaccine virus positive serum, PCV3 positive serum, and pig negative serum), respectively. The detection results are shown in Table 6.

[0143] Table 6 Specificity detection results table

[0144]

[0145] The specificities of the above detection devices 1-3 are good, which is greatly related to the fact that the recombinant fusion protein p54-F3 expressed by the application in combination with the epitope recognized by the neutralizing antibody is used as an antigen, and the recombinant antigen expressed by the application capable of recognizing the epitope of the neutralizing antibody adopts the SpyCatcher / SpyTag system, and the ferritin is fused, which improves the antigenicity of the recombinant antigen complex, and the SpyTag system displays multiple neutralizing antibody epitopes on the surface of the ferritin, thereby ensuring the high efficiency and specificity of the detection results.

[0146] The above only describes some embodiments of the application, and those skilled in the art can make several modifications and improvements without departing from the inventive concept, which are all within the protection scope of the application.

Claims

1. A portable immunochromatographic device for detecting neutralizing antibodies against African swine fever virus, characterized in that: The invention comprises a reading module, a sample preparation module and a detection module connected in an upper and lower integral manner, wherein the sample preparation module is provided with a sample addition hole, a filtration chromatography cavity and a conducting tube in order from top to bottom, the filtration chromatography cavity is provided with a filtration chromatography component, and the filtration chromatography component is a sheep anti-porcine fibrinogen antibody-magnetic nanocellulose chromatography pad, the detection module is provided with a fluorescent immunochromatographic test strip and a shell, the fluorescent immunochromatographic test strip is located in the shell, the fluorescent immunochromatographic test strip comprises a sample pad, a fluorescent marker pad, an NC membrane, a water-absorbing pad and a PVC bottom plate, the sample pad, the fluorescent marker The recording pad, NC membrane, and absorbent pad are overlapped and adhered to the PVC bottom plate in sequence. The detection line and quality control line are arranged in sequence on the NC membrane along the sample flow direction. The two ends of the conducting tube are respectively connected to the filtration chromatography cavity and the housing. One end of the conducting tube is located below the sheep anti-porcine fibrinogen antibody-magnetic nanocellulose chromatography pad, and the other end is located above the sample pad. The absorbent pad and NC membrane of the fluorescent immunochromatographic test strip are sequentially inserted into the reading module. The reading module is connected to the housing by a snap and reads the quality control line and detection line of the inserted NC membrane.

2. A portable immunochromatographic device for detecting neutralizing antibodies against African swine fever virus according to claim 1, characterized in that: The preparation method of the filtration chromatography component comprises the following steps: S1, preparing a uniform nanocellulose suspension with a concentration of 1-2 wt% by acid hydrolysis, wherein the molecular weight of the nanocellulose is 12-14 kDa; S2, adding FeCl3 and FeCl2 in a molar ratio of 2:1 to the uniformly dispersed nanocellulose suspension to load the iron salt, and then preparing magnetic nanocellulose by an in situ precipitation synthesis method; S3, performing amino modification and DEAE group grafting on the magnetic nanocellulose prepared in S2 to obtain DEAE-magnetic nanocellulose; S4, add the DEAE-magnetic nanocellulose and sheep anti-porcine fibrinogen antibody prepared in S3 into the PBS solution in a mass ratio of 1:5, gently stir to allow the sheep anti-porcine fibrinogen antibody to supersaturate with the DEAE-magnetic nanocellulose, then add Tween-20 to a final concentration of 0.1%, continue stirring, place the chromatography pad in the above mixed solution, soak it at 4°C overnight, and dry it at low temperature the next day to obtain a sheep anti-porcine fibrinogen antibody-magnetic nanocellulose chromatography pad, i.e., a filtration chromatography component, which is cut into appropriate sizes, sealed and stored for later use.

3. A portable immunochromatographic device for detecting neutralizing antibodies against African swine fever virus according to claim 1, characterized in that: The outer shell of the detection module is covered on the sample pad and the fluorescent marker pad. The detection line, quality control line and absorbent pad of the NC membrane are all located outside the outer shell. The outer shell is also provided with a clamping device, which clamps and fixes one end of the sample pad of the fluorescent immunochromatographic test strip. The buckle is located at the open end of the outer shell.

4. A portable immunochromatographic device for detecting neutralizing antibodies against African swine fever virus 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 of the fluorescent immunochromatographic test strip and the NC membrane are sequentially inserted into the test strip insertion hole. The reading module performs fluorescent readings on the test results displayed by the test line and the quality control line of the NC membrane, and displays the test values ​​on the display screen. The card slot is located outside the test strip insertion hole, and the buckle is correspondingly engaged with the card slot.

5. A portable immunochromatographic device for detecting neutralizing antibodies against African swine fever virus according to claim 1, characterized in that: The fluorescent marker pad contains 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 polyclonal antibody against p54-F3 recombinant fusion protein.

6. A portable immunochromatographic device for detecting neutralizing antibodies against African swine fever virus according to claim 5, characterized in that: 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 shown in SEQ ID NO: 18, and the amino acid sequence of the SC-F protein is shown in SEQ ID NO: 20, that is, the amino acid sequence shown in SEQ ID NO: 15 is fused to the amino acid sequence shown in SEQ ID NO: 14 through a flexible linker.

7. A portable immunochromatographic device for detecting neutralizing antibodies against African swine fever virus according to claim 6, characterized in that: 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 was sequentially connected to six repeats of the amino acid sequence shown in SEQ ID NO: 9, and then sequentially connected to six repeats of 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 was constructed into the pcDNA3.1 vector, and the plasmid was extracted. After sequencing and identification, the p54 recombinant plasmid was obtained. The p54 recombinant plasmid was transfected into 293F cells, cultured, and centrifuged. The supernatant of the transfected cells was collected to remove impurities and purified. After separation and purification, the p54-3 recombinant protein with the amino acid sequence shown in SEQ ID NO: 18 was obtained.

8. A portable immunochromatographic device for detecting neutralizing antibodies against African swine fever virus 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 was fused to the amino acid sequence shown in SEQ ID NO: 14 via a flexible linker to obtain the amino acid sequence shown in SEQ ID NO:

20. This amino acid sequence was then constructed into the pET28a prokaryotic expression vector and induced for expression using an Escherichia coli expression system. The Spycatcher-fused ferritin was then purified using a nickel column and molecular sieves. The protein concentration was determined by the BCA method and adjusted to 1 mg / mL with PBS for later use. The purified protein was designated as SC-F protein.

9. A portable immunochromatographic device for detecting neutralizing antibodies against African swine fever virus according to claim 6, characterized in that: The polyclonal antibody is prepared by immunizing animals with the p54-F3 recombinant fusion protein according to claim 6.

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

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