Monoclonal antibody for detecting African swine fever virus p72 protein, immunodetection test paper and application

Abstract

The invention relates to a monoclonal antibody and test paper for detecting African swine fever virus p72 protein as well as a preparation method and application of the monoclonal antibody and the test paper. The African swine fever virus p72 recombinant protein is used for an immunochromatography detection system marked by quantum dots, and the quantum dots are used for replacing colloidal gold particles to serve as a signal marker; the marker and the African swine fever virus p72 protein are coupled and then coated on the combination pad, and the staphylococcus A protein and the monoclonal antibody of another site corresponding to the conjugate are coated on the nitrocellulose membrane to form a detection line and a control line. The African swine fever virus antibody fluorescence immunochromatography test paper prepared by using the ASFV-p72 recombinant protein as the antigen can accurately and rapidly detect whether the African swine fever virus antibody exists in serum to be detected or not, is short in detection time, strong in specificity and high in sensitivity, greatly reduces the omission ratio, improves the detection accuracy and reduces the detection cost. The kit can be used for ASFV infection diagnosis, quarantine inspection and epidemiological investigation and research, and has good popularization value.

Description

Technical Field

[0001] The invention relates to a monoclonal antibody of African swine fever virus p72 protein, an immunoassay test paper, a preparation method and application thereof, and belongs to the technical field of immunoassay. Background Art

[0002] African swine fever is a highly contagious viral disease of pigs caused by African swine fever virus (ASFV), characterized by high fever and skin hemorrhage. The mortality rate of domestic pigs infected by the virus is close to 100%. The World Organization for Animal Health (OIE) has listed the disease as a notifiable animal disease and it has been listed as one of the key diseases to be prevented in my country.

[0003] The p72 protein is the main component of the ASFV icosahedral viral capsid, which is encoded by the B646L gene. In the early stage of virus particle formation, the newly synthesized p72 protein is distributed between the endoplasmic reticulum and the soluble cytoplasmic library binding membrane, and the ASFV core and the outer capsid are assembled in different regions of the ER membrane. In the late stage of ASFV virus infection, the B646L gene begins to be expressed, and the viral late promoter regulates and induces the synthesis of the late protein p72. The p72 protein is mainly involved in the assembly of the ASFV capsid and plays an important role in the adsorption and invasion of susceptible cells by the virus. It can induce the body to produce neutralizing antibodies. The B602L protein has the function of assisting the production of antitrypsin. When the p72 protein is expressed alone in HEK293F cells, soluble aggregation occurs and the correct three-dimensional structure cannot be formed. Only when the p72 protein and the B602L protein are expressed together in mammalian cells can the p72 protein be correctly folded and assembled, thereby exercising normal biological functions.

[0004] Compared with conventional techniques such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), quantum dot-labeled immunochromatography technology has the advantages of simple operation, high detection sensitivity, good specificity, and little influence by external factors, and has developed rapidly in recent years. In the prior art, there are no reports on quantum dot-labeled antibody detection strips based on African swine fever virus protein. Summary of the invention

[0005] In order to solve the problems existing in the detection of African swine fever virus, the present invention provides a monoclonal antibody for detecting the p72 protein of African swine fever virus.

[0006] The present invention also provides a fluorescent immunochromatographic test paper for detecting African swine fever virus antibodies. The test paper has the advantages of strong specificity, good sensitivity, accurate detection results, etc., and is suitable for rapid detection of clinical samples.

[0007] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0008] 1. A monoclonal antibody against African swine fever virus p72 protein, comprising a heavy chain variable region amino acid sequence and a light variable region amino acid sequence.

[0009] The amino acid sequence of the heavy chain variable region includes CDR-H1: LLFLD; the amino acid sequence of the light variable region includes CDR-L1, CDR-L2 and CDR-L3, and the corresponding amino acid sequences are: LTQSPASLAVSLGQR, YSYMHWN and TDFT, respectively.

[0010] The monoclonal antibody further comprises heavy chain variable framework region sequences FR-H1, FR-H2, FR-H3 and FR-H4; and light chain variable framework regions FR-L1, FR-L2, FR-L3, the sequences of which are:

[0011] FR-H1:MSNTDWKEGQRKTIHIYATGNNSSHAVLG;

[0012] FR-H2:IMAWVWTLLFLMAA;

[0013] FR-H3: AQSAQAQIQLVQSGPELKKPGETVKISCKASG;

[0014] FR-H4:YTFTNLGMNWVKQAPGKGLKWMGWINTYSGN;

[0015] FR-L1 WEFQETDTLLLWVLLLWVPGSTG;

[0016] FR-L2:ATISYRASKSVSTSG;

[0017] FR-L3: QQKPGQPPRLLIYLVSNLESGVPARFSGSGSG.

[0018] 2. The method for preparing the monoclonal antibody comprises the following steps:

[0019] (1) Preparation of p72 recombinant protein

[0020] According to the codon preference of the CHO expression system, the sequence of the B602L gene encoding the p72 protein of the African swine fever virus was optimized and synthesized; the synthesized gene was used as a template to obtain the target fragment by PCR amplification, and the target fragment was cloned into the expression vector pCGS3 to obtain the recombinant plasmid pCGS3-p72;

[0021] The recombinant plasmid pCGS3-p72 was transferred into Escherichia coli DH5α cells, and the plasmid was extracted after amplification culture. The recombinant plasmid and the B602L gene were double-digested and connected using T4 DNA to obtain the recombinant plasmid pCGS3-p72-B602L;

[0022] The recombinant plasmid pCGS3-p72-B602L was transferred into CHO cells, the cells were collected and lysed, and the impurities were removed using an Anti-Flag affinity chromatography column, eluted and purified, and concentrated by ultrafiltration and centrifugation to obtain the African swine fever virus p72 recombinant protein, which was stored for future use;

[0023] (2) Preparation of monoclonal antibodies

[0024] The p72 recombinant protein was used as an immunogen to immunize mice. The first immunization was performed with Freund's complete adjuvant emulsified antigen, which was injected subcutaneously at multiple points at a dose of 50 μg / mouse. The booster immunization was performed 2-3 weeks apart, with Freund's incomplete adjuvant emulsified antigen injected. One to two weeks after the last booster immunization, the p72 recombinant protein was used to coat an enzyme-linked immunosorbent assay plate, and the immune serum titer was determined by an indirect ELISA method. When the titer exceeded the expected value, the mouse spleen was collected for cell fusion, and positive hybridoma cells were obtained through multiple rounds of subcloning and indirect ELISA screening. Mouse ascites was obtained by an in vivo ascites induction method, and the monoclonal antibody against African swine fever virus p72 protein was obtained for purification using a protein A affinity chromatography column, and the monoclonal antibody was stored for future use.

[0025] 3. A fluorescent immunochromatographic test strip prepared using the African swine fever virus p72 protein antibody comprises a supporting base plate, on which a sample pad, a conjugation pad, a chromatographic detection membrane, and a water-absorbing pad are fixed in sequence; the conjugation pad is coated with glass fiber cotton, and a quantum dot-labeled p72 recombinant protein of African swine fever virus is fixed on the surface of the conjugation pad; the chromatographic detection membrane adopts a nitrocellulose membrane, and a detection line and a quality control line are provided on the chromatographic detection membrane, the detection line is coated with Staphylococcus a protein, and a monoclonal antibody against African swine fever virus p72 protein is fixed on the quality control line.

[0026] The fluorescent immunochromatographic test strip uses quantum dots as signal markers to convert the immune response of African swine fever virus antigens and antibodies into recognizable fluorescent signals; the quantum dots are connected to the p72 recombinant protein via 1-ethyl-(3-dimethylaminopropyl) carbodiimide.

[0027] The method for judging the test result of the fluorescent immunochromatographic test strip is as follows: if two fluorescent strips appear on the test strip under ultraviolet light, the test result is positive, indicating that the sample to be tested contains African swine fever virus antibodies; if only the fluorescent strip appears at the quality control line C position, the test result is negative, indicating that the sample to be tested does not contain African swine fever virus antibodies; if no fluorescent strip appears at the test line T or the quality control line C position, it indicates that the test result is invalid; and the depth of the color of the fluorescent strip is proportional to the content of African swine fever virus antibodies in the sample to be tested.

[0028] 4. The use of the monoclonal antibody in detecting the p72 protein of African swine fever virus; the reagent or kit for detecting the p72 protein of African swine fever virus prepared with the monoclonal antibody; the use of the fluorescent immunochromatographic test strip in detecting the African swine fever virus.

[0029] Beneficial technical effects of the present invention:

[0030] 1. The present invention uses ASFVp72 gene and B602L gene to obtain correctly folded p72 recombinant protein, uses Staphylococcus auricularia auricularia auriculariae protein for quantum dot labeling, uses quantum dots as labeling tracers, prepares quantum dot-labeled immunochromatographic test paper, and uses antigen-antibody reaction and lateral chromatography method to detect samples. The labeled complex and antibody bind stably, have bright colors, are visible to the naked eye under ultraviolet light, and are easy to use.

[0031] 2. The present invention selects ASFV-p72 recombinant protein as the detection target. Different from the protein obtained by the prokaryotic expression system, the recombinant protein has similar antigens to the viral protein that infects mammals, has good immunogenicity and high biological activity, and has significant advantages in terms of immunity and quantum dot labeling.

[0032] 3. The African swine fever virus antibody fluorescent immunochromatographic test paper prepared by the present invention using ASFV-p72 recombinant protein as an antigen can accurately and quickly detect whether African swine fever virus antibodies are present in the test serum. The detection time is short, the specificity is strong, and the sensitivity is high, which greatly reduces the missed detection rate and improves the accuracy of the detection. It can be used for ASFV infection diagnosis, quarantine inspection and epidemiological investigation and research, and has great promotion and application value.

[0033] 4. The monoclonal antibody prepared by the present invention using ASFV p72 recombinant protein as an antigen has high purity and strong specificity, and can specifically recognize p72 protein, thereby improving the sensitivity and accuracy of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the identification diagram of the purified ASFVp72 recombinant protein;

[0035] Among them, M: Marker; A: p72 recombinant protein purification SDS-PAGE; B: p72 recombinant protein and ASFV positive serum Western Blot results;

[0036] Figure 2 This is the identification diagram of the purified monoclonal antibody;

[0037] In the figure, A. SDS-PAGE identification of the purified anti-p72 recombinant protein monoclonal antibody; M is a marker, and 1 is the purified monoclonal antibody; B is a Western blot identification of the positive hybridoma cell supernatant and the p72 recombinant protein, where M is a marker and 1 is the p72 recombinant protein;

[0038] Figure 3 It is the specific identification diagram of monoclonal antibody;

[0039] Among them, A is the reactivity of anti-p72 protein monoclonal antibody and full-length p72 protein identified by IPMA; B is the positive control; C is the negative control; D is the blank cell control;

[0040] Figure 4 It is the affinity determination graph of monoclonal antibody;

[0041] Figure 5 This is a schematic diagram of the test results of the test paper for ASFV standard positive serum samples and negative serum samples;

[0042] Figure 6 It is the specific test result of lateral flow immunochromatography test paper;

[0043] Figure 7 This is the sensitivity test result of lateral flow immunochromatography test paper. DETAILED DESCRIPTION

[0044] The specific implementation modes of the present invention are further described in detail below in conjunction with the embodiments.

[0045] Materials and reagents that may be used in the present invention:

[0046] 1. Competent cells: Escherichia coli DH5α was purchased from Biomed Biotechnology Co., Ltd.; 2. Plasmid: pCGS3 plasmid was purchased from Wuhan Miaoling Biotechnology Co., Ltd.; 3. Cells and culture medium: CHO cells and culture medium were purchased from Beijing Sino Biological Technology Co., Ltd.; 4. Experimental animals: female BALB / c mice were purchased from the Experimental Animal Center of Zhengzhou University; 5. Kit: Endotoxin-free extraction kit was purchased from Kangwei Century Biotechnology Co., Ltd. The identification of recombinant plasmids was carried out by General Biotechnology (Anhui) Co., Ltd.

[0047] Unless otherwise specified, other instruments and equipment are conventional instruments and equipment available on the market; other reagents and materials are conventional reagents and materials available on the market.

[0048] Example 1. Preparation of ASFVp72 recombinant protein

[0049] 1. Construction of eukaryotic expression vector and preparation of African swine fever virus p72 recombinant protein

[0050] The gene sequence of p72 protein was obtained according to the China / 2018 / Anhui XCGQ strain (GenBank: AYW34030.1). Considering factors such as codon preference, balanced GC content, forward and reverse repeat sequences, mRNA secondary structure, expression vector construction, and restriction sites, the sequence of the African swine fever virus p72 protein encoding gene B602L was optimized using codon optimization software with reference to the codon usage frequency of Cricetulus griseus (Mamalian / CHO cells) to enable better expression in the CHO expression system.

[0051] Referring to the above codon optimized sequence, it was submitted to General Biotechnology (Anhui) Co., Ltd. for synthesis, and the pCGS3 vector was constructed. The synthesized gene was used as a template, and the target fragment was obtained by PCR amplification. The target fragment was cloned into the expression vector pCGS3, and a 10×His-3×Flag tag was added to the N segment of the p72 protein to obtain the recombinant plasmid pCGS3-p72;

[0052] The recombinant plasmid pCGS3-p72 was transformed into Escherichia coli DH5α cells, and the plasmid was extracted after expansion culture. The recombinant plasmid pCGS3-p72 and B602L gene were double-digested and connected using T4 DNA ligase (B602L gene was derived from its whole genome Genbank Accession: MK128995.1) to obtain a recombinant plasmid named pCGS3-p72-B602L.

[0053] The pCGS3-p72-B602L plasmid was transferred into CHO cells, the cells were collected and lysed, and the cells were purified by Anti-Flag affinity chromatography column. The African swine fever virus p72 recombinant protein was obtained by ultrafiltration and centrifugation concentration. The protein was packaged and stored at -80°C for subsequent use in the preparation of fluorescent immunochromatographic test strips.

[0054] 2. Result determination

[0055] Take appropriate amount of ASFV-p72 recombinant protein and perform SDS-PAGE and Western blot detection. Figure 1 As shown, Figure 1A The results showed that the recombinant protein was successfully expressed, and the eluted p72 protein showed a target band at around 72 kDa.

[0056] Take an appropriate amount of ASFV-p72 recombinant protein and react with positive pig serum for Western blot detection. The results are as follows Figure 1 As shown in B, it shows that the p72 recombinant protein has good reactivity with ASFV-positive pig serum.

[0057] Example 2. Preparation and identification of monoclonal antibodies

[0058] 1. Preparation of Monoclonal Antibodies

[0059] Mice were immunized with p72 recombinant protein as an immunogen. The first immunization was performed with Freund's complete adjuvant emulsified antigen and injected subcutaneously at multiple points at a dose of 50 μg / mouse. The booster immunization was performed 2-3 weeks apart and the antigen was emulsified with Freund's incomplete adjuvant for injection. The last booster immunization was performed 1-2 weeks later. The p72 recombinant protein was coated on an enzyme-linked immunosorbent assay plate at a concentration of 1-2 μg / mL and a dosage of 50-100 μL per well. The serum titer was determined by an indirect ELISA method. When the titer reached the expected value (1:102400) or above, the mouse spleen was collected for cell fusion. After multiple rounds of subcloning and indirect ELISA screening, positive hybridoma cells were obtained. Mouse ascites was obtained by the in vivo ascites induction method, and purified by a protein A affinity chromatography column to obtain monoclonal antibodies against African swine fever virus p72 protein. The antibodies were packaged and stored at -80°C for use in the preparation of quality control lines.

[0060] The obtained monoclonal antibodies were identified and the results were as follows Figure 2 As shown in A, the monoclonal antibody has only two bands, heavy chain and light chain, and no other mixed bands, indicating that the purity of the monoclonal antibody is relatively high.

[0061] Figure 2 B is the Western blot identification of positive hybridoma cell supernatant and p72 recombinant protein, indicating that the monoclonal antibody can react specifically with p72 protein.

[0062] 2. Specificity identification of monoclonal antibodies

[0063] The immunoperoxidase monolayer cell assay (IPMA) was used to identify the specificity of the monoclonal antibody. The specific operation process is as follows:

[0064] (1) Recovery of HEK-293T cells: 293T cells were cultured using complete DMEM medium until the cells were oval, transparent and healthy under a microscope;

[0065] (2) Treatment of cell plates: Use 1× poly-lysine solution to treat 96-well cell culture plates, 60 μL per well. Place the cell plates in a biosafety cabinet and let them stand for 8 min. Use a multi-channel pipette to aspirate the poly-lysine solution for subsequent recycling. Wash the cell plates three times with sterile deionized water and wait for the bottom of the plates to dry before plating.

[0066] (3) Plating: When the density of HEK-293T cells reached about 80%, HEK-293T cells were digested with trypsin and plated at 2×10 5 The density of cells / mL was added into the cell culture plate;

[0067] (4) Transfection: The next day, observe the cell density in the 96-well cell culture plate. 2 h before transfection, replace the complete DMEM medium with Opti-MEM complete medium. Be gentle to avoid blowing up the HEK-293T cells that have already adhered to the plate. Jet the plate at 0.3 μL / well for plasmid and 0.5 μL / well for transfection. regent, 12μL / well jet- Buffer and pcDNA3.1-p72 plasmid / pcDNA3.1 empty plasmid were vortexed for 15 seconds to mix, and finally jet- The transfection reagent was vortexed again for 15 seconds, and then allowed to stand in a biosafety cabinet for 10 minutes. The transfection complex was added dropwise to the cell wells. After 7 hours, the culture medium in each well was discarded along the well wall, and the same volume of fresh complete DMEM medium was added. Then, the cells were cultured in a carbon dioxide incubator for 48 hours.

[0068] (5) Fixing cells: After 48 h, discard the supernatant of each well and fix the cells on the bottom of the cell plate using 4% tissue cell fixative, 60 μL / well, and place at room temperature for 10 min;

[0069] (6) Adding primary antibody: discard the tissue fixative, wash three times with PBS, add 50 μL of diluted anti-p72 protein monoclonal antibody to each well, set up three replicate wells for each monoclonal antibody, and dilute the serum of non-immunized mice 200 times with PBS solution as a negative control, and the eyeball serum of fused mice 200 times with PBS solution as a positive control. The supernatant of cells transfected with pcDNA3.1 empty plasmid is used as a blank control. Place in a 37°C water-tight incubator for 1 h;

[0070] (7) Adding secondary antibody: discard the primary antibody, wash with PBS solution 5 times, then wash twice with sterile water to completely remove the salt solution ions, add 50 μL of goat anti-mouse IgG / HRP solution (1:2000 dilution) to each well, and place at 37°C for 1 h;

[0071] (8) Color development: discard the secondary antibody, wash with PBS solution for 5 times, and then wash twice with sterile water. Add 50 μL of AEC color development solution to each well and let it stand in a dark place to avoid light interference. After 10 minutes, aspirate the AEC color development solution and add sterile water to dilute the remaining color development solution to terminate the reaction. Observe the color development of each well under a microscope and take photos to record.

[0072] The immunized mouse eye serum and the unimmunized mouse serum were used as positive and negative controls. Figure 3 .

[0073] Figure 3 It is an identification diagram of the specificity of the monoclonal antibody; wherein, A is the reactivity of the anti-p72 protein monoclonal antibody with the full-length p72 protein identified by IPMA; B is the positive control; C is the negative control; and D is the blank cell control.

[0074] It can be seen that the monoclonal antibodies of the present invention react with the p72 recombinant protein expressed by eukaryotic cells, but do not react with irrelevant proteins expressed by eukaryotic cells, indicating that the monoclonal antibodies have strong specificity.

[0075] 3. Determination of Monoclonal Antibody Affinity

[0076] The p72 recombinant protein was diluted to 1 μg / mL and 2 μg / mL with CBS solution, coated on the bottom of a 96-well plate, and the corresponding OD450 reading was determined by indirect ELISA. The OD450 was read with a microplate reader, and a double reciprocal curve was drawn with 1 / CmAb and 1 / OD450 as the X-axis and Y-axis, respectively. The curve formula was obtained by curve fitting: y=0.0077x+0.236, R 2 =0.9975; y=0.0056x+0.26, R 2 =0.9968.

[0077] Find the absorbance value of the monoclonal antibody concentration corresponding to 50% OD450 under different coating concentrations, substitute it into the double reciprocal curve formula, calculate the corresponding monoclonal antibody concentration, convert it into molar concentration, and substitute it into the following formula to calculate the affinity constant Kaff of each monoclonal antibody.

[0078] Kaff=(n-1) / 2(n[Ab']t-[Ab]t)

[0079] Where n = [Ag]t / [Ag']t, [Ag]t is the protein coating concentration of 2 μg / mL, [Ag']t is the protein coating concentration of 1 μg / mL, [Ab']t and [Ab]t are the 50% OD when the p72 recombinant protein coating concentration is 2 μg / mL and 1 μg / mL, respectively. 450 The values ​​below correspond to the molar concentration of the monoclonal antibody.

[0080] from Figure 4 It can be seen that the affinity constant of the monoclonal antibody obtained is 7.143×10 9 The monoclonal antibodies screened have strong affinity and have great advantages in detecting African swine fever virus.

[0081] 4. Sequencing of Monoclonal Antibodies

[0082] The monoclonal antibody against the African swine fever virus p72 protein was sequenced to obtain the amino acid sequence of the heavy chain variable region and its framework region sequence, and the amino acid sequence of the light chain variable region and its framework region sequence.

[0083] The heavy chain variable region amino acid sequence of the monoclonal antibody includes CDR-H1, and the heavy chain variable region framework region sequence includes FR-H1, FR-H2, FR-H3 and FR-H4;

[0084] CDR-H1: LLFLD (SEQ ID NO. 1)

[0085] FR-H1:MSNTDWKEGQRKTIHIYATGNNSSHHAVLG(SEQ ID NO.2)

[0086] FR-H2:IMAWVWTLLFLLMAA(SEQ ID NO.3)

[0087] FR-H3:AQSAQAQIQLVQSGPELKKPGETVKISCKASG(SEQ ID NO.4)

[0088] FR-H4:YTFTNLGMNWVKQAPGKGLKWMGWINTYSGN(SEQ ID NO.5)

[0089] The amino acid sequence of the light chain variable region of the monoclonal antibody includes CDR-L1, CDR-L2 and CDR-L3, and the framework region sequence includes FR-L1, FR-L2 and FR-L3.

[0090] CDR-L1:LTQSPASLAVSLGQR(SEQ ID NO.6)

[0091] FR-L1:WEFQETDTLLLWVLLLWVPGSTG(SEQ ID NO.7)

[0092] CDR-L2: YSYMHWN (SEQ ID NO. 8)

[0093] FR-L2:ATISYRASKSVSTSG(SEQ ID NO.9)

[0094] CDR-L3: TDFT (SEQ ID NO. 10)

[0095] FR-L3: QQKPGQPPRLLIYLVSNLESGVPARFSGSGSG (SEQ ID NO.11) Example 3. Preparation of quantum dot solution and coupling with p72 recombinant protein

[0096] 1. Preparation of Quantum Dots

[0097] Weigh 0.26 g of CdO and place it in three-necked bottle A, add 3.2 mL of oleic acid and 6.8 mL of liquid paraffin, and heat to 150°C to completely dissolve CdO to obtain a Cd precursor stock solution. Weigh 0.81 g of zinc stearate and place it in three-necked bottle B, add 2 mL of TOP, and oscillate ultrasonically under nitrogen protection to completely dissolve zinc stearate to obtain a Zn precursor preparation solution.

[0098] Weigh 0.064g of Se powder and place it in a three-necked bottle C, add 15.4mL of liquid paraffin, 4.5mL of TOP and 0.9mL of oleylamine, the molar ratio of Se, TOP and oleylamine is 1:15:5, and ultrasonically vibrate under nitrogen protection to completely dissolve Se to obtain a Se precursor stock solution. Heat the Se precursor stock solution to 220°C under nitrogen protection. Quickly inject 2mL of Zn precursor solution and 1.6mL of Cd precursor solution into the high-temperature solution of Se precursor at the same time. In the mixed reaction solution, the molar concentration ratio of Cd precursor, Zn precursor and Se precursor is 4:1:2.5. At the same time, it is accompanied by strong mechanical stirring. React for 30 minutes and quickly cool the solution to room temperature;

[0099] Methanol was added to the solution after the reaction, and the solution was allowed to stand to form flocculent precipitates of CdZnSe quantum dots. After centrifugation and removal of the upper clear liquid, the fluid CdZnSe precipitate was dissolved in tetrahydrofuran, and the solution was centrifuged again to remove the lower precipitate, thereby obtaining CdZnSe quantum dots uniformly dispersed in tetrahydrofuran.

[0100] 2. Coupling of quantum dots with p72 recombinant protein

[0101] The obtained quantum dots were used to label the p72 recombinant protein, quantum dots: p72 recombinant protein: EDC (mass ratio) = 1: 6: 1500, pH: 7.5: 2.5 μL of quantum dots were placed in a 1.5 mL centrifuge tube, 9.6 μL of EDC solution was added, and the mixture was mixed by pipetting and placed in a 25°C shaker in the dark for 30 min, with a rotation speed of 220 rpm; 0.78 μL of p72 recombinant protein (protein concentration 2 mg / mL) was added to the Ep tube, and then 20.1 μL of BBS buffer was added, and the reaction was carried out in the dark on a shaker for 2.5 h to obtain the coupling product QDs-Ag, which was stored in the dark at 4°C for future use. Example 4, Preparation of Antibody Fluorescence Immunochromatography Test Strip

[0102] 1. Test strip structure

[0103] The invention discloses an immunochromatographic test paper for detecting quantum dot-labeled antibodies against p72 protein of African swine fever virus, comprising a supporting base plate, on which a sample pad, a conjugation pad, a chromatographic detection membrane and a water-absorbing pad are fixed in sequence; the conjugation pad is coated with glass fiber cotton and is fixed with p72 recombinant protein labeled with quantum dots; the chromatographic detection membrane is a nitrocellulose membrane, on which a detection line and a quality control line are arranged, the detection line T is made of Staphylococcus a protein and is used to capture antibodies in a solution to be tested; the quality control line C is fixed with a monoclonal antibody against p72 protein of African swine fever virus.

[0104] 2. Preparation method

[0105] (1) Preparation of chromatographic detection membrane

[0106] Cut the nitrocellulose test membrane into long strips, place it on the spraying instrument platform, and fix it; spray a 1 mg / mL staphylococcal protein A solution on the nitrocellulose membrane to obtain the test line T blot; dilute the monoclonal antibody solution against African swine fever virus p72 protein to a concentration of 1 mg / mL, spray it on the nitrocellulose membrane at an amount of 1 μL / cm, i.e., the quality control line C blot, dry it, seal it, and store it for later use;

[0107] (2) Preparation of sample pad

[0108] Cut the glass fiber cotton into strips, soak the glass fiber cotton strips in a PBS solution containing 10 g / L BSA, 5 g / L Tween20, and 0.1 mol / L NaNO3, and then dry them to make sample pads, which are sealed and stored at room temperature for later use;

[0109] (3) Preparation of conjugate pad

[0110] Cut the glass fiber cotton into long strips, place them on the spraying instrument platform and fix them; spray the Na2B407 buffer solution containing 1.0% BSA and 0.1% NaN3 on the glass fiber cotton strips, dry them and set them aside;

[0111] A 0.8 mg / mL quantum dot-labeled ASFVp72 recombinant protein solution (the coupling product QDs-Ag prepared in Example 3) was sprayed on a glass fiber cotton strip at a volume of 4 μL / cm, dried to prepare a conjugate pad, and sealed and stored at room temperature for later use;

[0112] (4) Preparation of absorbent pad

[0113] Cut the absorbent paper into long strips to make absorbent pads, dry them, and store them in a sealed container at room temperature for later use;

[0114] (5) Preparation of supporting base plate

[0115] Attach double-sided tape to the PVC board and cut it into long boards to prepare the supporting base plate;

[0116] (6) Assembly of test strips

[0117] Assemble the sample pad, conjugate pad, chromatography detection membrane and absorbent pad in order on the supporting base plate; overlap each layer by 2 mm, then cut into test strips, dry and seal for storage.

[0118] 3. Detection principle and result judgment method

[0119] The test sample is the separated serum of pigs. The sample is dripped onto the test strip for 10-15 minutes. The fluorescence intensity tends to be stable, and the test results are observed under ultraviolet light. When the sample contains antibodies against ASFVp72 protein, the antibodies will be captured by the QDs-p72 conjugate to form an antigen-antibody complex with fluorescence. It continues to swim forward due to capillary action, and the Fc segment of the antibody will bind to the SPA on the T line. The excess antigen-antibody complex further swims to the C line and binds to the monoclonal antibody against p72 protein on the C line. Under ultraviolet light, both the C line and the T line show fluorescence, which is determined to be an ASFV positive sample. On the contrary, if the sample does not contain antibodies against ASFVp72 protein, no fluorescent antigen-antibody complex will be formed, the T line will not show color, and the monoclonal antibody at the C line will combine with the fluorescent probe to show fluorescence, which is determined to be ASFV negative. If neither the C line nor the T line shows fluorescence, the test strip is determined to be invalid. For test results, see Figure 5 .

[0120] Example 5: Specificity and sensitivity of immunochromatographic test paper

[0121] To verify the specificity and sensitivity of the immunochromatographic test paper of Example 4, the following test was performed.

[0122] (1) Specificity

[0123] Serum samples to be tested: PCV2 positive serum, PRRSV positive serum, PRV positive serum, PPV positive serum, ASFV positive serum and ASFV negative serum, detection method:

[0124] The serum to be tested was diluted with sample diluent (PBS) at a ratio of 1:100. After sufficient mixing, 100 μl of sample was added to the sample well of each test strip, and the experimental results were observed after 10 minutes.

[0125] The test results are as follows Figure 6 The test strip can accurately detect ASFV-positive sera and has no cross-reaction with other swine pathogen antibody-positive sera, indicating good specificity.

[0126] (2) Sensitivity

[0127] Using ASFV negative serum as control, ASFV standard positive serum samples were selected and diluted in different gradients (1:10 2 , 1:10 3 , 1:10 4 , 1:10 5 , 1:10 6 ) to test the sensitivity of the test strip.

[0128] Test results such as Figure 7 The detection dilution factor was 1:10 6 When the ASFV positive serum was tested, two reaction bands still appeared at the test line and the quality control line at the same time, which was obviously different from the negative control and could be judged as positive. It can be seen that the minimum detection limit of the sensitivity of the standard positive serum sample of African swine fever virus to p72 antibody is 1:10 6 , indicating that the test strip has a higher sensitivity.

[0129] The above are only preferred embodiments of the present invention. Any equivalent changes or modifications to the present invention without departing from the principle of the present invention shall fall within the protection scope of the present invention.

Claims

1. A monoclonal antibody against African swine fever virus p72 protein, characterized in that: The monoclonal antibody comprises a heavy chain variable region amino acid sequence and a light variable region amino acid sequence. The amino acid sequence of the heavy chain variable region includes CDR-H1:LLFLD; the amino acid sequence of the light variable region includes CDR-L1, CDR-L2 and CDR-L3, and the corresponding amino acid sequences are: LTQSPASLAVSLGQR, YSYMHWN, and TDFT.

2. The monoclonal antibody according to claim 1, characterized in that The monoclonal antibody further comprises heavy chain variable framework region sequences FR-H1, FR-H2, FR-H3 and FR-H4; and light chain variable framework regions FR-L1, FR-L2, FR-L3, the sequences of which are: FR-H1: MSNTDWKEGQRKTIHIYATGNNSSHAVLG; FR-H2:IMAWVWTLLFLMAA; FR-H3: AQSAQAQIQLVQSGPELKKPGETVKISCKASG; FR-H4: YTFTNLGMNWVKQAPGKGLKWMGWINTYSGN; FR-L1 WEFQETDTLLLWVLLLWVPGSTG; FR-L2:ATISYRASKSVSTSG; FR-L3: QQKPGQPPRLLIYLVSNLESGVPARFSGSGSG.

3. A method for preparing the monoclonal antibody according to claim 1, characterized in that: The steps include: (1) Preparation of p72 recombinant protein According to the codon preference of the CHO expression system, the sequence of the B602L gene encoding the p72 protein of the African swine fever virus was optimized and synthesized; the synthesized gene was used as a template to obtain the target fragment by PCR amplification, and the target fragment was cloned into the expression vector pCGS3 to obtain the recombinant plasmid pCGS3-p72; The recombinant plasmid pCGS3-p72 was transferred into Escherichia coli DH5α cells, and the plasmid was extracted after amplification culture. The recombinant plasmid and the B602L gene were double-digested and connected using T4 DNA to obtain the recombinant plasmid pCGS3-p72-B602L; The recombinant plasmid pCGS3-p72-B602L was transferred into CHO cells, the cells were collected and lysed, and the impurities were removed using an Anti-Flag affinity chromatography column, eluted and purified, and concentrated by ultrafiltration and centrifugation to obtain the African swine fever virus p72 recombinant protein, which was stored for future use; (2) Preparation of monoclonal antibodies Mice were immunized with p72 recombinant protein as an immunogen, and the first immunization was performed with Freund's complete adjuvant emulsified antigen, which was injected subcutaneously at multiple points at a dose of 50 μg / mouse; booster immunization was performed 2-3 weeks apart, and antigen was emulsified with Freund's incomplete adjuvant for injection; 1-2 weeks after the last booster immunization, an enzyme-linked immunosorbent assay plate was coated with p72 recombinant protein, and the immune serum titer was determined by an indirect ELISA method. When the titer reached a value above the expected value, the mouse spleen was collected for cell fusion, and positive hybridoma cells were obtained through multiple rounds of subcloning and indirect ELISA screening. Mouse ascites was obtained by an in vivo ascites induction method, and the monoclonal antibody against African swine fever virus p72 protein was obtained for purification using a protein A affinity chromatography column, and the monoclonal antibody was stored for future use.

4. A fluorescent immunochromatographic test strip prepared using the African swine fever virus p72 protein antibody according to claim 1, characterized in that: The test strip comprises a supporting base plate, on which a sample pad, a conjugation pad, a chromatographic detection membrane and a water-absorbing pad are fixed in sequence; the conjugation pad is coated with glass fiber cotton, and a p72 recombinant protein of African swine fever virus marked with quantum dots is fixed on the surface of the conjugation pad; the chromatographic detection membrane adopts a nitrocellulose membrane, and a detection line and a quality control line are arranged on the chromatographic detection membrane, the detection line is coated with Staphylococcus A protein, and a monoclonal antibody against African swine fever virus p72 protein is fixed on the quality control line.

5. The fluorescent immunochromatographic test strip according to claim 4, characterized in that: Quantum dots are used as signal markers to convert the immune response of African swine fever virus antigens and antibodies into recognizable fluorescent signals; quantum dots are connected to p72 recombinant protein through 1-ethyl-(3-dimethylaminopropyl) carbodiimide.

6. The fluorescent immunochromatographic test strip according to claim 4, characterized in that: The method for judging the test results is: if two fluorescent strips appear on the test strip under ultraviolet light, the test result is positive, indicating that the sample to be tested contains African swine fever virus antibodies; if only a fluorescent strip appears at the quality control line C, the test result is negative, indicating that the sample to be tested does not contain African swine fever virus antibodies; if no fluorescent strips appear at either the test line T or the quality control line C, the test result is invalid; and the depth of the color of the fluorescent strip is proportional to the content of African swine fever virus antibodies in the sample to be tested.

7. Use of the monoclonal antibody according to claim 1 in detecting African swine fever virus p72 protein.

8. A reagent or kit for detecting African swine fever virus p72 protein prepared from the monoclonal antibody according to claim 1.

9. Use of the fluorescent immunochromatographic test strip according to claim 4 in detecting African swine fever virus.

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