African swine fever virus p30 protein antibody and application thereof in preparation of colloidal gold immunochromatography detection test strip

Abstract

The invention discloses an African swine fever virus p30 protein antibody and application thereof in preparation of a colloidal gold immunochromatography detection test strip, the antibody comprises a light chain and a heavy chain, the light chain and the heavy chain comprise a light chain variable region and a heavy chain variable region, amino acid sequences of three complementary determining regions in the light chain variable region are respectively shown as SEQ ID NO.1-3, and amino acid sequences of three complementary determining regions in the heavy chain variable region are respectively shown as SEQ ID NO.2-3. The amino acid sequences of the three complementary determining regions in the heavy chain variable region are respectively shown as SEQ ID NO.4-6. The amino acid sequence of the light chain is as shown in SEQ ID NO.7, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO.9. The African swine fever virus p30 protein antibody is applied to a colloidal gold immunochromatography test strip method, a serum sample with the p30 antibody after African swine fever virus infection can be rapidly and accurately detected, the detection time is shortened, the detection steps are simplified, and the method has the characteristics of being rapid, convenient and suitable for field detection, and has a good application prospect. And a reliable immunological technology is provided for rapidly and efficiently detecting African swine fever virus infection.

Description

Technical Field

[0001] The present invention relates to an antibody against African swine fever virus p30 protein and its application in the preparation of a colloidal gold immunochromatographic test strip, belonging to the field of biotechnology. Background Art

[0002] African swine fever (ASF) is listed as a notifiable animal disease by the World Organization for Animal Health (OIE), and China classifies it as a Class I animal infectious disease. This disease is caused by the African swine fever virus (ASFV), which has a large double-stranded DNA genome (170 - 193 kb) containing 151 - 167 genes, and the functions of most of the encoded genes are still unclear. So far, ASF has spread to most countries in the world, causing great trouble and huge economic losses to the pig farming industry.

[0003] At present, there is no effective commercial vaccine available, and the prevention and control work faces great challenges. Therefore, timely and accurate detection of ASFV-infected pigs is the top priority of the prevention and control work. Among the detection methods for ASF, the colloidal gold immunochromatographic test strip has attracted more and more attention due to its unique advantages. First of all, the colloidal gold immunochromatographic test strip has the characteristics of rapid detection, and usually can obtain results in a short time, which is crucial for the rapid response in the prevention and control work. Secondly, this detection method is easy to operate and does not require complex experimental equipment, and can be applied on-site or on the farm, greatly facilitating the daily detection of farmers. In addition, traditional nucleic acid detection and serological detection methods have certain limitations in terms of timeliness and sensitivity of monitoring. The colloidal gold immunochromatographic technique can identify infections by detecting specific antibodies, and can provide accurate detection results at an early stage, helping to identify potential infected individuals as early as possible, so as to implement effective isolation and control measures.

[0004] In short, the colloidal gold immunochromatographic test strip has a rapid, convenient and efficient application prospect in the detection of African swine fever virus, providing a new technical means for the prevention and control work of the pig farming industry. With the continuous development of technology, these test strips are expected to be further optimized in the future to improve the detection sensitivity and specificity, providing stronger support for the prevention and control of ASF. Summary of the Invention

[0005] Object of the Invention: The object of the present invention is to provide an African swine fever virus p30 antibody fragment and antibody for detecting African swine fever virus, and its application in the preparation of a colloidal gold immunochromatographic test strip.

[0006] Technical solution: The present invention provides an antibody fragment of African swine fever virus p30 protein. The antibody fragment includes a light chain and a heavy chain variable region. The amino acid sequences of the three complementarity-determining regions in the light chain variable region are respectively shown as SEQ ID NO.1-3, and the amino acid sequences of the three complementarity-determining regions in the heavy chain variable region are respectively shown as SEQ ID NO.4-6.

[0007] The present invention also provides an antibody of African swine fever virus p30 protein. The antibody includes a light chain and a heavy chain. The amino acid sequence of the light chain is shown as SEQ ID NO.7, and the amino acid sequence of the heavy chain is shown as SEQ ID NO.9.

[0008] The nucleotide sequence encoding the light chain is shown as SEQ ID NO.8, and the nucleotide sequence encoding the heavy chain is shown as SEQ ID NO.10.

[0009] The heavy chain and the light chain are connected by a disulfide bond.

[0010] The present invention also provides the application of the above-mentioned African swine fever virus p30 protein antibody fragment and African swine fever virus p30 protein antibody in detecting African swine fever virus antibody or preparing a reagent / strip for detecting African swine fever virus.

[0011] The present invention also provides a colloidal gold immunochromatographic strip for detecting African swine fever virus. The strip contains the above-mentioned African swine fever virus p30 protein antibody fragment or the above-mentioned African swine fever virus p30 protein antibody.

[0012] Furthermore, the strip also includes a gold-labeled conjugate pad labeled with colloidal gold particles of African swine fever virus p30 protein, an NC membrane sprayed with staphylococcal protein A and African swine fever virus p30 protein antibody, a sample pad, a water-absorbing pad, and a PCV bottom plate.

[0013] Furthermore, the colloidal gold particles of African swine fever virus p30 protein are prepared by the following method: adding 0.1 mol / L K2CO3 to the colloidal gold solution, adding African swine fever virus p30 protein under stirring conditions, and standing at room temperature for 30 min after stirring.

[0014] Furthermore, the dosage of African swine fever virus p30 protein is 17-83 μg added to every 1 mL of colloidal gold solution.

[0015] Furthermore, the dosage of K2CO3 is 12-20 μL added to every 1 mL of colloidal gold solution.

[0016] Furthermore, the concentration of the African swine fever virus p30 protein antibody is 1 mg / mL.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: The method using the African swine fever virus p30 protein antibody of the present invention for colloidal gold immunochromatographic test strips can be applied to quickly and conveniently detect serum samples with p30 antibody after African swine fever virus infection on site, shortening the detection time, simplifying the detection steps, and having the characteristics of rapid and convenient detection, providing a reliable immunological technology for rapid and efficient on-site detection of African swine fever virus infection. Brief Description of the Drawings

[0018] Figure 1 : Schematic diagram of the colloidal gold immunochromatographic test strip.

[0019] Figure 2 : Determination of the optimal pH value of colloidal gold-labeled recombinant His-p30 protein. Figure A shows the color change of the colloidal gold solution after adding different volumes of K2CO3 solution. Figure B shows the change of the absorbance value of the colloidal gold solution at OD520nm after adding different volumes of K2CO3 solution. Among them, the vertical coordinate represents the OD520 value detected by the microplate reader; the horizontal coordinate represents the amount of K2CO3 solution added.

[0020] Figure 3 : Determination of the optimal amount of colloidal gold-labeled His-p30 protein. Figure A shows the color change of the colloidal gold solution after adding different volumes of recombinant His-p30 protein. Figure B shows the change of the absorbance value of the colloidal gold solution at OD520nm after adding different volumes of recombinant His-p30 protein. Among them, the vertical coordinate represents the OD520 value detected by the microplate reader; the horizontal coordinate represents the amount of protein added.

[0021] Figure 4 : Identification of immunocolloidal gold. Among them, the vertical coordinate represents the OD value detected by the microplate reader; the horizontal coordinate represents different visible light wavelengths detected by the microplate reader.

[0022] Figure 5 : Optimization of the optimal conditions of the colloidal gold immunochromatographic test strip.

[0023] Figure 6 : Determination of the detection result of the colloidal gold immunochromatographic test strip. Figure A shows the detection result of the negative sample. Among them, the T line does not show color and the C line shows color. Figure B shows the detection result of the positive sample. Among them, both the T line and the C line show color.

[0024] Figure 7 : Specificity analysis result of the colloidal gold immunochromatographic test strip. Among them, the test samples of the test strip are ASFV, PDCoV, PEDV, PRRSV, PCV2 porcine sera and ASFV-negative porcine sera.

[0025] Figure 8: Results of the sensitivity analysis of the colloidal gold immunochromatographic test strip. The test samples for the test strip were ASFV positive sera diluted in 8 gradients from 1:50 and ASFV negative sera.

[0026] Figure 9 : Results of the repeatability analysis of the colloidal gold immunochromatographic test strip. Figures A and B are test strips prepared in different batches. The test samples were ASFV negative sera and 8 ASFV positive sera 1-8. Specific implementation manners

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0028] In order to make the invention object, technical solution and beneficial technical effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. Those skilled in the art can easily understand other advantages and effects of the invention of this application from the content disclosed in this specification. Without departing from the spirit and essence of the present invention, any modification or replacement made to the methods, steps or conditions of the present invention belongs to the scope of the present invention.

[0029] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt the conventional molecular biology, biochemistry, analytical chemistry, cell culture, recombinant DNA technology and related conventional technologies in the technical field. These technologies have been well described in the existing literature. For details, see Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (P.M. Wassarman and

[0030] A.P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (P.B. Becker, ed.) Humana Press, Totowa, 1999, etc.

[0031] Example 1: Obtaining Hybridoma Cell Lines

[0032] 1. Animal Immunization

[0033] Six-week-old female BALB / c mice were purchased from the Comparative Medicine Center of Yangzhou University. The specific immunization procedure was as follows: The recombinant His-p30 protein of African swine fever virus (the preparation method refers to the patent titled "An Antibody Against African Swine Fever Virus p30 Protein and Its Application", application number 2024116686838) was mixed with Freund's complete adjuvant at a ratio of 1:1 and fully emulsified, and then subcutaneously injected into 3 mice at multiple points (the back of the neck and abdomen). Fourteen days after the first immunization, the second immunization was carried out. Both immunizations were 200 μL per mouse (containing 100 μg of recombinant His-p30 protein). Fourteen days after the second immunization, the recombinant His-p30 protein without adjuvant was intraperitoneally injected into the mice, 100 μL per mouse (containing 100 μg of recombinant His-p30 protein), for booster immunization.

[0034] 2. Cell Fusion

[0035] Three days after booster immunization, the mice were bled, and the serum was collected and stored at -20°C for use as a positive control for subsequent screening. The mice were euthanized, disinfected with alcohol immersion, and the spleen cells were fused with the myeloma cell line SP2 / 0 in the logarithmic growth phase at a cell ratio of 1:5 under the action of 50% polyethylene glycol (PEG). ICR mouse peritoneal macrophages were used as feeder cells. The fused cells and feeder cells were suspended and mixed with HAT medium and plated in 96-well plates, and then cultured in a 37°C cell incubator. Five days later, HAT medium was supplemented, and after 9 days, HT medium was used for culture. When the single cell clusters grew to about 90% of the 96-well plates, the cell supernatant was collected for indirect ELISA detection.

[0036] 3. Establishment of Indirect ELISA Detection Method and Screening of Positive Clones

[0037] The indirect ELISA method was used to screen for positive cell clones. The specific method is as follows: The enzyme-linked immunosorbent assay (ELISA) plate was coated with the optimal coating concentration of the recombinant GST-p30 protein of African swine fever virus at 1 μg / mL (the preparation method refers to the patent titled "An Antibody Against African Swine Fever Virus p30 Protein and Its Application", application number 2024116686838), 100 μL per well, overnight at 4 °C; washed 3 times with PBST, added PBS blocking solution containing 1% BSA, 200 μL per well, incubated at 37 °C for 2 h; after blocking, washed 3 times with PBST, added the supernatant of hybridoma cells; at the same time, negative and positive control groups were set up, the supernatant of SP2 / 0 cells was used as the negative control, and the immune mouse polyclonal serum was used as the positive control, 100 μL per well, water bath at 37 °C for 2 h; washed 5 times with PBST; added goat anti-mouse IgG labeled with horseradish peroxidase (HRP) diluted 1:5000, 100 μL per well, water bath at 37 °C for 1 h; after washing 7 times, added TMB for color development for 10 min, after terminating the color development, the OD 450 value was detected by an enzyme-linked immunosorbent assay (ELISA) reader, and the experimental results were determined according to the following formula: OD 450 cell well / OD 450 negative well ≥ 2.1 was determined as a positive well. The screened positive clone was named 3E1.

[0038] 4. Cloning of positive hybridoma cells

[0039] The positive cell clone 3E1 screened was subcloned 3 times by the limiting dilution method and preserved.

[0040] Example 2: Preparation and identification of monoclonal antibody against African swine fever virus p30 protein

[0041] 1. Preparation of monoclonal antibody against African swine fever virus p30 protein

[0042] The in vivo ascites induction method was used: Healthy BALB / c mice aged 9 - 12 weeks were intraperitoneally injected with 0.5 mL of liquid paraffin per mouse. After 7 days, they were intraperitoneally inoculated with African swine fever virus p30 hybridoma cells 3E1 cultured to the logarithmic growth phase and diluted with PBS, 2 × 10 5 cells per mouse; after 7 days, the ascites was collected, the supernatant was collected by centrifugation, and stored at -70 °C.

[0043] The prepared ascites was purified by Protein A affinity chromatography and stored at -70 °C.

[0044] 2. Identification of monoclonal antibody against African swine fever virus p30 protein

[0045] The monoclonal antibody subclass was identified using a monoclonal antibody subclass kit (Beijing Boaolong BF16001). The culture supernatant of the African swine fever virus p30 hybridoma cell 3E1 prepared in Example 1 was added to an ELISA microplate pre-coated with 1 μg / mL African swine fever virus recombinant GST-p30 protein, 100 μL per well, and incubated at 37 °C for 2 h; washed 3 times with PBST; sheep anti-mouse IgA, IgG1, IgG2a, IgG2b, IgG3, and IgM diluted 1:1000 with PBS were added respectively, 100 μL per well, and left at room temperature for 30 min, then washed 3 times with PBST; HRP-rabbit anti-sheep IgG enzyme-labeled antibody diluted 1:5000 with PBS was added, 100 μL per well, and incubated at room temperature for 15 min; washed 5 times with PBST; TMB chromogenic solution was added, 100 μL per well, and left at room temperature for 5 min; 0.5 M H2SO4 was added to terminate the reaction, 50 μL per well, and the OD 450 value was detected with an ELISA reader. The subclass of the monoclonal antibody was determined based on the OD 450 value.

[0046] The results showed that the subclass of the monoclonal antibody 3E1 was IgG1.

[0047] Upon identification, the results indicated that the amino acid sequence of the complementarity-determining region 1 (CDR1) of the light chain variable region of the monoclonal antibody was as shown in SEQ ID NO.1, specifically: RASSSVSSSYFH.

[0048] The amino acid sequence of the complementarity-determining region 2 (CDR2) of the light chain variable region was as shown in SEQ ID NO.2, specifically: STSNLAS.

[0049] The amino acid sequence of the complementarity-determining region 3 (CDR3) of the light chain variable region was as shown in SEQ ID NO.3, specifically: QQYSGYPFT.

[0050] The amino acid sequence of the complementarity-determining region 1 (CDR1) of the heavy chain variable region was as shown in SEQ ID NO.4, specifically: NGYSWH.

[0051] The amino acid sequence of the complementarity-determining region 2 (CDR2) of the heavy chain variable region was as shown in SEQ ID NO.5, specifically: YIHYSGSTNYNPSLKS.

[0052] The amino acid sequence of the complementarity-determining region 3 (CDR3) of the heavy chain variable region was as shown in SEQ ID NO.6, specifically: SGITATTLDY.

[0053] The full-length amino acid sequence of the light chain is shown in SEQ ID NO.7: MDFLVQIFSFLLISASVAMSRGENVLTQSPAIMSASPGEKVTMTCRASSSVSSSYFHWYQQKSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYFCQQYSGYPFTFGSGTKLEIK.

[0054] The nucleotide sequence encoding the light chain is shown in SEQ ID NO.8: ATGGATTTTCTGGTGCAGATTTTCAGCTTCTTGCTAATCAGTGCCTCAGTTGCAATGTCCAGAGGAGAAAATGTGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAAAAGGTCACCATGACCTGCAGGGCCAGCTCAAGTGTAAGTTCCAGTTACTTTCACTGGTACCAGCAGAAGTCAGGTGCCTCCCCCAAACTCTGGATTTATAGCACATCCAACTTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGTGTGGAGGCTGAAGATGCTGCCACTTATTTCTGCCAACAATACAGTGGTTACCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA.

[0055] The full-length amino acid sequence of the heavy chain is shown in SEQ ID NO.9: MRVLILLCLFTAFPGILSDVQLQESGPDLVKPSQSLSLTCTVTGYSITNGYSWHWIRQFP GNKLEWMGYIHYSGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYNCARSGITATTLDYWGQGTSVTVSS.

[0056] The nucleotide sequence encoding the heavy chain is shown in SEQ ID NO.10: ATGAGAGTGCTGATTCTTTTGTGCCTGTTCACAGCCTTTCCTGGTATCCTGTCTGATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCACTGGCTACTCCATCACCAATGGTTATAGTTGGCACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAGTGGATGGGCTACATACACTACAGTGGTAGCACTAACTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATAACTGTGCAAGATCCGGTATTACGGCTACAACTCTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA。

[0057] Example 3: Preparation and Identification of Colloidal Gold Solution

[0058] The colloidal gold was prepared by the sodium citrate reduction method. Briefly described as: taking 100 mL as an example, measure 100 mL of deionized water into a 500 mL clean beaker, add 1 mL of 10 g / L chloroauric(III) hydrate (Sigma 254169) and boil; quickly add 1.6 mL of freshly prepared 10 g / L sodium citrate solution (Sinopharm 10019418) under heating and stirring, observe the color change from colorless → blue → dark red → bright red until the color no longer changes, after the solution cools, add deionized water to make up the volume to 100 mL, and store it in the dark at 4°C.

[0059] Observe the prepared colloidal gold solution under light to preliminarily identify its quality by checking for abnormal conditions such as turbidity, precipitation or floating matter in the solution; use a full-wavelength scanning multifunctional reader to scan the absorbance value of the colloidal gold solution in the wavelength range of 400 - 700 nm to determine its maximum absorption peak in this wavelength range, and observe the peak width, peak shape, etc. of the maximum absorption peak (the results are as Figure 4 , for comparison with the colloidal gold solution used to label the recombinant His-p30 protein in Example 5).

[0060] Example 4: Preparation of Gold-Labeled p30 Protein

[0061] 1. Pretreatment of African swine fever virus recombinant His-p30 protein

[0062] Dialyze and desalt the recombinant His-p30 protein solution of African swine fever virus to be labeled with PBS (0.01 mol / L, pH 7.4) solution, because high-concentration salts can cause the colloidal gold solution to aggregate, affecting the labeling results.

[0063] Preparation of dialysis bag: Cut a suitable length of dialysis bag, check for no leakage, and boil it in a solution containing 2% NaHCO3 and 1 mmol / L EDTA (ethylenediaminetetraacetic acid) for 10 min; wash it thoroughly with deionized water and boil it in 1 mmol / L EDTA solution for 10 min; take it out and cool it to room temperature, and store it at 4°C for later use; put the recombinant His-p30 protein solution of African swine fever virus to be labeled into the dialysis bag, place it in PBS solution (0.01 mol / L, pH 7.4), and dialyze at 4°C for 48 h, changing the dialysis solution midway; suck out the dialyzed His-p30 protein solution and store it at -20°C for later use; before labeling, centrifuge at 12,000 r / min at 4°C for 20 min to remove aggregates.

[0064] 2. Preparation of colloidal gold solution

[0065] Take an appropriate amount of colloidal gold solution and centrifuge it at a low speed of 1000 r / min at 4°C for 30 min to remove the aggregates that may form during storage.

[0066] 3. Selection of the optimal pH value for colloidal gold-labeled recombinant His-p30 protein

[0067] In this study, the determination of the optimal pH for colloidal gold solution labeling was adjusted by the amount of 0.1 mol / L K2CO3 added. If the pH of the colloidal gold solution is measured with a pH meter, its probe may be damaged; while using pH test paper to measure pH, there are inevitable human errors, so the amount of 0.1 mol / L K2CO3 added is used as the standard. Take 1.5 mL centrifuge tubes, add 0.5 mL of colloidal gold solution to each tube, and adjust the pH value with 0.1 mol / L K2CO3 solution; sequentially add an excessive amount of recombinant His-p30 protein to each tube, mix well, and let it stand at room temperature for 10 min; add 100 μL of 10% NaCl solution to each tube, mix well, and let it stand at room temperature for 2 h; observe the color change of the solution and record the tube numbers that remain red. Transfer 200 μL of the gold-labeled solution from each tube to a 96-well enzyme-linked immunosorbent assay (ELISA) plate respectively, mark it well, and measure the OD520nm value. Write down the tube number with the largest OD520 value. The pH value of the colloidal gold solution in this tube number is the optimal labeling pH. The specific addition amounts of each component in the system are shown in Table 1.

[0068] Table 1 Determination of the optimal pH in colloidal gold and protein

[0069]

[0070] The results are as Figure 2 shown in Figure A. Compared with the blank control group, when 0, 2, and 4 μL of K2CO3 were added to 0.5 mL of colloidal gold solution, the color of the colloidal gold solution was light gray and light purplish red, indicating that the gold-labeled protein was in an unstable state and there was a coagulation phenomenon of changing from red to blue. Therefore, the colloidal gold solution was not suitable for labeling in this pH range. However, when 6, 8, and 10 μL of K2CO3 were added to 0.5 mL of colloidal gold solution, there were almost no blue particles in the solution and the color was stable.

[0071] As Figure 2 shown in Figure B, when 6 μL of K2CO3 was added to 0.5 mL of colloidal gold solution, the OD520 value of the gold-labeled protein complex was the highest, indicating that the adsorption of colloidal gold particles and His-p30 protein was better and the optical density value was the highest. Therefore, the pH when 12 μL of 0.1 mol / L K2CO3 was added to 1 mL of colloidal gold solution was the optimal pH for labeling His-p30 protein with colloidal gold solution.

[0072] 4. Selection of the optimal amount of colloidal gold-labeled His-p30 protein

[0073] Colloidal gold is a hydrophobic particle with a negatively charged outer layer, which forms a stable colloid due to electrostatic interaction. When no protein is added or the amount of protein added is insufficient, the colloidal gold particles cannot be completely adsorbed. It will change the colloidal properties due to the salt ion effect. Therefore, when NaCl is added, a blue coagulation phenomenon will occur.

[0074] Take 1.5 mL centrifuge tubes and add 0.5 mL of colloidal gold solution with the optimal labeling pH respectively; then add 0, 4, 8, 10, 16, 20, and 32 μg of purified His-p30 protein in sequence, mix well and let stand at room temperature for 10 min; add 100 μL of 10% NaCl solution respectively, mix well and let stand at room temperature for 2 h; observe the color change and record the tube numbers that remain red; transfer 200 μL of the gold-labeled solution from each tube to a 96-well microplate, and repeat each tube 3 times; measure OD520nm and record the tube number at which the OD520 value starts to remain unchanged. The protein content added to this tube number is the minimum protein amount for colloidal gold labeling. On the basis of this amount, add 10% - 30%, which is the optimal protein labeling amount for 1 mL of colloidal gold solution. The specific addition amounts of each component in the system are shown in Table 2.

[0075] Table 2 Determination of the minimum addition amount of recombinant His-p30 protein labeled with colloidal gold

[0076]

[0077] The results are as Figure 3 shown in A. When 0 and 4 μg of protein were added to 0.5 mL of colloidal gold solution, obvious flocculation occurred. However, as the amount of added protein increased, the blue particle precipitate became less, and the color gradually turned red. When 8, 10, 16, 20, and 32 μg of protein were added to 0.5 mL of colloidal gold solution, the color was uniform and stable, and there was no blue particle precipitate, indicating that the amount of added protein reached or exceeded the minimum amount of protein required to stabilize the colloidal gold solution.

[0078] Combined with Figure 3 B, it can be seen that when 8 μg of protein was added to 0.5 mL of colloidal gold solution, the OD520 value was the highest, and the absorbance values of the adjacent wells changed little, indicating that the amount of added protein reached the minimum amount required to stabilize the colloidal gold solution at this time. Based on this, increasing by 30% is the actual dosage for labeling. That is, in 1 mL of colloidal gold solution, the optimal protein labeling amount is 20 μg.

[0079] Example 5: Identification and purification of immunogold

[0080] 1. Immunogold identification

[0081] According to the optimal pH value for colloidal gold labeling (i.e., adding 12 μL of 0.1 mol / L K2CO3 to 1 mL of colloidal gold solution) and the optimal protein concentration (i.e., adding 20 μg of His-p30 protein to 1 mL of colloidal gold solution), colloidal gold labeling was carried out. Separate treated beakers were placed on a magnetic stirrer, and 20 mL of pre-treated colloidal gold solution was added. The colloidal gold solution was adjusted to the optimal labeling pH with 0.1 mol / L K2CO3. Under stirring, the optimal amount of protein was slowly added to the colloidal gold solution at the corresponding pH. After stirring for 30 min, it was left to stand at room temperature for 10 min. 200 μL of immunogold solution was taken into a 96-well microplate. After making marks, the absorbance values were measured in the wavelength range of 400 nm - 700 nm, and the peak width and peak shape of the maximum absorption peak were observed. Comparative analysis was carried out with the corresponding absorption peak of the colloidal gold stock solution to preliminarily identify the labeling situation. In addition, under stirring, 10% BSA filtered through a microporous membrane was added to the immunogold solution to make the final concentration 1%. After 30 min, stirring was stopped, and it was stored in the dark at 4 °C for standby.

[0082] The results are as Figure 4As shown, after the colloidal gold particles are combined with proteins, the gold particles increase in size. Through visible light scanning, the maximum absorption peak shifts, indicating that the recombinant His-p30 protein is successfully labeled on the surface of the gold particles.

[0083] 2. Purification of immunogold

[0084] The low-temperature high-speed centrifugation method is used to remove unlabeled proteins, insufficiently labeled gold particles, and various polymers that may form during the labeling process to achieve the purification of the gold-labeled complex. The labeled immunogold solution is centrifuged at 4°C and 1500 r / min for 15 min, and the supernatant is aspirated into a new clean centrifuge tube; then centrifuged at 4°C and 12000 r / min for 40 min, and the supernatant is carefully aspirated away; the precipitate is resuspended in PBS (0.01 mol / L, pH 7.4) containing 1% BSA to the original volume, and the centrifugation is repeated 1 - 2 times; the precipitate of the gold-labeled complex is resuspended in the gold-labeled complex diluent to 10% of the original volume, aliquoted, and stored at 4°C for later use; observe whether there is agglomeration or precipitation the next day.

[0085] Example 6: Treatment of the gold conjugate pad and the sample pad

[0086] The sample pad (SB08 of Shanghai Gold Label Biotechnology Co., Ltd.) and the gold conjugate pad (RB45 of Shanghai Gold Label Biotechnology Co., Ltd.) of the test strip are glass fiber membranes with strong hydrophobicity and larger pore sizes. In order to reduce the background interference of the test strip and increase the release of the gold-labeled complex, the sample pad is treated with 0.1% Triton-X100 + 1% BSA + 10 mM PB, dried at room temperature, sealed, and stored at room temperature for later use.

[0087] The above-prepared gold-labeled protein is evenly spread on the treated gold conjugate pad (300×10 mm) at a rate of 10 μL / cm, dried at 37°C for 1 h. This pad is the colloidal gold conjugate pad, which is sealed with a desiccant and stored at room temperature.

[0088] Example 7: Preparation of the detection NC membrane

[0089] The NC test membrane (Sartorius WH00332-0001) was cut into strips with a specification of 300×25 mm. Protein A of Staphylococcus aureus SPA (Sigma P6031) and anti-p30 mAb 3E1 were respectively dispensed on the NC membrane as the test line (T line) and the control line (C line) of the test strip. The SPA protein was diluted to concentrations of 1.0, 1.5, and 2.0 mg / mL with 0.1 mol / L Tris-HCl buffer and sprayed onto the NC membrane at a rate of 1 μL / cm as the T line. The anti-p30 mAb 3E1 was diluted to a concentration of 1.0 mg / mL with PBS solution at pH 7.2 and sprayed onto the NC membrane at a rate of 1 μL / cm as the C line, 5 mm away from the T line, and dried at 37°C for 4 h. The sample pad, colloidal gold conjugate pad (prepared in Example 6), NC membrane, and absorbent pad (Shanghai Gold Label Biotechnology Co., Ltd. SX42) were assembled into a test strip to detect the ASFV antibody positive serum standard (National Veterinary Microbial Strain (Virus) Preservation Center, catalog number Z310). The concentration with the best interception effect and the least amount used was the spraying concentration of the test strip. The sprayed NC membrane was sealed and stored dry.

[0090] The results are as Figure 5 shown. When the spraying concentration of the T line is 1.5 mg / mL and the spraying concentration of the C line is 1 mg / mL, the band color development effect of the chromatography membrane is better. Therefore, it was determined that in this experiment, the T line was sprayed with SPA protein at a concentration of 1.5 mg / mL, and the C line was sprayed with anti-p30 mAb 3E1 at a concentration of 1 mg / mL for the quality control of the test strip.

[0091] Example 8: Preliminary assembly and identification of colloidal gold immunochromatographic test strip

[0092] The test strip was preliminarily assembled and used to detect anti-ASFV positive serum and negative serum, and the results were observed. The operation of preparing the test strip was as follows: The immunocolloidal gold solution was evenly spread on the gold conjugate pad and freeze-dried for later use; Protein A of SPA and anti-p30 mAb3E1 were respectively drawn lines (about 1 mm) on the chromatography membrane as the test line (T line) and the control line (C line), and the distance between the T line and the C line was about 5 mm, and then freeze-dried for later use. The colloidal gold conjugate pad, chromatography membrane (NC membrane), absorbent pad, and sample pad were assembled manually according to the schematic diagram ( Figure 1 ) Note that the pasting positions of each part should be appropriate, and there should be no air or bubbles at the pasting place; there should be a certain overlap at the connection between each membrane, and it should be pressed tightly. The assembled test strip board was placed in the slot of the strip cutter and cut into test strips with a width of about 4 mm, then packed into a plastic box and stored sealed and dry at 4°C for later use.

[0093] An appropriate amount of negative and positive samples were dropped onto the sample pad of the test strip, and the results were observed after 15 min.

[0094] The results are as follows Figure 6 shown. If the sample contains ASFV-specific antibodies, both the T and C lines will show color, indicating a positive result( Figure 6 B); if the T line does not show color and the C line shows color, it is a negative result( Figure 6 A); if the T line shows color while the C line does not show color, or neither of them shows color, the test strip is invalid or the operation is incorrect.

[0095] Example 9: Performance determination of colloidal gold immunochromatographic test strip

[0096] 1. Specificity evaluation of the test strip

[0097] The test strips developed in this application were used to detect ASFV and other porcine virus (PDCoV, PEDV, PRRSV, PCV2) antibody-positive sera and healthy porcine negative sera to evaluate the specificity of the test strips. During the detection, the serum samples were diluted with physiological saline at a dilution ratio of 1:200. 100 μL was taken and detected with the test strip. After about 15 minutes, the test strip was observed with the naked eye to determine the result.

[0098] The results are as follows Figure 7 shown. Consistent with the expectation, for the test strip with only ASFV-positive serum added, both the T and C lines showed color, indicating a positive result; for the groups with other serum samples added, the T line did not show color and the C line showed color, indicating a negative result. This shows that the test strip does not react with the positive sera of common pathogenic microorganisms such as PRRSV, PDCoV, PEDV, and PCV2, and has good specificity.

[0099] 2. Sensitivity evaluation of the test strip

[0100] The test strips developed in this application were used to detect the ASFV antibody-positive serum standard to evaluate the minimum detection limit of the colloidal gold test strip. Using physiological saline as the diluent, the sample was serially diluted from 1:50 to 1:12800. 100 μL was taken and detected with the test strip developed in this experiment. After about 15 minutes, the result was determined with the naked eye.

[0101] The results are as follows Figure 8 shown. When using the test strip to detect the serially diluted positive sera, the T line of the serum diluted at 1:3200 was weak, and for the subsequently diluted test strips, only the C line showed color. Its sensitivity can reach 1:3200.

[0102] 3. Repeatability evaluation of the test strip

[0103] Test strips made from different batches were used to detect porcine anti-ASFV negative and positive sera, and the results were observed.

[0104] The results are as follows Figure 9As shown, test strips prepared at different times were used to detect 9 ASFV positive and negative samples, and there were no significant differences in the detection results.

[0105] The above embodiments are only illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. An antibody fragment against African swine fever virus p30 protein, characterized in that, The antibody fragment comprises a light chain variable region and a heavy chain variable region. The amino acid sequences of the three complementarity-determining regions in the light chain variable region are shown in SEQ ID NO.1-3 respectively, and the amino acid sequences of the three complementarity-determining regions in the heavy chain variable region are shown in SEQ ID NO.4-6 respectively.

2. An antibody against African swine fever virus p30 protein, characterized in that, The antibody comprises a light chain and a heavy chain. The amino acid sequence of the light chain is shown in SEQ ID NO.7, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.

9.

3. The African swine fever virus p30 protein antibody according to claim 2, characterized in that, The nucleotide sequence encoding the light chain is shown in SEQ ID NO.8, and the nucleotide sequence encoding the heavy chain is shown in SEQ ID NO.

10.

4. Use of the African swine fever virus p30 protein antibody fragment according to claim 1 and the African swine fever virus p30 protein antibody according to any one of claims 2 to 3 in detecting African swine fever virus antibodies or preparing reagents / strips for detecting African swine fever virus.

5. A colloidal gold immunochromatographic test strip for detecting African swine fever virus, characterized in that, The strip contains the African swine fever virus p30 protein antibody fragment according to claim 1 or the African swine fever virus p30 protein antibody according to any one of claims 2 to 3.

6. The colloidal gold immunochromatographic test strip for detecting African swine fever virus according to claim 5, wherein The strip further comprises a gold-labeled conjugate pad labeled with colloidal gold particles of African swine fever virus p30 protein, an NC membrane coated with staphylococcal protein A and African swine fever virus p30 protein antibody, a sample pad, a water-absorbing pad and a PCV bottom plate.

7. The colloidal gold immunochromatographic strip for detecting African swine fever virus according to claim 6, wherein The colloidal gold particles of African swine fever virus p30 protein are prepared by the following method: 0.1mol / L K2CO3 is added to the colloidal gold solution, and African swine fever virus p30 protein is added under stirring conditions. After stirring for 30 min, it is allowed to stand at room temperature to obtain.

8. The colloidal gold immunochromatographic strip for detecting African swine fever virus according to claim 7, wherein, The dosage of the African swine fever virus p30 protein is 17-83 μg added to each 1 mL of colloidal gold solution.

9. The colloidal gold immunochromatographic strip for detecting African swine fever virus according to claim 7, wherein, The dosage of the K2CO3 is 12-20 μL added to each 1 mL of colloidal gold solution.

10. The colloidal gold immunochromatographic test strip for detecting African swine fever virus according to claim 7, wherein The concentration of the African swine fever virus p30 protein antibody is 1 mg / mL.

Images