Preparation method of colloidal gold test strip and application of colloidal gold test strip in detection of African swine fever antibody

Abstract

The invention relates to the technical field of molecular diagnosis, in particular to a preparation method of a colloidal gold test strip and application of the colloidal gold test strip to detection of an African swine fever antibody. B602L genes of ASFV and Fc genes of swine IgG are synthesized respectively, flexible protein genes are creatively added between the genes, and then an expression vector is constructed; the expression vector is converted into competent cells, then induced expression is carried out, the expressed ASFV recombinant protein is extracted and purified to serve as a detection band, staphylococcus aureus protein A serves as a quality control band, the sensitivity of the test strip can reach 1: 320, and the test strip is good in specificity and high in sensitivity. The kit has no cross reaction with pseudorabies virus, classical swine fever virus, encephalitis B virus, porcine reproductive and respiratory syndrome virus and porcine circovirus type 1 and type 2, has good stability, and can be used for detecting the African swine fever antibody in serum.

Description

Technical Field

[0001] The present invention relates to the field of molecular diagnosis technology, and in particular to a method for preparing a colloidal gold test strip and an application of the colloidal gold test strip in detecting African swine fever antibodies. Background Art

[0002] African swine fever (ASF) is a highly contagious disease of pigs caused by African swine fever virus (ASFV). ASF is currently the disease that causes the greatest losses to my country's pig farming industry. The virus genome is large, the structure is complex, and there are many immune escape mechanisms. At present, no effective vaccine or drug has been developed for the disease. Therefore, rapid and accurate diagnosis of the disease is particularly important for the prevention and control of the disease. With the emergence of low-virulence natural mutants of ASFV in my country, ASF presents the characteristics of persistent, non-lethal, subacute or chronic disease. ASFV infection can cause a strong humoral immune response in pigs and last for a long time. It is difficult to detect the virus in the blood of recovered pigs infected with ASFV, but the blood contains a high level of antibodies, which makes the research on ASF serum antibody detection methods particularly important for the prevention and control and purification of ASF.

[0003] The ASFV B602L protein has good antigenicity, and IgG Fc contributes to antigen presentation and phagocytosis. Previous studies have shown that B602L has strong antigenicity and can be a candidate antigen for the development of diagnostic methods for ASFV. Previously, an ELISA test based on B602L was used to detect ASFV serum antibodies, and the test results were basically consistent with the gold standard Western blot test results. Moreover, the test method can also distinguish pigs that are persistently infected with natural ASFV strains from pigs immunized with subunit vaccines of structural proteins. B602L is likely to be a suitable target for the development of diagnostic tools for evaluating humoral immune responses to these vaccines, because antibodies against this protein can only be produced after B602L is expressed in host cells. Based on the above, the current research on B602L is also very important, and this protein may become a candidate antigen for ASFV detection and subunit vaccines.

[0004] The p30 protein is one of the most antigenic proteins of ASFV. It can be expressed and secreted in the early stage of ASFV infection, and the antigen can be detected 4 hours after infection. Therefore, the p30 protein is an ideal antigen for early infection. As the main structural protein of ASFV, the p72 protein has strong antigenicity and immunogenicity, and the gene sequence of the protein is well conserved, which makes the p72 protein have relatively stable antigenicity. At present, a variety of ASFV serological detection methods have been established based on this protein, among which antibodies can be detected on the 7th day of virus infection. The CD2v protein expresses protein in the late stage of ASFV infection, and its function is to enable red blood cells to adsorb to the surface of virus-infected cells, thereby promoting the spread of the virus in the host. Studies have reported that a non-blood adsorption phenotype with a natural deletion of the EP402R gene has appeared in my country, with low virulence but high transmissibility. The diagnosis of ASF has become more difficult due to the emergence of this low-virulence natural mutant, which has also brought new challenges to the control of ASFV.

[0005] Staphylococcus aureus protein A is a 40–60 kDa surface protein consisting of 4 or 5 homologous immunoglobulin binding domains of 56–61 residues. This protein is a protein component isolated from the cell wall of the bacterium. Because this protein can bind to the Fc segment of IgG molecules of various mammals, but does not affect the binding of IgG to antigens, it is widely used in the field of immunology. The SPA is used to prepare SPA columns for plasma in vitro immunoadsorbents, and has achieved good therapeutic effects in the treatment of rheumatoid arthritis, idiopathic thrombocytopenic purpura, breast cancer, and neurological diseases. Since the protein is partially hydrophobic, there are still some difficulties in purifying SPA. At present, ion exchange chromatography, affinity chromatography and gel filtration are generally used for purification. Since the protein is stable in nature and can bind well to colloidal gold, it is widely used in colloidal gold lateral flow chromatography test strip detection. In the field of colloidal gold test strips, this protein is mainly used to bind to the IgG molecule Fc in the serum of various mammals, present the antibody to the test strip, and detect the antibody by reacting with the antigen marked on the test strip.

[0006] In summary, although the above-mentioned proteins show certain potential for serological detection, the current existing technology still lacks detection methods or materials for rapid diagnosis of African swine fever virus. Based on this, the present invention proposes a method for preparing a colloidal gold test strip and its application in detecting African swine fever antibodies. Summary of the invention

[0007] The present invention solves the problems in the related art and proposes a method for preparing a colloidal gold test strip. The method synthesizes the B602L gene of ASFV and the Fc gene of pig IgG respectively, constructs an expression vector after creatively adding a flexible protein gene between the genes, transforms the expression vector into competent cells and induces expression, extracts and purifies the expressed ASFV recombinant protein and uses it as a detection band, and Staphylococcus aureus protein A is used as a quality control band. The test strip has a sensitivity of 1:320 and good specificity. It has no cross reaction with pseudorabies virus, classical swine fever virus, Japanese encephalitis virus, porcine reproductive and respiratory syndrome virus, and porcine circovirus type 1 and type 2, has good stability, and can be used to detect African swine fever antibodies in serum.

[0008] In order to solve the above technical problems, the present invention is achieved through the following technical solutions: a method for preparing a colloidal gold test strip, comprising the following steps:

[0009] S1. Target gene fusion amplification:

[0010] Referring to the coding sequence of the ASFV (Pig / HLJ / 2018 strain) gene in GenBank, the B602L gene of ASFV and the Fc gene of porcine IgG were synthesized respectively, and the upstream primer and downstream primer of the target gene were designed to amplify the target gene, wherein the upstream primer of the B602L gene is shown in SEQ.No.1, the downstream primer of the B602L gene is shown in SEQ.No.2, the upstream primer of the Fc gene is shown in SEQ.No.3, and the downstream primer of the Fc gene is shown in SEQ.No.4. The composition of the amplification reaction system is: 2 μL of template DNA, 2 μL of upstream and downstream primers, 25 μL of Taq DNA polymerase mix, and 19 μL of ddH2O; the gene sequence of the B602L gene is shown in SEQ.No.5, and the gene sequence of the Fc gene of porcine IgG is shown in SEQ.No.6;

[0011] A flexible protein gene with a length of 15 amino acids was added between the Fc segment gene and the B602L gene for fusion PCR, wherein the gene sequence of the flexible protein gene is shown in SEQ.No.7, and the reaction system of the fusion PCR is: template DNA 2μL, Fc gene 2μL, Taq DNA polymerase mix 25μL, ddH2O 17μL; the fusion PCR amplification program is: 95℃5min, 95℃30s; 58℃60s; 72℃30-120s; 10 cycles, 72℃5min;

[0012] Add 2 μL of the upstream primer of the B602L gene and the downstream primer of the Fc fragment gene to the reaction system, and continue to amplify for 25 cycles according to the amplification procedure of the fusion PCR. After the reaction is completed, the amplified product is electrophoresed using 1% agarose gel to identify the amplified product, and the target gene is recovered and stored in a -20°C refrigerator after the concentration is determined;

[0013] S2. Construction and identification of recombinant plasmid: pCold I vector and recovered target gene were double-digested with Xba I and EcoR I endonucleases in a 37°C water bath for 4 h. The composition of the digestion system was as follows: 1 μg of vector and template gene, 1 μL of Xba I enzyme, 1 μL of EcoR I enzyme, 3 μL of 10×Cut Smart Buffer, and dd H2O to make up to 30 μL. After the digestion was completed, the digestion products were identified by electrophoresis on 1% agarose gel. After the target fragment was recovered, the concentration was measured and stored in a -20°C refrigerator.

[0014] Use T4 ligase to carry out ligation reaction between the target fragment and pCold I vector at 16°C overnight, wherein the composition of the ligation reaction system is as follows: T4 ligase 0.5μL, T4 buffer 1μL, the target fragment and the vector are added at a molar ratio of 1:3, and dd H2O is used to make up to 10μL; the ligation product is added to DH5α competent cells for transformation. After the transformation is completed, the bacterial liquid is spread on an ampicillin-resistant plate and cultured at 37°C overnight. A single colony is picked and placed in LB medium containing ampicillin resistance and cultured at 37°C incubator for 6 hours, and a positive single clone is selected.

[0015] Furthermore, the target fragment was transformed into DH5α competent cells, which specifically included the following steps: adding the ligation product to the DH5α competent cells and gently mixing, placing on ice for 30 minutes, then heat shocking at 42°C for 90 seconds, continuing to ice bath for 5 minutes, adding 700 μL of LB medium, placing in a 37°C incubator for 40 minutes (200 rpm / min), centrifuging and taking 100 μL of bacterial solution to spread on an ampicillin-resistant plate, placing in a 37°C incubator for overnight culture; picking a single colony to LB medium containing ampicillin resistance and placing in a 37°C incubator (200 rpm / min) for 6 hours; taking 2 μL of bacterial solution as a template, and using the designed primers to perform PCR identification to screen out the positive single clone.

[0016] The positive recombinant plasmids were extracted using a plasmid extraction kit and sent to Qingke Biotechnology Company for sequencing. The plasmids with correct sequencing results were named pCold I-B602L and pCold I-B602L-Fc.

[0017] Prokaryotic expression, purification and identification of S3, ASFV B602L and B602L-Fc proteins

[0018] The constructed positive recombinant plasmids (pCold I-B602L, pCold I-B602L-Fc) were transformed into competent cells BL21 (DE3), and monoclonal bacteria were selected and cultured overnight. 1 mL of bacterial solution was added to 200 mL of LB medium and cultured on a shaking table until the OD was 0.6-0.8, and IPTG was added to induce expression at a concentration of 1 mM IPTG. The bacterial solution was ultrasonically broken in an ice bath and then frozen centrifuged to extract inclusion body proteins. The precipitate was resuspended in 8 M urea lysis buffer and Ni was used to precipitate. 2+ The recombinant proteins (ASFV B602L and B602L-Fc proteins) were purified by affinity chromatography, and anti-His tag monoclonal antibody was used as the primary antibody. The recombinant proteins (ASFV B602L and B602L-Fc proteins) obtained after induced expression were identified by Western-blot using goat anti-mouse secondary antibody with HRP;

[0019] Furthermore, the extraction and purification step of inclusion body protein includes the following steps: centrifuging the cultured bacterial solution at 4°C and 6000rpm to discard the supernatant, washing twice with PBS, resuspending the bacterial solution with PBS according to a volume ratio of PBS to bacterial solution of 1:8, adding PMSF according to a volume ratio of PMSF to bacterial solution of 1:1000, ultrasonicating at 4°C and 8000rpm to obtain a precipitate, adding a lysis solution containing 8M urea to resuspend the precipitate, ultrasonicating at 4°C and 8000rpm to obtain a supernatant, and using Ni 2+ The recombinant protein was purified by affinity chromatography.

[0020] Furthermore, using Ni 2+ The specific steps for purifying recombinant proteins by affinity chromatography are as follows:

[0021] (1) Replace storage buffer: Ni 2+ Add 5 column volumes of deionized water to the affinity chromatography column to replace its storage buffer;

[0022] (2) Balanced Ni 2+ Column: Add 10 column volumes of lysate containing 8M urea to balance Ni 2+ Affinity chromatography columns;

[0023] (3) Protein over Ni 2+ Column: Add the treated supernatant into the column, control the flow rate at 1 drop / 3s, and collect the flow-through;

[0024] (4) Washing: Add 10-15 column volumes of a washing solution containing 8 M urea to the column and collect the washing solution;

[0025] (5) Use 250 mM imidazole eluent containing 8 M urea for elution. Add 10 times column volume of eluent, collect the eluent and divide it into smaller portions. After preparing the protein sample, observe the protein purification by SDS-PAGE electrophoresis. After the protein concentration of the purified protein is determined by the BCA method, divide it into smaller portions and store it at -80°C.

[0026] Furthermore, the steps of Western-blot identification of the recombinant protein are as follows:

[0027] (1) Prepare filter paper and PVDF membrane and soak them in transfer solution;

[0028] (2) Take out the protein gel, cut off the upper concentrated gel, and place them in the order of one layer of sponge pad, three layers of filter paper protein gel, PVDF membrane, three layers of filter paper, and sponge pad. Suppress the bubbles, put the black electrode at the bottom and the white electrode at the top, close the clamp and place it in the electrophoresis tank. Add transfer solution to the electrophoresis tank and place it in an ice-water mixture. Constant current 240A for 120 min.

[0029] (3) Take out the transferred membrane, add 15 mL of TBST containing 5% skim milk powder, block for 3 h at room temperature, discard the blocking solution, and wash with TBST three times;

[0030] (4) Add primary antibody (1:5000 dilution) and incubate overnight at 4°C. After recovering the primary antibody, wash with TBST three times, 15 min each time;

[0031] (5) Add HRP-labeled secondary antibody (1:5000 dilution), incubate at room temperature for 50 min, and then wash three times with TBST, each time for 15 min;

[0032] (6) Scanning: Take 500 μL of each of luminescent solution A and solution B, mix them evenly, and then add them to the nitrocellulose membrane in the dark. After they are fully combined, place the membrane in a membrane scanner for scanning and take a photo for preservation.

[0033] S4. Preparation of colloidal gold test strips for detecting African swine fever antibodies: soak the gold label pad with the heavy suspension in advance and place it in an oven to dry. Then assemble the PVC base plate, gold label pad, nitrocellulose membrane and absorbent paper together. Dilute the purified B602L-Fc protein and SPA to 0.5 mg / mL, and mark them on the detection line and quality control line of the nitrocellulose membrane respectively to obtain the colloidal gold test strips.

[0034] The present invention also provides an application of the colloidal gold test strip, namely, application of the colloidal gold test strip in detecting African swine fever antibodies in serum.

[0035] When the gold-labeled protein B602L-Fc does not bind to SPA, the quality control line C line has no band, indicating that the test strip is invalid; when the gold-labeled protein B602L-Fc can bind to the SPA of the quality control line C line, and the quality control line has a band, if there is B602L protein antibody in the serum, it will bind to the B602L-Fc of the gold-labeled protein, and bind to the labeled B602L protein on the detection line T line to form a double antigen sandwich, and the color development result of the T line is judged to be positive; when there is no band on the T line, the result is judged to be negative. Based on this, the judgment criteria of the colloidal gold test strip for detecting African swine fever antibodies prepared by the present invention are: no band on the quality control line, the test strip is invalid; there are bands on the quality control line and the detection line, positive; there are bands on the quality control line, no bands on the detection line, negative.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) The present invention fuses the antigen with porcine IgG Fc, uses a prokaryotic expression system to express and purify, and obtains a fusion protein that binds well to SPA. Functional verification shows that the protein without Fc fusion does not react with the SPA protein, while the B602L-Fc fusion protein can react with obvious color development;

[0038] (2) The detection method of ASFV antibodies based on nanogold particle visualization established by the present invention is established by combining Fc with SPA. It can detect serum antibodies without the need to prepare antibodies and has good specificity and sensitivity, providing a new idea for the construction of a rapid visualization detection method.

[0039] (3) The present invention uses the pCold I prokaryotic expression vector to express the B602L protein of ASFV and the B602L Fc fused with the Fc fragment. The results of induced expression show that the expression amount of the recombinant protein fused with the Fc protein is significantly increased compared with that of the recombinant protein not fused with the Fc protein. It is possible that the Fc protein plays a role in promoting expression. On the one hand, it can increase the expression amount of the target protein, and on the other hand, it can be purified using a SPA affinity chromatography column;

[0040] (4) It has no cross-reaction with pseudorabies virus, classical swine fever virus, Japanese encephalitis virus, porcine reproductive and respiratory syndrome virus, and porcine circovirus type 1 and type 2. It has good stability and can be used to detect African swine fever antibodies in serum. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is the electrophoresis result of the fusion of the ASFV B602L protein gene with the ASFVB602L protein gene and the Fc fragment of the present invention, wherein 1 in A: B602L-1; 2: B602L-2; M: DL2000; 1-2 in B: IgG Fc; M: DL2000;

[0042] Figure 2 It is the result of PCR electrophoresis identification of B602L-Fc of the present invention, 1: B602L-Fc; M: Maker IV;

[0043] Figure 3 The results of Western-blot identification of the recombinant proteins B602L and B602L-Fc of the present invention are expressed, A: anti-His; B: ASFV positive serum; C: ASFV positive serum;

[0044] Figure 4 The SDS-PAGE analysis of the B602L recombinant protein of the present invention shows the purification conditions, 1: B602L; 2: flow-through solution; 3: 40 mM washing solution; 4: 60 mM washing solution; 5-25: eluent;

[0045] Figure 5 The SDS-PAGE analysis of the B602L-Fc recombinant protein of the present invention shows the purification conditions, 1: B602L-Fc; 2: flow-through solution; 3: 40 mM washing solution; 4: 60 mM washing solution; 5-25: eluent;

[0046] Figure 6 This is a preliminary verification of the function of the recombinant protein of the present invention;

[0047] Figure 7 It is the sensitivity detection of the colloidal gold test strip of the present invention;

[0048] Figure 8 It is the specific detection of the colloidal gold test strip of the present invention. DETAILED DESCRIPTION

[0049] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0051] Unless otherwise specifically stated, the relative arrangement of the parts and steps described in these embodiments, numerical expressions and numerical values ​​do not limit the scope of the present invention. At the same time, it should be understood that, for ease of description, the sizes of the various parts shown in the accompanying drawings are not drawn according to the actual proportional relationship. The technology, method and equipment known to ordinary technicians in the relevant field may not be discussed in detail, but in appropriate cases, the technology, method and equipment should be regarded as a part of the authorization specification. In all examples shown and discussed here, any specific value should be interpreted as being merely exemplary, rather than as a limitation. Therefore, other examples of exemplary embodiments may have different values. It should be noted that similar reference numerals and letters represent similar items in the following drawings, so once a certain item is defined in an accompanying drawing, it does not need to be further discussed in subsequent drawings.

[0052] In the following examples, the pCold I vector was stored in this laboratory and is commercially available. BL21 (DE3) and DH5α competent cells were purchased from Tiangen Biochemical Technology Co., Ltd. The ASFV positive serum used in the present invention was provided by the China Animal Health and Epidemiology Center, and the ASFV negative serum, CSFV, PRV, PEDV, PRRSV, PCV1, PCV2, and JEV serum were stored in the laboratory of Shanghai Veterinary Research Institute.

[0053] The main reagents in the following examples are prepared according to the following composition:

[0054] 1% chloroauric acid solution: Add 1 g of chloroauric acid to a volume of 100 mL with ultrapure water and store in a clean glass container (after acidification and siliconization treatment) at 4°C away from light.

[0055] 1% trisodium citrate solution: 1 g of citric acid is diluted with ultrapure water to 100 mL. After dissolution, the solution is filtered using a 0.22 μm filter membrane and then stored at 4°C.

[0056] Blocking solution: BSA 2.5g, add PBS buffer to make up to 50mL, and store in a 4℃ refrigerator.

[0057] Resuspension: 50 g sucrose, 40 g trehalose, 20 g sorbitol, 10 g BSA, 0.026 g sodium azide, add PBS and make up to 1 L.

[0058] Example 1 Amplification of the target protein gene of the present invention and identification and expression of the recombinant plasmid

[0059] S1. Fusion amplification of target gene

[0060] The plasmid synthesized by the company was used as a template and the genes of B602L and Fc fragment were amplified using the synthesized primers. The PCR reaction system (50 μL) was as follows:

[0061]

[0062]

[0063] B602L and Fc fragments were successfully amplified, and the results were as follows Figure 1 .

[0064] The Fc target gene was fused and amplified with the African swine fever target gene, and a 15-amino acid long flexible protein was added in the middle to prevent the two segments from affecting each other. The Fc segment was fused with the gene of the African swine fever virus protein, and the fusion PCR reaction system was:

[0065]

[0066] The fusion PCR amplification procedure was as follows: pre-denaturation at 95°C for 5 min, followed by 1 cycle of 95°C for 30 s, 58°C for 60 s, and 72°C for 30-120 s, for 10 cycles, and finally extension at 72°C for 5 min. 2 μL of each fragment F primer and Fc-R were added to the reaction system, pre-denaturation at 95°C for 5 min, followed by 1 cycle of 95°C for 30 s, 58°C for 60 s, and 72°C for 30-120 s, for 25 cycles, and finally extension at 72°C for 5 min.

[0067] After the reaction was completed, the amplified product was identified by electrophoresis using 1% agarose gel. Figure 2 ), cut out the target fragment, and use a gel recovery kit to recover the target gene. The recovered DNA fragments were measured for concentration and stored in a -20°C refrigerator.

[0068] (2) Construction and identification of recombinant plasmids

[0069] The pCold I vector and the recovered target genes were double-digested with Xba I and EcoR I endonucleases in a 37°C water bath for 4 hours. The enzyme digestion system was as follows:

[0070]

[0071] After the enzyme digestion is completed, the enzyme digestion products are identified by electrophoresis using 1% agarose gel, the target fragments are cut out, and they are recovered using a gel recovery kit. The recovered DNA fragments are measured for concentration and stored in a -20°C refrigerator.

[0072] Use T4 ligase to connect the target fragment and pCold I vector at 16°C overnight. The reaction system is as follows:

[0073]

[0074] Add the ligation product to DH5α competent cells and mix gently, place on ice for 30 minutes, heat shock at 42°C for 90 seconds, and then place on ice for 5 minutes, add 700 μL of LB medium, and culture in a 37°C incubator for 40 minutes (200 rpm / min). Centrifuge and take 100 μL of the bacterial solution and apply it to an ampicillin-resistant plate, and culture in a 7°C incubator overnight.

[0075] Pick a single colony and place it in LB medium containing ampicillin resistance and culture it in a 37℃ incubator (200rpm / min) for 6h; take 2μL of bacterial solution as a template, use the designed primers for PCR identification, and screen out the positive single clone. Use a plasmid mini-extraction kit to extract the positive recombinant plasmid and send it to Qingke Biotechnology Company for sequencing. The plasmids with correct sequencing results were named pCold I-B602L and pCold I-B602L-Fc.

[0076] (3) Prokaryotic expression, purification and identification of ASFV recombinant protein

[0077] The constructed positive recombinant plasmid was transformed into competent cells BL21 (DE3), and a single clone was selected and cultured overnight. 1 mL of the bacterial solution was added to 200 mL of LB medium and shaken until the OD was 0.6-0.8. IPTG was added to induce expression at a concentration of 1 mM IPTG.

[0078] Purification of inclusion body protein: centrifuge the cultured bacterial solution at 4°C and 6000rpm, discard the supernatant, wash twice with PBS, resuspend the bacterial solution in PBS at a volume ratio of 1:8, add PMSF at a volume ratio of 1:1000, sonicate on ice for 30 minutes, and after ultrasonic disruption, centrifuge at 4°C and 8000rpm to obtain the precipitate, add lysis buffer containing 8M urea to resuspend the precipitate, sonicate on ice for 30 minutes, and after ultrasonic disruption, centrifuge at 4°C and 8000rpm to obtain the supernatant.

[0079] By using Ni 2+ The recombinant protein was purified by affinity chromatography. The specific steps of purification are as follows:

[0080] (1) Replace storage buffer: Ni 2+ Add 5 column volumes of deionized water to the affinity chromatography column to replace its storage buffer;

[0081] (2) Balanced Ni 2+ Column: Add 10 column volumes of lysate containing 8M urea to balance Ni 2+ column;

[0082] (3) Protein over Ni 2+Column: Add the treated supernatant to the column, control the flow rate at 1 drop / 3s, and collect the flow-through;

[0083] (4) Washing: Add 10-15 column volumes of a washing solution containing 8 M urea to the column and collect the washing solution;

[0084] (5) Use 250 mM imidazole eluent containing 8 M urea for elution. Add 10 times column volume of eluent, collect the eluent and divide it into smaller portions. After preparing the protein sample, observe the protein purification by SDS-PAGE electrophoresis. After the protein concentration of the purified protein is determined by the BCA method, divide it into smaller portions and store it at -80°C.

[0085] The bacterial suspension was purified by inclusion body purification method to obtain the eluate to prepare protein samples, which were then detected by SDS-PAGE. Figure 4 and Figure 5 As shown, all seven proteins were expressed in inclusion bodies and the target proteins were successfully purified.

[0086] The specific steps for recombinant protein Western-blot identification are as follows:

[0087] (1) Prepare filter paper and PVDF membrane and soak them in transfer solution;

[0088] (2) Take out the protein gel, cut off the upper concentrated gel, and place them in the order of one layer of sponge pad, three layers of filter paper protein gel, PVDF membrane, three layers of filter paper, and sponge pad. Suppress the bubbles, put the black electrode at the bottom and the white electrode at the top, close the clamp and place it in the electrophoresis tank. Add transfer solution to the electrophoresis tank and place it in an ice-water mixture. Constant current 240A for 120 min.

[0089] (3) Take out the transferred membrane, add 15 mL of TBST containing 5% skim milk powder, block for 3 h at room temperature, discard the blocking solution, and wash with TBST three times;

[0090] (4) Add primary antibody (1:5000 dilution) and incubate overnight at 4°C. After recovering the primary antibody, wash with TBST three times, 15 min each time;

[0091] (5) Add HRP-labeled secondary antibody (1:5000 dilution), incubate at room temperature for 50 min, and then wash three times with TBST, each time for 15 min;

[0092] (6) Scanning: Take 500 μL of each of luminescent solution A and solution B, mix them evenly, and then add them to the nitrocellulose membrane in the dark. After they are fully combined, place the membrane in a membrane scanner for scanning and take a photo for preservation.

[0093] Anti-His tag monoclonal antibody was used as primary antibody, and goat anti-mouse secondary antibody with HRP was used to detect the recombinant protein obtained after induced expression. Western-blot identification results (such as Figure 3 As shown in A), the fusion Fc proteins all carry His tags and can be correctly expressed in the prokaryotic expression system; ASFV positive serum and negative serum were used as primary antibodies, and sheep anti-swine antibody with HRP was used as secondary antibody to detect B602L protein. The Western-blot identification results are shown in ( Figure 3 As shown in B and C, compared with the control group, the four proteins were all recognized by ASFV positive serum antibodies, indicating that the B602L protein could be correctly expressed in the prokaryotic expression vector.

[0094] Example 2 ASFV recombinant protein and fusion protein Fc segment function verification

[0095] Take the prepared 4 test strips, and spot 0.4 μL of B602L-Fc fusion protein (0.4 mg / mL) and ASFV B602L protein (0.4 mg / mL) on the nitrocellulose membrane respectively. Insert it into the gold-labeled SPA solution to observe the test strip reaction results.

[0096] Take the 4 prepared test strips, spot 0.4 μL of ASFV B602L (0.4 mg / mL) on the nitrocellulose membrane, two strips for each protein, and insert them into the gold-labeled SPA with positive serum to observe the reaction results of the test strips.

[0097] Dilute the prepared proteins to a concentration of 0.4 mg / mL, spot 0.4 μL of B602L-Fc fusion protein and b602L protein on the nitrocellulose membrane, insert it into the prepared gold-labeled SPA solution, and observe the test strip results (such as Figure 6 As shown in A), the B602L-Fc protein can react with SPA to develop color, while the B602L protein without Fc fusion does not react with it. Use B602L and the resuspension solution to spot other test strips, and then insert them into the gold-labeled SPA solution with ASFV positive serum to observe the test strip results (such as Figure 6 As shown in B), the B602L protein can develop color, while the resuspension does not develop color, indicating that the prepared prokaryotic expression B602L protein can bind well to the antibodies of ASFV positive serum. Therefore, the B602L-Fc fusion protein can be used as a detection line.

[0098] Example 3 Determination of the optimal pH and optimal labeled protein amount of the colloidal gold labeled B602L-Fc protein prepared by the present invention

[0099] In this embodiment, the optimal pH determination method for colloidal gold labeled B602L-Fc is:

[0100] (1) Place 8 EP tubes on a plate rack and add 100 μL of the prepared colloidal gold into each tube;

[0101] (2) Add 0, 1, 2, 3, 4, 5, 6 and 8 μL of 0.1 M K2CO3 solution to each tube, mix well, add 10 μg of B602L-Fc protein, mix well and let stand at room temperature for 10 min;

[0102] (3) Add 10 μL of 10% NaCl solution to each tube, mix well, and let stand at room temperature for 2 h. Observe the color of the solution. The minimum amount of K2CO3 solution that can maintain the wine red color of the colloidal gold solution is the optimal labeled pH.

[0103] The method for determining the optimal protein labeling amount of colloidal gold labeled B602L-Fc is as follows:

[0104] (1) Take 800 μL of the prepared colloidal gold into a 1.5 mL EP tube, add 0.1 M K2CO3 solution required for the optimal marking pH and mix well;

[0105] (2) Place 8 EP tubes on a plate rack and add 100 μL of pH-adjusted colloidal gold to each tube;

[0106] (3) Add 1, 2, 3, 4, 6, 8, 10, and 12 μg of B602L-Fc protein to each tube, mix well, and let stand at room temperature for 10 min;

[0107] (4) Add 10 μL of 10% NaCl solution to each tube, mix well, and let stand at room temperature for 2 h. Observe the color of the solution. The minimum amount of B602L-Fc protein that can maintain the wine red color of the colloidal gold solution is the optimal amount of labeled protein.

[0108] Example 4 Preparation of colloidal gold test strips of the present invention and result judgment criteria

[0109] After the gold label pad was soaked with the resuspension solution in advance, it was placed in an oven to dry, and then the PVC base plate, gold label pad, nitrocellulose membrane and absorbent paper were assembled together. The purified B602L-Fc protein and SPA were diluted to 0.5 mg / mL and marked on the detection line and quality control line of the nitrocellulose membrane respectively. The diluted serum was added to the sample tank, and the experimental results were determined after 10 minutes.

[0110] When the gold-labeled protein B602L-Fc does not bind to SPA, there will be no band on the quality control line C, indicating that the test strip is invalid; when the gold-labeled protein B602L-Fc can bind to the SPA on the quality control line C and there is a band on the quality control line, if there is B602L protein antibody in the serum, it will bind to the gold-labeled protein B602L-Fc and bind to the labeled B602L protein on the test line T to form a double antigen sandwich. At this time, the color development result of the T line is judged to be positive; when there is no band on the T line, the result is judged to be negative.

[0111] Example 5 Performance measurement of colloidal gold test strips of the present invention

[0112] (1) Sensitivity detection of the colloidal gold test strip detection method of the present invention

[0113] The test strip prepared by the present invention was used to detect the sensitivity of ASFV positive serum, and the positive serum was diluted to 1:20, 1:40, 1:80, 1:160, 1:320, 1:640 and 1:1280 times before testing. The results are as follows Figure 7 As shown, when the serum is diluted to 1:320, the T-line band can still be seen on the colloidal gold test strip, and when the serum is diluted to 1:640, the T-line band is very faint and can hardly be seen by the naked eye.

[0114] (2) Specificity detection of the colloidal gold test strip detection method of the present invention

[0115] The test strip was used to detect ASFV, CSFV, PRV, CPRRSV, PRRSV, PCV1, PCV2, and JEV positive sera to evaluate the specificity of the test strip. Figure 8 As shown, the C line of the test strip has bands, proving that the test strip is effective; the T line of ASFV-positive serum has bands, while the T lines of other sera have no bands, and there is no non-specific situation, indicating that the test strip has good specificity.

[0116] (3) Determination of the compliance rate of the colloidal gold test strip detection method of the present invention

[0117] 100 clinical samples were tested using a test strip detection method and a commercial African swine fever virus ELISA antibody detection kit, respectively. The manufacturer of the commercial African swine fever virus ELISA antibody detection kit is Qingdao Lijian Biological Company.

[0118] The results of the two detection methods were compared and the coincidence rate of the two detection methods was calculated, and the coincidence rate (%) = (true positive + true negative) / (true positive + true negative + false positive + false negative) × 100%. The results are shown in Table 1. This method detected 32 positive samples and 68 ASFV negative samples, and the commercial African swine fever virus ELISA antibody detection kit detected 31 positive samples and 69 negative samples. The coincidence rate between this detection method and the commercial African swine fever virus ELISA antibody detection kit is 98%.

[0119] Table 1 Comparison results of the colloidal gold test strip detection method and the ASFV B-ELISA antibody detection kit detection method

[0120]

[0121] The above are preferred implementation modes of the present invention. Those skilled in the art to which the present invention belongs can also change and modify the above implementation modes. Therefore, the present invention is not limited to the above specific implementation modes. Any obvious improvements, substitutions or modifications made by those skilled in the art on the basis of the present invention belong to the protection scope of the present invention.

Claims

1. A method for preparing a colloidal gold test strip, characterized in that: Here are the steps: S1. Target gene fusion amplification: Referring to the coding sequence of the ASFV (Pig / HLJ / 2018 strain) gene in GenBank, the B602L gene of ASFV and the Fc gene of porcine IgG were synthesized respectively, and the upstream primers and downstream primers of the target gene were designed to amplify the target gene; the gene sequence of the B602L gene is shown in SEQ.No.5, and the gene sequence of the Fc gene of porcine IgG is shown in SEQ.No.6; A 15-amino-acid-long flexible protein gene was added between the Fc segment gene and the B602L gene for fusion PCR, wherein the gene sequence of the flexible protein gene is shown in SEQ.No.7; Add 2 μL of the upstream primer of the B602L gene and the downstream primer of the Fc fragment gene to the reaction system, and continue to amplify for 25 cycles according to the amplification procedure of the fusion PCR. After the reaction is completed, the amplified product is identified by electrophoresis using 1% agarose gel, the target gene is recovered, the concentration is determined, and then stored in a -20°C refrigerator; S2. Construction and identification of recombinant plasmid: pCold I vector and recovered target gene were subjected to double restriction enzyme digestion reaction with XbaI and EcoRI endonucleases in a 37°C water bath for 4 hours. After the restriction enzyme digestion was completed, the restriction enzyme digestion products were identified by electrophoresis using 1% agarose gel. After the target fragment was recovered, the concentration was measured and stored in a -20°C refrigerator; T4 ligase was used to perform a 16°C overnight ligation reaction on the target fragment and pCold I vector, and the ligation product was added to DH5α competent cells for transformation. After the transformation, the bacterial solution was applied to an ampicillin-resistant plate and cultured at 37°C overnight. A single colony was picked to LB medium containing ampicillin resistance and placed in a 37°C incubator for 6 hours, and a positive single clone was selected and a positive recombinant plasmid was extracted; Prokaryotic expression, purification and identification of S3, ASFV B602L and B602L-Fc proteins: The constructed positive recombinant plasmid was transformed into competent cells BL21 (DE3), monoclonal bacteria were selected and cultured overnight, 1 mL of bacterial solution was added to 200 mL of LB medium, and cultured on a shaking table until the OD was 0.6-0.8, IPTG was added to induce expression at a concentration of 1 mM IPTG; the bacterial solution was ultrasonically broken in an ice bath and then frozen centrifuged to extract inclusion body proteins, and the precipitate was resuspended in 8 M urea lysis buffer and Ni was used to precipitate. 2+ ASFV B602L and B602L-Fc proteins were purified by affinity chromatography, and anti-His tag monoclonal antibody was used as primary antibody, and goat anti-mouse with HRP was used as secondary antibody to identify ASFV B602L and B602L-Fc proteins obtained after induced expression by Western-blot. S4. Preparation of colloidal gold test strips: soak the gold label pad with heavy suspension in advance and place it in an oven to dry. Then assemble the PVC base plate, gold label pad, nitrocellulose membrane and absorbent paper together. Dilute the purified B602L-Fc protein and Staphylococcus aureus protein A to 0.5 mg / mL and mark them on the detection line and quality control line of the nitrocellulose membrane respectively to obtain colloidal gold test strips.

2. The method for preparing a colloidal gold test strip according to claim 1, wherein: In step S1, the upstream primer of the B602L gene is shown in SEQ.No.1, the downstream primer of the B602L gene is shown in SEQ.No.2, the upstream primer of the Fc gene is shown in SEQ.No.3, and the downstream primer of the Fc gene is shown in SEQ.No.

4. The composition of the amplification reaction system is: 2μL of template DNA, 2μL of upstream and downstream primers respectively, 25μL of Taq DNA polymerase mix, and 19μL of ddH2O; the reaction system of fusion PCR is: 2μL of template DNA, 2μL of Fc gene, 25μL of Taq DNA polymerase mix, and 17μL of ddH2O; the fusion PCR amplification program is: 95℃5min, 95℃30s; 58℃60s; 72℃30-120s; 10 cycles, 72℃5min.

3. The method for preparing a colloidal gold test strip according to claim 1, wherein: In step S2, the composition of the enzyme digestion system is as follows: 1 μg of vector and template gene, 1 μL of Xba I enzyme, 1 μL of EcoR I enzyme, 3 μL of 10×Cut Smart Buffer, and dd H2O is used to make up to 30 μL; the composition of the ligation reaction system is as follows: 0.5 μL of T4 ligase, 1 μL of T4 buffer, the target fragment and the vector are added at a molar ratio of 1:3, and ddH2O is used to make up to 10 μL.

4. The method for preparing a colloidal gold test strip according to claim 1, wherein: In step S2, the target fragment is transformed into DH5α competent cells, which specifically includes the following steps: adding the ligation product to the DH5α competent cells and gently mixing, placing on ice for 30 minutes, heat shocking at 42°C for 90 seconds, and continuing to ice bath for 5 minutes; adding 700 μL of LB culture medium, placing in a 37°C incubator for incubation for 40 minutes, centrifuging and taking 100 μL of bacterial solution to be smeared on an ampicillin-resistant plate, placing in a 37°C incubator for incubation overnight; picking a single colony into an LB culture medium containing ampicillin resistance and placing in a 37°C incubator for incubation for 6 hours; taking 2 μL of bacterial solution as a template, using the designed primers to perform PCR identification to screen out the positive single clone, and using a plasmid mini-extraction kit to extract the positive recombinant plasmid.

5. The method for preparing a colloidal gold test strip according to claim 1, wherein: In step S3, the extraction and purification of the inclusion body protein includes the following steps: centrifuging the cultured bacterial solution at 4°C and 6000rpm to discard the supernatant, washing twice with PBS, resuspending the bacterial solution with PBS according to a volume ratio of PBS to bacterial solution of 1:8, adding PMSF according to a volume ratio of PMSF to bacterial solution of 1:1000, ultrasonicating at 4°C and 8000rpm to obtain a precipitate, adding a lysis solution containing 8M urea to resuspend the precipitate, continuing ultrasonicating at 4°C and 8000rpm to obtain a supernatant, and using Ni 2+ The recombinant protein was purified by affinity chromatography.

6. The method for preparing the colloidal gold test strip according to claim 1, characterized in that: In step S3, Ni 2+ The specific steps for purifying recombinant proteins by affinity chromatography are as follows: (1) Replace storage buffer: Ni 2+ Add 5 column volumes of deionized water to the affinity chromatography column to replace its storage buffer; (2) Balanced Ni 2+ Column: Add 10 column volumes of lysate containing 8M urea to balance Ni 2+ Affinity chromatography columns; (3) Protein over Ni 2+ Column: Add the treated supernatant into the column, control the flow rate at 1 drop / 3s, and collect the flow-through; (4) Washing: Add 10-15 column volumes of a washing solution containing 8 M urea to the column and collect the washing solution; (5) Use 250 mM imidazole eluent containing 8 M urea for elution. Add 10 times column volume of eluent, collect the eluent and divide it into smaller portions. After preparing the protein sample, observe the protein purification by SDS-PAGE electrophoresis. After the protein concentration of the purified protein is determined by the BCA method, divide it into smaller portions and store it at -80°C.

7. The method for preparing the colloidal gold test strip according to claim 1, characterized in that: In step S3, the steps of Western-blot identification of the recombinant protein are as follows: (1) Prepare filter paper and PVDF membrane and soak them in transfer solution; (2) Take out the protein gel, cut off the upper concentrated gel, and place them in the order of one layer of sponge pad, three layers of filter paper protein gel, PVDF membrane, three layers of filter paper, and sponge pad. Suppress the bubbles, put the black electrode at the bottom and the white electrode at the top, close the clamp and place it in the electrophoresis tank. Add transfer solution to the electrophoresis tank and place it in an ice-water mixture. Constant current 240A for 120 min. (3) Take out the transferred membrane, add 15 mL of TBST containing 5% skim milk powder, block for 3 h at room temperature, discard the blocking solution, and wash with TBST three times; (4) Add primary antibody diluted 1:5000 and incubate at 4°C overnight. After recovering the primary antibody, wash with TBST three times, 15 min each time; (5) Add 1:5000 diluted HRP-conjugated secondary antibody, incubate at room temperature for 50 min, and then wash three times with TBST, each time for 15 min; (6) Scanning: Take 500 μL of each of luminescent solution A and solution B, mix them evenly, and then add them to the nitrocellulose membrane in the dark. After they are fully combined, place the membrane in a membrane scanner for scanning and take a photo for preservation.

8. Use of a colloidal gold test strip prepared by the method for preparing a colloidal gold test strip as described in any one of claims 1 to 7 in detecting African swine fever antibodies in serum.

9. The use of the colloidal gold test strip according to claim 8 in detecting African swine fever antibodies in serum, characterized in that: The judgment criteria of the colloidal gold test strip are: if there is no band on the quality control line, the test strip is invalid; if there are bands on the quality control line and the test line, it is positive; if there is a band on the quality control line and no band on the test line, it is negative.

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