A fusion protein GLuc-p30 and its preparation method and application

Abstract

The invention relates to a fusion protein GLuc-p30 and a preparation method and application thereof. With p30 protein of African swine fever as a diagnostic antigen, an African swine fever antibody detection method based on a luciferase immunoprecipitation system is established. In the method, a fusion antigen of luciferase and p30 is prepared by DNA transfection of eukaryotic cells, the antigen is secreted outside the cell, the fusion antigen is combined with African swine fever antibodies in a serum sample, and then captured by magnetic beads coupled with Staphylococcus aureus protein A, and the fusion antigen specifically combined with the magnetic beads is detected by a luciferase test; the virus antigen expressed by the eukaryotic cells used in the invention contains post-translational modifications such as phosphorylation, is closer to the virus protein expressed under infection, and does not need to be purified, thereby simplifying the preparation process of the antigen; compared with the existing indirect ELISA, the detection method of the invention has better detection sensitivity and specificity; the detection method of the invention has the advantages of simplicity, sensitivity, specificity, low price, etc., and can meet the requirements of large-scale detection.

Description

Technical Field

[0001] The present invention relates to a fusion protein GLuc-p30 and a preparation method and application thereof, belonging to the technical fields of biomedicine and immune diagnosis and detection, in particular to the technical field of virus detection. Background Art

[0002] African swine fever (ASF) is an acute, highly contagious pig disease caused by the African swine fever virus (ASFV), often accompanied by clinical symptoms such as high fever, extensive bleeding in internal organs, and respiratory and nervous system dysfunction. It has a short course and a mortality rate of up to 100%. ASF first appeared in Kenya in 1921 and gradually became prevalent in Europe, South America, and the Caucasus region of Russia. ASF is an animal disease that must be reported to the World Organization for Animal Health (OIE) and has been listed as a Class I animal disease in my country. To date, there is no effective vaccine or specific antiviral treatment on the market. Strict biosafety measures and precise detection and elimination are the core strategies for ASF prevention and control in my country.

[0003] ASFV is the only member of the Asfarviridae family and can infect various domestic and wild pigs. It is the only known insect-borne DNA virus, and soft ticks are its important transmission hosts and vectors. The ASFV genome is a single-stranded double-stranded DNA with a length of 170 kb to 190 kb, encoding 150 to 170 viral proteins. Among them, the p30 protein is an early protein encoded by the CP204L gene, which induces a high level of antibody response in the early stage of infection. Based on this, the protein is widely used to detect ASFV-specific antibody responses in serum. ASFV can be divided into 24 genotypes, and the strains prevalent in my country are mainly genotype II strains, although genotype I strains were also detected in my country last year. With the widespread prevalence of genotype II ASFV in my country, a variety of naturally mutated epidemic strains have been isolated clinically, including gene-deficient strains, natural variant strains, and naturally attenuated strains. Compared with traditional epidemic strains, pigs infected with mutant strains often show chronic or latent infection, prolonged incubation period, mild clinical symptoms and decreased mortality rate. These characteristics make the infection of mutant strains more hidden, not conducive to accurate detection and elimination, and bring new challenges to ASF prevention and control.

[0004] At present, there is no effective vaccine on the market for the prevention and control of ASF. The detection of ASF antigens is costly and slow, which is not suitable for large-scale rapid detection. In addition, false negatives may occur in the context of the prevalence of mutant weak strains. The serological detection method for specific antibody response to ASFV can make up for the shortcomings of molecular biological detection. At present, the p30-based ELISA detection method is widely used in the serological diagnosis of ASFV, but they have the disadvantages of low sensitivity and low detection specificity. Therefore, it is urgent to develop a sensitive, specific, efficient, low-cost and easy-to-implement ASFV serological detection method.

[0005] The luciferase immunoprecipitation system (LIPS) is a liquid-phase immunoassay that uses luciferase fusion antigens to capture antigen-specific antibodies. It has been widely used in serological detection of various pathogens, such as parasites, viruses, and some autoimmune diseases. In LIPS-based detection methods, fusion antigens are usually expressed in eukaryotic cells, and they maintain their native conformation. Due to the low natural background of bioluminescence, eukaryotic expressed fusion antigens do not need to be purified and can be directly used in LIPS, which greatly simplifies the process of antigen preparation. Compared with ELISA, LIPS has better detection sensitivity and specificity and can be used for ASFV antibody detection. Summary of the invention

[0006] In view of the problems existing in the above-mentioned prior art, the present invention provides a fusion protein GLuc-p30 and a preparation method and application thereof, and the application method thereof can quickly and accurately detect ASFV-specific antibodies in pig serum.

[0007] The objective of the present invention is achieved through the following technical scheme: a fusion protein GLuc-p30, the amino acid sequence of the fusion protein GLuc-p30 is shown in SEQ ID No.1; SEQ ID No.1: MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLE PMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGDDYKDDDDKGSGDIMDFILNISMKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTA IKTLLSTVKYDIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIYGTP LKEEEKEVVRLMVIKLLKKK.

[0008] Among them, SEQ ID No.1 consists of 392 amino acids, the 199th to 392th amino acids of SEQ ID No.1 are the amino acid sequence of ASFV p30 antigen, the 1st to 185th amino acids of SEQ ID No.1 are the amino acid sequence of the luciferase GLuc, and the 186th to 198th amino acids of SEQ ID No.1 are the amino acid sequence of the connecting peptide.

[0009] The coding gene sequence of the fusion protein GLuc-p30 is shown in SEQ ID No.2, SEQ ID No.2: ATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGAT CTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGATGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCC CACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGA GATTCCTGGGTTCAAGGACTTGGAGCCCATGGAGCAGTTCATCGCACAGGTCGATCTGT GTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAG GTGGACAAGATCAAGGGGGCCGGTGGTGACGATTACAAGGATGACGACGATAAGGGGAGTGGCGATATCATGGACTTCATCCTGAACATCAGCATGAAAATGGAAGTGATTTTCAAGA CAGATCTGAGAAGCTCTTCTCAGGTGGTGTTCCACGCCGGATCTCTGTACAACTGGTTC AGCGTGGAAATCATCAACAGCGGCAGAATCGTGACCACCGCCATCAAGACACTGCTGAGCACCGTGAAGTACGACATCGTGAAAAGCGCTAGAATCTACGCCGGCCAGGGCTACAC CGAGCACCAGGCCCAGGAGGAATGGAATATGATCCTGCACGTGCTGTTCGAGGAAGAGACAGAGAGCTCCGCCAGCTCCGAGAACATCCACGAGAAGAACGACAATGAAACCAACGAGTGCACCAGCTCCTTCGAAACCCTGTTTGAGCAGGAGCCAAGCTCTGAGGTCCCCAAGGACAGCAAGCTGTACATGCTGGCTCAAAAGACAGTGCAGCATATCGAGCAGTACGG CAAGGCCCCTGATTTTAACAAGGTGATCCGGGCCCACAACTTCATCCAGACCATCTATGG CACCCCTCTGAAGGAAGAAGAGAAAGAGGTTGTGCGGCTGATGGTGATTAAGCTGCTCAAGAAAAAGTGA.

[0010] Among them, SEQ ID No. 2 consists of 1179 nucleotides, and the encoded amino acid sequence is the protein of SEQ ID No. 1. The nucleotides at positions 595 to 1179 of SEQ ID No. 2 encode the African swine fever virus p30 antigen in SEQ ID No. 1; the nucleotides at positions 1 to 555 of SEQ ID No. 2 encode the GLuc in SEQ ID No. 1; and the nucleotides at positions 556 to 594 of SEQ ID No. 2 encode the connecting peptide in SEQ ID No. 1.

[0011] A recombinant plasmid pCAGGS-GLuc-p30, wherein the recombinant plasmid has a gene as shown in SEQ ID No. 2, and the vector is a eukaryotic expression vector pCAGGS.

[0012] The preparation method of the recombinant plasmid pCAGGS-GLuc-p30 is as follows: the GLuc gene is fused with the codon-optimized African swine fever virus p30 protein nucleotide sequence and cloned into the eukaryotic expression vector pCAGGS; the codon-optimized African swine fever virus p30 protein nucleotide sequence is used as a template, and the XhoⅠ and EcoRⅤ double restriction sites are used for connection to construct the pCAGGS-GLuc-p30 expression plasmid.

[0013] A method for preparing fusion protein GLuc-p30, comprising the following steps:

[0014] S1: Construction of recombinant plasmid pCAGGS-GLuc-p30;

[0015] S2-1: HEK-293T cells were seeded in a 6-well cell culture plate, and DNA transfection experiments were performed when the cell density was about 80%;

[0016] S2-2: HEK-293T cells were transfected using Lipofectamine 2000 transfection reagent (ThermoFisher Scientific). According to the instructions of the kit, 1 μg of pCAGGS-GLuc-p30 plasmid was transfected into each well.

[0017] S2-3: The cells were cultured at 37° C. and 5% CO 2 for 48 hours, and the transfected supernatant was collected to obtain a solution containing the GLuc-p30 antigen. The protein expression level was determined by measuring the luciferase activity in the solution.

[0018] A non-diagnostic application of the fusion protein GLuc-p30 in the detection of African swine fever antibodies.

[0019] The application method is as follows: a pig serum sample diluted 1:50-1:3200 and a luciferase activity gradient of 10 8 -10 5 The photon-number fusion protein GLuc-p30 was added to phosphate buffer and incubated overnight at 4°C; 5 μL of Protein A Magbeads was added to each reaction, incubated at room temperature for 1 hour, and then the magnetic beads were collected by magnetic separation stand; after washing the magnetic beads with PBST to remove non-specifically bound luciferase, GLuc substrate was added to incubate and luciferase activity was detected.

[0020] A system for detecting ASFV infection, including reagents and instruments for detecting whether ASFV is infected using luciferase immunoprecipitation technology. Specifically, the system for detecting ASFV infection can be composed of GLuc-p30 fusion protein, Protein A MagBeads, luciferase substrate, and other reagents and instruments required for detecting whether ASF is infected using luciferase immunoprecipitation technology. The Protein A MagBeads can be a product of GenScript, with the item number L00273. The luciferase substrate can be Coelenterazine h, a product of Maokang Biological Company, with the item number MX4608. The chemiluminescence detector can be SuPerMax 3000FL of Shanghai Flash Spectrum.

[0021] The system for detecting ASFV also includes a carrier recording the following content: a LIPS experiment is performed with known positive pig serum and negative pig serum to obtain a critical value of S / N. If the S / N of the serum to be tested is greater than or equal to the critical value, the serum to be tested is positive for African swine fever; if the S / N of the serum to be tested is less than the critical value, the serum to be tested is negative for African swine fever.

[0022] In the above-mentioned system for detecting African swine fever virus infection, the eukaryotic cells may be 293T cells.

[0023] In the above-mentioned system for detecting African swine fever virus infection, the culture is carried out for 48 hours.

[0024] Furthermore, the serum dilution factor is 1:50-1:3200, and more preferably the serum dilution factor is 1:100.

[0025] Furthermore, the luciferase activity gradient of the fusion protein GLuc-p30 was 10 8 -10 5 The photon number is preferably 10 7 Number of photons.

[0026] A kit for detecting African swine fever antibodies, comprising the following components: 10 7 Luciferase-active GLuc-p30 protein, PBS buffer, PBST, Protein A Magbeads, GLuc substrate.

[0027] The present invention has the following beneficial effects: 1. The present invention fuses ASFV p30 with GLuc luciferase for expression, and GLuc-p30 is secreted outside the cell by the secretion signal of GLuc, and the cell culture supernatant can be directly used for LIPS detection. The fusion protein GLuc-p30 is expressed by transfected eukaryotic cells, contains post-translational modifications such as phosphorylation of eukaryotic organisms, and is consistent with the protein conformation expressed under viral infection. Compared with serological detection methods such as ELISA using purified antigens, the background of bioluminescence in LIPS detection is low, and there is no need to purify antigens, which reduces the detection cost and improves the detection sensitivity.

[0028] 2. This method is simple to operate and can be mastered in a short time without professional training. The porcine serum sample and fusion antigen can be incubated in an eight-tube with a volume of 200 μL. The amount of serum sample required is small, the operation is convenient, the possibility of cross contamination is reduced, and the cost of consumables is low. After adding magnetic beads for incubation, the magnetic beads can be quickly washed and collected by a magnetic separator and a spray gun.

[0029] 3. The ASFV serological detection method based on different types of ELISA has the disadvantage of high false positives. In comparison, the ASFV serological detection method of the present invention has a specificity and sensitivity of 100%. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Figure 2 is a map of the expression plasmid pCAGGS-GLuc-p30.

[0031] Figure 2 To investigate the specificity and sensitivity of LIPS method for detecting African swine fever antibodies.

[0032] Figure 3 Cross-reactivity of LIPS method for detecting African swine fever antibodies. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0034] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0035] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0036] In the following embodiments, the African swine fever-positive pig sera are PCR-positive, and the African swine fever-negative pig sera are all African swine fever-free pig sera.

[0037] Example 1: Preparation of fusion protein GLuc-p30

[0038] 1. Construction of recombinant plasmid

[0039] The codon-optimized African swine fever virus p30 protein nucleotide sequence was fused with the GLuc gene and cloned into the eukaryotic expression vector pCAGGS; the codon-optimized African swine fever virus p30 protein nucleotide sequence was used as a template and connected with XhoⅠ and EcoRⅤ double restriction sites to construct the pCAGGS-GLuc-p30 expression plasmid.

[0040] 2. DNA transfection of HEK-293T cells to express fusion protein

[0041] 1) 293T cells were seeded in a 6-well cell culture plate, and DNA transfection experiments were performed when the cell density was about 80%;

[0042] 2) HEK-293T cells were transfected using Lipofectamine 2000 transfection reagent (ThermoFisher Scientific). According to the kit instructions, 1 μg of pCAGGS-GLuc-p30 plasmid was transfected into each well;

[0043] 3) The transfected supernatant was collected after culturing at 37° C. and 5% CO 2 for 48 hours to obtain a solution containing the GLuc-p30 antigen. The protein expression level was determined by measuring the luciferase activity in the solution (as shown in Table 1 below).

[0044] Table 1

[0045] serial number Detection volume (uL) Fluorescence intensity value 1 20 303415666.7 2 20 224510000 3 20 185869666.7

[0046] Example 2: Establishment and optimization of LIPS for ASFV antibody detection

[0047] 1. Optimization of the amount of fusion protein GLuc-p30 added for LIPS detection

[0048] 10 8 , 10 7 , 10 6 , 10 5 The fusion antigen with luciferase activity (photon count) was subjected to LIPS with ASFV positive (P) and negative serum (N), and the amount of fusion antigen used when P / N was the largest was the optimal addition amount. 7 The P / N value is the largest when 7 Enzyme activities / reactions are shown in Table 2.

[0049] Table 2 Optimization results of the amount of fusion protein GLuc-p30 used

[0050]

[0051] 2. Optimization of serum dilution for LIPS detection

[0052] One clinically confirmed ASFV-positive serum and one ASFV-negative serum were serially diluted at 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, and 1:3200, respectively, and the dilution was performed using a 10 7 The fusion protein GLuc-p30 with luciferase activity was tested by LIPS to determine the serum dilution multiple. By adding luciferase substrate to the collected magnetic beads, the fluorescence intensity value was read by a chemiluminescence detector, and the final serum dilution multiple was determined by the change in the P / N value. The results showed that the P / N value was the largest at a dilution of 1:100, so the serum dilution was determined to be 1:100. As shown in Table 3.

[0053] Table 3 Determination of serum dilution for LIPS detection

[0054]

[0055] 3. Optimized ASFV antibody detection LIPS test procedure

[0056] (1) Mix 10uL of 1:100 diluted porcine serum with 10uL of 10 7 The luciferase-active fusion protein GLuc-p30 was added to 130uL of PBS to form a 150uL final system, which was incubated overnight at 4°C with shaking in an eight-tube strip with a volume of 200uL;

[0057] (2) After washing Protein A Magbeads three times with PBST, add 5uL of magnetic beads to the system in step (1); wash three times with LIPS Buffer, and incubate at room temperature with shaking for 1 hour;

[0058] (3) After the incubation, the magnetic beads were precipitated using a magnetic separator, the supernatant was removed, 150 mL of PBS was added for washing, and the magnetic beads were precipitated using a magnetic separator again, and the washing solution was removed. After washing for a total of 3 times, the beads were washed once with PBS, and the supernatant was removed to obtain a magnetic bead precipitate;

[0059] (4) Use 20uL of PBS to transfer the magnetic beads from the eight-well tube strip to a 96-well microplate;

[0060] (5) Add 50 μl of luciferase substrate (Coelenterazine h, MX4608) to the magnetic bead precipitate transferred to the 96-well microplate in step (4), place in a chemiluminescence detector, set the integration time to 1 second, the delay time to 1 second, and select photon counting as the test method. Obtain the fluorescence intensity value of the serum sample detected by the LIPS method and record the data.

[0061] Example 3: Sensitivity and specificity of ASFV antibody detection LIPS

[0062] The 37 ASFV-positive pig sera and 81 ASFV-negative pig sera used in this study were verified by commercial African swine fever indirect ELISA. The samples were diluted 1:100 and mixed with 10 7 The fusion protein GLuc-p30 with luciferase activity was incubated and the LIPS detection procedure optimized in Example 2 was used. The S / N values ​​of all samples were calculated, the detection cutoff value was determined by the receiver operating characteristic curve (ROC curve), and the detection sensitivity and specificity of LIPS were calculated, as shown in FIG. Figure 1 .

[0063] When S / N≥30.59, the serum sample was judged to be ASFV positive; when S / N<30.59, the serum sample was judged to be ASFV negative. 37 ASFV positive pig sera were detected from the 37 clinically confirmed ASFV positive pig serum samples, with a detection sensitivity of 100%; 81 ASFV clinically negative pig sera were detected from the 81 clinically confirmed ASFV negative pig serum samples using LIPS, with a detection specificity of 100% ( Figure 2 ).

[0064] Example 4: Validation of cross-reactivity of ASFV antibody detection LIPS

[0065] In order to verify the cross-reactivity of the ASFV antibody detection method of the present invention to other pathogens, we selected 17 specific pathogen-positive pig sera, including 2 porcine epidemic diarrhea virus-positive sera, 3 porcine deltacoronavirus-positive sera, 5 porcine Seneca virus-positive sera, 2 swine fever virus-positive sera, and 5 porcine reproductive and respiratory syndrome virus-positive sera. The test results of LIPS were all negative ( Figure 3 A). At the same time, the 17 specific pathogen-positive pig sera were tested using a commercial African swine fever indirect ELISA antibody detection kit based on p30 antigen (Beijing Yisenbao, catalog number FZE024). The test result of one porcine reproductive and respiratory syndrome virus-positive serum was a false positive ( Figure 3 B), indicating that the detection method of the present invention has better specificity in comparison.

[0066] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents. Sequence Listing <110> Yangzhou University <120> A fusion protein GLuc-p30 and its preparation method and application <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 392 <212> PRT <213> Artificial Sequence <400> 1 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu 1 5 10 15 Ala Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala 20 25 30 Ser Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro 35 40 45 Gly Lys Lys Leu Pro Leu Glu Val Leu Lys Glu Met Glu Ala Asn Ala 50 55 60 Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile 65 70 75 80 Lys Cys Thr Pro Lys Met Lys Lys Phe Ile Pro Gly Arg Cys His Thr 85 90 95 Tyr Glu Gly Asp Lys Glu Ser Ala Gln Gly Gly Ile Gly Glu Ala Ile 100 105 110 Val Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Met Glu 115 120 125 Gln Phe Ile Ala Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys 130 135 140 Leu Lys Gly Leu Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp 145 150 155 160 Leu Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val 165 170 175 Asp Lys Ile Lys Gly Ala Gly Gly Asp Asp Tyr Lys Asp Asp Asp Asp 180 185 190 Lys Gly Ser Gly Asp Ile Met Asp Phe Ile Leu Asn Ile Ser Met Lys 195 200 205 Met Glu Val Ile Phe Lys Thr Asp Leu Arg Ser Ser Ser Gln Val Val 210 215 220 Phe His Ala Gly Ser Leu Tyr Asn Trp Phe Ser Val Glu Ile Ile Asn 225 230 235 240 Ser Gly Arg Ile Val Thr Thr Ala Ile Lys Thr Leu Leu Ser Thr Val 245 250 255 Lys Tyr Asp Ile Val Lys Ser Ala Arg Ile Tyr Ala Gly Gln Gly Tyr 260 265 270 Thr Glu His Gln Ala Gln Glu Glu Trp Asn Met Ile Leu His Val Leu 275 280 285 Phe Glu Glu Glu Thr Glu Ser Ser Ala Ser Ser Glu Asn Ile His Glu 290 295 300 Lys Asn Asp Asn Glu Thr Asn Glu Cys Thr Ser Ser Phe Glu Thr Leu 305 310 315 320 Phe Glu Gln Glu Pro Ser Ser Glu Val Pro Lys Asp Ser Lys Leu Tyr 325 330 335 Met Leu Ala Gln Lys Thr Val Gln His Ile Glu Gln Tyr Gly Lys Ala 340 345 350 Pro Asp Phe Asn Lys Val Ile Arg Ala His Asn Phe Ile Gln Thr Ile 355 360 365 Tyr Gly Thr Pro Leu Lys Glu Glu Glu Lys Glu Val Val Arg Leu Met 370 375 380 Val Ile Lys Leu Leu Lys Lys Lys 385 390 <210> 2 <211> 1179 <212> DNA <213> Artificial Sequence <400> 2 atgggagtca aagttctgtt tgccctgatc tgcatcgctg tggccgaggc caagcccacc 60 gagaacaacg aagacttcaa catcgtggcc gtggccagca acttcgcgac cacggatctc 120 gatgctgacc gcgggaagtt gcccggcaag aagctgccgc tggaggtgct caaagagatg 180 gaagccaatg cccggaaagc tggctgcacc aggggctgtc tgatctgcct gtcccacatc 240 aagtgcacgc ccaagatgaa gaagttcatc ccaggacgct gccacaccta cgaaggcgac 300 aaagagtccg cacagggcgg cataggcgag gcgatcgtcg acattcctga gattcctggg 360 ttcaaggact tggagcccat ggagcagttc atcgcacagg tcgatctgtg tgtggactgc 420 acaactggct gcctcaaagg gcttgccaac gtgcagtgtt ctgacctgct caagaagtgg 480 ctgccgcaac gctgtgcgac ctttgccagc aagatccagg gccaggtgga caagatcaag 540 ggggccggtg gtgacgatta caaggatgac gacgataagg ggagtggcga tatcatggac 600 ttcatcctga acatcagcat gaaatgaa gtgattttca agacagatct gagaagctct 660 tctcaggtgg tgttccacgc cggatctctg tacaactggt tcagcgtgga aatcatcaac 720 agcggcagaa tcgtgaccac cgccatcaag acactgctga gcaccgtgaa gtacgacatc 780 gtgaaaagcg ctagaatcta cgccggccag ggctacaccg agcaccaggc ccaggagagaa 840 tggaatatga tcctgcacgt gctgttcgag gagagacag agagctccgc cagctccgag 900 960 tttgagcagg agccaagctc tgaggtcccc areacagca agctgtacat gctggctcaa 1020 aagacagtgc agcatatcga gcagtacgg aaggcccctg attttaacaa ggtgatccgg 1080 1140 gtgcggctga tggtgattaa gctgctcaag aaaaagtga 1179

Claims

1. A fusion protein GLuc-p30, characterized in that: The amino acid sequence of the fusion protein GLuc-p30 is shown in SEQ ID No. 1; the fusion protein GLuc-p30 is secreted through the strong secretion signal peptide of GLuc, and more than 95% of it is secreted outside the cell.

2. The fusion protein GLuc-p30 according to claim 1, characterized in that: The coding gene sequence of the fusion protein GLuc-p30 is shown in SEQ ID No.

2.

3. A recombinant plasmid pCAGGS-GLuc-p30, characterized in that: The recombinant plasmid contains the gene sequence shown in SEQ ID No. 2, and the vector is a eukaryotic expression vector pCAGGS.

4. The method for preparing the recombinant plasmid pCAGGS-GLuc-p30 according to claim 3, characterized in that: The method is as follows: the codon-optimized African swine fever virus p30 protein nucleotide sequence is fused with the GLuc gene and cloned into the eukaryotic expression vector pCAGGS; the codon-optimized African swine fever virus p30 protein nucleotide sequence is used as a template, and the XhoⅠ and EcoRⅤ double restriction sites are used for connection to construct a pCAGGS-GLuc-p30 expression plasmid.

5. A method for preparing a fusion protein GLuc-p30, characterized in that: The steps of the method are as follows: S1: construct the recombinant plasmid pCAGGS-GLuc-p30 as described in claim 3; S2: Transfecting the recombinant plasmid into eukaryotic cells, culturing the cells to express the encoding gene, and obtaining the fusion protein GLuc-p30.

6. The method for preparing the fusion protein GLuc-p30 according to claim 5, characterized in that: The specific steps of S2 are as follows: S2-1: HEK-293T cells were seeded in a 6-well cell culture plate, and DNA transfection experiments were performed when the cell density reached 80%; S2-2: HEK-293T cells were transfected using Lipofectamine 2000 transfection reagent. According to the instructions of the kit, 1 μg of pCAGGS-GLuc-p30 plasmid was transfected into each well. S2-3: The transfected supernatant was collected after culturing for 48 hours at 37° C. and 5% CO 2 to obtain a solution containing the GLuc-p30 antigen. The protein expression level was determined by measuring the luciferase activity in the solution.

7. Use of the fusion protein GLuc-p30 according to claim 1 or 2 for non-diagnostic purposes in the detection of African swine fever antibodies.

8. The use according to claim 7, characterized in that: The application method is as follows: a pig serum sample diluted 1:50-1:3200 and a luciferase activity gradient of 10 8 -10 5 The fusion protein with the photon number was added to the phosphate buffer and incubated overnight at 4°C; 5 μL of Protein A Magbeads was added to each reaction, incubated at room temperature for 1 hour, and then the magnetic beads were collected by a magnetic separation stand; after washing the magnetic beads with PBST to remove non-specifically bound luciferase, GLuc substrate was added to incubate and luciferase activity was detected.

9. The use according to claim 8, characterized in that: The dilution of the porcine serum sample is 1:100, and the fusion protein contains 10 7 Luciferase-active fusion protein.

10. A kit for detecting African swine fever antibodies, characterized in that: It includes the following components: 10 7 The fusion protein GLuc-p30 according to claim 1 or 2, PBS buffer, PBST, Protein A Magbeads, and GLuc substrate with luciferase activity.

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