Multi-epitope fusion protein and preparation method thereof and use in detecting African swine fever virus antibodies

Abstract

The present application relates to the technical field related to the detection of African swine fever virus antibodies, and specifically to a multi-epitope fusion protein and a preparation method thereof and its use in the detection of African swine fever virus antibodies. The multi-epitope fusion protein is obtained by fusion of the amino acid sequence shown in SEQ ID NO.15, the amino acid sequence shown in SEQ ID NO.16 and the amino acid sequence shown in SEQ ID NO.17. The present application uses the multi-epitope fusion protein as an antigen, which can specifically bind and detect African swine fever virus antibodies, improve the detection sensitivity, and shorten the detection time.

Description

Technical Field

[0001] The present application relates to the technical field related to the detection of African swine fever virus antibodies, and specifically to a multi-epitope fusion protein and a preparation method thereof and use thereof in the detection of African swine fever virus antibodies. Background Art

[0002] African swine fever (ASF) is an acute and severe infectious disease of pigs caused by African swine fever virus (ASFV). The mortality rate of highly pathogenic strains of African swine fever can be as high as 90% to 100%. The World Organization for Animal Health (OIE) has listed it as an animal disease that must be reported, and my country has listed it as a Class I animal infectious disease that requires key prevention.

[0003] Due to the large size of the ASFV genome, the numerous encoded proteins, and the complex viral structure, there is still no safe and effective vaccine for epidemic prevention and control. Therefore, prevention and diagnosis remain the main means of epidemic prevention and control of African swine fever. Summary of the invention

[0004] In view of this, the present application provides a multi-epitope fusion protein and a preparation method thereof and use thereof in detecting antibodies to African swine fever virus. The present application uses a multi-epitope fusion protein as an antigen, which can specifically bind and detect antibodies to African swine fever virus, thereby improving the detection sensitivity and shortening the detection time.

[0005] In the first aspect of the present application, the present application provides a multi-epitope fusion protein, which is obtained by fusing the amino acid sequence shown in SEQ ID NO.15, the amino acid sequence shown in SEQ ID NO.16 and the amino acid sequence shown in SEQ ID NO.17.

[0006] In some optional embodiments, the multi-epitope fusion protein has the general formula shown in formula (I):

[0007]

[0008] AAS1, AAS2 and AAS3 each independently represent the amino acid sequence shown in SEQ ID NO.15, the amino acid sequence shown in SEQ ID NO.16 or the amino acid sequence shown in SEQ ID NO.17, and AAS1, AAS2 and AAS3 represent different amino acid sequences;

[0009] L represents the amino acid sequence GGGGS;

[0010] Each n is independently selected from 1, 2, 3 or 4.

[0011] In some optional embodiments, AAS1 represents the amino acid sequence shown in SEQ ID NO.15, AAS2 represents the amino acid sequence shown in SEQ ID NO.14, and AAS3 represents the amino acid sequence shown in SEQ ID NO.17;

[0012] Or AAS1 represents the amino acid sequence shown in SEQ ID NO.16, AAS2 represents the amino acid sequence shown in SEQ ID NO.15, and AAS3 represents the amino acid sequence shown in SEQ ID NO.17;

[0013] Or AAS1 represents the amino acid sequence shown in SEQ ID NO.17, AAS2 represents the amino acid sequence shown in SEQ ID NO.15, and AAS3 represents the amino acid sequence shown in SEQ ID NO.14.

[0014] In some optional embodiments, it is characterized in that n is selected from 2.

[0015] In some optional embodiments, the amino acid sequence of the multi-epitope fusion protein is shown in any one of SEQ ID NOs. 19-22.

[0016] In the second aspect of the present application, the present application provides a gene encoding the multi-epitope fusion protein as described in the first aspect of the present application, wherein the gene encoding is obtained by recombination of the nucleotide sequence shown in SEQ ID NO.2, the nucleotide sequence shown in SEQ ID NO.4, and the nucleotide sequence shown in SEQ ID NO.5;

[0017] Wherein, the nucleotide sequence shown in SEQ ID NO.2 encodes the amino acid sequence shown in SEQ ID NO.15;

[0018] The nucleotide sequence shown in SEQ ID NO.4 encodes the amino acid sequence shown in SEQ ID NO.16;

[0019] The nucleotide sequence shown in SEQ ID NO.5 encodes the amino acid sequence shown in SEQ ID NO.17.

[0020] In some optional embodiments, the encoding gene has the general formula shown in formula (II):

[0021]

[0022] Wherein, NAS1, NAS2 and NAS3 each independently represent the nucleotide sequence shown in SEQ ID NO.2, the nucleotide sequence shown in SEQ ID NO.4 or the nucleotide sequence shown in SEQ ID NO.5, and NAS1, NAS2 and NAS3 represent different nucleotide sequences;

[0023] Each L' independently represents the nucleotide sequence GGTGGTGGTGGTTCT, and the nucleotide sequence GGTGGTGGTGGTTCT encodes the amino acid sequence GGGGS;

[0024] Each n' is independently selected from 1, 2, 3 or 4;

[0025] E1 and E2 are each independently selected from the nucleotide sequence of the restriction site.

[0026] In some optional embodiments, NAS1 represents the nucleotide sequence shown in SEQ ID NO.2, NAS2 represents the nucleotide sequence shown in SEQ ID NO.4, and NAS3 represents the nucleotide sequence shown in SEQ ID NO.5;

[0027] Alternatively, NAS1 represents the nucleotide sequence shown in SEQ ID NO.4, NAS2 represents the nucleotide sequence shown in SEQ ID NO.2, and NAS3 represents the nucleotide sequence shown in SEQ ID NO.5;

[0028] Alternatively, NAS1 represents the nucleotide sequence shown in SEQ ID NO.5, NAS2 represents the nucleotide sequence shown in SEQ ID NO.2, and NAS3 represents the nucleotide sequence shown in SEQ ID NO.4.

[0029] In some optional embodiments, the nucleotide sequence of the encoding gene is shown in any one of SEQ ID NOs. 9 to 12.

[0030] In the third aspect of the present application, the present application provides a recombinant plasmid comprising the encoding gene described in the second aspect of the present application.

[0031] In a fourth aspect of the present application, the present application provides a method for preparing the multi-epitope fusion protein according to the first aspect of the present application, the preparation method comprising the following steps:

[0032] S201, introducing the recombinant plasmid described in the third aspect of the present application into competent cells of the expression bacteria;

[0033] S202, inducing competent cells to express proteins to obtain induced products;

[0034] S203. The induced product is purified, renatured and dialyzed to obtain the multi-epitope fusion protein described in the first aspect of the present application.

[0035] In the fifth aspect of the present application, the present application provides a use of the multi-epitope fusion protein described in the first aspect of the present application in detecting African swine fever virus antibodies.

[0036] In a sixth aspect of the present application, the present application provides a kit for detecting African swine fever virus antibodies based on an enzyme-linked immunosorbent assay, comprising a coating antigen and a competing antigen;

[0037] The coating antigen is the multi-epitope fusion protein described in the first aspect of the present application;

[0038] The competing antigen is the multi-epitope fusion protein described in the first aspect of the present application labeled with HRP.

[0039] In a seventh aspect of the present application, the present application provides a kit for detecting African swine fever virus antibodies based on magnetic microparticle chemiluminescence, characterized in that it comprises a coating antigen and a competing antigen;

[0040] The coating antigen is a biotin-labeled multi-epitope fusion protein as described in the first aspect of the present application;

[0041] The competing antigen is the multi-epitope fusion protein described in the first aspect of the present application labeled with an acridinium ester.

[0042] This application has the following beneficial effects:

[0043] First, the present application uses a multi-epitope fusion protein as an antigen, which can specifically bind to and detect African swine fever virus antibodies, thereby improving detection sensitivity and shortening detection time.

[0044] Second, compared with the fusion protein formed by the fusion of two amino acid sequences, SEQ ID NO.15 and SEQ ID NO.17, and the fusion protein formed by the fusion of two amino acid sequences, SEQ ID NO.16 and SEQ ID NO.17, the fusion of three amino acid sequences, SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO.17, can significantly improve the detection sensitivity of African swine fever virus antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1It is the Native-PAGE electrophoresis identification result of the multi-epitope fusion protein SEQ ID NO.19 expressed by the coding gene SEQ ID NO.9, the multi-epitope fusion protein SEQ ID NO.20 expressed by the coding gene SEQ ID NO.10 and the multi-epitope fusion protein SEQ ID NO.21 expressed by the coding gene SEQ ID NO.11 in Example 3 of the present application.

[0046] Figure 2 It is the Native-PAGE electrophoresis identification result of the multi-epitope fusion protein SEQ ID NO.23 expressed by the coding gene SEQ ID NO.13 and the multi-epitope fusion protein SEQ ID NO.24 expressed by the coding gene SEQ ID NO.14 in Example 3 of the present application. DETAILED DESCRIPTION

[0047] The present application discloses a multi-epitope fusion protein of African swine fever virus and its preparation method and use. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The methods and applications of this application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of this application to implement and apply the technology of this application.

[0048] Multi-epitope fusion protein:

[0049] In the first aspect of the present application, the present application provides a multi-epitope fusion protein, which is obtained by fusing the amino acid sequence shown in SEQ ID NO.15, the amino acid sequence shown in SEQ ID NO.16 and the amino acid sequence shown in SEQ ID NO.17.

[0050] In some optional embodiments, the multi-epitope fusion protein has the general formula shown in formula (I):

[0051]

[0052] AAS1, AAS2 and AAS3 each independently represent the amino acid sequence shown in SEQ ID NO.15, the amino acid sequence shown in SEQ ID NO.16 and the amino acid sequence shown in SEQ ID NO.17, and AAS1, AAS2 and AAS3 represent different amino acid sequences;

[0053] L represents the amino acid sequence GGGGS;

[0054] Each n is independently selected from 1, 2, 3 or 4.

[0055] In some specific embodiments, AAS1 represents the amino acid sequence shown in SEQ ID NO.15, AAS2 represents the amino acid sequence shown in SEQ ID NO.16, and AAS3 represents the amino acid sequence shown in SEQ ID NO.17.

[0056] In some specific embodiments, AAS1 represents the amino acid sequence shown in SEQ ID NO.16, AAS2 represents the amino acid sequence shown in SEQ ID NO.15, and AAS3 represents the amino acid sequence shown in SEQ ID NO.17.

[0057] In some specific embodiments, AAS1 represents the amino acid sequence shown in SEQ ID NO.17, AAS2 represents the amino acid sequence shown in SEQ ID NO.15, and AAS3 represents the amino acid sequence shown in SEQ ID NO.16.

[0058] In some specific embodiments, each n is selected from 2.

[0059] In some specific embodiments, Represents the amino acid sequence shown in SEQ ID NO.18.

[0060] In some optional embodiments, the amino acid sequence of the multi-epitope fusion protein is shown in any one of SEQ ID NOs. 19-22.

[0061] In some optional embodiments, the amino acid sequence of the multi-epitope fusion protein is shown in any one of SEQ ID NOs. 19-21.

[0062] In some specific embodiments, the amino acid sequence of the multi-epitope fusion protein is shown in SEQ ID NO.19.

[0063] In some specific embodiments, the amino acid sequence of the multi-epitope fusion protein is shown in SEQ ID NO.20.

[0064] In some specific embodiments, the amino acid sequence of the multi-epitope fusion protein is shown in SEQ ID NO.21.

[0065] Coding genes:

[0066] In a second aspect of the present application, the present application provides a gene encoding the multi-epitope fusion protein as described in the first aspect of the present application, wherein the gene encoding the protein is obtained by recombination of the nucleotide sequence shown in SEQ ID NO.2, the nucleotide sequence shown in SEQ ID NO.4, and the nucleotide sequence shown in SEQ ID NO.5;

[0067] Wherein, the nucleotide sequence shown in SEQ ID NO.2 encodes the amino acid sequence shown in SEQ ID NO.15;

[0068] The nucleotide sequence shown in SEQ ID NO.4 encodes the amino acid sequence shown in SEQ ID NO.16;

[0069] The nucleotide sequence shown in SEQ ID NO.5 encodes the amino acid sequence shown in SEQ ID NO.17.

[0070] In some optional embodiments, the encoding gene has the general formula shown in formula (II):

[0071]

[0072] Wherein, NAS1, NAS2 and NAS3 each independently represent the nucleotide sequence shown in SEQ ID NO.2, the nucleotide sequence shown in SEQ ID NO.4 and the nucleotide sequence shown in SEQ ID NO.5, and NAS1, NAS2 and NAS3 represent different nucleotide sequences;

[0073] Each L' independently represents the nucleotide sequence GGTGGTGGTGGTTCT, and the nucleotide sequence GGTGGTGGTGGTTCT encodes the amino acid sequence GGGGS;

[0074] Each n' is independently selected from 1, 2, 3 or 4;

[0075] E1 and E2 are each independently selected from the nucleotide sequence of the restriction site.

[0076] In some specific embodiments, NAS1 represents the nucleotide sequence shown in SEQ ID NO.2, NAS2 represents the nucleotide sequence shown in SEQ ID NO.4, and NAS3 represents the nucleotide sequence shown in SEQ ID NO.5.

[0077] In some specific embodiments, NAS1 represents the nucleotide sequence shown in SEQ ID NO.4, NAS2 represents the nucleotide sequence shown in SEQ ID NO.2, and NAS3 represents the nucleotide sequence shown in SEQ ID NO.5.

[0078] In some specific embodiments, NAS1 represents the nucleotide sequence shown in SEQ ID NO.5, NAS2 represents the nucleotide sequence shown in SEQ ID NO.2, and NAS3 represents the nucleotide sequence shown in SEQ ID NO.4.

[0079] In some optional embodiments, NAS1 represents the nucleotide sequence shown in SEQ ID NO.5, NAS2 represents the nucleotide sequence shown in SEQ ID NO.4, and NAS3 represents the nucleotide sequence shown in SEQ ID NO.2.

[0080] In some specific embodiments, each n' is selected from 2.

[0081] In some specific embodiments, Represents the nucleotide sequence shown in SEQ ID NO.6.

[0082] In some optional embodiments, E1 and E2 are each independently selected from a NotI cleavage site or an Xho cleavage site.

[0083] The NotⅠ restriction site comprises the nucleotide sequence shown in SEQ ID NO. 7. The Xho restriction site comprises the nucleotide sequence shown in SEQ ID NO. 8.

[0084] In some specific embodiments, E1 is selected from the NotI cleavage site.

[0085] In some specific embodiments, E2 is selected from the Xho cleavage site.

[0086] In some optional embodiments, the nucleotide sequence of the encoding gene is shown in any one of SEQ ID NOs. 9 to 12.

[0087] In some optional embodiments, the nucleotide sequence of the encoding gene is shown in any one of SEQ ID NOs. 9 to 11.

[0088] In some specific embodiments, the nucleotide sequence of the encoding gene is shown as SEQ ID NO.9.

[0089] In some specific embodiments, the nucleotide sequence of the encoding gene is shown as SEQ ID NO.10.

[0090] In some specific embodiments, the nucleotide sequence of the encoding gene is shown as SEQ ID NO.11.

[0091] Recombinant plasmid:

[0092] In the third aspect of the present application, the present application provides a recombinant plasmid comprising the encoding gene described in the second aspect of the present application.

[0093] In some optional embodiments, the plasmid vector of the recombinant plasmid is selected from the group consisting of a pPICZαA plasmid vector and a PUC57 plasmid vector.

[0094] In some specific embodiments, the plasmid vector of the recombinant plasmid is selected from pPICZαA plasmid vector.

[0095] Preparation method:

[0096] In a fourth aspect of the present application, the present application provides a method for preparing the multi-epitope fusion protein according to the first aspect of the present application, the preparation method comprising the following steps:

[0097] S201, introducing the recombinant plasmid described in the third aspect of the present application into competent cells of the expression bacteria;

[0098] S202, inducing competent cells to express proteins to obtain induced products;

[0099] S203. The induced product is purified, renatured and dialyzed to obtain the multi-epitope fusion protein described in the first aspect of the present application.

[0100] In some optional embodiments, methods for introducing the recombinant plasmid into the expression bacteria include but are not limited to heat shock method, electroporation method, microinjection method, etc.

[0101] In some optional embodiments, the expression bacteria are selected from Escherichia coli, such as Escherichia coli DH5α or Escherichia coli BL21 (DE3) and the like.

[0102] use:

[0103] In the fifth aspect of the present application, the present application provides a use of the multi-epitope fusion protein described in the first aspect of the present application in detecting African swine fever virus antibodies.

[0104] Enzyme-linked immunosorbent assay (ELISA) kit for detecting antibodies to African swine fever virus:

[0105] In a sixth aspect of the present application, the present application provides a kit for detecting African swine fever virus antibodies based on an enzyme-linked immunosorbent assay, comprising a coating antigen and a competitive antigen;

[0106] The coating antigen is the multi-epitope fusion protein described in the first aspect of the present application;

[0107] The competing antigen is the multi-epitope fusion protein described in the first aspect of the present application labeled with HRP.

[0108] In some specific embodiments, the coated antigen is bound to the surface of the ELISA plate to form a coated ELISA plate.

[0109] In some optional embodiments, the method for preparing the coated ELISA plate comprises the following steps:

[0110] S301, antigen coating: dilute the multi-epitope fusion protein into coating solution using CBS buffer (pH=9.6), add the coating solution to the ELISA plate for coating, and wash;

[0111] S302, blocking: adding blocking solution to the ELISA plate obtained in step S301 for blocking, washing, and obtaining the coated ELISA plate.

[0112] In some optional embodiments, the final concentration of the coating solution is 1-4 μg / mL, such as 1.5 μg / mL, 2 μg / mL, 2.5 μg / mL, 3 μg / mL or 3.5 μg / mL.

[0113] In some optional embodiments, the amount of the coating solution is 50-150 μL / well, such as 60 μL / well, 70 μL / well, 80 μL / well, 90 μL / well, 100 μL / well, 110 μL / well, 120 μL / well, 130 μL / well or 140 μL / well, etc.

[0114] In some optional embodiments, the coating conditions are: temperature of 1-10°C (such as 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C or 9°C, etc.), time of 12-18h (such as 13h, 14h, 15h, 16h or 17h, etc.).

[0115] In some optional embodiments, in step S301, the washing process is: adding PBST buffer (0.05% Tween-20, pH=7.4) to the ELISA plate for washing, 300 L / well, 5 times, 3 min / time.

[0116] In some specific embodiments, the blocking solution is selected from BSA blocking solution.

[0117] In some optional embodiments, the BSA blocking solution is prepared by diluting bovine serum albumin (BSA) with PBS buffer (pH=7.4). Further, the concentration of the BSA blocking solution is 1-3% (mass volume ratio w / v), such as 1.5%, 2% or 2.5%.

[0118] In some optional embodiments, the amount of the blocking solution is 150-250 μL / well, such as 160 μL / well, 170 μL / well, 180 μL / well, 190 μL / well, 200 μL / well, 210 μL / well, 220 μL / well, 230 μL / well, or 240 μL / well, etc.

[0119] In some optional embodiments, the sealing conditions are: temperature of 30-40°C (such as 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, or 39°C, etc.), time of 1-3h (such as 1.5h, 2h or 2.5h, etc.).

[0120] In some optional embodiments, in step S302, the washing process is: adding PBST buffer (0.05% Tween-20, pH=7.4) to the ELISA plate for washing, 300 L / well, 5 times, 3 min / time.

[0121] In some optional embodiments, the kit for detecting African swine fever virus antibodies based on enzyme-linked immunosorbent assay further comprises a negative control and a positive control.

[0122] In some optional embodiments, the kit for detecting African swine fever virus antibodies based on enzyme-linked immunosorbent assay further comprises a color developer. Specifically, the color developer is selected from a two-component TMB color developer.

[0123] In some optional embodiments, the working process of the kit for detecting African swine fever virus antibodies based on enzyme-linked immunosorbent assay specifically includes the following steps:

[0124] S401. Preparation of the sample to be tested: diluting the serum sample to be tested by 5 to 20 times (e.g., 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times or 19 times, etc.) with PBST buffer (0.05 volume % Tween-20, pH=7.4);

[0125] S402, incubation: add negative control, positive control and sample to be tested to the coated ELISA plate, 100 μL / well, and mix well; then add HRP-labeled multi-epitope fusion protein solution, 50 μL / well, and mix well; seal the ELISA plate with a sealing film, and incubate at 37°C for 10 min;

[0126] The HRP-labeled multi-epitope fusion protein solution is prepared by diluting the HRP-labeled multi-epitope fusion protein with PBST buffer (0.05 volume % Tween-20, pH=7.4), with a dilution ratio of 1:10000 (mass to volume ratio w / v);

[0127] S403, washing: discard the solution in the wells of the ELISA plate, add PBST buffer (0.05% Tween-20, pH=7.4) for washing, 300 μL / well, wash 5 times, 3 min / time;

[0128] S404, color development and termination: add two-component TMB color development solution to the ELISA plate, 100 μL / well, mix well, and color develop at 37°C in the dark for 10 min; then add 2M concentrated sulfuric acid, 50 μL / well, and gently shake the ELISA plate until the color develops evenly;

[0129] S405, reading: put the ELISA plate into the ELISA instrument, and perform dual wavelength measurement and read the absorbance OD value under the conditions of a test wavelength of 450nm and a reference wavelength of 630nm.

[0130] In some optional embodiments, the result of the kit for detecting African swine fever virus antibodies by enzyme-linked immunosorbent assay is determined as follows:

[0131] The OD value of the negative control product is recorded as ODN, the OD value of the positive control product is recorded as ODP, and the OD value of the sample to be tested is recorded as ODS; Cut Off value (threshold) = ODN + 0.15;

[0132] Test establishment conditions: ODN value ≤ 0.1 and ODP value ≥ 0.5, the test result is valid, otherwise retest;

[0133] Result determination: If the ODS value of the sample to be tested is ≤ the Cut Off value, the result is judged as negative; if the ODS value of the sample to be tested is greater than the Cut Off value, the result is judged as positive.

[0134] Kit for detecting African swine fever virus antibodies based on magnetic particle chemiluminescence:

[0135] In a seventh aspect of the present application, the present application provides a kit for detecting African swine fever virus antibodies based on a magnetic microparticle chemiluminescence method, which comprises a coating antigen and a competitive antigen;

[0136] The coating antigen is a biotin-labeled multi-epitope fusion protein as described in the first aspect of the present application;

[0137] The competing antigen is the multi-epitope fusion protein described in the first aspect of the present application labeled with an acridinium ester.

[0138] In order to make the purpose, technical solutions and advantages of the present application clearer, the implementation scheme of the present application will be further described in detail below in conjunction with examples.

[0139] Example 1 (Preparation of encoding gene):

[0140] The preparation method of the coding gene comprises the following steps:

[0141] Screening and codon optimization of antigen epitope sequences of S101 and B646L genes encoding capsid protein p72

[0142] The full-length sequences of the B646L gene of 45 strains of classical swine fever virus retrieved from the DNA sequence database Genbank were analyzed, and the gene sequence of ASFVWuhan2019-1 strain B646L (104788-106728 bp; accession number MN393476.1) was screened. The antigenic epitope prediction analysis was performed using the biological online software ABCPred and DNAStar-Protean software, and two nucleotide sequences in the nucleotide sequence of the dominant linear antigenic epitope were selected. The two nucleotide sequences were optimized according to the preferred codons of Pichia pastoris.

[0143] Among them, the nucleotide sequence of a dominant linear antigen epitope is shown in SEQ ID NO.1, and its nucleotide sequence after codon optimization is shown in SEQ ID NO.2.

[0144] The dominant linear antigen epitope nucleotide sequence SEQ ID NO.1 is as follows:

[0145] AGCATCCTGCCAGGATGAATGACATGCACCCAATATATGATGGCCCACC ATATCATGGAAAAAGTCTCCGTACTGGGGAATACCAAAGGTAAGCTTGTTTT CCCAAGGTGGGGGTACCCGTATGCGGGCGTACTTTATT (from 5' end to 3' end direction).

[0146] The linear antigen epitope nucleotide sequence SEQ ID NO.2 after codon optimization is as follows:

[0147] AGCATCCTGCCAGGATGAATGACAAGCACCCAAAATATGATGGCCCAC CATATCATGGAAAAAATCTCCGTACTGGGGAATACCAAAGGTCAACTTGTT TCCCAAGGTGGGGGTACCCGTATGCGGGCGTACCTTATT (from 5' end to 3' end direction).

[0148] Among them, the nucleotide sequence of another dominant linear antigen epitope is shown in SEQ ID NO.3, and its nucleotide sequence after codon optimization is shown in SEQ ID NO.4.

[0149] The dominant linear antigen epitope nucleotide sequence SEQ ID NO.3 is as follows:

[0150] GCCTCCGTAGTGGAAGGGTATGTAAGAGCTGCAGAACTTTGATGGAAA TTTATCGATAAGATTGATACCATGAGCAGTTACGGAAATGTTTTTAATAATAG GTAATGTGATCGGATACGTAACGGGGCTAATATCAGATATAGATGA (from 5' to 3' direction).

[0151] The codon-optimized linear antigen epitope nucleotide sequence SEQ ID NO.4 is as follows:

[0152] GCCTCCGTAGTGGAAGGGAATGTAAGAGCTGCAGAACTTTGATGGAA ACTTATCAATCAAATTAATACCATGAGCAGTTACGGAAATGTTCTTAATAATA GGCAATGTAATCGGATACGTAACGGGGCTAATATCAGAAATAGATGA (from 5' to 3' direction).

[0153] Screening and codon optimization of antigen epitope sequences of S102 and CP204L genes encoding p30 protein

[0154] The full-length sequences of the CP204L gene of 45 strains of classical swine fever virus retrieved from the DNA sequence database Genbank were analyzed, and the CP204L gene of ASFVWuhan2019-1 strain (125994-126554 bp; accession number MN393476.1) was screened and optimized according to the codons preferred by Pichia pastoris. The nucleotide sequence after codon optimization is shown in SEQ ID NO.5.

[0155] The codon-optimized nucleotide sequence SEQ ID NO.5 is as follows:

[0156] ATGAAAATGGAAGTCATCTTCAAAACGGATTTAAGATCATCTTCACAAGTTGTGTTTCATGCGGGTAGCCTGTATAATTGGTTTTCTGTTGAAATTATCAATAGCGGTAGAATTGTTACGACCGCTATAAAAACATTGCTTAGTACTGTTAAGTATGATATTGTGAAATCTGCTCGTAT ATATGCAGGGCAAGGGTATACTGAACATCAGGCTCAAGAAGAATGGAATATGATTCTGCATGTGCTGTTTGAAGAAGAAACGGAATCCTCAGCATCTTCGGAAAACATTCATGAAAAAAATGATAATGAAACCAATGAATGCACATCCTCCTTTGAAACGTTGTTTGAACAAGAACCCTC ATCGGAGGTACCTAAAGATCCAAGCTGTATATGCTTGCACAAAAGACTGTGCAACATATTGAACAATATGGAAAGGCACCTGATTTTAACAAGGTTATTAGAGCACATAATTTTATTCAAACCATTTATGGAACCCCTCTAAAGGAAGAAGAAAAAGAGGTGGTAAGACTCATGGTCATTAAACTTTTAAAAAAAAAATAA (from 5' to 3' direction).

[0157] S103, Synthesis of coding genes

[0158] In the process of recombining nucleotide sequences, the nucleotide sequence SEQ ID NO.6 encoding the flexible connecting peptide is inserted into two adjacent nucleotide sequences, and the nucleotide sequence SEQ ID NO.7 of the NotⅠ restriction site and the nucleotide sequence SEQ ID NO.8 of the Xho restriction site are respectively inserted into both ends of the recombined nucleotide sequence.

[0159] The nucleotide sequence SEQ ID NO.6 encoding the flexible connecting peptide is as follows: GGTGGTGGTGGTTCTGGTGGTGGTGGTTCT (from 5' end to 3' end).

[0160] The nucleotide sequence of the NotⅠ restriction site, SEQ ID NO.7, is as follows: AAATATGCGGCCGCTATAAA (from 5' to 3').

[0161] The nucleotide sequence of the Xho restriction site, SEQ ID NO. 8, is as follows: CCGCTCGAGCGG (from the 5' end to the 3' end).

[0162] The nucleotide sequence SEQ ID NO.2, the nucleotide sequence SEQ ID NO.4 and the nucleotide sequence SEQ ID NO.5 after codon optimization in step S101 are recombined, and there are four recombination orders:

[0163] The first recombinant sequence is: nucleotide sequence SEQ ID NO.2, nucleotide sequence SEQ ID NO.4 and nucleotide sequence SEQ ID NO.5, and forms a coding gene of the nucleotide sequence shown in SEQ ID NO.9;

[0164] The second recombinant sequence is: nucleotide sequence SEQ ID NO.4, nucleotide sequence SEQ ID NO.2 and nucleotide sequence SEQ ID NO.5, and forms a coding gene of the nucleotide sequence shown in SEQ ID NO.10;

[0165] The third recombinant sequence is: nucleotide sequence SEQ ID NO.5, nucleotide sequence SEQ ID NO.2 and nucleotide sequence SEQ ID NO.4, and forms a coding gene of the nucleotide sequence shown in SEQ ID NO.11;

[0166] The fourth recombination sequence is: nucleotide sequence SEQ ID NO.5, nucleotide sequence SEQ ID NO.4 and nucleotide sequence SEQ ID NO.2, and a coding gene of the nucleotide sequence shown as SEQ ID NO.12 is formed.

[0167] The nucleotide sequence SEQ ID NO.2 and the nucleotide sequence SEQ ID NO.5 after codon optimization in step S101 are recombined in the order of: nucleotide sequence SEQ ID NO.2 and nucleotide sequence SEQ ID NO.5, and a coding gene of the nucleotide sequence shown in SEQ ID NO.13 is formed.

[0168] The nucleotide sequence SEQ ID NO.4 and the nucleotide sequence SEQ ID NO.5 after codon optimization in step S101 are recombined in the order of: nucleotide sequence SEQ ID NO.4 and nucleotide sequence SEQ ID NO.5, and a coding gene of the nucleotide sequence shown in SEQ ID NO.14 is formed.

[0169] The nucleotide sequence of the coding gene is shown in SEQ ID NO.9:

[0170] AAATATGCGGCCGCTATAAA AGCATCCTGCCAGGATGAATGACAAGCACCCAAAATATGATGGCCCACCATATCATGGAAAAAATCTCCGTACTGGGGAATACCAAAGGTCAACTTGTTTCCCAAGGTGGGGGTACCCGTATGCGGGCGTACCTTATT GGTGGTGGTGGTTCTGGTGGTGGTGGTTCT GCCTCCGTAGTGGAAGGGAATGTAAGAGCTGCAGAACTTTGATGGAAACTTATCAATCAAATTAATACCATGAGCAGTTACGGAAATGTTCTTAATAATAGGCAATGTAATCGGATACGTAACGGGGCTAATATCAGAAATAGATGA GGTGGTGGTGGTTCTGGTGGTGGTGGTTCT ATGAAAATGGAAGTCATCTTCAAAACGGATTTAAGATCATCTTCACAAGTTGTGTTTCATGCGGGTAGCCTGTATAATTGGTTTTCTGTTGAAATTATCAATAGCGGTAGAATTGTTACGACCGCTATAAAAACATTGCTTAGTACTGTTAAGTATGATATTGTGAAATCTGCTCGTATATATGCAGGGCAAGGGTATACTGAACATCAGGCTCAAGAAGAATGGAATATGATTCTGCATGTGCTGTTTGAAGAAGAAACGGAATCCTCAGCATCTTCGGAAAACATTCATGAAAAAAATGATAATGAAACCAATGAATGCACATCCTCCTTTGAAACGTTGTTTGAACAAGAACCCTCATCGGAGGTACCTAAAGATCCAAGCTGTATATGCTTGCACAAAAGACTGTGCAACATATTGAACAATATGGAAAGGCACCTGATTTTAACAAGGTTATTAGAGCACATAATTTTATTCAAACCATTTATGGAACCCCTCTAAAGGAAGAAGAAAAAGAGGTGGTAAGACTCATGGTCATTAAACTTTTAAAAAAAAAATAA CCGCTCGAGCGG (In the 5'-to-3' direction).

[0171] The nucleotide sequence of the coding gene, SEQ ID NO.10, is as follows:

[0172] AAATATGCGGCCGCTATAAAGCCTCCGTAGTGGAAGGGAATGTAAGAGCTGCAGAACTTTGATGGAAACTTATCAATCAAATTAATACCATGAGCAGTTACGGAAATGTTCTTAATAATAGGCAATGTAATCGGATACGTAACGGGGCTAATATCAGAAATAGATGA GGTGGTGGTGGTTCTGGTGGTGGTGGTTCT AGC ATCCTGCCAGGATGAATGACAAGCACCCAAAATATGATGGCCCACCATATCATGGAAAAAATCTCCGTACTGGGGAATACCAAAGGTCAACTTGTTTCCCAAGGTGGGGGTACCCGTATGCGGGCGTACCTTATT GGTGGTGGTGGTTCTGGTGGTGGTGGTTCT ATGAAAATGGAAGTCATCTTCAAAACGGATTTAAGATCATCTTCACAAGTTGTGTTTCATGCGGGTAGCCTGTATAATTGGTTTTCTGTTGAAATTATCAATAGCGGTAGAATTGTTACGACCGCTATAAAAACATTGCTTAGTACTGTTAAGTATGATATTGTGAAATCTGCTCGTATATATGCAGGGCAAGGGTATACTGAACATCAGGCTCAAGAAGAATGGAATATGATTCTGCATGTGCTGTTTGAAGAAGAAACGGAATCCTCAGCATCTTCGGAAAACATTCATGAAAAAAATGATAATGAAACCAATGAATGCACATCCTCCTTTGAAACGTTGTTTGAACAAGAACCCTCATCGGAGGTACCTAAAGATCCAAGCTGTATATGCTTGCACAAAAGACTGTGCAACATATTGAACAATATGGAAAGGCACCTGATTTTAACAAGGTTATTAGAGCACATAATTTTATTCAAACCATTTATGGAACCCCTCTAAAGGAAGAAGAAAAAGAGGTGGTAAGACTCATGGTCATTAAACTTTTAAAAAAAAAATAA CCGCTCGAGCGG (In the 5'-to-3' direction).

[0173] The nucleotide sequence of the coding gene, SEQ ID NO.11, is as follows:

[0174] AAATATGCGGCCGCTATAAAATGAAAATGGAAGTCATCTTCAAAACGGATTTAAGATCATCTTCACAAGTTGTGTTTCATGCGGGTAGCCTGTATAATTGGTTTTCTGTTGAAATTATCAATAGCGGTAGAATTGTTACGACCGCTATAAAAACATTGCTTAGTACTGTTAAGTATGATATTGTGAAATCTGCTCGTATATATGCAGGGCAAGGGTATACTGAACATCAGGCTCAAGAAGAATGGAATATGATTCTGCATGTGCTGTTTGAAGAAGAAACGGAATCCTCAGCATCTTCGGAAAACATTCATGAAAAAAATGATAATGAAACCAATGAATGCACATCCTCCTTTGAAACGTTGTTTGAACAAGAACCCTCATCGGAGGTACCTAAAGATCCAAGCTGTATATGCTTGCACAAAAGACTGTGCAACATATTGAACAATATGGAAAGGCACCTGATTTTAACAAGGTTATTAGAGCACATAATTTTATTCAAACCATTTATGGAACCCCTCTAAAGGAAGAAGAAAAAGAGGTGGTAAGACTCATGGTCATTAAACTTTTAAAAAAAAAATAA GGTGGTGGTGGTTCTGGTGG TGGTGGTTCT AGCATCCTGCCAGGATGAATGACAAGCACCCAAAATATGATGGCCCACCATATCATGGAAAAAATCTCCGTACTGGGGAATACCAAAGGTCAACTTGTTTCC CAAGGTGGGGGTACCCGTATGCGGGCGTACCTTATT GGT GGTGGTGGTTCTGGTGGTGGTGGTTCT GCCTCCGTAGTGGAAGGGAATGTAAGAGCTGCAGAACTTTGATGGAAACTTATCAATCAAATTAATACCATGAGCAGTTACGGAAATGTTCTTAATAATAGGCAATGTAATCGGATACGTAACGGGGCTAATATCAGAAATAGATGA CCGCTCGAGCGG (From the 5'-end to the 3'-end direction).

[0175] The nucleotide sequence of the coding gene SEQ ID NO.12 is shown below:

[0176] AAATATGCGGCCGCTATAAA ATGAAAATGGAAGTCATCTTCAAAACGGATTTAAGATCATCTTCACAAGTTGTGTTTCATGCGGGTAGCCTGTATAATTGGTTTTCTGTTGAAATTATCAATAGCGGTAGAATTGTTACGACCGCTATAAAAACATTGCTTAGTACTGTTAAGTATGATATTGTGAAATCTGCTCGTATATATGCAGGGCAAGGGTATACTGAACATCAGGCTCAAGAAGAATGGAATATGATTCTGCATGTGCTGTTTGAAGAAGAAACGGAATCCTCAGCATCTTCGGAAAACATTCATGAAAAAAATGATAATGAAACCAATGAATGCACATCCTCCTTTGAAACGTTGTTTGAACAAGAACCCTCATCGGAGGTACCTAAAGATCCAAGCTGTATATGCTTGCACAAAAGACTGTGCAACATATTGAACAATATGGAAAGGCACCTGATTTTAACAAGGTTATTAGAGCACATAATTTTATTCAAACCATTTATGGAACCCCTCTAAAGGAAGAAGAAAAAGAGGTGGTAAGACTCATGGTCATTAAACTTTTAAAAAAAAAATAA GGTGGTGGTGGTTCTGGTGG TGGTGGTTCT GCCTCCGTAGTGGAAGGGAATGTAAGAGCTGCAGAACTTTGATGGAAACTTATCAATCAAATTAATACCATGAGCAGTTACGGAAATGTTCTTAATAATAGGCAATGTAATCGGATACGTAACGGGGCTAATATCAGAAATAGATGA GGTGGTGGTGGTTCTGGTGGTGGTGGTTCT AGCATCCTGCCAGGATGAATGACAAGCACCCAAAATATGATGGCCCACCATATCATGGAAAAAATCTCCGTACTGGGGAATACCAAAGGTCAACTTGTTTCCCAAGGTGGGGGTACCCGTATGCGGGCGTACCTTATT CCGCTCGAGCGG (From 5'-end to 3'-end direction).

[0177] The nucleotide sequence of the coding gene SEQ ID NO.13 is as follows:

[0178] AAATATGCGGCCGCTATAAA AGCATCCTGCCAGGATGAATGACAAGCACCCAAAATATGATGGCCCACCATATCATGGAAAAAATCTCCGTACTGGGGAATACCAAAGGTCAACTTGTTTCCCAAGGTGGGGGTACCCGTATGCGGGCGTACCTTATT GGTGGTGGTGGTTCTGGTGGTGGTGGTTCT ATGAAAATGG AAGTCATCTTCAAAACGGATTTAAGATCATCTTCACAAGTTGTGTTTCATGCGGGTAGCCTGTATAATTGGTTTTCTGTTGAAATTATCAATAGCGGTAGAATTGTTACGACCGCTATAAAAACATTGCTTAGTACTGTTAAGTATGATATTGTGAAATCTGCTCGTATATATGCAGGGCAAGGGTATACTGAACATCAGGCTCAAGAAGAATGGAATATGATTCTGCATGTGCTGTTTGAAGAAGAAACGGAATCCTCAGCATCTTCGGAAAACATTCATGAAAAAAATGATAATGAAACCAATGAATGCACATCCTCCTTTGAAACGTTGTTTGAACAAGAACCCTCATCGGAGGTACCTAAAGATCCAAGCTGTATATGCTTGCACAAAAGACTGTGCAACATATTGAACAATATGGAAAGGCACCTGATTTTAACAAGGTTATTAGAGCACATAATTTTATTCAAACCATTTATGGAACCCCTCTAAAGGAAGAAGAAAAAGAGGTGGTAAGACTCATGGTCATTAAACTTTTAAAAAAAAAATAA CCG CTCGAGCGG (From the 5'-end to the 3'-end direction).

[0179] The nucleotide sequence of the coding gene SEQ ID NO.14 is shown below:

[0180] AAATATGCGGCCGCTATAAAGCCTCCGTAGTGGAAGGGAATGTAAGAGCTGCAGAACTTTGATGGAAACTTATCAATCAAATTAATACCATGAGCAGTTACGGAAATGTTCTTAATAATAGGCAATGTAATCGGATACGTAACGGGGCTAATATCAGAAATAGATGA GGTGGTGGTGGTTCTGGTGGTGGTGGTTCT ATGAAAATGGAAGTCATCTTCAAAACGGATTTAAGATCATCTTCACAAGTTGTGTTTCATGCGGGTAGCCTGTATAATTGGTTTTCTGTTGAAATTATCAATAGCGGTAGAATTGTTACGACCGCTATAAAAACATTGCT TAGTACTGTTAAGTATGATATTGTGAAATCTGCTCGTATATATGCAGGGCAAGGGTATACTGAACATCAGGCTCAAGAAGAATGGAATATGATTCTGCATGTGCTGTTTGAAGAAGAAACGGAATCCTCAGCATCTTCGG AAAACATTCATGAAAAAAATGATAATGAAACCAATGAATGCACATCCTCCTTTGAAACGTTGTTTGAACAAGAACCCTCATCGGAGGTACCTAAAGATCCAAGCTGTATATGCTTGCACAAAAGACTGTGCAACATATTG AACAATATGGAAAGGCACCTGATTTTAACAAGGTTATTAGAGCACATAATTTTATTCAAACCATTTATGGAACCCCTCTAAAGGAAGAAGAAAAAGAGGTGGTAAGACTCATGGTCATTAAACTTTTAAAAAAAAAATAA CCGCTCGAGCGG (from 5' to 3' direction).

[0181] Unless otherwise specified, the coding gene of the present application was commissioned to be synthesized by General Biotechnology (Anhui) Co., Ltd. However, in the actual synthesis, the nucleotide sequence of the coding gene SEQ ID NO.12 was not successfully synthesized.

[0182] Among them, the amino acid sequence expected to be expressed by the linear antigen epitope nucleotide sequence SEQ ID NO.2 is shown in SEQ ID NO.15: NKVRPHTGTPTLGNKLTFGIPQYGDFFHDMVGHHILGACHSSWQDA (from N-terminus to C-terminus).

[0183] The amino acid sequence expected to be expressed by the linear antigen epitope nucleotide sequence SEQ ID NO.4 is shown in SEQ ID NO.16: SSISDISPVTYPITLPIIKNISVTAHGINLIDKFPSKFCSSYIPFHYGG (from N-terminus to C-terminus).

[0184] The amino acid sequence expected to be expressed by the nucleotide sequence SEQ ID NO.5 is shown in SEQ ID NO.17: MKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDI VKSARIYAGQGYTEHQAQEEWNMILHVLFEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVI (from N-terminus to C-terminus).

[0185] The amino acid sequence of the flexible connecting peptide expected to be expressed by the nucleotide sequence SEQ ID NO.6 is shown in SEQ ID NO.18: GGGGSGGGGS (from N-terminus to C-terminus).

[0186] The nucleotide sequence of the coding gene is SEQ ID NO.9 and the amino acid sequence of the multi-epitope fusion protein expected to be expressed is shown in SEQ ID NO.19:

[0187] NKVRPHTGTPTLGNKLTFGIPQYGDFFHDMVGHHILGACHSSWQDA GGGGSGGGGS SSISDISPVTYPITLPIIKNISVTAHGINLIDKFPSKFCSSYIPFHYGG GGGGSGGGGS MKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVI (from N end to C end).

[0188] The nucleotide sequence of the coding gene is SEQ ID NO.10, and the amino acid sequence of the multi-epitope fusion protein expected to be expressed is shown in SEQ ID NO.20:

[0189] SSISDISPVTYPITLPIIKNISVTAHGINLIDKFPSKFCSSYIPFHYGG GGGGSGGGGSNKVRPHTGTPTLGNKLTFGIPQYGDFFHDMVGHHILGACHSSWQDA GGGGSGGGGS MKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEEETESSASSENI HEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVI (from N end to C end).

[0190] The nucleotide sequence of the coding gene is SEQ ID NO.11, and the amino acid sequence of the multi-epitope fusion protein expected to be expressed is shown in SEQ ID NO.21:

[0191] MKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVI GGGGSGGGGS SSISDISPVTYPITLPIIKNISVTAHGINLIDKFPSKFCSSYIPFHYGG GGGGSGGG GS NKVRPHTGTPTLGNKLTFGIPQYGDFFHDMVGHHILGACHSSWQDA (from N-terminus to C-terminus).

[0192] The amino acid sequence of the multi-epitope fusion protein expected to be expressed by the coding gene SEQ ID NO.12 is shown in SEQ ID NO.22:

[0193] MKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVI GGGGSGGGGS NKVRPHTGTPTLGNKLTFGIPQYGDFFHDMVGHHILGACHSSWQDA GGGGSGGGGSSSISDISPVTYPITLPIIKNISVTAHGINLIDKFPSKFCSSYIPFHYGG (from N-terminus to C-terminus).

[0194] The amino acid sequence of the multi-epitope fusion protein encoded by the coding gene SEQ ID NO.13 is shown in SEQ ID NO.23:

[0195] NKVRPHTGTPTLGNKLTFGIPQYGDFFHDMVGHHILGACHSSWQDA GGGGSGGGGS MKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVI (from N end to C end).

[0196] The amino acid sequence of the multi-epitope fusion protein encoded by the coding gene SEQ ID NO.14 is shown in SEQ ID NO.24:

[0197] SSISDISPVTYPITLPIIKNISVTAHGINLIDKFPSKFCSSYIPFHYGG GGGGSGGGGS MKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEEETESSASSENIHEKNDN ETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVI (from N end to C end).

[0198] Example 2 (Construction of recombinant plasmid): In this example, the method for constructing the recombinant plasmid is as follows: the coding genes SEQ ID NOs. 9 to 9 and 11 to 12 in Example 1 are respectively inserted into the pPICZαA plasmid vector by double restriction enzyme digestion vector construction technology to obtain five recombinant plasmids.

[0199] Example 3 (multi-epitope fusion protein):

[0200] In this embodiment, the method for preparing the multi-epitope fusion protein comprises the following steps:

[0201] S201, respectively introducing the five recombinant expression plasmids in Example 2 into Escherichia coli DH5α competent cells;

[0202] S202, inducing Escherichia coli to express and obtain an induced product;

[0203] S203. The induced products were purified, renatured and dialyzed to obtain five multi-epitope fusion proteins.

[0204] Native-PAGE electrophoresis was used to detect the multi-epitope fusion protein SEQ ID NO.19 expressed by the coding gene SEQ ID NO.9, the multi-epitope fusion protein SEQ ID NO.20 expressed by the coding gene SEQ ID NO.10, and the multi-epitope fusion protein SEQ ID NO.21 expressed by the coding gene SEQ ID NO.11. Figure 1 shown.

[0205] Figure 1 In the figure, from left to right are the 34 kDa protein band appearing in the multi-epitope fusion protein SEQ IDNO.19 expressed by the coding gene SEQ ID NO.9, the 34 kDa protein band appearing in the multi-epitope fusion protein SEQ IDNO.20 expressed by the coding gene SEQ ID NO.10, and the 34 kDa protein band appearing in the multi-epitope fusion protein SEQ IDNO.21 expressed by the coding gene SEQ ID NO.11; thus confirming that the product fragment size is in line with expectations; and the expression amount of the multi-epitope fusion protein expressed by the coding gene SEQ IDNO.9 is better than the expression amounts of the multi-epitope fusion proteins expressed by the coding genes SEQ ID NO.10 and SEQ IDNO.11.

[0206] The multi-epitope fusion protein SEQ ID NO.23 expressed by the coding gene SEQ ID NO.13 and the multi-epitope fusion protein SEQ ID NO.24 expressed by the coding gene SEQ ID NO.14 were determined by Native-PAGE electrophoresis. Figure 2 shown.

[0207] Figure 2 In the assay, the coding gene SEQ ID NO.13 expressed the multi-epitope fusion protein SEQ ID NO.23 and showed an obvious protein band of 23 kDa; the coding gene SEQ ID NO.14 expressed the multi-epitope fusion protein SEQ ID NO.24 and showed an obvious protein band of 23.3 kDa; thus, it was confirmed that the product fragment size was in line with expectations.

[0208] Example 4 (Kit for detecting African swine fever virus antibodies based on enzyme-linked immunosorbent assay):

[0209] In this embodiment, the main components of the kit for detecting African swine fever virus antibodies based on enzyme-linked immunosorbent assay are: coated ELISA plate, horseradish peroxidase-labeled multi-epitope fusion protein (HRP-labeled antigen), positive quality control, negative quality control, diluent, washing solution, luminescent substrate solution A, luminescent substrate solution B, and stop solution.

[0210] The method for preparing the coated ELISA plate specifically comprises the following steps:

[0211] S301. Antigen coating: Use CBS buffer (pH=9.6) to dilute the multi-epitope fusion protein into a coating solution with a final concentration of 2 μg / mL, add the coating solution to the ELISA plate at 100 μL / well, coat at 4°C for 12-18 hours, and discard the solution in the wells of the ELISA plate; add PBST buffer (0.05% Tween-20, pH=7.4) for washing, 300 L / well, wash 5 times, 3 min / time;

[0212] S302, blocking: add BSA blocking solution to the ELISA plate obtained in step S301, 200 μL / well, block at 37°C for 2 hours, discard the solution in the wells of the ELISA plate; add PBST buffer (0.05% Tween-20, pH = 7.4) for washing, 300 L / well, wash 5 times, 3 min / time; obtain a coated ELISA plate.

[0213] The BSA blocking solution is prepared by diluting bovine serum albumin (BSA) with PBS buffer (pH=7.4), and the concentration of the BSA blocking solution is 2% (mass volume ratio w / v).

[0214] Luminescent substrate solution A: prepared from 3,3',5,5'-tetramethylbenzidine (200 mg) and anhydrous ethanol (100 ml).

[0215] Luminescent substrate solution B: Add water for injection to 900 ml of Na2HPO4 (14.6 g) and citric acid (9.33 g), adjust the pH to 5.0-5.4, and add more water for injection to make up to 1000 ml.

[0216] In this embodiment, the working principle of the kit for detecting African swine fever virus antibodies based on enzyme-linked immunosorbent assay is: the double antigen sandwich method is used to determine whether the sample contains African swine fever virus (ASFV) antibodies. The ASFV antigen (i.e., the multi-epitope fusion protein of the present application) is coated on the ELISA plate to prepare a stationary phase; the sample is added to the microwells of the coated ELISA plate, and then combined with the HRP-labeled ASFV antigen to form an antigen-antibody-enzyme-labeled antigen complex, and after washing, a two-component 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric solution is added; TMB is converted into blue under the catalysis of HRP, and finally converted into yellow under the action of acid. The depth of color is positively correlated with the content of ASFV antibodies in the sample; the absorbance (OD value) is measured at a wavelength of 450nm using an ELISA instrument, and the OD value is used to calculate whether the sample contains ASFV antibodies.

[0217] The working process of the kit for detecting African swine fever virus antibodies based on enzyme-linked immunosorbent assay specifically includes the following steps:

[0218] S401. Preparation of the sample to be tested: dilute the serum sample to be tested 10 times with PBST buffer (0.05% by volume Tween-20, pH=7.4); specifically: 20 μL of the serum sample to be tested, 180 μL of PBST buffer;

[0219] S402, incubation: add negative control, positive control and sample to be tested to the coated ELISA plate, 100 μL / well, and mix well; then add HRP-labeled multi-epitope fusion protein solution, 50 μL / well, and mix well; seal the ELISA plate with a sealing film, and incubate at 37°C for 10 min;

[0220] The HRP-labeled multi-epitope fusion protein solution is prepared by diluting the HRP-labeled multi-epitope fusion protein with PBST buffer (0.05 volume % Tween-20, pH=7.4), with a dilution ratio of 1:10000 (mass to volume ratio w / v);

[0221] S403, washing: discard the solution in the wells of the ELISA plate, add PBST buffer (0.05% Tween-20, pH=7.4) for washing, 300 μL / well, wash 5 times, 3 min / time;

[0222] S404, color development and termination: add two-component TMB color development solution (prepared with luminescent substrate solution A and luminescent substrate solution B in a volume ratio of 1:1) to the ELISA plate, 100 μL / well, mix well, and color develop at 37°C in the dark for 10 min; then add 2M concentrated sulfuric acid, 50 μL / well, and gently shake the ELISA plate until the color is uniform;

[0223] S405, reading: put the ELISA plate into the ELISA instrument, and perform dual wavelength measurement and read the absorbance OD value under the conditions of a test wavelength of 450nm and a reference wavelength of 630nm.

[0224] Result judgment:

[0225] The OD value of the negative control product is recorded as ODN, the OD value of the positive control product is recorded as ODP, and the OD value of the sample to be tested is recorded as ODS; Cut Off value (threshold) = ODN + 0.15;

[0226] Test establishment conditions: ODN value ≤ 0.1 and ODP value ≥ 0.5, the test result is valid, otherwise retest;

[0227] Result determination: If the ODS value of the sample to be tested is ≤ the Cut Off value, the result is judged as negative; if the ODS value of the sample to be tested is greater than the Cut Off value, the result is judged as positive.

[0228] Example 5 (Kit for detecting African swine fever virus antibodies based on magnetic microparticle chemiluminescence method):

[0229] In this embodiment, the working principle of the kit for detecting African swine fever virus antibodies based on magnetic particle chemiluminescence is as follows: the sandwich immunological principle is combined with magnetic particle separation technology, the sample to be tested is mixed and incubated with biotin-labeled multi-epitope fusion protein, acridinium ester-labeled multi-epitope fusion protein and streptavidin magnetic beads to form an immune complex, and after the unbound impurities are removed by washing with solid phase carrier magnetic particles, a luminescent substrate is added to promote luminescence, and the relative luminescence intensity (RLU) is measured. Within a certain range, RLU is proportional to the African swine fever virus antibody titer, and RLU=corresponding African swine fever virus antibody titer value is output through the built-in standard curve of the instrument.

[0230] In this embodiment, the main components of the kit for detecting African swine fever virus antibodies based on the magnetic particle chemiluminescence method are: magnetic bead working solution, antigen working solution, acridinium ester marker working solution, luminescent substrate solution A, luminescent substrate solution B, calibrator, positive quality control product, and negative quality control product.

[0231] Magnetic bead working solution: prepared from 225ul of streptavidin affinity magnetic beads and 4275μl of PBS-BSA solution (pH=7.4).

[0232] Antigen working solution: prepared from 5.5ug of biotin-labeled multi-epitope fusion protein and 10994.5μl of PBS-BSA solution (pH=7.4).

[0233] Acridinium ester label working solution: prepared from 1.1 ug of acridinium ester labeled multi-epitope fusion protein and 10998.9 μl of PBS-BSA solution (pH=7.4).

[0234] Luminescent substrate solution A: prepared from 3,3',5,5'-tetramethylbenzidine (200 mg) and anhydrous ethanol (100 ml).

[0235] Luminescent substrate solution B: Add water for injection to 900 ml of Na2HPO4 (14.6 g) and citric acid (9.33 g), adjust the pH to 5.0-5.4, and add more water for injection to make up to 1000 ml.

[0236] The working process of the kit for detecting African swine fever virus antibodies based on magnetic particle chemiluminescence method specifically includes the following steps:

[0237] Sample (50 μL) and antigen working solution (50 μl) + magnetic bead working solution (20 μl) + acridinium ester marker working solution (50 μl) were reacted at 37°C for 5-30 min, washed with 0.1 mol / L PBS buffer solution, enzyme marker solution (100 μl) was added, reacted at 37°C for 10-20 min, washed with 0.1 mol / L PBS buffer solution, luminescent substrate solution A (100 μl) and luminescent substrate solution B (100 μl) were added, reacted at 37°C for 5 min, and the luminescence value was detected.

[0238] The establishment of the standard curve: First, add sterile injection water to the African swine fever positive serum (lyophilized) and mix thoroughly; then, use PBST buffer (0.05 volume % Tween-20, pH = 7.4) as a diluent to perform a 2-fold gradient dilution to obtain 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 128-fold, 256-fold, 512-fold, 1024-fold, 2048-fold, 4096-fold, 8192-fold, 16384-fold diluted positive serum samples, and select 512-fold, 1024-fold, 2048-fold, 4096-fold, 8192-fold, 16384-fold diluted positive serum samples as calibrators, named as calibrators 1, 2, 3, 4, 5, 6 respectively. When testing, add them in order, the instrument automatically recognizes the test, and generates a standard curve.

[0239] Example 6 (Verification of Sensitivity):

[0240] Preparation of sensitive samples: Select African swine fever positive serum (lyophilized) (CVCC deposit number: Z310) as the standard, with a specification of 1 ml / tube; the serum is positive for African swine fever virus antibodies by indirect immunofluorescence (IFA), and the IFA titer is 1:4000. First, add sterile injection water to the African swine fever positive serum (lyophilized) and mix thoroughly; then, use PBST buffer (0.05 volume% Tween-20, pH = 7.4) as a diluent to perform a 2-fold gradient dilution to obtain 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 128-fold, 256-fold, 512-fold, 1024-fold, 2048-fold, 4096-fold, 8192-fold, and 16384-fold diluted positive serum samples.

[0241] Based on the above sensitivity samples, the sensitivity of the enzyme-linked immunosorbent assay and the magnetic particle chemiluminescence method of the present application for detecting African swine fever virus antibodies was verified.

[0242] The sensitivity of the enzyme-linked immunosorbent assay for detecting antibodies to African swine fever virus was verified. The results are shown in Table 1.

[0243] Table 1 The results of the enzyme-linked immunosorbent assay of this application:

[0244]

[0245] As can be seen from Table 1, the maximum detection limit of the enzyme-linked immunosorbent assay of the present application can reach 4096 times.

[0246] By comparing the multi-epitope fusion proteins SEQ ID NO.19 to SEQ ID NO.21 and SEQ ID NO.23 to 24, it can be seen that compared with the multi-epitope fusion proteins SEQ ID NO.23 and SEQ ID NO.24, the multi-epitope fusion proteins SEQ ID NO.19 to SEQ ID NO.21 can significantly improve the detection sensitivity of antibodies to African swine fever virus, especially the multi-epitope fusion protein SEQ ID NO.19 can significantly improve the detection sensitivity of antibodies to African swine fever virus. It can be seen that compared with the fusion protein of the two amino acid sequences SEQ ID NO.15 and SEQ ID NO.17, and the fusion protein of the two amino acid sequences SEQ ID NO.16 and SEQ ID NO.17, the fusion of the three amino acid sequences SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO.17 can significantly improve the detection sensitivity of antibodies to African swine fever virus.

[0247] By comparing the multi-epitope fusion proteins SEQ ID NO.19 to SEQ ID NO.21, it can be seen that the multi-epitope fusion proteins SEQ ID NO.19 to SEQ ID NO.21 are all obtained by fusing the three amino acid sequences of SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO.17, among which the multi-epitope fusion protein SEQ ID NO.19 has the most significant improvement in the detection sensitivity of African swine fever virus antibodies. It can be seen that the connection order of the three amino acid sequences of SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO.17 affects the detection sensitivity of the multi-epitope fusion protein to African swine fever virus antibodies.

[0248] The sensitivity of the magnetic microparticle chemiluminescence method for detecting antibodies against African swine fever virus was verified. The results of the assay are shown in Table 2.

[0249] Table 2 The measurement results of the magnetic particle chemiluminescence method of this application:

[0250]

[0251] It can be seen from Table 2 that the detection limit of the magnetic particle chemiluminescence method of the present application can reach up to 8192 times.

[0252] By comparing the multi-epitope fusion proteins SEQ ID NO.19 to SEQ ID NO.21 and SEQ ID NO.23 to 24, it can be seen that compared with the multi-epitope fusion proteins SEQ ID NO.23 and SEQ ID NO.24, the multi-epitope fusion proteins SEQ ID NO.19 to SEQ ID NO.21 can significantly improve the detection sensitivity of African swine fever virus antibodies, especially the multi-epitope fusion protein SEQ ID NO.19 can significantly improve the detection sensitivity of African swine fever virus antibodies. It can be seen that compared with the fusion protein of the two amino acid sequences SEQ ID NO.15 and SEQ ID NO.17, and the fusion protein of the two amino acid sequences SEQ ID NO.16 and SEQ ID NO.17, the fusion of the three amino acid sequences SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO.17 can significantly improve the detection sensitivity of African swine fever virus antibodies.

[0253] By comparing the multi-epitope fusion proteins SEQ ID NO.19 to SEQ ID NO.21, it can be seen that the connection order of the three amino acid sequences of SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO.17 affects the detection sensitivity of the multi-epitope fusion protein to African swine fever virus antibodies; among them, the improvement of the detection sensitivity of African swine fever virus antibodies is from best to worst: "SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO.17" is better than "SEQ ID NO.16, SEQ ID NO.15 and SEQID NO.17" is better than "SEQ ID NO.17, SEQ ID NO.16 and SEQ ID NO.15".

[0254] Based on the multi-epitope fusion protein SEQ ID NO.19 of the present application, the sensitivity of the enzyme-linked immunosorbent assay of the present application, the magnetic microparticle chemiluminescence method of the present application, a domestic Elisa kit, and a domestic colloidal gold kit for detecting antibodies against African swine fever virus was compared. The results of the assay are shown in Table 3.

[0255] Table 3 Comparison of the sensitivity of the enzyme-linked immunosorbent assay of this application, the magnetic particle chemiluminescence method of this application, a domestic Elisa kit, and a domestic colloidal gold kit:

[0256]

[0257] It can be seen from Table 3 that the sensitivity of the enzyme-linked immunosorbent assay and the magnetic particle chemiluminescence assay of the present application are much higher than the sensitivity of a domestic Elisa kit and a domestic colloidal gold kit.

[0258] The above is a detailed introduction to the multi-epitope fusion protein provided by the present application, its preparation method, and its use in detecting African swine fever virus antibodies. This article uses specific examples to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method of the present application and its core idea. It should be pointed out that for those skilled in the art, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. A multi-epitope fusion protein, characterized in that: The multi-epitope fusion protein is selected from the general formula shown in formula (I): Wherein, L represents the amino acid sequence GGGGS; Each n is independently selected from 1, 2, 3 or 4; AAS1 represents the amino acid sequence shown in SEQ ID NO.15, AAS2 represents the amino acid sequence shown in SEQ ID NO.16, and AAS3 represents the amino acid sequence shown in SEQ ID NO.17; Or AAS1 represents the amino acid sequence shown in SEQ ID NO.16, AAS2 represents the amino acid sequence shown in SEQ ID NO.15, and AAS3 represents the amino acid sequence shown in SEQ ID NO.17; Alternatively, AAS1 represents the amino acid sequence shown in SEQ ID NO.17, AAS2 represents the amino acid sequence shown in SEQ ID NO.15, and AAS3 represents the amino acid sequence shown in SEQ ID NO.

16.

2. The multi-epitope fusion protein according to claim 1, characterized in that: n is selected from 2.

3. The multi-epitope fusion protein according to claim 1, characterized in that: The amino acid sequence of the multi-epitope fusion protein is shown in any one of SEQ ID NOs. 19 to 21.

4. The gene encoding the multi-epitope fusion protein according to any one of claims 1 to 3, characterized in that: The coding gene is selected from the general formula shown in formula (II): wherein each L' independently represents the nucleotide sequence GGTGGTGGTGGTTCT, and the nucleotide sequence GGTGGTGGTGGTTCT encodes the amino acid sequence GGGGS; Each n' is independently selected from 1, 2, 3 or 4; E1 and E2 are each independently selected from the nucleotide sequence of the restriction site; NAS1 represents the nucleotide sequence shown in SEQ ID NO.2, NAS2 represents the nucleotide sequence shown in SEQ ID NO.4, and NAS3 represents the nucleotide sequence shown in SEQ ID NO.5; or, NAS1 represents the nucleotide sequence shown in SEQ ID NO.4, NAS2 represents the nucleotide sequence shown in SEQ ID NO.2, and NAS3 represents the nucleotide sequence shown in SEQ ID NO.5; or, NAS1 represents the nucleotide sequence shown in SEQ ID NO.5, NAS2 represents the nucleotide sequence shown in SEQ ID NO.2, and NAS3 represents the nucleotide sequence shown in SEQ ID NO.4; Wherein, the nucleotide sequence shown in SEQ ID NO.2 encodes the amino acid sequence shown in SEQ ID NO.15; The nucleotide sequence shown in SEQ ID NO.4 encodes the amino acid sequence shown in SEQ ID NO.16; The nucleotide sequence shown in SEQ ID NO.5 encodes the amino acid sequence shown in SEQ ID NO.

17.

5. The coding gene according to claim 4, characterized in that The nucleotide sequence of the coding gene is shown in any one of SEQ ID NOs. 9 to 11.

6. A recombinant plasmid comprising the coding gene according to any one of claims 4 to 5.

7. A method for preparing a multi-epitope fusion protein, characterized in that: The preparation method comprises the following steps: S201, introducing the recombinant plasmid according to claim 6 into competent cells of expression bacteria; S202, inducing competent cells to express proteins to obtain induced products; S203. The induced product is purified, renatured and dialyzed to obtain the multi-epitope fusion protein according to any one of claims 1 to 3.

8. Use of the multi-epitope fusion protein according to any one of claims 1 to 3 in the preparation of a kit for detecting African swine fever virus antibodies.

9. A kit for detecting African swine fever virus antibodies based on enzyme-linked immunosorbent assay, characterized in that: It contains coating antigen and competing antigen; The coating antigen is the multi-epitope fusion protein according to any one of claims 1 to 3; The competing antigen is the multi-epitope fusion protein according to any one of claims 1 to 3 labeled with HRP.

10. A kit for detecting African swine fever virus antibodies based on magnetic particle chemiluminescence, characterized in that: It contains coating antigen and competing antigen; The coating antigen is a biotin-labeled multi-epitope fusion protein according to any one of claims 1 to 3; The competing antigen is the multi-epitope fusion protein according to any one of claims 1 to 3 labeled with an acridinium ester.

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