Polypeptide of ASFV pE248R protein B cell epitope, monoclonal antibody, kit and application

Abstract

The invention relates to the technical field of molecular immunology, in particular to a polypeptide of ASFVpE248R protein B cell epitope, a monoclonal antibody, a kit and application. The amino acid sequence of the polypeptide is as shown in SEQ ID No.1, and the nucleotide sequence of the encoding gene of the polypeptide is as shown in SEQ ID No.2. According to the invention, through screening and truncation identification, the screened monoclonal antibody can identify 87QEVALTQWMDAG98, and the polypeptide reacts with ASFV standard positive pig serum. The polypeptide is a B cell epitope region of pE248R, the epitope region is displayed in a pE248R protein three-dimensional structure, and it is found that the polypeptide is located on the surface of the protein structure. The B cell epitope of the ASFVpE248R protein is reported for the first time, and a material basis is provided for further research on an ASFV immune escape mechanism and a diagnostic reagent.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular immunology, and in particular to a polypeptide, a monoclonal antibody, a kit and applications of an ASFVpE248R protein B cell epitope. Background Art

[0002] African swine fever (ASF) is a highly contagious viral disease caused by the African swine fever virus (ASFV). Its main characteristics are clinical symptoms such as high fever and visceral bleeding. The mortality rate of clinically infected pigs can be as high as 100%. It was first introduced to China in 2018, causing huge economic losses to China's pig farming industry. ASFV is a large double-stranded DNA virus with an envelope. It is the only member of the African swine fever virus family and the only DNA virus transmitted by arthropods. ASFV is an icosahedral multilayer structure containing more than 60 proteins, and the diameter of the virus particle is about 200-300nm. The inner core of ASFV consists of a nucleoid, including the viral genome and some nucleoproteins, and its exterior is successively wrapped by the core shell, inner membrane, capsid and outer membrane. The genome of ASFV is a genome with a length of 170-194kb. When ASFV enters the cell, the viral genome is delivered to the cytoplasm, and then the transcription of early viral genes is initiated. The ASFV genome contains more than 150 open reading frames, encoding a variety of proteins, including more than 60 structural proteins and more than 100 non-structural proteins.

[0003] The pE248R protein is a structural component of late ASFV particles and one of the substrates for other virally encoded proteins involved in redox reactions, the formation of which requires disulfide bonds. The amino acid sequence of pE248R contains a myristoylation site and a hydrophobic transmembrane region near its carboxyl terminus. The pE248R protein has similarities in amino acid sequence to the L1 protein of vaccinia virus (VACV). The L1 protein is a component of the vaccinia virus polyprotein invasion and fusion complex, essential for normal membrane fusion or core penetration, and is an important target protein for neutralizing antibodies.

[0004] The pE248R protein plays a key role in the early steps after the virus infects the cell and is an essential protein for the fusion of the virus and the cell. The pE248R protein is not required for viral disintegration during the infection process, but is used for membrane fusion and release of the viral core into the cytoplasm. If pE248R is missing, early and late gene expression is impaired, and infectivity is reduced a hundredfold. The pE248R protein also plays an important role in virus reproduction and can significantly promote the replication of ASFV in vitro. In addition, pE248R also plays an important role in immune escape. The E248R gene inhibits the innate immune response mediated by the cGAS-STING signaling pathway by reducing the expression of the STING protein. In a 2023 study, an ASFV vaccine was prepared by fusing amino acids 1-198 of the pE248R protein with other ASFV proteins or epitopes. Experiments showed that this vaccine can induce strong ASFV-specific humoral immunity and cellular immune responses in pigs.

[0005] Vaccines are the most economical and effective means of preventing and controlling infectious diseases, and are crucial to the prevention and control of ASFV. However, due to the complexity of ASFV, only Vietnam has launched a live attenuated ASFV vaccine (ASFV-G-ΔI177-L) based on genetic engineering in the country, and there is no commercial ASFV vaccine that can provide broad protection. In-depth research on the pathogenicity, immunogenicity and antigenic epitopes of ASFV is of great significance in guiding the development of ASFV vaccines. The pE248R protein is an ASFV inner envelope protein that mediates the membrane fusion process and plays an important role in the replication and immune escape of the virus. It can be used as an important target antigen for vaccine research. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a polypeptide, a monoclonal antibody, a kit and an application of the B cell epitope of ASFVpE248R protein. The present invention identifies the African swine fever virus pE248R epitope peptide and prepares a monoclonal antibody that specifically binds to the African swine fever virus pE248R protein, laying a foundation for the preparation of an African swine fever detection kit.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a polypeptide of an ASFVpE248R protein B cell epitope, and the amino acid sequence of the polypeptide is shown in SEQ ID No.1.

[0009] Preferably, the nucleotide sequence of the gene encoding the polypeptide is shown as SEQ ID No.2.

[0010] The present invention also provides the use of the polypeptide described in the above technical scheme in the preparation of drugs for preventing and treating African swine fever infection and reagents for detecting or diagnosing African swine fever virus infection.

[0011] The present invention also provides an African swine fever pE248R monoclonal antibody, including monoclonal antibody 1E3;

[0012] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1E3 is shown in SEQ ID No.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.4;

[0013] Preferably, the heavy chain variable region nucleotide sequence of the monoclonal antibody 1E3 is shown as SEQ ID No.5, and the light chain variable region nucleotide sequence is shown as SEQ ID No.6.

[0014] The present invention also provides the use of the African swine fever pE248R monoclonal antibody described in the above technical scheme in the preparation of a reagent for detecting African swine fever.

[0015] The present invention also provides a kit for detecting African swine fever, comprising the African swine fever pE248R monoclonal antibody described in the above technical solution.

[0016] Beneficial effects of the present invention:

[0017] In this study, the pE248R recombinant protein was expressed and purified using a prokaryotic expression system. The pE248R recombinant protein was used as an immunogen to immunize mice, and mice with high serum titers were selected for cell fusion. After screening and identification, five specific monoclonal cell lines were obtained, and the B cell epitopes of the ASFVpE248R protein were further identified. The expression and purification of the ASFVpE248R protein, the preparation of monoclonal antibodies, and the identification of its B cell epitopes can provide a deep understanding of the immunological characteristics of ASFV. At the same time, by revealing the B cell epitopes of the pE248R protein, it will also help to enhance the understanding of the ASFV infection mechanism and immune escape strategy, and provide a scientific basis for responding to the threats of related pathogens such as ASFV.

[0018] The present invention screened out a monoclonal antibody through screening and truncation identification, and all of them can recognize D2 ( 87 QEVALTQWMDAG 98 ), and the peptide reacted with ASFV standard positive pig serum. 87 QEVALTQWMDAG 98 The epitope region is the B cell epitope region of pE248R, and the epitope region is displayed in the three-dimensional structure of the pE248R protein, and it is found to be located on the surface of the protein structure. The present invention provides a new monoclonal antibody source for preparing an African swine fever detection kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0020] Figure 1 To construct and identify the pE248R prokaryotic expression vector;

[0021] Figure 2 For the purification and identification of pE248R protein;

[0022] Figure 3 Immunize animals with pE248R recombinant protein;

[0023] Figure 4 For the screening and identification of monoclonal antibodies against pE248R protein;

[0024] Figure 5 To express and identify the truncated ASFVpE248R protein;

[0025] Figure 6 To identify the reactivity of truncated proteins with monoclonal antibodies;

[0026] Figure 7 To preliminarily identify the B cell epitopes of pE248R protein by scanning peptide method;

[0027] Figure 8 To identify the antigenic epitope of pE248R protein;

[0028] Fig. 9 Bioinformatics analysis of the antigenic epitope of pE248R protein. DETAILED DESCRIPTION

[0029] The present invention provides a polypeptide of an ASFVpE248R protein B cell epitope, and the amino acid sequence of the polypeptide is shown in SEQ ID No.1.

[0030] SEQ ID No.1:

[0031] MGGSTSSKNSFKNTTNIISNSIFNQMQSCISMLDGKNYIGVFGDGNILNHVFQD

[0032] LNLSLNTSCVQKHVNEENFITNLSNQITQNLKDQEVALTQWMDAGTHDQKT

[0033] DIEENIKVNLTTTLIQNCVSSLSGMNVLVVKGNGNIVENATQKQSQQIISNCLQ

[0034] GSKQAIDTTTGITNTVNQYSHYTSKNFFDFIADAISAVFKNIMVAAVVIVLIIVGFIAVFYFLHSRHRHEEEEEAEPLISNKVLKNAAVS。

[0035] In the present invention, the nucleotide sequence of the coding gene of the polypeptide is as shown in SEQ ID No.2.

[0036] SEQ ID No.2:

[0037] aagcttATGGGCGGCTCTACCTCTAAGAACTCCTTCAAGAACACCACCAACATCATCTCTAACTCTATCTTTAACCAGATGCAGTCCTGTATCTCTATGCTGGATGGAAAGAACTATATCGGCGTGTTCGGCGACGGCAACATCCTGAATCACGTGTTTCAGGATCTGAACCTGTCTCTGAACACCTCCTGTGTGCAGAAGCATGTGAATGAGGAGAATTTTATCACCAATCTGAGCAATCAGATCACACAGAACCTGAAGGACCAGGAAGTGGCCCTGACCCAGTGGATGGACGCTGGCACCCACGATCAGAAAACAGACATTGAGGAGAACATCAAGGTGAACCTGACCACCACCCTGATCCAGAACTGCGTGTCTAGCCTGTCTGGCATGAACGTGTTGGTGGTGAAGGGCAACGGCAACATCGTGGAGAACGCTACCCAGAAGCAGTCCCAGCAGATCATCTCTAATTGTCTGCAGGGCTCTAAGCAGGCCATCGATACCACCACCGGCATCACCAATACCGTGAATCAGTACTCTCACTATACCAGCAAGAATTTCTTTGATTTTATCGCTGATGCCATCTCTGCTGTGTTTAAGAATATCATGGTGGCTGCTGTCGTGATCGTGCTGATCATCGTGGGCTTTATCGCCGTGTTTTACTTTCTGCATAGCCGCCATCGGCATGAGGAAGAGGAAGAGGCCGAGCCTCTGATCTCTAACAAGGTGCTGAAGAACGCCGCCGTGAGCTGAttaattaa。

[0038] The present invention also provides the use of the polypeptide described in the above technical scheme in the preparation of drugs for preventing and treating African swine fever infection and reagents for detecting or diagnosing African swine fever virus infection.

[0039] The present invention also provides an African swine fever pE248R monoclonal antibody, including monoclonal antibody 1E3; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1E3 is shown in SEQ ID No.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.4.

[0040] SEQ ID No.3:

[0041] QVQLQQSGAELVKPGASVKLSCKASGYTFTSYYMYWVKQRPGQGLEWIGEIKPSNGDTNVSDKFKRKATLTVDKSSSTAYMQLSTLTSEDSAVYYCSSTATAYWGQGTLVTVSA

[0042] The amino acid sequence of the heavy chain variable region of monoclonal antibody 1E3 was further analyzed and is shown in Table 1.

[0043] Table 1 Heavy chain variable region of monoclonal antibody 1E3

[0044]

[0045]

[0046] SEQ ID No.4:

[0047] DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWK

[0048] The amino acid sequence of the light chain variable region of monoclonal antibody 1E3 was further analyzed and is shown in Table 2.

[0049] Table 2 Light chain variable region of monoclonal antibody 1E3

[0050]

[0051] In the present invention, the heavy chain variable region nucleotide sequence of the monoclonal antibody 1E3 is shown as SEQ ID No.5, and the light chain variable region nucleotide sequence is shown as SEQ ID No.6.

[0052] SEQ ID No.5:

[0053] TCTCACCATGGGATGCAGCTGGGTCATCCTCTTTTTGGTTGCAACAGCAACAGGTGTCCACTCCCAGGTCCAACTGCAGCAGTCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTATATGTACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAAACCTAGTAATGGTGATACTAACGTCAGTGACAAGTTCAAGAGGAAGGCCACACTGACTGTTGACAAATCCTCCAGCACAGCATACATGCAACTCAGCACCCTGACATCTGAGGACTCTGCGGTCTATTACTGTTCCTCTACGGCTACCGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCCGCAGCCAAAACA。

[0054] SEQ ID No.6:

[0055] TCCCAAGCTTTACTGGATGGTGGGAAGATGGATACAGTTGGTGCAGCATCAGCCCGTTTTATTTCCAGCTTGGTCCCCCCTCCGAACGTGTAAGCTCCCTAATGTGCTGACAGTAATAGGTTGCAGCATCCTCCTCCTCCACAGGATGGATGTTGAGGGTGAAGTCTGTCCCAGACCCACTGCCACTGAACCTGGCAGGGACCCCAGATTCTAGGTTGGATACAAGATAGATGAGGAGTCTGGGTGGCTGTCCTGGTTTCTGTTGGTTCCAGTGCATATAACTATAGCCAGATGTACTGACACTTTTGCTGGCCCTGTATGAGATGGTGGCCCTCTGCCCCAGAGATACAGCTAAGGAAGCAGGAGACTGTGTCAGCACAATGTCACCAGTGGAACCTGGAACCCAGAGCAGCAGTACCCATAGCAGGAGTGTGTCTGTCTCCATGAATTCCCATCTCCAT。

[0056] The present invention also provides the use of the African swine fever pE248R monoclonal antibody described in the above technical scheme in the preparation of a reagent for detecting African swine fever.

[0057] The present invention also provides a kit for detecting African swine fever, comprising the African swine fever pE248R monoclonal antibody described in the above technical solution.

[0058] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0059] Unless otherwise specified, the experimental methods used in the following embodiments and experimental examples are all conventional methods; the raw materials (including biological materials), reagents, culture media, instruments, etc. used are all commonly used in the art, available to the public or available through commercial channels unless otherwise specified; the terms and abbreviations involved have the conventional meanings in the art, such as PBS is phosphate buffered saline, and PBST is phosphate-tween buffered saline.

[0060] Example 1

[0061] Construction and identification of prokaryotic expression vector of ASFV pE248R

[0062] 1.1 Amplification of the ASFV pE248R protein target gene

[0063] After optimizing the E248R gene sequence in the GenBank database (GenBank: AYW34102.1), specific primers were designed using SnapGene software, BamH I and Xho I were selected as restriction sites, the primer sequences are shown in Table 3, and the PCR reaction system is shown in Table 4, which was synthesized by General Bio (Anhui) Co., Ltd. The ASFVpE248R gene was used for PCR amplification, and the PCR amplification product was verified by agarose gel electrophoresis. The target band was recovered using a universal DNA purification and recovery kit, and the concentration was determined, marked, and then stored at -20°C.

[0064] Table 3 Primer sequences

[0065] Primer name Sequence (5'-3') BamHI-F SEQ ID No.18: CGCGGATCCATGGGCGGCTCTACCTCTAAGAAC XhoI-R SEQ ID No:19:CCGCTCGAGTTATTAGCTCACGGCGGCGTTC

[0066] Table 4 PCR reaction system

[0067] sample volume BamHI-F 1μL XhoI-R 1μL PrimeSTAR HS DNA Polymerase 1μL 5× PrimeStar Buffer 10μL dNTP 4μL ASFVE248R gene 1μL Sterile water 32μL

[0068] 1.2 Double restriction enzyme digestion of target gene and vector

[0069] The target gene ASFV E248R and the pET-28a vector were double-digested with BamH I and Xho I restriction endonucleases. The digestion system is shown in Table 5. After digestion at 37°C for 3 hours, the digestion product was recovered using the steps of a universal DNA purification and recovery kit.

[0070] Table 5 E248R gene double restriction enzyme digestion reaction system

[0071] sample volume B H 1μL XOt 1μL Cutsmartbuffer 5μL Recycling E248R gene / pET-28a plasmid by enzyme digestion 3μg / 2μg Sterile water Make up to 50 μL

[0072] 1.3 Connection between target gene and vector

[0073] The concentrations of the E248R gene fragment recovered after double enzyme digestion and the pET-28a vector were measured and mixed at a molar ratio of 4:1. The corresponding volumes were calculated and ligase was added for reaction. For details, see Table 6. The ligation was carried out at 16°C for 9 hours.

[0074] Table 6 Enzyme-linked system

[0075] sample volume E248R gene digestion products 6μL pET-28a vector digestion products 2μL T4 DNA ligase 1μL 10×T4 DNA Ligase Buffer 1μL

[0076] 1.4 Preparation and transformation of competent cells

[0077] To transform competent cells, follow these steps:

[0078] (1) Take out the competent cells and put them in ice for about three minutes to slowly thaw them;

[0079] (2) Add 6 μL of the ligation product to the competent cells in a clean bench, mix gently with a 10 μL pipette, and place on ice for 20 min;

[0080] (3) After 20 min, place 50 μL of competent cells in the centrifuge tube at 42°C for 90 s and then place in ice water for 5 min;

[0081] (4) Add 800 μL LB liquid culture medium to the centrifuge tube in the clean bench, incubate on a shaker for 50 min, and then centrifuge at 4000 rpm for 4 min;

[0082] (5) Pipette 60 μL of supernatant and discard it, then resuspend the precipitate with the supernatant. Place the resuspended liquid on LB solid medium (kanamycin resistance) for coating;

[0083] (6) Incubate the cells in an inverted position at 37°C in a constant temperature incubator overnight.

[0084] 1.5 Screening of positive clones by PCR

[0085] (1) Select several relatively large and round single colonies from the plate and inoculate them into 1 mL of kanamycin-resistant LB medium. After culturing for about 4 h, perform PCR on the bacterial solution. The PCR system is shown in Table 7.

[0086] (2) After agarose gel electrophoresis, observe the results and identify the band positions, then send the bacterial culture with the correct bands to the company for sequencing.

[0087] (3) The correctly sequenced strain was inoculated into 6 mL of culture medium for overnight expansion culture, the plasmid was extracted according to the kit steps, and transformed into BL21 competent cells, following the same steps as above.

[0088] (4) The plasmid was double-digested and successfully sequenced, indicating that the pET-28a-pE248R-BL21 expression bacteria were successfully constructed. A small amount of sterile glycerol was added to the bacterial solution and then frozen at -80°C.

[0089] Table 7 Bacterial liquid PCR reaction system

[0090] sample volume BamHI-F 1μL XhoI-R 1μL Sterile deionized water 2μL PremixrTaqTM Enzyme 5μL Bacterial liquid 1μL

[0091] The results of this embodiment are described as follows:

[0092] The target gene of ASFV E248R is 747 bp in size. After PCR amplification, there is an obvious band near 750 bp (e.g. Figure 1 Middle A), indicating that the E248R target gene was amplified successfully.

[0093] The pE248R protein gene was inserted between the BamH I and Xho I restriction endonuclease sites of the pET-28a vector and then transformed into E. coli DH5α and BL21 competent cells. Figure 1 In B), a bright band was observed at 747 bp, consistent with the expected size of the gene.

[0094] The constructed recombinant expression vector was double-digested and verified. The verification results showed that there was an obvious band at 5000-7500 bp and 747 bp, which was consistent with expectations (such as Figure 1 At the same time, the sequencing results were consistent with the known sequences, indicating that the pET-28a-pE248R recombinant expression vector was successfully constructed.

[0095] Example 2

[0096] Expression and identification of ASFV pE248R protein in Escherichia coli expression system

[0097] 2.1 Inducible expression

[0098] The obtained pET-28a-pE248R-BL21 expression bacteria were initially induced to express:

[0099] (1) First activation: Add 6 mL of Kan + Add 6 μL of BL21(DE3)-pET-28a-pE248R bacterial solution to the resistant LB medium and culture overnight.

[0100] (2) Second activation: Add 150 mL of Kan + Add 200 mL of bacterial solution to the culture medium and culture for about 4 hours until the OD600 value is 0.6.

[0101] (3) Induction: Add IPTG solution to the bacterial solution with a final concentration of 0.1 mM, 37°C, 220 rpm, and induce expression for 7 h;

[0102] (4) Ultrasonic disruption: Centrifuge and resuspend the cells with 10 mL of PBS buffer. Turn on the ultrasonic machine, clean the probe of the ultrasonicator with alcohol, sterile water and PBS buffer, place the resuspended cells in ice water, start ultrasonic disruption, and stop for 5 seconds after every 3 seconds of ultrasonication. Stop after 20 minutes, centrifuge at 12,000 rpm for 15 minutes, take the supernatant, and resuspend the precipitate with the same volume of PBS buffer as the supernatant.

[0103] 2.2 Preliminary analysis by SDS-PAGE

[0104] (1) Analysis of the expression results of ASFVpE248R protein by SDS-PAGE:

[0105] Sample preparation: Add the supernatant and precipitate before and after induction into the loading buffer and boil for 8 min.

[0106] (2) Electrophoresis: Load the protein marker and the sample from the previous step separately and run at 150 V for 55 min.

[0107] (3) Staining and destaining: Use Coomassie Brilliant Blue staining solution to stain the protein gel and then destain it.

[0108] (4) Analysis: Observe the results using a gel imager and keep them.

[0109] The results of this example are as follows: the pET-28a-pE248R-BL21 expression bacteria were activated and then induced to express. The analysis results showed that a band appeared near 35 kDa in the ultrasonic precipitation, which was consistent with the expected size (such as Figure 2 (A) Analysis of the ultrasonic supernatant and precipitate after induction showed that the pET-28a-pE248R-BL21 expression bacteria were successfully expressed, but in the form of inclusion bodies.

[0110] Example 3

[0111] Purification and identification of ASFV pE248R protein

[0112] 3.1 Denaturation and purification of ASFV pE248R protein

[0113] Using the optimized conditions, the ASFVpE248R protein was induced to express in large quantities. After centrifuging 800 mL of bacterial solution, the cells of the expressing bacteria were collected, resuspended in PBS buffer, and ultrasonically broken in an ice water bath. The ultrasonicator was set to stop for 5 seconds after every 3 seconds of ultrasonication, and the breaking time was 1 hour. After breaking, the liquid was placed at 4°C, centrifuged at 13000rpm for 15 minutes, and the ultrasonic precipitate was collected. Washed with inclusion body washing buffer, stirred at room temperature for 30 minutes each time, and repeated three times. Centrifuged at 13000rpm for 15 minutes at 4°C, and the washing supernatant was collected. At the same time, the precipitate was added to the inclusion body dissolution buffer, stirred and dissolved at room temperature for 30 minutes, and then placed in a 4°C refrigerator overnight. The next day, the dissolved solution was taken out, centrifuged at 4°C and 13000rpm for 15 minutes, and the supernatant and precipitate were collected.

[0114] ASFVpE248R protein was purified using a nickel column as follows:

[0115] (1) Sample treatment: All loaded samples were filtered to avoid clogging of the nickel column.

[0116] (2) Equilibration of nickel column: After flushing with ddw, the nickel column is washed with ten times column volume of inclusion body solution to provide an environment for subsequent purification.

[0117] (3) pE248R protein binding: After equilibration of the nickel column, the supernatant of the denatured solution was filtered and passed through the column at a slow drip rate. This was repeated three times to bind the protein to the nickel column. The nickel column was then washed with inclusion body solubilization buffer to remove excess impurities.

[0118] (3) Elution of pE248R protein: The column was eluted with a gradient of buffer containing different imidazole concentrations. During the elution, the eluted liquid was mixed with G250 protein indicator and the color change was observed. When there was no obvious color change after mixing, the next higher concentration was replaced and elution was continued. The tubes of liquid with the darkest color change were collected.

[0119] (4) Nickel column storage: The nickel column was washed with a 500 mmol / L imidazole buffer and ddw, and then sealed with 20% alcohol for storage.

[0120] (5) Collect the proteins in the tubes with the deepest color changes and analyze them by SDS-PAGE. Select the proteins with the correct band position and high purity for renaturation: The purified pE248R protein is dialyzed against a renaturation buffer containing different urea concentrations (6, 4, 2, 1.5, 1, 0 mol / L) at 4°C for 8 h each time. The renaturation must be slow so that the pE248R recombinant protein can be fully renatured.

[0121] 3.2 Identification of ASFV pE248R protein

[0122] 3.2.1SDS-PAGE identification:

[0123] Take the bacterial solution before purification and the protein after purification for sample processing respectively. For specific methods, refer to 2.2.

[0124] 3.2.2 Western Blot Identification:

[0125] The purified pE248R protein sample was subjected to SDS-PAGE and transferred to the membrane for 55 minutes. After the transfer, it was blocked for 2 hours. Then anti-His monoclonal antibody (1:4000) or ASFV standard positive pig serum (1:4000) was added at 37°C for 1 hour. The primary antibody was recovered, and Goat Anti-Mouse IgG / HRP (1:4000) or Goat Anti-Swine IgG / HRP (1:4000) was placed after washing the membrane, and incubated at 37°C for 1 hour. The membrane was washed with PBST solution, then developed with ECL chemiluminescence kit, and the results were saved.

[0126] 3.2.3 Indirect ELISA identification

[0127] (1) Coating: Dilute the pE248R recombinant protein with CBS solution, coat each well with 200 ng, and add to the ELISA plate for coating at 37°C for 2 h.

[0128] (2) Blocking: Discard the coating solution, wash three times with 1×PBST solution and pat dry, add 200 μL of 5% skim milk solution to each well, and block at 37°C for 2 h.

[0129] (3) Primary antibody: Discard the blocking solution in the wells, wash the plate three times, add 200 μL ASFV-positive pig serum (1:1000) to the first well, add 100 μL PBS to the remaining wells, and then dilute the plate in multiples starting from the first well. Discard the last 100 μL of the dilution solution and incubate at 37°C for 1 h.

[0130] (4) Secondary antibody: Discard the primary antibody, wash the plate three times, add 100 μL Goat Anti-Swine IgG / HRP (1:4000) to each well, and incubate at 37°C for 1 h.

[0131] (5) Color development: After washing the plate five times, add 100 μL of TMB color development solution to each well and react in the dark for 6 min. Then add 100 μL of stop solution to terminate the reaction.

[0132] (6) Reading: Turn on the microplate reader and read the OD 450 When the reading is twice greater than the reading of the negative well, it indicates a valid titer.

[0133] The results of this example are described as follows: urea was used to renature the protein to make it soluble, and a target protein band was found in the dissolution buffer. The protein was then purified by nickel affinity chromatography. After purification, the standard protein and pE248R protein were measured at different dilutions using the BCA protein concentration assay kit to obtain the OD values ​​of each dilution. 562 The formula of the standard curve is y = 0.0092x + 0.0964, and the correlation coefficient R2 = 0.99. The OD of the pE248R recombinant protein at different dilutions was measured. 562 The corresponding ASFV pE248R protein concentration was calculated and the average value was finally taken. After calculation, the concentration of pE248R protein after purification and renaturation was about 0.3 mg / mL.

[0134] The purified protein was analyzed by SDS-PAGE ( Figure 2 Middle B) shows that the purified renatured protein has a band at around 35 kDa. Through Western Blot identification, it was observed that the renatured pE248R recombinant protein reacted with anti-His monoclonal antibody and ASFV antibody positive serum (such as Figure 2 (C, D), indicating that the renatured pE248R recombinant protein has good reactivity.

[0135] Example 4

[0136] Animal immunization with ASFV pE248R protein

[0137] ASFVpE248R protein was used as an immunogen to immunize two BALB / c mice, and the subcutaneous injection method was used to inject multiple points on the back of the mice. For the first immunization, Freund's complete adjuvant was mixed with an equal volume of protein for emulsification. The immunization dose for each mouse was 50μg, and immunization was performed once every 14 days. After three immunizations, the mouse tail blood was diluted with PBS, and the supernatant was retained after centrifugation to determine the mouse serum titer. After three immunizations, the mouse tail venous blood was taken to determine the titer. Mice with high titers were selected for booster immunization, and only 100μg of protein was directly injected into the mouse through the abdominal cavity. After 3-5 days, the cells fused. The titer of mouse polyclonal antibody serum was determined by indirect ELISA, and the pE248R protein was used to coat the plate. The mouse tail serum after three immunizations was used as the primary antibody, and Goat Anti-Mouse IgG / HRP (1:4000) was used as the secondary antibody.

[0138] The results of this example are described as follows: The titer of mouse serum was detected by indirect ELISA (such as Figure 3 A) All mice produced a good immune response, and the serum titer of mouse No. 1 was 1:5.12×10 5 The serum titer of mouse No. 2 was 1:2.56×10 5 This time, mouse No. 1 with a higher titer was selected for cell fusion.

[0139] Example 5

[0140] Monoclonal antibody preparation and screening

[0141] Take blood from the eyeball of the super-immunized mouse, collect the serum after centrifugation, and store it at -20°C. Subsequently, the mouse was killed, soaked in 75% alcohol for 5 minutes for disinfection, and the spleen was removed. The spleen was placed on a 200-mesh nylon net and slowly ground with scissors. While grinding, GNK solution was continuously added, and the spleen cells flowed through the nylon net into the sterile beaker below. Cut off the rope of the fixed nylon net, pour the resuspension into a centrifuge tube, centrifuge, discard the supernatant, gently shake the bottom cells, then add 20mL GNK, mix well, and count. Then perform cell fusion. First, treat and collect SP2 / 0 cells, and then add 20mL GNK. Use a pipette to mix the mixture and count the cells. After calculation, mix the myeloma cells and spleen cells in a ratio of 1:6, and centrifuge at 1100rpm for 5 minutes after mixing. After centrifugation, discard the supernatant and flick the cells with your fingers. Place the centrifuge tube in a 37°C warm water environment. Pipette 1mL of preheated PEG 1500 and slowly add it to the bottom of the centrifuge tube. Add 1mL, 3mL and 11mL of GNK to the cell mixture at a speed of 30 seconds, respectively. Add the mixture within 90 seconds, and shake the centrifuge tube gently during the process. After the addition is completed, let it stand in a 37°C incubator for 5 minutes, and then add 25mL of GNK again. After the addition, centrifuge and discard the supernatant, gently flick the cells, and resuspend them with HAT medium. Finally, add 100μL of the resuspension to each well of a 96-well cell culture plate with feeder cells, and place it in a cell culture incubator at 37°C for culture.

[0142] After cell fusion, observe whether the cell plate has cell clusters. After about a week, half of the wells of the cell plate have cell clusters, and the medium is changed by indirect ELISA to screen the positive hybridoma cell wells. The pE248R recombinant protein and the pET-28a vector expression protein are used as coating sources, the cell supernatant is used as the primary antibody, and the mouse eye blood (1:1000 dilution) is used as the positive control. Select the cell wells that react well with the pE248R recombinant protein and do not react with the pET-28a vector expression protein. Then subclone the positive hybridoma cell wells screened for the first time to make them into single cluster cells, and then screen and subclone this cell line twice.

[0143] Description of the results of this example: 8 positive hybridoma cell lines (such as Figure 3 (B) These 8 hybridoma cell lines all reacted positively to the pE248R recombinant protein and did not react to the protein expressed by the pET-28a empty vector expression bacteria.

[0144] Example 6

[0145] Characterization of monoclonal antibodies

[0146] The eukaryotic expression system was used to transiently transfect the pCAGGS-pE248R plasmid to express the eukaryotic pE248R protein. The reaction between the eukaryotic expressed pE248R protein and the supernatant of the positive hybridoma cell line was identified by IFA and IPMA. Specificity: The pE248R protein of ASFV was used as a positive control, and the supernatants of 5 positive hybridoma cells (1E3, 2H1, 4G11, 5C3 and 5H3) that reacted with the eukaryotic protein were used for indirect ELISA and Western Blot experiments with porcine pseudorabies virus protein, porcine delta coronavirus protein, and porcine respiratory coronavirus protein. Monoclonal antibodies were prepared by the induction method in animals, and the ascites was purified by ProteinA affinity chromatography column. The titer and affinity of the monoclonal antibodies were determined by indirect ELISA.

[0147] The results of this example are described as follows: IFA and IPMA tests were used to identify the reaction between the pE248R protein expressed in the eukaryotic system and the cell supernatants of 8 hybridoma cell lines. The positive control was the serum of mice immunized with the pE248R protein, and the negative control was the serum of unimmunized mice. Figure 4 Middle A) The results showed that 1E3, 2H1, 4G11, 5C3, and 5H3 all reacted with the pE248R protein expressed in eukaryotic cells, among which 1E3, 4G11, and 5H3 reacted relatively well, and the results of IFA and IPMA were consistent.

[0148] Taking the results of ASFV-pE248R-1E3 as an example (the reaction results of the other four monoclonal antibodies were the same as ASFV-pE248R-1E3), the supernatants of five hybridoma cells (1E3, 2H1, 4G11, 5C3 and 5H3) only produced specific bands with ASFV pE248R protein, and did not react with other swine disease proteins. The results of indirect ELISA and Western Blot were consistent, indicating that these five monoclonal antibodies had good specificity.

[0149] The hybridoma cell lines with higher cell supernatant titers were selected and combined with the results of IFA and IPMA experiments, 1E3 and 5H3 cell lines were comprehensively selected to prepare ascites. Figure 4 In B), it can be seen that the purified monoclonal antibodies have two obvious target bands and no other obvious mixed bands, indicating that the monoclonal antibodies with high purity were successfully obtained. The antibody concentrations of 1E3 and 5H3 after purification were determined by NanoDrop 2000 to be 5 mg / mL and 1 mg / mL respectively.

[0150] The titers of the purified monoclonal antibodies were determined by indirect ELISA. The titers of 1E3 and 5H3 after purification were both 1:2.048×10 6 (like Figure 4 C). The affinity constant regression curves of the two monoclonal antibodies were established respectively (such as Figure 4 D), the affinity constant of 1E3 was calculated to be 1.578×10 10 L / mol, the affinity constant of 5H3 is 3.273×10 9 L / mol.

[0151] Example 7

[0152] Expression and identification of truncated ASFV pE248R protein

[0153] 7.1 Design of truncated expression of ASFV pE248R protein

[0154] The overlapping peptide method was used for truncation design, and two truncations were performed. The first truncated fragments were P1 (aa 1-62), P2 (aa 42-124), P3 (aa 104-186), and P4 (aa 166-248). The P1 polypeptide contained 62 amino acids, and each of the P2, P3, and P4 polypeptides contained 82 amino acids. There were 20 amino acids overlapping between adjacent polypeptides. The second time, aa 62-104 was truncated again, with 15 amino acids in each segment, and each segment overlapped 6 amino acids. The truncated fragments were P2-1 (aa 62-77), P2-2 (aa 72-86), P2-3 (aa 81-95), and P2-4 (aa 90-104).

[0155] 7.2 Construction of truncated fragment expression vector

[0156] 7.2.1 Primer design

[0157] Through the analysis of the P1-P4 and P2-1-P2-4 truncated fragments and the pET-32a vector, the primers for the P1-P4 and P2-1-P2-4 truncated fragments were designed using SnapGene with BamH I and Xho I as restriction sites. The sequences are shown in Table 8 and were synthesized by General Biotechnology (Anhui) Co., Ltd.

[0158] Table 8 Primers

[0159] Primer name Primer sequence (5'-3') P1-F SEQ ID No:20:CGCGGATCCATGGGCGGCTCTACCTC P1-R SEQ ID No:21:CCGCTCGAGTTAACAGGAGGTGTTCAGAGA P2-F SEQ ID No:22:CGCGGATCCATGGGCGACGGCAACATCC P2-R SEQ ID No:23:CCGCTCGAGTTAAGACACGCAGTTCTGGAT P3-F SEQ ID No:24:CGCGGATCCATGACAGACATTGAGGAGAAC P3-R SEQ ID No:25:CCGCTCGAGTTAAAAGAAATTCTTGCTGGT P4-F SEQ ID No:26:CGCGGATCCATGACCACCACCGGCATCA P4-R SEQ ID No:27:CCGCTCGAGTTAGCTCACGGGCGGCGTT P2-1-F SEQ ID No:28:CGCGGATCCATGGTGCAGAAGCATGTGA P2-1-R SEQ ID No:29:CCGCTCGAGTTAGCTCAGATTGGTGATAAA P2-2-F SEQ ID No:30:CGCGGATCCATGTTTATCACCAATCTGAGC P2-2-R SEQ ID No:31:CCGCTCGAGTTAGTCCTTCAGGTTCTGT P2-3-F SEQ ID No:32:CGCGGATCCATGACACAGAACCTGAAGG P2-3-R SEQ ID No:33:CCGCTCGAGTTACATCCACTGGGTCAG P2-4-F SEQ ID No:34:CGCGGATCCATGGCCCTGACCCAGTG P2-4-R SEQ ID No:35:CCGCTCGAGTTATTTCTGATCGTGGGTGC

[0160] 7.2.2 Construction of truncated expression vector

[0161] Using the gene sequence of pE248R protein as a template, using the primers in Table 8, PCR amplified each truncated fragment, after DNA was recovered, double enzyme digestion and T4 ligation (conditions are the same as Table 5 and Table 6), the truncated fragment was constructed into the prokaryotic expression vector pET-32a, and identified by bacterial liquid PCR. The strain with positive identification results and correct sequencing alignment was expanded and cultured, the plasmid was extracted, and transformed into BL21 (DE3) competent cells, and it was also identified by bacterial liquid PCR.

[0162] 7.2.3 Expression and identification of truncated proteins

[0163] The plasmid was transformed into BL21 expression bacteria, and the constructed truncated protein expression bacteria were induced to express in large quantities at 26°C until the next day. The next day, the bacteria were harvested, resuspended in PBS, ultrasonically broken and centrifuged, and then the P1~P4 and P2-1~P2-4 truncated proteins were analyzed by SDS-PAGE and identified by Western Blot.

[0164] The results of this example are described as follows: the extracted recombinant expression vector was transformed into BL21 competent cells to induce expression, and the truncated proteins P1 to P4 were analyzed and identified. Figure 5 As shown, the size of the P1 recombinant protein is about 27 kDa, and the size of the P2-P4 recombinant proteins is about 29 kDa (as shown in Figure 5 A), which is consistent with expectations, and P1 to P4 can specifically bind to the anti-His monoclonal antibody ( Figure 5 (B) This indicates that the recombinant proteins P1 to P4 were successfully obtained in this study.

[0165] Preliminary screening of antigen epitope regions was performed by indirect ELISA and Dot-Blot, such as Figure 6As shown in A, these five monoclonal antibodies can all recognize the truncated second segment P2 (aa42-124). After removing the overlapping part, it was determined that the epitope region recognized by the monoclonal antibodies was located at 64-104 amino acids.

[0166] After the truncated proteins were induced to express, P2-1 to P2-4 were analyzed and identified, such as Figure 5 As shown, the size of P2-1 to P2-4 recombinant proteins is approximately 21 kDa ( Figure 5 C), which is consistent with expectations, and the recombinant proteins P2-1 to P2-4 can specifically bind to the anti-His monoclonal antibody ( Figure 5 D). This indicates that the recombinant proteins P2-1 to P2-4 were successfully obtained in this study.

[0167] Based on the results of the first truncated protein and monoclonal antibody reaction, the amino acid sequence at positions 62 to 104 was further truncated into P2-1, P2-2, P2-3, and P2-4 fragments. After induced expression, the reaction of monoclonal antibodies with truncated proteins P2-1 to P2-4 was identified by indirect ELISA and Dot-Blot. It was identified that all five monoclonal antibodies could recognize P2-3 and P2-4. Among them, 4G11 reacted more strongly with P2-3, and the remaining four reacted more strongly with P2-4 (e.g. Figure 6 (middle B).

[0168] Example 8

[0169] Preliminary identification of B cell epitopes of pE248R protein by scanning peptide method

[0170] 8.1 Indirect ELISA Identification

[0171] Indirect ELISA identification: Truncated protein and vector-expressed protein were used as coating agents, the culture supernatant of the monoclonal antibody cell line was used as the primary antibody, and GoatAnti-Mouse IgG / HRP (1:4000) was used as the secondary antibody to identify the B cell epitope region recognized by the monoclonal antibody.

[0172] 8.2 Dot-Blot Identification

[0173] Dot-Blot was used to identify the epitope regions recognized by the supernatants of 5 hybridoma cells: the NC membrane was cut into strips of appropriate size, and a hole of appropriate size was gently printed with a hole puncher. The NC membrane was soaked in ddw and dried naturally. The pE248R protein (positive control), vector-expressed protein (negative control), and the ultrasonic supernatant of each truncated protein were spotted into each round hole with a pipette, 0.8 μL each time, and spotted again after drying, and the operation was repeated three times. After the spotting was completed, wait for the membrane to be completely dried and place it in a 5% skim milk solution, and block it at 37°C for 2h. Pour off the blocking solution and wash the membrane with PBST solution for 15min. Mark the NC membrane, place it in the corresponding cell supernatant, and incubate it at 37°C for 1h. Recover the cell supernatant, wash the membrane with PBST solution for 15min, add GoatAnti-Mouse IgG / HRP (1:4000), and incubate it at 37°C for 1h. The NC membrane was washed in PBST solution for 20 min, developed with ECL chemiluminescence kit, and photographed and analyzed.

[0174] Description of the results of this example: The epitope region recognized by the monoclonal antibody was identified by indirect ELISA and Dot-Blot, and it was found that the screened monoclonal antibody can recognize the amino acid sites between 81 and 104 in the pE248R protein. Synthetic peptides were designed and coupled with BSA. Peptide-ELISA and Dot-Blot were combined to identify the reactivity of 1E3, 2H1, 4G11, 5C3, 5H3 monoclonal antibodies with short peptides D1, D2, and D3. The results are shown in the figure. Figure 7 As shown, these five monoclonal antibodies can recognize the D2 (aa87-98) polypeptide sequence.

[0175] Example 9

[0176] Identification of Antigen Epitopes Recognized by Monoclonal Antibodies

[0177] The overlapping peptide method was used to truncate the reaction area, and the peptide was coupled with BSA. 2.2 mg of BSA was added to 1 mL of PBS, and 1 mg of synthetic peptide was added after dissolving. The insoluble peptide was dissolved with a small amount of DMSO and then added. Then 2.5 μL of 50% glutaraldehyde was added, stirred at room temperature overnight, and then placed at 4°C for use. Then, the monoclonal antibodies (1E3, 2H1, 4G11, 5C3 and 5H3) were identified for their recognition of antigenic epitopes by Peptide-ELISA and Dot-Blot.

[0178] Peptide-ELISA and Dot-Blot were combined to identify the reactivity of short peptide D2 (aa 87-98) with ASFV standard positive pig serum. Short peptide D2 (SEQ ID NO.3) reacted well with positive pig serum.

[0179] The results of this example are described as follows: Peptide-ELISA and Dot-Blot were used to identify the reactivity of short peptide D2 (aa 87-98) with ASFV standard positive pig serum. Figure 8 As shown, the short peptide D2 reacted well with positive pig serum.

[0180] Example 10

[0181] Epitope spatial structure analysis

[0182] The pE248R protein was homologously modeled using the SWISS-MODEL software. The identified epitope sequence was displayed on the simulated structure of the pE248R protein using Pymol. The epitope region was located on the surface of the pE248R protein. The green part was the pE248R protein, and the orange part represented the 87QEVALTQWMDAG98 epitope region recognized by the monoclonal antibody.

[0183] Description of the results of this example: The pE248R protein was homologously modeled using the SWISS-MODEL software. Fig. 9 As shown in A. The identified epitope sequence was displayed on the simulated structure of the pE248R protein by Pymol. The epitope region is located on the surface of the pE248R protein, as shown in Fig. 9 As shown in B, the green part is the pE248R protein, and the orange part represents the 87QEVALTQWMDAG98 epitope region recognized by the monoclonal antibody.

[0184] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A polypeptide of the B cell epitope of ASFVpE248R protein, characterized in that: The amino acid sequence of the polypeptide is shown as SEQ ID No.

1.

2. The polypeptide according to claim 1, characterized in that The nucleotide sequence of the gene encoding the polypeptide is shown as SEQ ID No.

2.

3. Use of the polypeptide according to claim 1 or 2 in the preparation of drugs for preventing and treating African swine fever infection, and reagents for detecting or diagnosing African swine fever virus infection.

4. An African swine fever pE248R monoclonal antibody, characterized in that Includes monoclonal antibody 1E3. The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1E3 is shown in SEQ ID No.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

4.

5. The African swine fever pE248R monoclonal antibody according to claim 4, characterized in that The nucleotide sequence of the heavy chain variable region of the monoclonal antibody 1E3 is shown in SEQ ID No.5, and the nucleotide sequence of the light chain variable region is shown in SEQ ID No.

6.

6. Use of the African swine fever pE248R monoclonal antibody according to any one of claims 4 to 5 in the preparation of a reagent for detecting African swine fever.

7. A kit for detecting African swine fever, characterized in that: Comprising the African swine fever pE248R monoclonal antibody as described in any one of claims 4 to 5.

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