A porcine epidemic diarrhea virus polyepitope fusion protein, and a preparation method and application thereof

Abstract

The present application relates to the technical field of genetic engineering, and provides a porcine epidemic diarrhea virus (PEDV) multi-epitope fusion protein, a preparation method and application thereof.The present application links multiple B lymphocyte epitopes and T lymphocyte epitopes of a PEDV spike protein by using a GGSSGG polypeptide as a linker, and uses a fusion expression technology to embed the same on a ferritin nanoparticle to construct a PEDV multi-epitope recombinant protein.The PEDV multi-epitope fusion protein designed in the present application can be efficiently expressed and can be effectively recognized by a PEDV specific antibody.The PEDV multi-epitope fusion protein provided in the present application effectively solves the problems of complex serum types of the PEDV, weak cross-protection of existing vaccines and low protein expression.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a multi-epitope fusion protein of porcine epidemic diarrhea virus, its preparation method, and its application. Background Technology

[0002] Porcine epidemic diarrhea (PED) is a highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV), characterized by diarrhea, vomiting, and dehydration. Studies have shown that the PEDV positivity rate can reach 25%–72.72%. The disease spreads very rapidly, with suckling piglets under one week old being most susceptible. Morbidity is extremely high, and without timely control, mortality can reach 100%. Currently, there is no mature and effective treatment. In recent years, PED outbreaks in piglets have spread to almost all Asian countries, causing enormous economic losses.

[0003] The PEDV gene is approximately 28 kb in size, with cap structures at both ends and a poly(A) tail. The genome contains four structural proteins (S, E, M, and N proteins) and three non-structural proteins (replicaase 1a, replicaase 1b, and ORF3). ORF3 encodes a polymorphic product with an unknown function. S is the largest structural protein, containing neutralizing antibody epitopes and specific receptor-binding sites for viral entry.

[0004] Multi-epitope fusion antigens are a vaccine technology based on antigen structure and epitopes, providing a new platform for the development of multivalent vaccines. By mimicking the natural epitopes of antigens and presenting multiple heterologous neutralizing epitopes, multi-epitope fusion antigens allow a single immunogen (protein) to carry antigenic elements (epitopes or peptides) from multiple virulence determinants, thus possessing broad-spectrum immunogenicity, which is of great significance for the development of PEDV multivalent vaccines. Summary of the Invention

[0005] This invention designs PEDV multi-epitope antigens using synthetic biology design principles and ferritin self-assembly nanotechnology, and obtains recombinant proteins. This can effectively solve the problems of complex circulating viral strains and poor immunization effects of conventional vaccines, and is expected to improve the current situation of frequent diarrhea, high mortality, and poor vaccine protection efficiency in piglets.

[0006] More specifically, the present invention provides a porcine epidemic diarrhea virus multi-epitope fusion protein, its preparation method, and its application.

[0007] In a first aspect, the present invention provides a porcine epidemic diarrhea virus (PEDV) multi-epitope fusion protein, wherein the PEDV multi-epitope fusion protein is any one of the following:

[0008] (1) A protein with the amino acid sequence shown in SEQ ID NO.1;

[0009] (2) A protein with the same function as the amino acid sequence shown in SEQ ID NO.1, which has been substituted and / or deleted and / or added to one or more amino acid residues;

[0010] (3) The protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein described in (1) or (2).

[0011] Secondly, the present invention provides nucleotides encoding a multi-epitope fusion protein of porcine epidemic diarrhea virus.

[0012] The nucleotides of the porcine epidemic diarrhea virus multi-epitope fusion protein provided by the present invention include the nucleotide sequences described in SEQ ID NO.3-10;

[0013] Preferably, the sequence of the nucleotide is as shown in SEQ ID NO.2.

[0014] Thirdly, the present invention provides biological materials containing the above-mentioned nucleotides, wherein the biological materials are recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

[0015] When the biological material is a pET plasmid, it includes, but is not limited to: pET-28a, pET-30a, and pET-32a.

[0016] The present invention also provides the use of the above-mentioned porcine epidemic diarrhea virus multi-epitope fusion protein or the above-mentioned nucleotides in the preparation of porcine epidemic diarrhea virus vaccines or porcine epidemic diarrhea virus antibodies.

[0017] Fourthly, the present invention provides a porcine epidemic diarrhea virus vaccine, wherein the porcine epidemic diarrhea virus vaccine contains the above-mentioned porcine epidemic diarrhea virus multi-epitope fusion protein.

[0018] Fifthly, the present invention provides a porcine epidemic diarrhea virus antibody, which is obtained by immunization with the above-mentioned porcine epidemic diarrhea virus multi-epitope fusion protein as an immunogen.

[0019] In a sixth aspect, the present invention provides a method for preparing the above-mentioned porcine epidemic diarrhea virus multi-epitope fusion protein, using GGSSGG polypeptide as a linker to link the B lymphocyte epitope protein and the T lymphocyte epitope protein of the porcine epidemic diarrhea virus spike protein.

[0020] The nucleotide sequences encoding the B lymphocyte epitope proteins are shown in SEQ ID NO.3-8;

[0021] The nucleotide sequences encoding the T lymphocyte epitope proteins are shown in SEQ ID NO. 9-10.

[0022] More specifically, the method for producing a multi-epitope fusion protein of porcine epidemic diarrhea virus provided by the present invention includes:

[0023] S1. Porcine epidemic diarrhea virus B lymphocyte and T lymphocyte epitope sequences were infused with self-assembled ferritin nanoantigen particles to obtain PEDV-MEFA-Fe.

[0024] The porcine epidemic diarrhea virus B lymphocyte epitope sequences are KRSFIEDLLFNKV, GPRLQPY, TSLLASACTIDLFGYP, SSTFNSTREL, MQYVYEPTYYML, and YSNIGVCK;

[0025] The porcine epidemic diarrhea virus T lymphocyte epitope sequences are AKFVAAWTLKAAA and TAKSKKFPSYTATYQF;

[0026] The nucleotide sequence encoding the ferritin is shown in SEQ ID NO.11;

[0027] S2. The PEDV-MEFA-Fe obtained in S1 was cloned into the vector through BamHI and EcoRI restriction enzyme sites to construct a recombinant plasmid, which was then transformed into competent cells.

[0028] S3. The recombinant plasmid was induced in competent cells, the bacterial cells were collected, the bacterial cells were broken up using lysis buffer to collect inclusion bodies, the inclusion bodies were washed in inclusion body washing buffer, and the porcine epidemic diarrhea virus multi-epitope fusion protein was purified.

[0029] The competent cells used in this invention are Escherichia coli competent cells, including but not limited to: BL21, DH5α, and JM109.

[0030] In the method for producing a multi-epitope fusion protein of porcine epidemic diarrhea virus provided by the present invention, the induction conditions in step S3 are: 0.1-1.0 mmol / L IPTG and 16-37°C.

[0031] The beneficial effects of this invention are as follows:

[0032] The method for preparing PEDV multi-epitope fusion protein provided by this invention effectively presents the B and T lymphocyte epitopes of the spike protein; the fusion protein has good specificity and can significantly improve the efficiency of traditional methods, which is of great significance for solving the common clinical problem of multiple serum in PEDV. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0034] Figure 1 This section describes the preparation and identification of PEDV multi-epitope recombinant proteins. A) Identification of PEDV multi-epitope recombinant proteins: Line 1, uninduced bacterial culture; Line 2, induced supernatant bacterial culture; Line 3, induced bacterial culture precipitate; Line 4, treated recombinant protein; Line M, protein marker. B) Identification of PEDV multi-epitope recombinant proteins: Line 1, PEDV multi-epitope recombinant protein; Line M, protein marker. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0036] Unless otherwise specified, all examples were performed under standard experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2001), or as recommended by the manufacturer’s instructions.

[0037] The lysis buffer consisted of 50 mM Tris-HCl buffer (pH 8.0) and 0.2 M NaCl; the inclusion body washing buffer I consisted of 20 mM Tris-HCl buffer (pH 8.0), 0.2 M NaCl, and 1% Triton X-100; the inclusion body washing buffer II consisted of 20 mM Tris-HCl buffer (pH 8.0), 0.2 M NaCl, 1% Triton X-100, and 2 M urea; the urea NTA-0, urea NTA-25, urea NTA-50, urea NTA-100, urea NTA-250, and urea NTA-500 buffers consisted of 20 mM Tris-HCl buffer (pH 8.0) and 5% glycerol, with imidazole concentrations of 0 mM, 25 mM, 50 mM, 100 mM, 250 mM, and 500 mM, respectively; the refolding buffer consisted of 20 mM Tris-HCl buffer (pH 8.0) and 5% glycerol. Tris-HCl (pH 8.0), 10% sucrose, 0.6 mM Arg, 0.2 mM EDTA and urea, wherein the urea is 6 M, 4 M, 2 M, 1 M or 0 M.

[0038] Example 1

[0039] (1) Design of PEDV multi-epitope antigens based on self-assembled ferritin nanoantigen particles: B cell linear epitopes on PEDV S structural proteins:

[0040] KRSFIEDLLFNKV, amino acid sequence shown in SEQ ID NO.12 (nucleotide sequence of the encoding gene shown in SEQ ID NO.3); GPRLQPY, amino acid sequence shown in SEQ ID NO.13 (nucleotide sequence of the encoding gene shown in SEQ ID NO.4); TSLLASACTIDLFGYP, amino acid sequence shown in SEQ ID NO.14 (nucleotide sequence of the encoding gene shown in SEQ ID NO.5); SSTFNSTREL, amino acid sequence shown in SEQ ID NO.15 (nucleotide sequence of the encoding gene shown in SEQ ID NO.6); MQYVYEPTYYML, amino acid sequence shown in SEQ ID NO.16 (nucleotide sequence of the encoding gene shown in SEQ ID NO.7); and YSNIGVCK, amino acid sequence shown in SEQ ID NO.17 (nucleotide sequence of the encoding gene shown in SEQ ID NO.8).

[0041] Universal T-cell epitopes: AKFVAAWTLKAAA, amino acid sequence shown in SEQ ID NO.18 (nucleotide sequence of the encoding gene shown in SEQ ID NO.9) and TAKSKKFPSYTATYQF, amino acid sequence shown in SEQ ID NO.19 (nucleotide sequence of the encoding gene shown in SEQ ID NO.10), were linked using GGSSGG as a linker, and the optimal combination was obtained through molecular docking and molecular dynamics simulations.

[0042] To improve the expression efficiency of the above sequence fused with ferritin nanoparticles, the asparagine (N) at position 19 of the Helicobacter pylori ferritin amino acid sequence (NCBI GenBank sequence number: WP000949190) was mutated to glutamine (Q) to eliminate the glycosylation site. Then, the first four amino acids of the ferritin amino acid sequence were removed. The optimized ferritin sequence (the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 11) had the PEDV antigen sequence linked to its N-terminus by a linker peptide (SGG). The designed sequences (SEQ ID NO.3-SEQ ID NO.11) were optimized using optimalGene™ technology. The optimized sequences were then modified according to the codon bias of *E. coli*. Optimization was performed on various parameters affecting gene transcription efficiency, translation efficiency, and protein folding, including GC content, CG dinucleotide content, codon bias, mRNA secondary structure, mRNA free energy stability, RNA instability gene sequences, and repetitive sequences, while maintaining the final translated protein sequence. The sequence was named PEDV-MEFA-Fe (its amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence encoding the gene is shown in SEQ ID NO.2). It was cloned into the pET-28a vector using BamHI and EcoRI restriction enzyme sites.

[0043] Example 2: Construction of recombinant plasmid pET-28a-PEDV-MEFA-Fe

[0044] The pET28a vector and the target gene were double-digested using the following system, as shown in Table 1.

[0045] Table 1. Double enzyme digestion system of pET28a vector and target gene

[0046]

[0047] Enzyme digestion conditions: 37℃, water bath digestion for 3 hours.

[0048] The products of double enzyme digestion were purified and recovered, and ligated using T4 ligase. The system is shown in Table 2.

[0049] Table 2. T4 ligase ligation system

[0050]

[0051] Connection conditions: After mixing, connect at 16°C overnight.

[0052] Take competent cells stored at -80℃, add 5μL of the above ligation product, and place on ice for 25min; place in a 42℃ water bath for 45s, then quickly place on ice for 2min; add about 700μL of LB medium to the tube, and incubate at 37℃ and 220rpm for 60min; centrifuge, discard most of the supernatant, and leave 100μL to spread on LB plates containing kanamycin (50μg / mL), and incubate in an inverted 37℃ incubator overnight.

[0053] Pick a single colony from the plate, inoculate it into LB liquid medium (50 μg / mL, Kana), place it in a 37℃ constant temperature shaking incubator, set the rotation speed to 220 rpm, and incubate for 14 h. Extract the plasmid and perform double enzyme digestion.

[0054] The enzyme digestion and sequencing identification of positive clones are shown in Table 3.

[0055] Table 3 Enzyme digestion system

[0056]

[0057] After enzyme digestion at 37℃ for 3 hours, 7 μL of the reaction solution was taken for electrophoresis detection. Plasmids with correct enzyme digestion were sequenced, and the results showed consistency with the target gene sequence. The recombinant plasmid was named pET28a-PEDV-MEFA-Fe.

[0058] Example 3: Expression, purification, and validation of PEDV-MEFA-Fe recombinant protein

[0059] (1) Recombinant plasmid was induced to express in Escherichia coli.

[0060] The correctly identified recombinant expression plasmid pET-28a-PEDV-MEFA-Fe was transformed into *E. coli* BL21(DE3) competent cells. After recovery, the cells were plated on solid medium and cultured overnight. The next day, a single clone was picked and inoculated into 10 mL of LB liquid medium (100 μg / mL, Kana) and cultured overnight at 37°C. The following day, 1% of the bacterial culture was transferred to 100 mL of LB liquid medium (100 μg / mL, Kana) and cultured with shaking at 37°C. When the OD600 value was approximately 0.6, IPTG (final concentration 1 mmol / L) was added, and the cells were cultured at 37°C for another 8 h. The cells were then collected by centrifugation at 4°C, 5000 rpm, for 10 min, and the bacterial pellet was washed with sterile PBS. Resuspend the bacterial cells in total cell protein extract (Thermo Scientific, 78248) at a volume of 4 mL / g of cells and incubate at room temperature for 15 min to lyse the cells (or resuspend the cells in lysis buffer at a volume of 100 μL lysis buffer / mL of bacterial culture, incubate on ice for 30 min, and then sonicate on ice to lyse the cells); centrifuge at 12,000 rpm for 10 min at 4 °C, discard the supernatant, and the precipitate is the recombinant protein inclusion bodies; wash the precipitate with appropriate amounts of inclusion body washing buffer I and II, discard the supernatant; resuspend the precipitate with an appropriate amount of urea NTA-O buffer and incubate overnight at 4 °C with stirring to dissolve.

[0061] (2) Purification of recombinant protein by nickel column affinity chromatography

[0062] Take the inclusion body solution dissolved overnight, centrifuge at 12,000 rpm for 15 min at 4 °C, collect the supernatant and filter it through a 0.45 μm membrane; purify the expressed protein using a Ni-NTA resin chromatography column, collecting the eluent at five gradients: urea NTA-25, urea NTA-50, urea NTA-100, urea NTA-250, and urea NTA-500.

[0063] After purification, the recombinant protein was observed to be a single, correctly sized band after SDS-PAGE electrophoresis. The purified protein sample was then placed in a dialysis bag and annealed in annealing buffers of different urea concentrations (6M, 4M, 2M, 1M, 0M) at 4°C for approximately 6 hours at each urea concentration. High-purity PEDV multi-epitope fusion protein was obtained by dialysis, with a concentration of approximately 70.87 μg / mL determined using the BCA method. The amino acid sequence of the fusion protein was identified by gel mass spectrometry and is shown in SEQ ID NO. 1.

[0064] (3) Verification of the results of PEDV-MEFA-Fe neutralization epitopes using Western blotting

[0065] After separation of the fusion protein by SDS-PAGE, the gel was rinsed with double-distilled water and transferred to electrophoresis buffer. The PVDF membrane was immersed in methanol (100%) for 30 seconds. From bottom to top, the following components were placed: sponge, thick filter paper, PVDF membrane, gel block, thick filter paper, and sponge. The assembled gel block rack was placed in the inner tank of the electrophoresis apparatus. The electrophoresis apparatus was placed in a container filled with an ice-water mixture to prepare for electrophoresis. Transfer parameters: 100V, approximately 60 minutes. After transfer, the gel block was transferred to a tray containing gel staining. Blocking buffer (TBS, 5% skim milk) was added to the container containing the PVDF membrane to ensure the membrane was submerged and free of air bubbles. The mixture was incubated on a shaker at room temperature for 1 hour. After electroporation, clean the instrument, sponge, filter paper, etc., and place them in an empty place to air dry. Discard the blocking solution, wash the PVDF membrane 4 times on a shaker with TBST for 5 min each time, add anti-PEDV IgY antibody diluted with 5% skim milk (1:100), remove air bubbles, place it flat on the shaker, and incubate at 4°C for 12 h. Collect the primary antibody in a centrifuge tube for future use, wash the PVDF membrane 5 times (TBST, 0.5% Tween-20) for 5 min each time to wash away excess or non-specifically bound antibodies. Add goat anti-chicken IgY secondary antibody (abcam, ab6753, 1:5000) and incubate at room temperature for 1 h. Then wash again using the above washing method, and discard the TBST rinse solution. Develop the color using ECL chemiluminescence solution (Beyotime, P0018AS).

[0066] Test results as follows Figure 1 As shown in the figure, the experimental results indicate that the PEDV multi-epitope antigen based on self-assembled ferritin nanoantigen particles can react with PEDV-specific IgY.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A multi-epitope fusion protein of porcine epidemic diarrhea virus, characterized in that, The porcine epidemic diarrhea virus multi-epitope fusion protein is any one of the following: (1) A protein with the amino acid sequence shown in SEQ ID NO.1; (2) A protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus of the protein described in (1).

2. Nucleotides encoding the porcine epidemic diarrhea virus multi-epitope fusion protein of claim 1.

3. The nucleotide according to claim 2, characterized in that, The sequence of the nucleotide is shown in SEQ ID NO.

2.

4. A biomaterial, characterized in that, The biological material contains the nucleotides described in claim 2 or 3, wherein the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria, or transgenic cell line.

5. The use of the porcine epidemic diarrhea virus multi-epitope fusion protein of claim 1 or the nucleotide of claim 2 or 3 in the preparation of a porcine epidemic diarrhea virus vaccine.

6. A porcine epidemic diarrhea virus vaccine, characterized in that, The porcine epidemic diarrhea virus vaccine contains the porcine epidemic diarrhea virus multi-epitope fusion protein as described in claim 1.

7. A method for preparing the porcine epidemic diarrhea virus multi-epitope fusion protein according to claim 1, characterized in that, include: S1, PEDV-MEFA-Fe with the nucleotide sequence shown in SEQ ID NO.2 is passed through... BamH I and EcoR The restriction enzyme site was cloned into the vector to construct a recombinant plasmid, which was then transformed into competent cells; S2. The recombinant plasmid was induced in competent cells, the bacterial cells were collected, the bacterial cells were broken up using lysis buffer to collect inclusion bodies, the inclusion bodies were washed in inclusion body washing buffer, and the porcine epidemic diarrhea virus multi-epitope fusion protein was purified.

8. The method according to claim 7, characterized in that, The induction conditions described in step S2 are: 0.1~1.0 mmol / L IPTG, 16℃~37℃.

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