Recombinant protein of classical swine fever virus and application thereof

Abstract

The present disclosure provides a recombinant E2 protein of classical swine fever virus (CSFV) comprising an amino acid sequence as set forth in SEQ ID NO: 2, or an amino acid sequence having at least 85% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2. The recombinant E2 protein of the present disclosure can comprise a point mutation at one or more of amino acids at positions 48, 63, 77, 81, 86, 95, 103, and 129. The present disclosure also provides a nucleic acid molecule encoding the recombinant E2 protein, a host cell comprising the nucleic acid molecule, an immunogenic composition comprising the recombinant E2 protein, and uses thereof.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of veterinary vaccines, and particularly relates to an immunogenic E2 recombinant protein against classical swine fever virus and use thereof. BACKGROUND

[0002] Classical swine fever (CSF) is a severe infectious disease caused by classical swine fever virus (CSFV) and is listed in the OIE (World Organization for Animal Health) as a disease that must be reported. China is a large pig-raising country, and CSF has been an important infectious disease that seriously threatens the pig industry.

[0003] CSFV is a single-stranded positive-sense RNA virus with a capsid, and the full-length genome is about 12.3 kb, encoding a polyprotein composed of 3,898 amino acid residues. The polyprotein is processed into 4 structural proteins (C, E rns , E1 and E2) and 8 non-structural proteins (N pro , p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) by proteases encoded by the virus and host cell proteases during and after translation. E1, E2 and E rns are important virulence factors and major protective antigens. E2 is the capsid protein of CSFV and is the most important structural glycoprotein of the virus, which can induce strong neutralizing antibodies to resist the attack of lethal CSFV virulent strains and is an important target protein for the development of a genetically engineered vaccine against swine fever.

[0004] The combination of preventive vaccination and culling strategy is the main means to control the outbreak of swine fever, and it is of great significance to provide a vaccine that can produce good immune protection against the currently widespread swine fever virus. Although there are currently E2 antigen subunit vaccines expressed by E. coli and baculovirus, mammalian cells with perfect post-translational modification function are still the first choice for most biological protein expression hosts. SUMMARY

[0005] To solve one of the above technical problems in the prior art, the present application provides a recombinant E2 protein of classical swine fever virus, which can effectively improve the protein expression amount by deleting the transmembrane domain and mutating at specific sites, and does not affect the immunogenicity of the E2 protein.

[0006] According to one aspect, a recombinant E2 protein of classical swine fever virus is provided, which lacks the transmembrane domain at the N-terminus and / or C-terminus relative to the wild-type E2 protein. In some embodiments, the wild-type E2 protein includes the amino acid sequence shown in SEQ ID NO: 1.

[0007] In some embodiments, the recombinant E2 protein comprises an amino acid sequence as set forth in SEQ ID NO: 2, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2.

[0008] In some embodiments, the recombinant E2 protein has a mutation at one or more of amino acid positions 48, 63, 77, 81, 86, 95, 103, and 129 based on the amino acid sequence as set forth in SEQ ID NO: 4.

[0009] In some specific embodiments, the cysteine at position 48 (C48) of the recombinant E2 protein can be mutated to any one of serine (S), alanine (A), glycine (G), threonine (T), and proline (P).

[0010] In some specific embodiments, the arginine at position 63 (R63) of the recombinant E2 protein can be mutated to any one of alanine (A), glutamine (Q), and glutamic acid (E).

[0011] In some specific embodiments, the serine at position 77 (S77) of the recombinant E2 protein can be mutated to any one of alanine (A), threonine (T), and glycine (G).

[0012] In some specific embodiments, the glutamic acid at position 81 (E81) of the recombinant E2 protein can be mutated to arginine (R) or lysine (K).

[0013] In some specific embodiments, the glycine at position 86 (G86) of the recombinant E2 protein can be mutated to any one of alanine (A), arginine (R), and glutamine (Q).

[0014] In some specific embodiments, the glycine at position 95 (G95) of the recombinant E2 protein can be mutated to any one of alanine (A), arginine (R), and glutamine (Q).

[0015] In some specific embodiments, the cysteine at position 103 (C103) of the recombinant E2 protein can be mutated to any one of serine (S), alanine (A), glycine (G), threonine (T), and proline (P).

[0016] In some embodiments, the cysteine at position 129 (C129) of the recombinant E2 protein can be mutated to any one of serine (S), alanine (A), glycine (G), threonine (T), and proline (P).

[0017] In some embodiments, the recombinant E protein can comprise any one of C48S, C48A, C48G, C48T, and C48P; any one of R63A, R63Q, and R63E; any one of S77A, S77T, and S77G; E81R or E81K; any one of G86A, G86R, and G86Q; any one of G95A, G95R, and G95Q; any one of C103S, C103A, C103G, C103T, and C103P; and / or, any one of C129S, C129A, C129G, C129T, and C129P. In some embodiments, the recombinant E protein can comprise any combination of the above mutations.

[0018] In some embodiments, the recombinant E2 protein can comprise one or more of the following mutations: C48P, R63E, S77A, G86A, G95R, G95Q, C103S, C103A, R63A / S77A, R63E / S77A, R63Q / S77T, R63E / S77T, E81K / G86R, and E81K / G86Q.

[0019] In some embodiments, the recombinant E2 protein can have an amino acid sequence as set forth in any one of SEQ ID NOs: 5-48, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity thereto.

[0020] The numbering of the mutation sites referred to in the present disclosure is based on the amino acid sequence set forth in SEQ ID NO: 2.

[0021] In some embodiments, the N-terminus or C-terminus of the recombinant E2 protein can have one or more tags selected from His, c-myc, FLAG, Strep-tag.

[0022] In some embodiments, the N-terminus of the recombinant E2 protein can have a signal peptide. In preferred embodiments, the signal peptide can have an amino acid sequence as set forth in SEQ ID NO: 49.

[0023] According to another aspect, there is provided a nucleic acid molecule encoding the recombinant E2 protein of the present disclosure. According to another aspect, there is provided a nucleic acid molecule encoding the recombinant E2 protein of the present disclosure.

[0024] According to yet another aspect, there is provided a host cell comprising a nucleic acid molecule encoding the recombinant E2 protein of the present disclosure. In some embodiments, the host cell can be selected from the group consisting of E. coli, yeast, mammalian cells and insect cells. In specific embodiments, the host cell can be selected from the group consisting of mammalian cells. In preferred embodiments, the host cell can include, but not limited to, human embryonic kidney (HEK) cells, Chinese hamster ovary (CHO) cells and African green monkey kidney cells (Vero cells). In preferred embodiments, the host cell can be selected from the group consisting of CHO-K1, CHO-S, CHO-DXB11, CHO-DG44, CHOZN GS, CHOK1SV GS-KO cells.

[0025] According to yet another aspect, there is provided an immunogenic composition comprising the recombinant E2 protein as described above and a pharmaceutically acceptable carrier. In some embodiments, the carrier can include, for example, aluminum salts (e.g. aluminum hydroxide, aluminum phosphate and potassium aluminum sulfate), oil adjuvants (e.g. MF59), nucleic acid adjuvants (e.g. CpG), protein adjuvants, liposomes.

[0026] In some embodiments, the immunogenic composition can be used to immunize a suid (e.g. a pig) against at least one disease associated with a classical swine fever virus.

[0027] In some embodiments, the immunogenic composition can be used for administration by injection, aerosol delivery, intranasal administration, oral, topical administration or a combination thereof.

[0028] According to yet another aspect, there is provided use of the immunogenic composition of the present disclosure in the treatment or prevention of a disease associated with a classical swine fever virus (CSFV).

[0029] According to yet another aspect, there is provided use of the immunogenic composition of the present disclosure in the manufacture of a medicament for the treatment or prevention of a disease associated with a classical swine fever virus (CSFV).

[0030] The present disclosure improves the expression of E2 protein by deleting the transmembrane domain of the N-terminus and C-terminus of the classical swine fever virus E2 protein, and has a better immunization effect than commercially available swine fever vaccines. By mutating some specific sites of the truncated E2 protein, the expression of the E2 protein can be further improved, and the immunogenicity of the E2 protein is not affected. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 SDS-Page detection results of Truncated_E2 protein according to embodiments of the present disclosure are shown.

[0032] Figure 2Detection results of antibody titers after immunization of Truncated_E2 protein mutants according to embodiments of the present disclosure are shown.

[0033] Figure 3 Detection results of antibody titers after immunization of Truncated_E2 protein mutants according to embodiments of the present disclosure are shown.

[0034] Figure 4 Detection results of antibody titers after immunization of Truncated_E2 protein mutants according to embodiments of the present disclosure are shown. DETAILED DESCRIPTION

[0035] In order to make the objects, technical solutions, and advantages of the present application clearer, the following further describes the present application in conjunction with embodiments. The specific embodiments described herein are only used to explain the present application and do not constitute any limitation on the present application. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following definitions are applied to the descriptions and claims herein, and throughout this document, unless otherwise expressly provided herein. Further, the terms "a" and "an" used herein include plural references unless the context clearly dictates otherwise. As used herein, the indefinite article "a" is intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0037] Unless the context clearly indicates otherwise, as used herein the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0038] As used herein, the term "about" means ± the range of values recited. In some embodiments, the term "about" means ± the range of values recited. In some embodiments, the term "about" means ± the range of values recited.

[0039] "Percent (%) sequence identity" with respect to a reference amino acid sequence means the percentage of amino acid residues in a candidate sequence that have the same residue as the reference amino acid sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, exclusive of any conservative substitutions that are part of the sequence identity. To determine percent amino acid sequence identity, the sequences can be aligned using a variety of methods within the skill in the art, such as using BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0040] The term "classical swine fever virus" or "CSFV" as used herein refers to all viruses belonging to the classical swine fever virus (CSFV) species of the Pestivirus genus in the Flaviviridae family. The E2 protein of the porcine pestivirus is an important envelope glycoprotein of CSFV, also known as gp55, and is the major antigenic protein of the virus. The E2 protein is able to induce neutralizing antibodies against the virus and is the major immunoprotective antigen of the porcine pestivirus and an important target protein for the development of genetically engineered vaccines against porcine pestivirus.

[0041] In order to make the objectives, technical solutions, and advantages of the present application clearer, the present application will be described in further detail below with reference to the embodiments. The specific embodiments described herein are intended for illustration only and are not intended to be limiting in any way. Moreover, in the following embodiments, the description of well-known structures and techniques has been omitted in order to avoid obscuring the concept of the present disclosure. Such structures and techniques are described in numerous publications such as Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press.

[0042] The following examples and drawings are provided to aid understanding of the present application. It should be understood, however, that these examples and drawings are set forth only for illustration and that the present application is not limited thereto. The actual scope of the present application is set forth in the appended claims. It should be understood that any modification and alteration can be made without departing from the spirit of the present application.

[0043] Example

[0044] Example 1. Expression of E2 protein and Truncated_E2 protein in CHO-K1 cells

[0045] 1) Construction of recombinant plasmid

[0046] The nucleic acid sequence encoding wild type E2 (WT-E2) protein (SEQ ID NO: 3) and Truncated_E2 protein (SEQ ID NO: 4) was synthesized by Suzhou Jinyuzhi Biotechnology Co., Ltd. The N-terminus of WT-E2 and Truncated_E2 protein both contained a signal peptide sequence (MDWTWRVFCLLAVAPGVHS). The gene was synthesized by conventional means, and the nucleic acid sequence encoding WT-E2 and Truncated_E2 was cloned into the vector pUC-GW-Kan (Jinyuzhi Biotechnology Co., Ltd.) respectively, to prepare recombinant plasmids pUC-GW-Kan-WT-E2 and pUC-GW-Kan-Truncated_E2 respectively. Then the expression plasmid of the target protein was constructed. Briefly, the expression vector pCDNA3.1(+) (Invitrogen), recombinant plasmids pUC-GW-Kan-WT-E2 and pUC-GW-Kan-Truncated_E2 were digested by Hind III and BamH I respectively, and the recovered target fragments WT-E2 and Truncated_E2 were ligated with the linearized expression vector pCDNA3.1(+), to obtain the expression plasmids pCDNA3.1(+)-E2 and pCDNA3.1(+)-Truncated_E2 of E2 protein and Truncated_E2 protein respectively, which were sequenced correctly. For the convenience of purification, a His tag was added at the C-terminus of the recombinant protein.

[0047] 2) CHO-K1 cell expressed protein

[0048] Chinese hamster ovary cells CHO-K1 (Merck) were used as host cells for expression of wild type E2 (WT-E2) protein and Truncated_E2 protein. The constructed expression plasmids pCDNA3.1(+)-E2 and pCDNA3.1(+)-Truncated_E2 were transfected into CHO cells respectively. After 48 hours of transfection, complete medium containing 600 μg / mL G418 (+ 4 mM L-Glutamine) was added for culture, and the complete medium containing G418 was replaced every 3 to 6 days until the survival rate recovered to more than 90%, the selection pressure was removed, and stable cell pools were obtained, which could stably express E2 protein.

[0049] 3) Purification and detection

[0050] (1) The cell culture supernatant was treated by centrifugation at 5000 rpm for 20 min, and then filtered through a 0.22 μm filter membrane (Millipore).

[0051] (2) The culture supernatant was purified by Ni Sephorase Excel (Cytiva) filler. The E2 protein was specifically captured by the specific binding of Ni2+ and His-tag, and then eluted by a buffer containing 250 mM imidazole.

[0052] (3) The pore size of the ultrafiltration tube (Millipore) was 30KD. The PBS buffer at pH 8.0 was replaced, and the PBS solution was added in equal volume to dilute and concentrate one time. This was repeated 5 times. The A280 value was detected by spectrophotometer, and the detection results are shown in Table 1.

[0053] Table 1.

[0054] Protein A280 E2 0.21 mg / mL Truncated_E2 3.20 mg / mL

[0055] According to the expression results shown in Table 1, it can be seen that the expression amount of WT-E2 is low, but Truncated_E2 obtains a higher expression amount by removing the N-terminal and C-terminal transmembrane region, and the expression amount in the cell pool stage can reach 3.2g / L.

[0056] (4) The sample was mixed with 5X protein loading buffer (Biyun Tian) to make a loading system at a ratio of 4:1, and the Truncated_E2 protein was denatured and combined with SDS at 100℃ for 10min. The sample was spotted into the well of 4-20% SurePAGE, and the gel was run at a voltage of 140V for 60min. Then, Coomassie brilliant blue was used for staining and acetic acid was used for decolorization. The SDS-Page detection results are shown in Figure 1 .

[0057] 4) Preparation and effect detection of subunit vaccine

[0058] After the Truncated_E2 protein was diluted with PBS, the adjuvant Gel 02 (SEPPIC company) was used to prepare the vaccine according to the adjuvant instruction. 15 weaned piglets were randomly divided into 3 groups, 5 in each group, of which 1 group was injected with Truncated_E2 protein vaccine 50μg / head, 2 group was injected with commercial swine fever vaccine, and 3 group was injected with PBS. The second immunization was performed 21 days after the first immunization. After the pigs were immunized, the body temperature of the pigs was monitored and recorded daily, and the diet and mental state were observed. ELISA antibody was detected by blood sampling on the 7th, 14th and 21st day after the first immunization, and ELISA antibody was detected by blood sampling on the 7th, 14th and 21st day after the second immunization.

[0059] The blocking rate of the antibody was detected by using the swine fever virus antibody detection kit (IDEXX) according to the instruction. The immunization results are shown in Table 2:

[0060]

[0061] Figure 2The antibody titer results of each group after immunization are shown in Table 1.

[0062] As can be seen from Table 2 and Figure 2 As can be seen from the results, the Truncated_E2 subunit vaccine had a 100% positive rate after immunization, and the pigs were healthy during the test period. The blocking rates at 14 days after the first immunization and 21 days after the first immunization were higher than those of the commercially available vaccine group. The antibody blocking rates of the Truncated_E2 subunit vaccine at 21 days after the first immunization and 7 days after the second immunization were both greater than 70%, and the antibody blocking rates at 21 days after the second immunization were all greater than 80%, indicating that it is sufficient to protect the pig population from infection of the classical swine fever virus and can be used for prevention and control of domestic classical swine fever.

[0063] Example 2. Construction of Truncated_E2 protein mutants and detection of expression amounts

[0064] 1) Construction of recombinant plasmid

[0065] The coding nucleic acid sequence of the Truncated_E2 protein mutant (N-terminal with signal peptide sequence MDWTWRVFCLLAVAPGVHS) was synthesized by Suzhou Jinyuzhi Biotechnology Co., Ltd. The gene was cloned into the vector pUC-GW-Kan by conventional synthesis to prepare the Truncated_E2 protein mutant recombinant plasmid. Subsequently, the protein expression plasmid was constructed, the expression vector pCDNA3.1(+) and the protein mutant recombinant plasmid DNA were double-digested by Hind III and BamH I, and the recovered Truncated_E2 protein mutant fragments were respectively ligated with the linearized expression vector pCDNA3.1(+), to obtain the expression plasmid of each Truncated_E2 protein mutant. After sequencing, it was used for CHO-K1 (Merck) cell transfection. In order to facilitate purification, a His tag was added at the C-terminal end of the recombinant protein. The mutation site and sequence of the Truncated_E2 protein mutant are shown in Table 3.

[0066] Table 3. Mutation sites of each Truncated_E2 protein mutant.

[0067]

[0068]

[0069] 2) CHO-K1 cell expression protein

[0070] Chinese hamster ovary cells CHO-K1 (Merck) were used as host cells for expression of the Truncated_E2 protein mutants. The expression plasmids of each Truncated_E2 protein mutant were transfected into CHO cells, respectively. After 48 hours of transfection, complete medium containing 600 μg / mL G418 (+ 4 mM L-glutamine) was added for culture, and the complete medium containing G418 was replaced every 3 to 6 days until the viability was recovered to more than 90%, the selection pressure was removed, and stable cell pools were obtained, which could stably express E2 protein.

[0071] 3) Purification and detection

[0072] (1) The culture supernatant was treated by centrifugation at a speed of 5000 rpm for 20 min and then filtered through a 0.22 μm filter membrane (Millipore).

[0073] (2) The culture supernatant was purified by Ni Sephorase Excel filler (Cytiva) to specifically capture E2 protein through the specific binding of Ni2+ to His-tag, and then eluted with a buffer containing 250 mM imidazole.

[0074] (3) The ultrafiltration tube (Millipore) with a pore size of 30 KD was replaced with PBS buffer at pH 8.0, and the PBS solution was added in equal volume to dilute and concentrate one time, and this was repeated 5 times. The A280 value was detected by spectrophotometer, and the detection results are shown in Table 4.

[0075] Table 4. Detection values of each Truncated E2 protein mutant at A280.

[0076]

[0077]

[0078] According to the detection results shown in Table 4, Truncated_E2_M05, Truncated_E2_M08, Truncated_E2_M09, Truncated_E2_M14, Truncated_E2_M18, Truncated_E2_M19, Truncated_E2_M20, Truncated_E2_M21, Truncated_E2_M30, Truncated_E2_M32, Truncated_E2_M34, Truncated_E2_M35, Truncated_E2_M42, and Truncated_E2_M44 were selected for SDS-Page detection.

[0079] (4) After diluting the sample 3 times, a sample loading system is prepared with 5X protein loading buffer at a ratio of 4:1, and the system is incubated at 100°C for 10 min to denature the protein and bind it to SDS. The sample is loaded into the wells of a 4-20% SurePAGE, and the gel is run at a voltage of 140V for 60 min. Coomassie brilliant blue is used for staining, and acetic acid is used for decolorization. The results of SDS-Page detection are shown in Figure 3 .

[0080] 4) Preparation of subunit vaccine and detection of effects

[0081] The Truncated_E2 protein mutants Truncated_E2_M14, Truncated_E2_M32, and Truncated_E2_M44 are diluted with PBS, and a vaccine is prepared using the adjuvant Gel 02 (SEPPIC) according to the instructions of the adjuvant. Twenty weaned piglets are randomly divided into four groups, with five piglets in each group. Groups 1, 2, and 3 are injected with 50 μg of the Truncated_E2 protein mutant per piglet, and group 4 is injected with PBS. The piglets are immunized twice, with the second immunization being performed 21 days after the first immunization. After immunization, the body temperature of the piglets is monitored and recorded daily, and the diet and mental state of the piglets are observed. Blood samples are collected 7, 14, and 21 days after the first immunization for ELISA antibody detection, and blood samples are collected 7, 14, and 21 days after the second immunization for ELISA antibody detection.

[0082] The blocking rate of the antibodies is detected using a porcine pestivirus antibody detection kit (IDEXX) according to the instructions. The results of the immunization are shown in Figure 4 , Figure 4 The results of detection of the antibody titers after immunization of the three Truncated_E2 protein mutants are given. As can be seen from Figure 4 , the Truncated_E2 protein mutants do not significantly affect the immunogenicity of the E2 protein.

[0083] The technical solutions of the present application are not limited to the above specific embodiments, and any technical variations made according to the technical solutions of the present application fall within the scope of protection of the present application.

Claims

1. A recombinant E2 protein of classical swine fever virus (CSFV), characterized in that, The amino acid sequence of the recombinant E2 protein is shown in any one of SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 36 and SEQ ID NO:

48.

2. The recombinant E2 protein according to claim 1, characterized in that, The recombinant E2 protein has one or more tags selected from His, c-myc, FLAG, and Strep-tag at its N-terminus or C-terminus.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the recombinant E2 protein as described in claim 1 or 2.

4. A host cell, characterized in that, The host cell includes the nucleic acid molecule as described in claim 3.

5. The host cell according to claim 4, characterized in that, The host cells are selected from Escherichia coli, yeast, mammalian cells, and insect cells.

6. The host cell according to claim 4, characterized in that, The host cell is selected from mammalian cells.

7. The host cell according to claim 4, characterized in that, The host cells were selected from human embryonic kidney (HEK) cells, Chinese hamster ovary (CHO) cells, and African green monkey kidney cells (Vero cells).

8. An immunogenic composition, characterized in that, The immunogenic composition comprises the recombinant E2 protein as described in claim 1 or 2 and a pharmaceutically acceptable carrier.

9. The use of the immunogenic composition of claim 8 in the preparation of a medicament for the prevention of classical swine fever virus (CSFV).

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