CN115925828A - Classical swine fever virus recombinant protein and application thereof - Google Patents

Abstract

The present disclosure provides a recombinant E2 protein of Classical Swine Fever Virus (CSFV), said recombinant E2 protein 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 an amino acid sequence as set forth in SEQ ID NO: 2. Recombinant E2 proteins of the present disclosure may include point mutations at one or more of amino acids 48, 63, 77, 81, 86, 95, 103, and 129. The disclosure also provides nucleic acid molecules encoding the recombinant E2 proteins, host cells containing the nucleic acid molecules, immunogenic compositions containing the recombinant E2 proteins, and uses thereof.

Description

Classical swine fever virus recombinant protein and application thereof

Technical Field

The invention belongs to the field of veterinary vaccines, and particularly relates to an E2 recombinant protein with immunogenicity for swine fever viruses and application thereof.

Background

Classical Swine Fever (CSF) is a virulent infectious disease caused by Classical Swine Fever Virus (CSFV), and is listed as an animal epidemic disease name list that must be declared by the world animal health Organization (OIE). China is a big pig-raising country, and CSF has always been an important infectious disease that seriously threatens the pig-raising industry.

CSFV is a enveloped, single-stranded, positive-stranded RNA virus with a genome of about 12.3kb in length, which encodes a polyprotein consisting of 3,898 amino acid residues. The polyprotein is processed and hydrolyzed into 4 structural proteins (C, E) under the action of protease encoded by virus and host cell protease during and after translation ^rns E1 and E2) and 8 non-structural proteins (N) ^pro P7, NS2, NS3, NS4A, NS4B, NS5A and NS 5B). E1, E2 and E ^rns Are important virulence factors and major protective antigens. E2 is envelope protein of CSFV, is the most important structural glycoprotein of virus, can induce stronger neutralizing antibody to resist the attack of lethal CSFV virulent strain, and is an important target protein for developing hog cholera genetic engineering vaccine.

The combination of implementation of preventive vaccination and a killing strategy is a main means for controlling the outbreak of the swine fever, and the provision of a vaccine which can generate a good immune protection effect on the swine fever viruses which are widely prevalent at present has important significance. Although there are swine fever E2 antigen subunit vaccines expressed by Escherichia coli, baculovirus and the like, mammalian cells with complete post-translational modification functions are still the first choice for most biological drug protein expression hosts.

Disclosure of Invention

In order to solve one of the technical problems in the prior art, the invention provides a recombinant E2 protein of classical swine fever virus, which can effectively improve the expression level of the protein without influencing the immunogenicity of the E2 protein by deleting a transmembrane domain and mutation at a specific site.

According to one aspect, there is provided a recombinant E2 protein of a classical swine fever virus lacking the N-terminal and/or C-terminal transmembrane domain relative to the wild-type E2 protein. In some embodiments, the wild-type E2 protein comprises the amino acid sequence set forth as SEQ ID NO 1.

In some embodiments, the recombinant E2 protein comprises an amino acid sequence 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 set forth in SEQ ID No. 2.

In some embodiments, the recombinant E2 protein has a mutation at one or more of amino acids 48, 63, 77, 81, 86, 95, 103, and 129 based on the amino acid sequence shown as SEQ ID No. 4.

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

In some embodiments, the arginine 63 (R63) of the recombinant E2 protein may be mutated to any one of alanine (a), glutamine (Q), and glutamic acid (E).

In some embodiments, the serine at position 77 (S77) of the recombinant E2 protein may be mutated to any one of alanine (a), threonine (T), and glycine (G).

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

In some embodiments, the glycine at position 86 (G86) of the recombinant E2 protein may be mutated to any one of alanine (a), arginine (R) and glutamine (Q).

In some embodiments, the glycine at position 95 (G95) of the recombinant E2 protein may be mutated to any one of alanine (a), arginine (R) and glutamine (Q).

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

In some embodiments, the cysteine 129 (C129) of the recombinant E2 protein may be mutated to any one of serine (S), alanine (a), glycine (G), threonine (T) and proline (P).

In some embodiments, the recombinant E protein may 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 of C129S, C129A, C129G, C129T and C129P. In some embodiments, the recombinant E protein may include any combination of the above mutations.

In some embodiments, the recombinant E2 protein may include 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.

In some embodiments, the recombinant E2 protein may 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.

The numbering of the mutation sites mentioned in this disclosure is based on the amino acid sequence shown in SEQ ID NO. 2.

In some embodiments, the recombinant E2 protein may have one or more tags selected from His, C-myc, FLAG, strep-tag at the N-terminus or C-terminus.

In some embodiments, the N-terminus of the recombinant E2 protein may have a signal peptide. In a preferred embodiment, the signal peptide may have an amino acid sequence as shown in SEQ ID NO. 49.

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

According to yet another aspect, a host cell is provided that includes a nucleic acid molecule encoding a recombinant E2 protein of the disclosure. In some embodiments, the host cell may be selected from the group consisting of escherichia coli, yeast, mammalian cells, and insect cells. In particular embodiments, the host cell may be selected from mammalian cells. In preferred embodiments, the host cells may include, but are not limited to, human Embryonic Kidney (HEK) cells, chinese Hamster Ovary (CHO) cells, and Vero cells. In a preferred embodiment, the host cell may be a CHO-K1, CHO-S, CHO-DXB11, CHO-DG44, CHOZN GS, CHOK1SV GS-KO cell.

According to yet another aspect, an immunogenic composition is provided comprising the above recombinant E2 protein and a pharmaceutically acceptable carrier. In some embodiments, the carrier can include, for example, aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate), oil adjuvants (e.g., MF 59), nucleic acid adjuvants (e.g., cpG), protein-based adjuvants, liposomes.

In some embodiments, the immunogenic composition can be used to immunize a porcine animal (e.g., a pig) against at least one swine fever virus-associated disease.

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

According to a further aspect, there is provided the use of an immunogenic composition of the disclosure in the treatment or prevention of a disease associated with Classical Swine Fever Virus (CSFV).

According to a further aspect, there is provided the use of an immunogenic composition of the disclosure in the manufacture of a medicament for the treatment or prevention of a disease associated with Classical Swine Fever Virus (CSFV).

According to the invention, the expression level of the E2 protein is improved by deleting the transmembrane domains at the N end and the C end of the classical swine fever virus E2 protein, and the vaccine has a better immune effect compared with a commercially available swine fever vaccine. The expression level of the E2 protein can be further improved by mutating some specific sites of the truncated E2 protein, and the immunogenicity of the E2 protein is not influenced.

Drawings

Fig. 1 shows SDS-Page detection results of a Truncated _ E2 protein according to an embodiment of the present disclosure.

Fig. 2 shows the detection results of antibody titers after immunization with a Truncated _ E2 protein according to embodiments of the present disclosure.

Fig. 3 shows SDS-Page detection results of Truncated _ E2 protein mutants according to embodiments of the present disclosure.

Fig. 4 shows the detection results of the antibody titer after immunization of the Truncated _ E2 protein mutant according to the embodiment of the present disclosure.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. The specific embodiments described herein are merely illustrative of the invention and do not constitute any limitation thereon. Moreover, in the following description, descriptions of well-known structures and techniques are omitted so as to not unnecessarily obscure the concepts of the present disclosure. Such structures and techniques are also described in numerous publications.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purpose of explaining the present specification, the following definitions will apply and, where appropriate, terms used in the singular will also include the plural and vice versa.

As used herein, the terms "a" and "an" include plural references 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.

The term "about" as used herein means the range of values thereafter used within the text. In some embodiments, the term "about" means ± range of the numerical value thereafter. In (c) is used. In some embodiments, the term "about" denotes the range of ± range of the numerical value following it.

"percent (%) sequence identity" with respect to a reference amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in the reference amino acid sequence, after aligning the sequences and (if necessary) introducing gaps to achieve the maximum percent sequence identity, but without regard to any conservative substitutions as part of the sequence identity. To determine percent amino acid sequence identity, alignments can be performed in a variety of ways within the art, for example, using BLAST, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared.

The term "classical swine fever virus" or "CSFV" as used herein refers to all viruses of the Classical Swine Fever Virus (CSFV) species belonging to the genus pestivirus in the family flaviviridae. The E2 protein of classical swine fever virus is an important envelope glycoprotein of CSFV, also called gp55, and is the main antigen protein of virus. The E2 protein can induce and generate a virus neutralizing antibody, is a main immunoprotective antigen of the swine fever virus, and is an important target protein for researching a swine fever genetic engineering vaccine.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. The specific embodiments described herein are merely illustrative of the invention and do not constitute any limitation on the invention. Furthermore, in the following embodiments, descriptions of well-known structures and techniques are omitted so as to not unnecessarily obscure the concepts of the present disclosure. Such structures and techniques are also described in a number of publications, such as Sambrook, j., fritsch, e.f., and maniis, t. (1989) Molecular Cloning: a Laboratory Manual,2nd edition, cold spring Harbor Laboratory Press.

The following examples and figures are provided to aid in the understanding of the present invention. It is to be understood that these examples and drawings are illustrative of the invention and are not to be construed as limiting in any way. The actual scope of the invention is set forth in the following claims. It is to be understood that any modifications and variations may be made without departing from the spirit of the invention.

Examples

Example 1 CHO-K1 cells express E2 protein and Truncated _ E2 protein

1) Construction of recombinant plasmid

The nucleic acid sequences encoding the wild-type E2 (WT-E2) protein (SEQ ID NO: 3) and the Truncated _ E2 protein (SEQ ID NO: 4) were synthesized by Kinzhi Biotech, suzhou, and the N-terminus of both WT-E2 and Truncated _ E2 proteins contained a signal peptide sequence (MDWTWWRVFCLLAVAPGVHS). Genes are synthesized by a conventional method, coding nucleic acid sequences of WT-E2 and Truncated _ E2 are respectively cloned into a vector pUC-GW-Kan (Kingzhi Biotechnology Co., ltd.), and recombinant plasmids pUC-GW-Kan-WT-E2 and pUC-GW-Kan-Truncated _ E2 are respectively prepared. Subsequently, an expression plasmid for the target protein is constructed. Briefly, expression vectors pCDNA3.1 (+) (Invitrogen), recombinant plasmids pUC-GW-Kan-WT-E2 and pUC-GW-Kan-Truncated _ E2 are subjected to double enzyme digestion by Hind III and BamH I respectively, and recovered target fragments WT-E2 and Truncated _ E2 are respectively connected with a linearized expression vector pCDNA3.1 (+), so as to obtain expression plasmids pCDNA3.1 (+) -E2 and pCDNA3.1 (+) -Truncated _ E2 of E2 protein and Truncated _ E2 protein, and the sequencing is correct. For purification, a His tag was added to the C-terminus of the recombinant protein.

2) Protein expressed by CHO-K1 cell

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. 48 hours after transfection, complete medium (+ 4mM L-Glutamine) containing 600. Mu.g/mL G418 was added for culture, the complete medium containing G418 was replaced every 3 to 6 days until the survival rate was recovered to 90% or more, and the selection pressure was removed to obtain a stable cell pool that stably expresses E2 protein.

3) Purifying and detecting

(1) The cell culture supernatant was centrifuged at 5000rpm for 20min and then filtered through a 0.22 μm filter (Millipore).

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

(3) The mixture was diluted with PBS buffer (pH8.0) in an ultrafiltration tube (Millipore) having a pore size of 30kD, diluted with an equal volume of PBS solution, and then concentrated twice, and the procedure was repeated 5 times. The A280 value was measured using a spectrophotometer, and the results are shown in Table 1 below.

Table 1.

Protein	A280
		E2	0.21mg/mL
Truncated_E2	3.20mg/mL

As can be seen from the expression results shown in Table 1, the expression level of WT-E2 is very low, but the Truncated _ E2 obtains a higher expression level by removing the N-terminal and C-terminal transmembrane regions, and the expression level at the cell pool stage can reach 3.2g/L.

(4) Samples were prepared into loading systems with 5X protein loading buffer (pelyun day) at a ratio of 4. Spotting to 4-20% of the channels of SurePAGE, running the gel at 140V for 60min, and then staining with Coomassie Brilliant blue and destaining with acetic acid. The results of SDS-Page examination are shown in FIG. 1.

4) Preparation and effect detection of subunit vaccine

After dilution of the Truncated _ E2 protein with PBS, the vaccine was formulated using adjuvant Gel 02 (SEPPIC corporation) according to the adjuvant instructions. 15 weaned piglets were randomly divided into 3 groups of 5 piglets, wherein 50. Mu.g of Truncated _ E2 protein vaccine was injected into 1 group, commercial swine fever vaccine was injected into 2 groups, PBS was injected into 3 groups, and secondary immunization was performed 21 days after primary 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. The ELISA antibodies were detected by blood collection 7, 14 and 21 days after the first immunization, and by blood collection 7, 14 and 21 days after the second immunization.

The blocking rate of the antibody was determined using the classical swine fever virus antibody detection kit (IDEXX) according to the instructions. The immunization results are shown in table 2 below:

the results of the antibody titers of the groups after immunization are given in FIG. 2.

As can be seen from the results in table 2 and fig. 2, the conversion rate to positive of the Truncated _ E2 subunit vaccine is 100% after immunization, and the blocking rate 14 days after one-time immunization and 21 days after one-time immunization of healthy pigs during the test period is higher than that of the commercial vaccine group. The antibody blocking rate of the Truncated _ E2 subunit vaccine is more than 70% at 21 days after primary immunization and 7 days after secondary immunization, and the antibody blocking rate is more than 80% at 21 days after secondary immunization, which indicates that the Truncated _ E2 subunit vaccine can sufficiently protect swinery from the infection of classical swine fever virus and can be used for preventing and controlling domestic classical swine fever.

Example 2 construction of Truncated (u) E2 protein mutant and detection of expression amount

1) Construction of recombinant plasmid

The coding nucleic acid sequence of the Truncated _ E2 protein mutant (with the N-terminal signal peptide sequence MDWTWVFCLLAVAPGVHS) is synthesized by Suzhou Jinzhi biotechnology, inc., and the gene is cloned into a vector pUC-GW-Kan through conventional synthesis to prepare the Truncated _ E2 protein mutant recombinant plasmid. Then constructing a target protein expression plasmid, carrying out double enzyme digestion on an expression vector pCDNA3.1 (+) and a protein mutant recombinant plasmid DNA through Hind III and BamH I, respectively connecting each recovered Truncated _ E2 protein mutant fragment with a linearized expression vector pCDNA3.1 (+) to obtain an expression plasmid of each Truncated _ E2 protein mutant protein, and carrying out transfection on CHO-K1 (Merck) cells after correct sequencing. For purification, a His tag was added to the C-terminus of the recombinant protein. The mutation sites and sequences of the Truncated _ E2 protein mutants are shown in Table 3 below.

TABLE 3 mutation sites of each Truncated E2 protein mutant.

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2) Protein expressed by CHO-K1 cell

Chinese hamster ovary cells CHO-K1 (Merck) were used as host cells for the expression of the Truncated _ E2 protein mutant. Expression plasmids of the respective Truncated _ E2 protein mutant proteins were transfected into CHO cells, respectively. 48 hours after transfection, complete medium (+ 4mM L-glutamine) containing 600. Mu.g/mL G418 was added for culture, the complete medium containing G418 was replaced every 3 to 6 days until the survival rate was restored to 90% or more, and the selection pressure was removed to obtain a stable cell pool which stably expresses E2 protein.

3) Purifying and detecting

(1) The culture supernatant was centrifuged at 5000rpm for 20min and then filtered through a 0.22 μm filter (Millipore).

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

(3) The mixture was diluted with PBS buffer (pH8.0) and replaced with 30kD pore size ultrafiltration tube (Millipore), and the dilution was repeated 5 times by adding PBS solution at the same volume and then concentrating the solution twice. The A280 value was measured using a spectrophotometer, and the results are shown in Table 4 below.

TABLE 4 measurement of A280 for each Truncated E2 protein mutant.

According to the detection results shown in table 4, the 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 with significantly higher expression are selected for SDS-Page detection.

(4) After diluting the sample 3-fold, a loading system was prepared with 5X protein loading buffer at a ratio of 4. Spotting to 4-20% of the pore passage of SurePAGE, running at 140V for 60min, and then staining with Coomassie brilliant blue and decolorizing with acetic acid. The results of SDS-Page examination are shown in FIG. 3.

4) Preparation and effect detection of subunit vaccine

After the Truncated _ E2 protein mutants, truncated _ E2_ M14, truncated _ E2_ M32, and Truncated _ E2_ M44, were diluted with PBS, the vaccine was formulated using adjuvant Gel 02 (SEPPIC corporation) according to the adjuvant instructions. 20 weaned piglets were randomly divided into 4 groups of 5 piglets, wherein 50. Mu.g of Truncated _ E2 protein mutant was injected into 1, 2 and 3 groups, respectively, and PBS was injected into 4 groups, and secondary immunization was performed 21 days after primary immunization. After the pigs are immunized, the body temperature of the pigs is monitored and recorded every day, and the diet and mental states are observed. The ELISA antibodies were detected by blood collection 7, 14 and 21 days after the first immunization, and by blood collection 7, 14 and 21 days after the second immunization.

The blocking rate of the antibody was determined using the classical swine fever virus antibody detection kit (IDEXX) according to the instructions. The immunization results are shown in FIG. 4, and FIG. 4 shows the detection results of the antibody titer of the three Truncated _ E2 protein mutants after immunization. As can be seen from fig. 4, the mutant of the Truncated _ E2 protein had no significant effect on the immunogenicity of the E2 protein.

The technical solution of the present invention is not limited to the limitations of the above specific embodiments, and all technical modifications made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims (10)

1. A recombinant E2 protein of Classical Swine Fever Virus (CSFV), wherein the recombinant E2 protein comprises the amino acid sequence depicted in SEQ ID No. 2, or an amino acid sequence having at least 85% sequence identity to the amino acid sequence depicted in SEQ ID No. 2.

2. The recombinant E2 protein of claim 1, wherein the recombinant E2 protein comprises point mutations at one or more of amino acids 48, 63, 77, 81, 86, 95, 103, and 129,

preferably, in the recombinant E2 protein:

cysteine at position 48 is mutated into any one of serine, alanine, glycine, threonine and proline;

arginine at position 63 is mutated into any one of alanine, glutamine and glutamic acid;

serine at position 77 is mutated into any one of alanine, threonine and glycine;

the 81 st glutamic acid is mutated into arginine or lysine;

the 86 th glycine is mutated into any one of alanine, arginine and glutamine;

glycine at position 95 is mutated into any one of alanine, arginine and glutamine;

cysteine 103 is mutated into any one of serine, alanine, glycine, threonine and proline; and/or

Cysteine 129 is mutated to any of serine, alanine, glycine, threonine and proline.

3. The recombinant E2 protein of claim 1 or 2, wherein the recombinant E protein comprises one or more mutations in C48P, R63E, S77A, G86A, G95R, G95Q, C103S, C103A, R63A/S77A, R63E/S77A, R63Q/S77T, R63E/S77T, E81K/G86R, and E81K/G86Q.

4. The recombinant E2 protein of any one of claims 1 to 3, wherein said recombinant E protein comprises 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.

5. The recombinant E2 protein according to claim 1, characterized in that the recombinant E protein has one or more tags selected from His, C-myc, FLAG, strep-tag at the N-terminus or C-terminus.

6. A nucleic acid molecule encoding the recombinant E2 protein of any one of claims 1 to 5.

7. A host cell comprising the nucleic acid molecule of claim 6.

8. The host cell according to claim 7, wherein the host cell is selected from the group consisting of E.coli, yeast, mammalian cells and insect cells, preferably from the group consisting of mammalian cells, more preferably from the group consisting of Human Embryonic Kidney (HEK) cells, chinese Hamster Ovary (CHO) cells and African green monkey kidney cells (Vero cells).

9. An immunogenic composition comprising the recombinant E2 protein of any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

10. Use of an immunogenic composition according to claim 9 in the manufacture of a medicament for the treatment or prevention of a disease associated with Classical Swine Fever Virus (CSFV).

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