A recombinant 2.1d subtype swine fever E2 protein, subunit vaccine and application thereof

Abstract

The application discloses a recombinant 2.1d subtype swine fever E2 protein, a subunit vaccine and application thereof, and belongs to the technical field of genetic engineering. By removing amino acids at positions 344-375 of the 2.1d subtype swine fever E2 protein and retaining amino acids at positions 1-343, the most important antigen epitope of the 2.1d subtype swine fever E2 protein having a protective effect can be retained, the spatial structure of the recombinant 2.1d subtype swine fever E2 protein is basically not affected, and the immunogenicity of the recombinant 2.1d subtype swine fever E2 protein can be retained to the maximum extent. In addition, the recombinant 2.1d subtype swine fever E2 protein can be efficiently expressed in a prokaryotic expression system. Further, by co-expressing a molecular chaperone, the soluble expression level of the recombinant 2.1d subtype swine fever E2 protein can be significantly improved. After the recombinant 2.1d subtype swine fever E2 protein is prepared into a subunit vaccine, the subunit vaccine has good protective efficacy on infection of a 2.1d subtype swine fever virus.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a recombinant 2.1d subtype classical swine fever E2 protein, subunit vaccine and its application. Background Technology

[0002] Classical swine fever (CSF) is a contagious disease caused by the classical swine fever virus (CSFV). It is characterized by immunosuppression, persistent high fever, widespread hemorrhage in multiple organs, and a high morbidity and mortality rate. In my country, it is classified as a Class A animal disease. Although no large-scale CSF outbreaks have occurred in my country in recent years, the increasingly complex clinical symptoms, infection patterns, and epidemiological modes of CSF virus, the rising number of chronic and subclinical infections, and the presence of mixed infections with multiple diseases have significantly increased the difficulty of diagnosing and controlling CSF.

[0003] The E2 protein is a membrane glycoprotein of approximately 55 kDa. It is the most important protective antigenic protein of CSFV and the main antigen that induces the production of neutralizing antibodies. The E2 protein has low conservation and a high coefficient of variation, possessing four distinct antigenic regions (A, B, C, and D). Region A is further divided into A1, A2, and A3 subregions. A1 and A2 are relatively conserved and less prone to mutation, while A3, B, C, and D are more susceptible to mutation. The antigenic epitopes in regions A1, B, and C are crucial factors in generating a protective immune response. Based on the E2 gene sequence, CSFV is classified into three genotypes (1, 2, and 3) and eleven genotypes (1.1, 1.2, 1.3, 1.4, 2.1, 2.2, 2.3, 3.1, 3.2, 3.3, and 3.4). Subtype 2.1 can be further divided into 2.1a, 2.1b, 2.1c, and 2.1d. The 2.1d subtype of CSFV was first reported in China in 2015 and has gradually become one of the main circulating strains in the country. The 2.1d subtype E2 protein is present in R... 32 S 35 W 183 K 207 K 306 The five amino acid sites share common molecular characteristics and show low homology with the standard virulent strain Shimen and the attenuated vaccine strain C in my country. This difference in antigenicity allows the 2.1d subtype of CSFV to escape the immune protection of the C strain vaccine, which is an important reason for the frequent outbreaks of swine fever in the 2.1d subtype in recent years.

[0004] Vaccines are a crucial means of preventing and controlling CSFV. The HCLV (swine fever rabbit-adapted attenuated live vaccine) developed in my country is one of the most widely used vaccine strains currently. However, this vaccine cannot effectively distinguish between wild-type and wild-type infections after immunization, and it also suffers from insufficient cross-protection against the 2.1d subtype CSFV. Recombinant E2 protein expressed through genetic engineering has advantages such as good antigenicity, high expression levels, easy purification, scalable production, and the ability to perform serotype labeling. The most commonly used system is the insect cell-baculovirus expression system, but this system suffers from high production costs, stringent production technology requirements, and long production cycles. Producing E2 antigens using E. coli expression systems, especially targeting the currently prevalent 2.1d subtype CSFV E2 protein, is more suitable for commercial production and utilization. However, achieving efficient and soluble expression of the 2.1d subtype CSFV E2 in E. coli with the correct conformation and immunogenicity remains a significant challenge for the entire industry. Summary of the Invention

[0005] The purpose of this invention is to provide a recombinant 2.1d subtype classical swine fever E2 protein, subunit vaccine, and its application. This addresses the problems of poor solubility, low expression levels, and poor immunogenicity of the E2 antigen protein expressed in the *E. coli* expression system during the preparation of existing E2 protein genetically engineered subunit vaccines.

[0006] In a first aspect, the present invention provides a recombinant 2.1d subtype classical swine fever E2 protein, the recombinant 2.1d subtype classical swine fever E2 protein being selected from any of the following: A1) having an amino acid sequence as shown in SEQ ID NO:3; A2) having an amino acid sequence with one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence defined in A1); A3) having an amino acid sequence with more than 80% sequence identity compared to the amino acid sequence defined in A1) or A2); A4) an amino acid sequence obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of the amino acid sequence defined in A1) or A2) or A3).

[0007] The recombinant 2.1d subtype classical swine fever E2 protein provided by the present invention can be a natural, recombinant, or synthetic active polypeptide. The active polypeptide can be a naturally purified product, a chemically synthesized product, or a product produced from a prokaryotic host (e.g., Escherichia coli) or a eukaryotic host (e.g., yeast, higher plants) using recombinant technology.

[0008] In this invention (A4), the linkage can be achieved through direct peptide bond connection or through a linker, using methods conventional in the art. The tags include, but are not limited to: GST (glutathione thioredoxin) tag protein, Trx (thioredoxin) tag protein, His tag protein (His-tag), Flag tag protein, LacZ tag protein, GFP (green fluorescent protein), sfGFP (hyperfolded green fluorescent protein), and HA tag (hemagglutinin tag). Those skilled in the art can select appropriate tag proteins according to actual needs. The use of tags does not alter the function of the target protein (recombinant 2.1d subtype classical swine fever E2 protein); its purpose is for separation, purification, detection, or tracing. The tags can be separated from the target protein (recombinant 2.1d subtype classical swine fever E2 protein) using chemical cleavage methods or enzymatic methods (such as introducing protease cleavage sites and using TEV protease to remove the tag) known in the art.

[0009] In this invention, by removing amino acids 344-375 of the 2.1d subtype classical swine fever E2 protein and retaining amino acids 1-343, not only can the most important protective antigenic epitopes of the 2.1d subtype classical swine fever E2 protein be preserved, but the spatial structure of the recombinant 2.1d subtype classical swine fever E2 protein is also basically not affected, thus maximizing its immunogenicity. In addition, the recombinant 2.1d subtype classical swine fever E2 protein can be expressed efficiently in a prokaryotic expression system.

[0010] In a second aspect, the present invention provides a nucleic acid molecule encoding the aforementioned recombinant 2.1d subtype classical swine fever E2 protein.

[0011] The nucleic acid molecules provided by this invention can be DNA, such as cDNA, genomic DNA, or recombinant DNA; or RNA, such as mRNA or hnRNA; and these nucleic acid molecules can usually be obtained by PCR amplification or artificial synthesis.

[0012] In some embodiments, the nucleic acid molecule is selected from any of the following: B1) a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO:4; B2) a nucleic acid molecule that hybridizes with the nucleic acid molecule defined in B1) under stringent conditions and encodes the above-mentioned recombinant 2.1d subtype classical swine fever E2 protein; B3) a nucleic acid molecule that has more than 90% sequence identity with the nucleic acid molecule defined in B1) or B2) and encodes the above-mentioned recombinant 2.1d subtype classical swine fever E2 protein.

[0013] As used herein, the term "hybridization under stringent conditions" refers to the hybridization of two nucleic acid fragments under standard hybridization conditions as described in the section "Expression of Cloned Genes in E. coli" of *Molecular Cloning: A Laboratory Manual* (1989) (Cold Spring Lane Laboratory, New York, USA). Such conditions include hybridization in 6.0 × SSC at 45 °C, followed by washing in 2 × SSC at 50 °C. To select stringency, the salt concentration in the washing step can be chosen, for example, between 2.0 × SSC at 50 °C for low stringency and 2.0 × SSC at 50 °C for high stringency. Additionally, the temperature in the washing step can be varied between room temperature (approximately 22 °C) for low stringency and 65 °C for high stringency.

[0014] As used herein, the term “sequence identity” can be evaluated by the naked eye or by computer software (such as the software program described in Current Protocols in Molecular Biology by Ausubel et al. eds. (2007)). Molecules are identical at that position when positions in the compared sequences are occupied by the same bases or amino acids. Identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. “Sequence identity” of a polynucleotide or amino acid sequence with another sequence having a certain percentage (e.g., 90%, 95%, 98%, or 99%) means that when the sequences are aligned, that percentage of bases or amino acids are the same in the two compared sequences.

[0015] In a third aspect, the present invention provides a recombinant vector comprising the aforementioned nucleic acid molecules.

[0016] The recombinant vectors in this invention include cloning vectors and expression vectors. The cloning vector is used to replicate the relevant sequence, and the expression vector is used to express the relevant gene. The vector used to construct the expression vector can be the pET28a vector.

[0017] In a fourth aspect, the present invention provides a recombinant cell comprising the above-mentioned nucleic acid molecule or the above-mentioned recombinant vector, and at least one of pTf16 and pG-KJE8 vectors.

[0018] In this invention, the inventors further discovered that by introducing at least one of the pTf16 and pG-KJE8 vectors into recombinant cells for expression, a molecular chaperone can be obtained, which can significantly improve the soluble expression level of recombinant 2.1d subtype classical swine fever E2 protein.

[0019] In some implementations, the molecular chaperone expressed by the pTf16 vector includes the tig protein.

[0020] In some implementations, the molecular chaperones expressed by the pG-KJE8 vector include dnaK, dnaJ, grpE, groES, and groEL proteins.

[0021] In some implementations, the method for preparing recombinant cells includes the step of converting the recombinant vector into expression host cells.

[0022] In this invention, the expression host cell is a conventional host cell in the art, as long as the recombinant vector can stably replicate itself and the gene it carries can be effectively expressed. It can be a prokaryotic cell or a eukaryotic cell, such as Escherichia coli or yeast. For example, Escherichia coli can be E. coli BL21(DE3).

[0023] In a fifth aspect, the present invention provides a method for preparing recombinant 2.1d subtype classical swine fever E2 protein, comprising the following steps: culturing the above-mentioned recombinant cells, inducing expression to obtain a culture; and isolating the above-mentioned recombinant 2.1d subtype classical swine fever E2 protein from the culture.

[0024] In this invention, there are no special requirements for the culture method and culture conditions; it is sufficient to ensure the normal growth of the recombinant cells. Furthermore, the methods for isolating the aforementioned recombinant 2.1d subtype classical swine fever E2 protein from the culture are all conventional methods in the art.

[0025] In some embodiments, the culture medium used in the preparation of recombinant 2.1d subtype classical swine fever E2 protein is a protein-expressing culture medium in the art, preferably LB medium.

[0026] In some implementations, the method also includes a step of purifying the recombinant 2.1d subtype classical swine fever E2 protein. The purification steps specifically include: first, ultrasonic lysis, then removal of endotoxin, and finally purification using molecular sieve chromatography.

[0027] In some preferred embodiments, ultrasonic lysis specifically includes: working at 0-4°C for 2-4 seconds, stopping for 2-4 seconds, with a total working time of 80-100 minutes.

[0028] In some preferred embodiments, endotoxin removal specifically includes treatment with Triton X-114 at a final concentration of 0.5-1.5%.

[0029] In some preferred embodiments, molecular sieve chromatography specifically includes: using Sepharose 6Fast Flow as the chromatography medium, a column height of 60-80 cm, and a maximum sample loading volume of 10-20% of the column volume.

[0030] In a sixth aspect, the present invention provides a recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine, comprising the above-mentioned recombinant 2.1d subtype classical swine fever E2 protein and an adjuvant.

[0031] The recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine provided by this invention has good protective efficacy against infection with 2.1d subtype classical swine fever virus.

[0032] In a seventh aspect, the present invention provides a method for preparing the recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine as described above, comprising the following steps: adding an inactivating agent to the recombinant 2.1d subtype classical swine fever E2 protein for inactivation treatment to obtain inactivated recombinant 2.1d subtype classical swine fever E2 protein; mixing the inactivated recombinant 2.1d subtype classical swine fever E2 protein with an adjuvant, and emulsifying the mixture to obtain the recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine.

[0033] The recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine provided by this invention has a simple preparation method, uses inexpensive and readily available raw materials, and is suitable for large-scale industrial production.

[0034] In some implementations, the inactivating agent includes formaldehyde, with a final concentration of 0.05-0.15%, and the inactivation treatment includes inactivation at a temperature of 0-4°C for 60-80 hours; the adjuvant includes the biphasic adjuvant ISA 201; and the recombinant 2.1d subtype classical swine fever E2 protein content in the recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine is 200-400 μg / mL.

[0035] In some preferred embodiments, the final concentration of the inactivating agent is 0.1%, and the inactivation treatment includes: inactivation at a temperature of 4°C for 72 h; the adjuvant includes the biphasic adjuvant ISA 201; and the content of recombinant 2.1d subtype classical swine fever E2 protein in the recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine is 300 μg / mL.

[0036] Understandably, the inactivating agent and adjuvant can be selected from conventional inactivating agents and adjuvants in the prior art according to actual needs, as long as they can produce a vaccine with excellent performance. For example, in this invention, the inactivating agent preferably includes formaldehyde, and the adjuvant preferably includes the biphasic adjuvant ISA 201.

[0037] In an eighth aspect, the present invention provides the use of the recombinant 2.1d subtype classical swine fever E2 protein, any of the above-mentioned nucleic acid molecules, the above-mentioned recombinant vector, the above-mentioned recombinant cells, the recombinant 2.1d subtype classical swine fever E2 protein prepared by the above-mentioned preparation method, the above-mentioned recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine, and the recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine prepared by any of the above-mentioned preparation methods in the preparation of a medicament for the prevention and / or treatment of 2.1d subtype classical swine fever virus infection.

[0038] The beneficial effects of this invention are as follows: Unlike existing technologies, this invention removes amino acids 344-375 from the 2.1d subtype classical swine fever E2 protein, retaining amino acids 1-343. This not only preserves the most important protective epitopes of the 2.1d subtype classical swine fever E2 protein but also largely does not affect the spatial structure of the recombinant 2.1d subtype classical swine fever E2 protein, thus maximizing its immunogenicity. Furthermore, this recombinant 2.1d subtype classical swine fever E2 protein can be efficiently expressed in prokaryotic expression systems. Moreover, through co-expression of molecular chaperones, the soluble expression level of the recombinant 2.1d subtype classical swine fever E2 protein can be significantly improved. When this recombinant 2.1d subtype classical swine fever E2 protein is prepared into a subunit vaccine, it exhibits good protective efficacy against infection with the 2.1d subtype classical swine fever virus. Attached Figure Description

[0039] Figure 1 This is the SDS-PAGE result of the protein expressed by the BL21(DE3) / pET28a-CSFV E2 engineered bacteria in Example 1 of the present invention. Lane M: pre-stained protein standard molecular weight; Lane 1: negative control; Lane 2: total protein expressed by the BL21(DE3) / pET28a-CSFV E2 engineered bacteria; Lane 3: soluble protein expressed by the BL21(DE3) / pET28a-CSFV E2 engineered bacteria; Lane 4: insoluble protein expressed by the BL21(DE3) / pET28a-CSFV E2 engineered bacteria.

[0040] Figure 2 This is the SDS-PAGE result of the protein expressed by the BL21(DE3) / pET28a-CSFV E2+pTf16 or pG-KJE8 engineered bacteria in Example 1 of the present invention. Lane M: pre-stained protein standard molecular weight; Lane 1: negative control; Lane 2: total protein expressed by the BL21(DE3) / pET28a-CSFV E2+pTf16 engineered bacteria; Lane 3: soluble protein expressed by the BL21(DE3) / pET28a-CSFV E2+pTf16 engineered bacteria; Lane 4: total protein expressed by the BL21(DE3) / pET28a-CSFV E2+pG-KJE8 engineered bacteria; Lane 5: soluble protein expressed by the BL21(DE3) / pET28a-CSFV E2+pG-KJE8 engineered bacteria.

[0041] Figure 3The SDS-PAGE results for the protein expressed by the BL21(DE3) / pET28a-CSFV E2+pG-KJE8 engineered bacteria in Example 1 of this invention are shown below. Lane M: pre-stained protein standard molecular weight; Lane 1: negative control; Lane 2: total protein expressed by the BL21(DE3) / pET28a-CSFV E2+pG-KJE8 engineered bacteria; Lane 3: total protein expressed by the BL21(DE3) / pET28a-CSFV E2+pG-KJE8 engineered bacteria after endotoxin removal; Lane 4: total protein expressed by the BL21(DE3) / pET28a-CSFV E2+pG-KJE8 engineered bacteria after molecular sieve purification.

[0042] Figure 4 The Western Blot results of the purified recombinant 2.1d subtype classical swine fever E2 protein in Example 1 of this invention are shown. Lane M: pre-stained protein standard molecular weight; Lane 1: negative control; Lane 2: purified recombinant 2.1d subtype classical swine fever E2 protein; Lane 3: 2.1d subtype CSFV tissue homogenate. Detailed Implementation

[0043] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0044] Experimental methods not specifically described in the examples are generally performed according to conventional experimental methods in the field of molecular biology, including but not limited to those described in *Molecular Cloning: A Laboratory Manual* by M.R. Green and *Molecular Biology* by Robert F. Weaver, or according to the experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, all reagents and biological materials used in the examples are commercially available.

[0045] Example 1: Modification of the 2.1d subtype classical swine fever E2 gene and construction of prokaryotic expression engineered bacteria.

[0046] 1.1 2.1 Obtaining the E2 gene of the 2.1d subtype classical swine fever

[0047] Gene mining was performed on NCBI (https: / / www.ncbi.nlm.nih.gov / ) to obtain the 2.1d subtype classical swine fever E2 gene sequence (accession number: PV648296.1), the nucleotide sequence of which is shown in SEQ ID NO:2, and the amino acid sequence of the 2.1d subtype classical swine fever E2 protein is shown in SEQ ID NO:1.

[0048] Further sequence alignment revealed that the amino acid sequence (SEQ ID NO:1) of the 2.1d subtype classical swine fever E2 protein was 88.06% similar to the amino acid sequence (accession number: ABI93151) of the existing conventional HCLV vaccine strain E2 protein, with 42 amino acid residues differing, as shown in Table 1.

[0049] Table 1. Analysis of amino acid sequence differences between the 2.1d subtype classical swine fever E2 protein and the HCLV vaccine strain E2 protein.

[0050]

[0051] After the 2.1d subtype classical swine fever E2 gene sequence was artificially synthesized, it was subcloned into the pTOPOT vector, transformed into DH5α competent cells, and the strain that was correctly identified by PCR was named E. coli DH5α / pTOPOT-CSFV E2 full-length.

[0052] 1.2 2.1 Modification of the d subtype classical swine fever E2 gene, construction of prokaryotic expression vector and induction of expression

[0053] Analysis of the structure of the 2.1d subtype classical swine fever E2 protein in step 1.1 revealed that amino acids 1-343aa are located outside the envelope and contain the main protective epitopes; amino acids 344-375aa are located across the transmembrane region, exhibiting an α-helix structure and containing a large number of hydrophobic amino acids. The abundance of hydrophobic amino acids in the 344-375aa region of the E2 protein prevents the full-length E2 gene from being expressed in the *E. coli* expression system. Deleting the 344-375aa region while retaining the 1-343aa region of the E2 protein not only preserves the most important protective epitopes but also largely maintains the spatial structure of the 1-343aa region, thus maximizing the preservation of its immunogenicity.

[0054] Therefore, the E2 gene of the 2.1d subtype classical swine fever was modified using the following method:

[0055] Extract the pTOPOT-CSFV E2 full-length plasmid from the E. coli DH5α / pTOPOT-CSFV E2 full-length strain in step 1.1, and then use this pTOPOT-CSFV E2 full-length plasmid as a template to generate CSFV E2 F (5'- CCC GGATCC ATGGGCCGGCTGTCCTGT-3' (SEQ ID NO:5)) and CSFV E2 R (5'- CCC CTCGAG Using primers TTCGGCGAAGTAGTCTGTGTG-3' (SEQ ID NO:6) to amplify the E2 (1-343aa) gene fragment. The italicized portions of the primer sequences represent introduced protective bases, and the underlined portions represent the introduced BamHI and XhoI restriction sites, respectively.

[0056] After agarose gel electrophoresis, the E2 (1-343aa) DNA fragment was recovered by gel excision and digested with BamHI and XhoI restriction endonucleases. The pET28a plasmid was also treated with the same endonucleases. After agarose gel electrophoresis, the digested products were further excision and recovery of the gene and vector fragments, which were then ligated using T4 DNA ligase. The ligation products were transformed into BL21(DE3) competent cells and screened using kanamycin. Single clones were selected and cultured. Correct clones were identified by PCR and sequenced for verification. The nucleotide sequence of the recombinant 2.1d subtype classical swine fever E2 gene is shown in SEQ ID NO:4, and the amino acid sequence of the recombinant 2.1d subtype classical swine fever E2 protein is shown in SEQ ID NO:3. The strain was named BL21(DE3) / pET28a-CSFVE2.

[0057] The above-mentioned BL21(DE3) / pET28a-CSFV E2 seed culture was inoculated into 50 mL of kanamycin-resistant LB medium at an inoculation rate of 5% (v / v) and cultured at 37°C and 200 rpm for 3 h. Then, α-lactose with a final concentration of 30 mM was added, the temperature was adjusted to 32°C, and the culture was induced at 200 rpm for 15 h. The cells were collected by centrifugation, and 10 times their weight of PBS solution was added. After resuspending the cells thoroughly, the cells were lysed using an ultrasonic disruptor.

[0058] After disruption, the protein was centrifuged at 12000g for 10 min at 4℃ to separate the supernatant (soluble protein). Protein content was determined using the BCA method, followed by SDS-PAGE analysis. The results are as follows: Figure 1 As shown.

[0059] from Figure 1As can be seen, the recombinant 2.1d subtype classical swine fever E2 protein has a high expression level, but poor solubility.

[0060] 1.3 Co-expression of molecular chaperones assists in the folding of recombinant 2.1d subtype classical swine fever E2 protein

[0061] As shown in section 1.2, after deleting the E2 (344-375aa) region from BL21(DE3) / pET28a-CSFV E2, it can be efficiently expressed in the *E. coli* system, but the protein has poor solubility, with only about 10% of the target protein being soluble and able to form the correct spatial conformation. Analysis of the amino acid composition of the recombinant 2.1d subtype classical swine fever E2 protein revealed that it contains 15 cysteine ​​residues. Excessive cysteine ​​residues may form intramolecular or intermolecular disulfide bonds at random positions, leading to incorrect tertiary and quaternary structures and ultimately precipitation.

[0062] To achieve soluble expression of the recombinant 2.1d subtype classical swine fever E2 protein, this invention utilizes molecular chaperones for co-expression. Molecular chaperones are proteins that assist in molecular assembly and protein folding. During nascent peptide chain synthesis, molecular chaperones can recognize and stabilize the partially folded conformation of the nascent peptide chain, refolding misfolded or folded proteins into their functional conformation and decomposing potentially toxic protein aggregates formed due to protein misfolding. By transforming the BL21(DE3) / pET28a-CSFV E2 engineered bacterial strain with pTf16 or pG-KJE8 plasmids capable of co-expressing molecular chaperones, the correct folding of the recombinant 2.1d subtype classical swine fever E2 protein is assisted, thereby improving its solubility.

[0063] Specifically, the method is as follows:

[0064] Take BL21(DE3) / pET28a-CSFV E2 seed culture and inoculate it into 50 mL of LB medium at an inoculation rate of 1% (v / v). Incubate at 37°C with shaking for 3 h until OD reaches 1. 600 The bacterial cells were centrifuged at approximately 0.6 g for 10 min at 4000 g to collect the cells. The cells were washed twice with sterile pure water. Finally, the cells were resuspended in 1 mL of sterile solution containing 10% glycerol and 100 mM CaCl2, aliquoted, and stored at -80°C. This bacterial culture is the BL21(DE3) / pET28a-CSFV E2 chemical transformation competent cell.

[0065] Take one tube of the above competent cells, add 1 μL of pTf16 or pG-KJE8 plasmid, transform, and finally select using 50 μg / mL kanamycin and 34 μg / mL chloramphenicol. The obtained monoclonal strains were named BL21(DE3) / pET28a-CSFV E2+pTf16 and BL21(DE3) / pET28a-CSFV E2+pG-KJE8, respectively.

[0066] The BL21(DE3) / pET28a-CSFV E2+pTf16 engineered bacterial strain was activated; it was inoculated at a 5% (v / v) inoculation rate into 50 mL of LB medium containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol, and L-arabinose was added to a final concentration of 1 mg / mL to induce the expression of the molecular chaperone tig. The culture was incubated at 37℃ and 200 rpm with shaking for 3 h; then, lactose was added to a final concentration of 30 mM to induce the expression of the target protein, and the culture was incubated at 32℃ and 150 rpm for 15 h. After induction, the bacterial cells were collected by centrifugation.

[0067] The engineered bacterial strain BL21(DE3) / pET28a-CSFV E2+pG-KJE8 was activated; it was inoculated at a 5% (v / v) inoculation rate into 50 mL of LB medium containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol, and L-arabinose and tetracycline were added to a final concentration of 1 mg / mL to induce the expression of molecular chaperones dnaK, dnaJ, grpE, groES, and groEL. The culture was incubated at 37℃ and 200 rpm with shaking for 3 h; then lactose was added to a final concentration of 30 mM to induce the expression of the target protein, and the culture was incubated at 32℃ and 150 rpm for 15 h. After induction, the bacterial cells were collected by centrifugation.

[0068] Ten times their weight of PBS solution were added to the bacterial cells, and after thorough resuspending, the cells were lysed using an ultrasonic homogenizer. The cells were centrifuged at 12000g, 4℃ for 10 min, and the supernatant was collected; this supernatant contained the soluble protein. Protein content was determined using the BCA method, and the solubility of the target protein was analyzed using SDS-PAGE. The results are as follows: Figure 2 As shown.

[0069] from Figure 2 As can be seen, co-expression of molecular chaperones pTf16 or pG-KJE8 can improve the solubility of recombinant 2.1d subtype classical swine fever E2 protein to varying degrees. Among them, the engineered bacteria co-expressing pG-KJE8 plasmid showed the highest solubility of recombinant 2.1d subtype classical swine fever E2 protein.

[0070] 1.4 Purification and identification of recombinant 2.1d subtype classical swine fever E2 protein

[0071] 1.4.1 Purification of recombinant 2.1d subtype classical swine fever E2 protein

[0072] Take 100g of BL21(DE3) / pET28a-CSFV E2+pG-KJE8 engineered bacterial cells from step 1.3, add 800mL of PBS, resuspend thoroughly, and then perform ultrasonic disruption. The ultrasonic disruption conditions are: 4℃, 3s operation, 3s pause, and a total operation time of 90min to obtain the bacterial cell lysis solution.

[0073] Add 1% Triton X-114 directly to the above bacterial lysis solution and stir at 4°C for 2 hours; dispense into horizontal rotor centrifuge bottles, transfer to a 28°C water bath, and let stand for 2 hours; centrifuge (4000g) for 30 minutes at 28°C; after centrifugation, carefully remove the upper aqueous phase. Repeat this process once. This yields a recombinant 2.1d subtype classical swine fever E2 protein solution free of endotoxin.

[0074] Next, molecular sieve chromatography purification was performed. A molecular sieve column was initially packed with Sepharose 6 FastFlow medium, with a column diameter of 10 cm and a height of 70 cm. A vacuum fiber column with a pore size of 0.45 μm was used to clarify the endotoxin-free recombinant 2.1d subtype classical swine fever E2 protein solution, and the resulting sample was loaded into the column, with a maximum loading volume of 15% of the column volume. Elution was performed using an industrial-grade protein purification system, and the protein in the aqueous peak was collected. The protein content was determined using the BCA method, and the endotoxin content in the antigen was determined using an endotoxin assay kit to be less than 50 EU / mL. The results are as follows: Figure 3 As shown.

[0075] from Figure 3 As can be seen, the purified recombinant 2.1d subtype classical swine fever E2 protein has high purity. The above process has high purification efficiency, is simple to operate, and is easy to scale up.

[0076] 1.4.2 Identification of recombinant 2.1d subtype classical swine fever E2 protein

[0077] To verify the antigenicity of the soluble recombinant 2.1d subtype classical swine fever E2 protein expressed in prokaryotes, it was identified using classical swine fever standard positive serum from the China Institute of Veterinary Drug Control. The specific procedure is as follows:

[0078] 1) SDS-PAGE: The negative control protein, the purified recombinant 2.1d subtype classical swine fever E2 protein (0.5 μg loading amount), and the tissue homogenate of 2.1d subtype CSFV were subjected to SDS-PAGE electrophoresis;

[0079] 2) Transfer and blocking: Using a semi-dry transfer apparatus, the protein on the gel was transferred onto the NC membrane; at 37°C, it was blocked with 5% skim milk powder for 1 hour.

[0080] 3) Primary antibody incubation: Dilute the standard positive serum for classical swine fever 500 times with blocking solution and incubate overnight at 4°C; wash the membrane 3 times with PBST solution for 5 min each time;

[0081] 4) Secondary antibody incubation: Dilute the HPR-labeled goat anti-pig IgG antibody 20,000 times with blocking buffer and incubate at 37°C for 2 hours; wash the membrane 3 times with PBST solution for 5 minutes each time;

[0082] 5) Color development and imaging: ECL color development solution was used for color development, and Bio Rad gel imaging was used for exposure and imaging.

[0083] Western Blot results are as follows: Figure 4 As shown, from Figure 4 The results show that the recombinant 2.1d subtype classical swine fever E2 protein expressed in prokaryotes exhibits a specific reaction with positive serum at 45 kDa, and the 2.1d subtype CSFV samples also show a specific reaction with positive serum at approximately 55 kDa (full-length glycosylated E2). These results indicate that the recombinant 2.1d subtype classical swine fever E2 protein possesses good antigenicity.

[0084] Example 2 Evaluation of Immunization Challenge with Recombinant 2.1d Subtype Classical Swine Fever E2 Protein Subunit Vaccine

[0085] 2.1 Preparation of recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine

[0086] The recombinant 2.1d subtype classical swine fever E2 protein purified in Example 1 was diluted, and formaldehyde was added to a final concentration of 0.1%. After mixing, the mixture was placed at 4°C for 72 hours to inactivate the protein. An equal mass of biphasic adjuvant ISA 201 was added, and the mixture was sheared at 31°C for 10 minutes at a low speed to form a stable emulsion. Finally, sterility testing was performed.

[0087] After passing the sterility test, the emulsion is a recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine with an antigen content of 300 μg / mL.

[0088] 2.2 Immunization and challenge with recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine

[0089] Ten-week-old Japanese white rabbits were divided into three groups: A, B, and C. Group A was the immune challenge group, Group B was the challenge control group, and Group C was the blank control group; each group contained 6 experimental animals.

[0090] The challenged group (Group A) received multiple subcutaneous injections of recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine on their backs, with each animal receiving 1 mL (300 μg antigen). A booster immunization of 1 mL was administered two weeks after the initial immunization. The challenged control group and the blank control group were not immunized with the vaccine but received only 1 mL of physiological saline. The animals were continuously observed for symptoms after immunization. No significant adverse reactions were observed in the rabbits; their mental state was good, and no induration, abscess, or ulceration was found at the injection sites upon necropsy.

[0091] Two weeks after the second immunization, animals in both the immunized and control groups were challenged with the virus via marginal ear vein injection, with each animal receiving 1×10⁻⁶ mmol / L. 6 Copies of 2.1d subtype CSFV virus fluid. Five days after challenge, the experimental rabbits were euthanized and necropsy was performed.

[0092] 2.3 Monitoring of rectal temperature in experimental animals

[0093] CSFV can induce a typed fever response in rabbits. Therefore, the rectal temperature of the experimental animals was measured every 12 hours after challenge. If the rectal temperature was ≥38.5℃, it was considered a fever. If fever occurred at two or more consecutive time points, it was considered a typed fever response, indicating that the animals were affected by the pathogenicity of the classical swine fever virus. The rectal temperature monitoring results are shown in Table 2 below.

[0094] Table 2 Results of rabbit rectal temperature monitoring and typological assessment after viral challenge

[0095]

[0096] As shown in Table 2, the six rabbits in the challenge control group developed fever 36 hours after challenge and it persisted until 96 hours. The rectal temperature reached its maximum at 60 hours (exceeding 41℃), exhibiting typical fever and type I fever, indicating that the challenge induced disease in the experimental rabbits. In contrast, the six rabbits in the immunized challenge group only experienced a brief period of fever at 48 hours, but did not develop type I fever. No fever was observed in the blank control group.

[0097] 2.4 Viral load determination

[0098] After necropsy, 0.3 g of rabbit spleen tissue was placed in a 1.5 mL centrifuge tube, and 1.5 mL of 0.05 mol / L PBS was added. The tissue was homogenized using a high-throughput cryogenic homogenizer at 50 Hz and -30°C for 90 s, followed by a 160 s pause, repeated three times. The homogenized tissue was then centrifuged at 4000 g for 3 min, and the supernatant was separated. Total RNA was extracted from the homogenate using an RNA extraction kit, and cDNA first-strand synthesis was performed using a reverse transcription kit.

[0099] The artificially synthesized CSFV RT F (5'-GGGTTACCAGTTGCTCCG-3'(SEQ ID NO:7)) and CSFV RT R (5'-TTACTCCTTTCACCACGA-3'(SEQ ID NO:8)) primers were used for real-time quantitative PCR according to the system in Table 3 below.

[0100] Table 3. Quantitative Real-Time PCR Amplification Reaction System

[0101]

[0102] A standard quality plasmid was constructed, and a standard curve of nucleic acid copy number versus Ct was established using the above-described quantitative real-time PCR method. The viral load of the 2.1d subtype CSFV in rabbit spleen was calculated using this standard curve, and the viral load results are shown in Table 4 below.

[0103] Table 4. Results of viral load analysis after infection

[0104]

[0105] As shown in Table 4, all six rabbits in the challenge control group were infected with classical swine fever virus, while neither the immune challenge group nor the blank control group were infected with classical swine fever virus.

[0106] 2.5 Specific antibody detection

[0107] Serum samples were collected from rabbits in each group before challenge, and the level of specific antibodies after immunization was measured using a classical swine fever (CSF) blocking ELISA detection kit. In CSF antibody detection, the blocking rate is directly related to the titer of specific antibodies; generally, a higher blocking rate indicates a higher antibody titer in the sample. When the blocking rate is below 30%, the recombinant 2.1d subtype CSF E2 protein antibody is considered negative; when the blocking rate is above 40%, the recombinant 2.1d subtype CSF E2 protein antibody is considered positive. The test results are shown in Table 5 below.

[0108] Table 5 Specific antibody detection results

[0109]

[0110] As shown in Table 5, among the three groups of experimental animals, only the immune challenge group had the highest blocking rate, exceeding 30%, indicating that the recombinant 2.1d subtype classical swine fever E2 protein antibody titer was the highest. In contrast, the blocking rates in the challenge control group and the blank control group were both below 30%, and no classical swine fever antibodies were detected in either group. The results indicate that immunization with the recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine can induce high-titer antibodies and provide 100% protection against challenge in the experimental animals.

[0111] In summary, the recombinant 2.1d subtype classical swine fever E2 protein provided by this invention has a high soluble expression level. When this recombinant 2.1d subtype classical swine fever E2 protein is prepared into a subunit vaccine, it has good protective efficacy against infection with the 2.1d subtype classical swine fever virus.

[0112] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing recombinant 2.1d subtype classical swine fever E2 protein, characterized in that, Includes the following steps: The recombinant cells were cultured and induced to express the desired expression, resulting in a culture. Recombinant 2.1d subtype classical swine fever E2 protein was isolated from the culture; The recombinant cells contain a nucleic acid molecule encoding the recombinant 2.1d subtype classical swine fever E2 protein or a recombinant vector containing a nucleic acid molecule encoding the recombinant 2.1d subtype classical swine fever E2 protein, and at least one of pTf16 and pG-KJE8 vectors; The recombinant 2.1d subtype classical swine fever E2 protein is selected from any of the following: A1) The amino acid sequence is shown in SEQ ID NO:3; A4) An amino acid sequence obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of the amino acid sequence defined in A1).

2. The preparation method according to claim 1, characterized in that, The nucleic acid molecule is selected from any of the following: B1) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO:

4.

3. A recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine, characterized in that, It includes the recombinant 2.1d subtype classical swine fever E2 protein prepared by the preparation method described in claim 1 and the adjuvant.

4. A method for preparing a recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine as described in claim 3, characterized in that, Includes the following steps: Inactivation agent was added to the recombinant 2.1d subtype classical swine fever E2 protein for inactivation treatment to obtain the inactivated recombinant 2.1d subtype classical swine fever E2 protein; The inactivated recombinant 2.1d subtype classical swine fever E2 protein was mixed with an adjuvant and emulsified to obtain a recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine.

5. The method for preparing the recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine according to claim 4, characterized in that, The inactivating agent includes formaldehyde, and the final concentration of the inactivating agent added is 0.05-0.15%. The inactivation treatment includes inactivation for 60-80 hours at a temperature of 0-4°C. The adjuvant includes the biphasic adjuvant ISA 201. The recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine contains 200-400 μg / mL of recombinant 2.1d subtype classical swine fever E2 protein.

6. The use of the recombinant 2.1d subtype classical swine fever E2 protein prepared by the preparation method of any one of claims 1-2, the recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine of claim 3, and the recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine prepared by the preparation method of any one of claims 4-5 in the preparation of a drug for the prevention and / or treatment of 2.1d subtype classical swine fever virus infection.

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