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

Abstract

The invention discloses a recombinant 2.1 d subtype swine fever E2 protein, a subunit vaccine and application of the subunit vaccine, and belongs to the technical field of genetic engineering. According to the 2.1 d subtype swine fever E2 protein, 344-375 amino acids of the 2.1 d subtype swine fever E2 protein are removed, and 1-343 amino acids of the 2.1 d subtype swine fever E2 protein are reserved, so that the most important antigenic epitope with a protective effect of the 2.1 d subtype swine fever E2 protein can be reserved, the space structure of the recombinant 2.1 d subtype swine fever E2 protein is basically not influenced, and the immunogenicity of the 2.1 d subtype swine fever E2 protein can be reserved to the greatest extent; in addition, the recombinant 2.1 d subtype swine fever E2 protein can be efficiently expressed in a prokaryotic expression system; furthermore, the soluble expression level of the recombinant 2.1 d subtype swine fever E2 protein can be remarkably improved through co-expression of the molecular chaperone, and after the recombinant 2.1 d subtype swine fever E2 protein is prepared into a subunit vaccine, the subunit vaccine has a good protection effect on 2.1 d subtype swine fever virus infection.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and in particular relates to a recombinant 2.1d subtype classical swine fever E2 protein, a subunit vaccine and applications thereof. Background Art

[0002] Classical swine fever (CSF) is caused by the classical swine fever virus (CSFV). It is a contagious disease characterized by immunosuppression, prolonged high fever, widespread hemorrhage in multiple organs, and high morbidity and mortality. It is classified as a Category I animal disease in my country. Although there have been no large-scale outbreaks of CSF in my country in recent years, the increasing complexity of clinical symptoms, infection patterns, and epidemic patterns of CSFV, along with the increasing number of chronic and subclinical infections and the presence of multiple co-infections, have significantly increased the difficulty of CSF diagnosis and prevention.

[0003] The E2 protein, an approximately 55 kDa envelope glycoprotein, is the primary protective antigen of CSFV and the primary antigen responsible for inducing neutralizing antibodies. The E2 protein is poorly conserved and has a high coefficient of variation. It contains four distinct antigenic regions (A, B, C, and D). Region A is further divided into subregions A1, A2, and A3. A1 and A2 are relatively conserved and resistant to variation, while A3, B, C, and D are more variable. The antigenic epitopes in regions A1, B, and C are crucial for generating protective immune responses. Based on the E2 gene sequence, CSFV is divided into three genotypes (1, 2, and 3) and 11 subtypes (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 is further divided into 2.1a, 2.1b, 2.1c, and 2.1d. In 2015, the 2.1d subtype of CSFV was first reported in China and gradually became one of the main prevalent strains in my country. 32 、S 35 、W 183 , K 207 , K 306 The five amino acid residues share common molecular features and have low homology with the Shimen strain, a standard virulent strain in my country, and the attenuated vaccine strain C. This antigenic difference allows the 2.1d subtype of CSFV to escape the immune protection of the C strain vaccine and is a major factor in the frequent outbreaks of the 2.1d subtype of swine fever in recent years.

[0004] Vaccines are an important means of preventing and treating CSFV. The HCLV (attenuated lapinized classical swine fever vaccine) developed in my country is currently one of the most widely used vaccine strains. However, this vaccine does not effectively discriminate against wild-type infection and lacks cross-protection against CSFV subtype 2.1d. Recombinant E2 protein expressed through genetic engineering offers advantages such as good antigenicity, high expression levels, ease of purification, scalable production, and the ability to serotype. The insect cell-baculovirus expression system is the most commonly used, but it suffers from high production costs, stringent technical requirements, and long production cycles. The E. coli expression system for E2 antigen production, particularly for the currently prevalent 2.1d CSFV subtype E2 protein, is more suitable for commercial production and utilization. However, efficient and soluble expression of 2.1d CSFV E2 in E. coli, with the correct conformation and immunogenicity, remains a major challenge for the industry. Summary of the Invention

[0005] The present invention aims to provide a recombinant 2.1d subtype classical swine fever E2 protein, a subunit vaccine, and its application. This invention aims to address the problems of poor solubility, low expression level, and poor immunogenicity of E2 antigen proteins expressed in Escherichia coli expression systems during the preparation of existing E2 protein genetically engineered subunit vaccines.

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

[0007] The above-mentioned 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 using recombinant technology from a prokaryotic host (such as Escherichia coli) or a eukaryotic host (such as yeast, higher plants).

[0008] In A4) of the present invention, the connection can be achieved through direct peptide bonds or through linkers, using methods conventional in the art. Tags include, but are not limited to, GST (glutathione sulfhydryltransferase)-tagged proteins, Trx (thioredoxin)-tagged proteins, His-tagged proteins (His-tag), Flag-tagged proteins, LacZ-tagged proteins, GFP (green fluorescent protein), sfGFP (superfolded green fluorescent protein), and HA tag (hemagglutinin tag). Those skilled in the art can select an appropriate tag protein based on actual use. The use of tags does not alter the function of the target protein (recombinant 2.1d subtype classical swine fever E2 protein) and is intended for isolation, 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 known in the art (e.g., introducing a protease cleavage site to remove the tag using TEV protease).

[0009] In the present 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 antigenic epitopes with protective effects of the 2.1d subtype classical swine fever E2 protein be retained, but the spatial structure of the recombinant 2.1d subtype classical swine fever E2 protein is basically not affected, and its immunogenicity can be retained to the greatest extent; in addition, the recombinant 2.1d subtype classical swine fever E2 protein can be efficiently expressed in a prokaryotic expression system.

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

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

[0012] In some embodiments, the nucleic acid molecule is selected from any one of the following: B1) a nucleic acid molecule having a nucleotide sequence as 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 a sequence identity of more than 90% 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 "hybridize under stringent conditions" refers to the hybridization of two nucleic acid molecule fragments under standard hybridization conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (1989) (Cold Spring Lane Laboratories, New York, USA), in the section "Expression of Cloned Genes in Escherichia coli." Such conditions include, for example, hybridization in 6.0×SSC at 45°C, followed by a wash step at 2×SSC at 50°C. To select the stringency, the salt concentration in the wash step can be selected, for example, between 2.0×SSC at 50°C for low stringency and 2.0×SSC at 50°C for high stringency. In addition, the temperature in the wash step can be varied between room temperature of approximately 22°C for low stringency and 65°C for high stringency.

[0014] As used herein, the term "sequence identity" can be assessed visually or using computer software (e.g., the software program described in Ausubel et al., eds. (2007), in Current Protocols in Molecular Biology). When a position in the compared sequences is occupied by the same base or amino acid, the molecules are identical at that position. The identity between two or more sequences can be expressed as a percentage (%), which can be used to assess the identity between related sequences. A polynucleotide sequence or amino acid sequence that has a certain percentage (e.g., 90%, 95%, 98%, or 99%) of "sequence identity" with another sequence means that, when the sequences are aligned, that percentage of bases or amino acids are the same in the two sequences being compared.

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

[0016] The recombinant vectors in the present invention include cloning vectors and expression vectors. The cloning vectors are used to replicate related sequences, and the expression vectors are used to express related genes. The vector used in constructing the expression vector can be a pET28a vector.

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

[0018] In the present 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 increase the soluble expression level of the recombinant 2.1d subtype classical swine fever E2 protein.

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

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

[0021] In some embodiments, the method for preparing a recombinant cell comprises the step of transforming the above-mentioned recombinant vector into an expression host cell.

[0022] In the present 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 genes it carries can be effectively expressed. It can be a prokaryotic cell or a eukaryotic cell, such as Escherichia coli, yeast, etc., and Escherichia coli can be, for example, E. coli BL21 (DE3).

[0023] In a fifth aspect, the present invention provides a method for preparing a 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 the present invention, there are no special requirements for the culture method and culture conditions, as long as the recombinant cells grow normally. The methods for isolating the 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 method for preparing the recombinant 2.1d subtype classical swine fever E2 protein is a culture medium that can express proteins in the art, preferably LB culture medium.

[0026] In some embodiments, the step of purifying the recombinant 2.1d subtype classical swine fever E2 protein is also included. The purification step specifically includes: first performing ultrasonic lysis, then removing endotoxins, and finally purifying using molecular sieve chromatography.

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

[0028] In some preferred embodiments, removing endotoxins specifically comprises: treating with Triton X-114 at a final concentration of 0.5-1.5%.

[0029] In some preferred embodiments, the molecular sieve chromatography specifically includes: the chromatography medium used is Sepharose 6 Fast Flow, the column height is 60-80 cm, and the maximum sample loading amount is 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 the present invention has good protective efficacy against infection by 2.1d subtype classical swine fever virus.

[0032] In the seventh aspect, the present invention provides a method for preparing the above-mentioned recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine, comprising the following steps: adding an inactivator to the recombinant 2.1d subtype classical swine fever E2 protein for inactivation treatment to obtain an 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 a recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine.

[0033] The preparation method of the recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine provided by the present invention is simple, the raw materials used are cheap and easily available, and the vaccine is suitable for industrial large-scale production.

[0034] In some embodiments, the inactivator includes formaldehyde, and the final concentration of the inactivator 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 content of the recombinant 2.1d subtype classical swine fever E2 protein 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 added is 0.1%, and the inactivation treatment includes: inactivation for 72 hours at a temperature of 4°C; the adjuvant includes the biphasic adjuvant ISA 201; and the content of the 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] It is understood that the inactivating agent and adjuvant can be selected from conventional inactivating agents and adjuvants in the prior art according to actual use needs, as long as a vaccine with excellent performance can be prepared. For example, in the present invention, the inactivating agent preferably includes formaldehyde, and the adjuvant preferably includes the biphasic adjuvant ISA 201.

[0037] In the eighth aspect, the present invention provides the use of the above-mentioned 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 cell, 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 drugs for preventing and / or treating 2.1d subtype classical swine fever virus infection.

[0038] The beneficial effects of the present invention are as follows: different from the prior art, the present invention removes amino acids 344-375 of the 2.1d subtype classical swine fever E2 protein and retains amino acids 1-343, thereby not only retaining the most important antigenic epitopes with protective effects of the 2.1d subtype classical swine fever E2 protein, but also substantially does not affect the spatial structure of the recombinant 2.1d subtype classical swine fever E2 protein, thereby retaining its immunogenicity to the greatest extent; in addition, the recombinant 2.1d subtype classical 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 classical swine fever E2 protein can be significantly improved, and after the 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The SDS-PAGE results of the protein expressed by the BL21(DE3) / pET28a-CSFV E2 engineering bacteria in Example 1 of the present invention are shown, wherein lane M: prestained protein standard molecular weight; lane 1: negative control; lane 2: total protein expressed by the BL21(DE3) / pET28a-CSFV E2 engineering bacteria; lane 3: soluble protein expressed by the BL21(DE3) / pET28a-CSFV E2 engineering bacteria; lane 4: insoluble protein expressed by the BL21(DE3) / pET28a-CSFV E2 engineering bacteria; Figure 2 The SDS-PAGE results of the proteins expressed by the BL21(DE3) / pET28a-CSFV E2+pTf16 or pG-KJE8 engineering bacteria in Example 1 of the present invention are shown, wherein lane M: prestained protein standard molecular weight; lane 1: negative control; lane 2: total protein expressed by the BL21(DE3) / pET28a-CSFV E2+pTf16 engineering bacteria; lane 3: soluble protein expressed by the BL21(DE3) / pET28a-CSFVE2+pTf16 engineering bacteria; lane 4: total protein expressed by the BL21(DE3) / pET28a-CSFV E2+pG-KJE8 engineering bacteria; lane 5: soluble protein expressed by the BL21(DE3) / pET28a-CSFV E2+pG-KJE8 engineering bacteria; Figure 3This is the SDS-PAGE result of the protein expressed by the BL21(DE3) / pET28a-CSFV E2+pG-KJE8 engineering bacteria in Example 1 of the present invention, wherein lane M: prestained protein standard molecular weight; lane 1: negative control; lane 2: total protein expressed by the BL21(DE3) / pET28a-CSFV E2+pG-KJE8 engineering bacteria; lane 3: total protein expressed by the BL21(DE3) / pET28a-CSFV E2+pG-KJE8 engineering bacteria after endotoxin removal; lane 4: total protein expressed by the BL21(DE3) / pET28a-CSFV E2+pG-KJE8 engineering bacteria purified by molecular sieves; Figure 4 This is the Western Blot result of the recombinant 2.1d subtype classical swine fever E2 protein purified in Example 1 of the present invention, wherein lane M: prestained 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 diseased tissue grinding fluid. DETAILED DESCRIPTION

[0040] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0041] Experimental procedures in the examples, where specific conditions are not specified, were generally performed in accordance with conventional methods in molecular biology, including but not limited to those described in M.R. Green's Molecular Cloning: A Laboratory Manual and Robert F. Weaver's Molecular Biology, or according to the recommendations of kit and instrument manufacturers. Unless otherwise specified, reagents and biological materials used in the examples were commercially available.

[0042] Example 1 Modification of the 2.1d subtype swine fever E2 gene and construction of prokaryotic expression engineered bacteria 1.1 Acquisition of the 2.1d subtype swine fever E2 gene 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 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.

[0043] Furthermore, sequence comparison revealed that the amino acid sequence of the 2.1d subtype classical swine fever E2 protein (SEQ ID NO: 1) was 88.06% similar to the amino acid sequence of the E2 protein of the existing conventional HCLV vaccine strain (accession number: ABI93151), with differences in 42 amino acid residues, as shown in Table 1.

[0044] Table 1 Analysis of amino acid sequence differences between the 2.1d subtype swine fever E2 protein and the HCLV vaccine strain E2 protein

[0045] The 2.1d subtype CSFV E2 gene sequence was artificially synthesized and subcloned into the pTOPOT vector, which was then transformed into DH5α competent cells. The correct strain identified by PCR was named E. coli DH5α / pTOPOT-CSFV E2 full-length.

[0046] 1.2 Modification of the 2.1d subtype swine fever E2 gene, construction of a prokaryotic expression vector, and induction of expression Structural analysis of the 2.1d subtype CSF E2 protein from step 1.1 revealed that regions 1-343aa of the E2 protein constitute the extracellular domain, containing the primary protective epitope. Regions 344-375aa of the E2 protein are transmembrane domains, forming an α-helix and containing numerous hydrophobic amino acids. The abundance of hydrophobic amino acids in the 344-375aa region of the E2 protein prevents expression of the full-length E2 gene in the E. coli expression system. Deleting the 344-375aa region while retaining the 1-343aa region not only preserves the most important protective epitope of the E2 protein, but also largely leaves the spatial structure of the 1-343aa region intact, maximizing its immunogenicity.

[0047] Therefore, the 2.1d subtype swine fever E2 gene was modified, and the specific method is as follows: 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 the pTOPOT-CSFV E2 full-length as a template to clone the CSFV E2 F (5'- CCC GGATCC ATGGGCCGGCTGTCCTGT-3' (SEQ ID NO:5)) and CSFV E2 R (5'- CCC CTCGAG The primers TTCGGCGAAGTAGTCTGTGTG-3' (SEQ ID NO: 6) were used to amplify the E2 gene fragment (1-343 aa). The italicized portions of the primer sequence represent the introduced protective bases, and the underlined portions represent the introduced restriction sites for BamH I and Xho I, respectively.

[0048] After agarose gel electrophoresis, the E2 DNA fragment (1-343 aa) was recovered and digested with BamH I and Xho I restriction enzymes. The pET28a plasmid was also treated with the same enzymes. Following agarose gel electrophoresis, the digested product was excised and recovered from the gel, followed by ligation with T4 DNA ligase. The ligated product was transformed into a BL21(DE3) competent cell line and selected with kanamycin. Single colonies were selected for culture, and correct clones were identified by PCR and verified by sequencing. The nucleotide sequence of the recombinant 2.1d subtype CSF E2 gene is shown in SEQ ID NO:4, and the amino acid sequence of the recombinant 2.1d subtype CSF E2 protein is shown in SEQ ID NO:3. The strain was named BL21(DE3) / pET28a-CSFVE2.

[0049] The BL21(DE3) / pET28a-CSFV E2 seed culture was inoculated into 50 mL of LB medium containing kanamycin resistance at a 5% (v / v) inoculum and cultured at 37°C and 200 rpm for 3 h. α-lactose was then added to a final concentration of 30 mM, the temperature was adjusted to 32°C, and the cells were induced at 200 rpm for 15 h. The cells were collected by centrifugation, 10 times their weight of PBS solution was added, the cells were fully resuspended, and then lysed using an ultrasonic disruptor.

[0050] After the fragmentation was completed, the supernatant (soluble protein) was separated by centrifugation at 4°C and 12000g for 10 min. The protein content was determined by BCA method and then analyzed by SDS-PAGE. The results were as follows: Figure 1 shown.

[0051] from Figure 1It can be seen that the recombinant 2.1d subtype classical swine fever E2 protein has a high expression level, but poor solubility.

[0052] 1.3 Co-expression of molecular chaperones to assist the folding of recombinant 2.1d subtype CSF E2 protein As shown in 1.2, deletion of the E2 (344-375aa) region in BL21(DE3) / pET28a-CSFV E2 allows for efficient expression in E. coli. However, the protein solubility is poor, with only approximately 10% of the target protein soluble and able to form the correct spatial conformation. Amino acid analysis of the recombinant 2.1d subtype CSFV E2 protein revealed that it contains 15 cysteines. Excessive cysteines may form intramolecular or intermolecular disulfide bonds at random locations, leading to incorrect tertiary and quaternary structures and ultimately precipitation.

[0053] To enable soluble expression of the recombinant 2.1d subtype classical swine fever E2 protein, the present invention uses molecular chaperones for co-expression. Molecular chaperones are a class of proteins that assist in molecular assembly and protein folding. During the synthesis of nascent peptide chains, molecular chaperones can recognize and stabilize the partially folded conformation of nascent peptide chains, refold misfolded or unfolded proteins into functional conformations, and decompose potentially toxic protein aggregates formed due to protein misfolding. By introducing the pTf16 or pG-KJE8 plasmids capable of co-expressing molecular chaperones into the BL21(DE3) / pET28a-CSFV E2 engineered strain, the correct folding of the recombinant 2.1d subtype classical swine fever E2 protein is assisted, thereby improving its solubility.

[0054] Specifically, the method is as follows: Take BL21(DE3) / pET28a-CSFV E2 seed solution and inoculate it into 50 mL LB medium at a 1% (v / v) inoculum. Incubate at 37°C with shaking for 3 h until the OD 600 The cell suspension was approximately 0.6, centrifuged at 4000g for 10 minutes, and the cells were harvested and washed twice with sterile water. Finally, the cells were resuspended in 1 mL of sterile 10% glycerol and 100 mM CaCl₂ solution, aliquoted, and stored at -80°C. This bacterial suspension is the competent BL21(DE3) / pET28a-CSFV E2 cell suspension for chemical transformation.

[0055] Take one tube of competent cells and add 1 μL of pTf16 or pG-KJE8 plasmid for transformation. Finally, select using 50 μg / mL kanamycin and 34 μg / mL chloramphenicol. The resulting monoclonal strains were designated BL21(DE3) / pET28a-CSFV E2 + pTf16 and BL21(DE3) / pET28a-CSFV E2 + pG-KJE8, respectively.

[0056] The engineered BL21(DE3) / pET28a-CSFV E2+pTf16 strain was activated and inoculated at a 5% (v / v) inoculum into 50 mL of LB medium containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol. L-arabinose was added to a final concentration of 1 mg / mL to induce expression of the molecular chaperone tig. The culture was shaken at 37°C and 200 rpm for 3 hours. Lactose was added to a final concentration of 30 mM to induce expression of the target protein. The culture was incubated at 32°C and 150 rpm for 15 hours. After induction, the cells were harvested by centrifugation.

[0057] The engineered BL21(DE3) / pET28a-CSFV E2+pG-KJE8 strain was activated and inoculated at a 5% (v / v) inoculum into 50 mL of LB medium containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol. L-arabinose (1 mg / mL) and tetracycline (10 ng / mL) were added to induce expression of the molecular chaperones dnaK, dnaJ, grpE, groES, and groEL. The culture was shaken at 37°C and 200 rpm for 3 hours. Lactose (30 mM) was added to induce expression of the target protein, and the culture was incubated at 32°C and 150 rpm for 15 hours. After induction, the cells were harvested by centrifugation.

[0058] Add 10 times the weight of PBS solution to the above bacteria, resuspend them thoroughly, and then use an ultrasonic disruptor to lyse the bacteria. Centrifuge at 12000g and 4℃ for 10 minutes, collect the supernatant, which is the soluble protein. Use the BCA method to determine the protein content, and use SDS-PAGE to analyze the solubility of the target protein. The results are as follows Figure 2 shown.

[0059] from Figure 2 It can be seen that co-expression of molecular chaperones pTf16 or pG-KJE8 can improve the solubility level of recombinant 2.1d subtype classical swine fever E2 protein to varying degrees. Among them, the engineered bacteria co-expressing pG-KJE8 plasmid has the highest solubility of recombinant 2.1d subtype classical swine fever E2 protein.

[0060] 1.4 Purification and identification of recombinant 2.1d subtype classical swine fever E2 protein 1.4.1 Purification of recombinant 2.1d subtype classical swine fever E2 protein Take 100 g of BL21(DE3) / pET28a-CSFV E2+pG-KJE8 engineered bacteria from step 1.3, add 800 mL of PBS, resuspend thoroughly, and then ultrasonicate. The ultrasonication conditions are: 4°C, 3 s on, 3 s off, for a total of 90 min, to obtain a bacterial lysis solution.

[0061] Add 1% Triton X-114 directly to the bacterial lysis solution and stir at 4°C for 2 hours. Aliquot into centrifuge bottles in a swinging bucket rotor, transfer to a 28°C water bath, and let stand for 2 hours. Centrifuge at 4000g for 30 minutes at 28°C. After centrifugation, carefully remove the upper aqueous phase. Repeat this process once to obtain a recombinant 2.1d subtype CSF E2 protein solution free of endotoxin.

[0062] Next, molecular sieve chromatography purification is performed. First, a molecular sieve column is filled with Sepharose 6 FastFlow as the medium. The column diameter is 10 cm and the height is 70 cm. A vacuum fiber column with a pore size of 0.45 μm is used to clarify the above-mentioned recombinant 2.1d subtype swine fever E2 protein solution that has been endotoxin-free. The sample is loaded onto the column with a maximum load of 15% of the column volume. An industrial-grade protein purification system is used for elution, and the protein in the external water peak is collected. The protein content is determined using the BCA method, and the endotoxin content in the antigen is determined using an endotoxin assay kit to be less than 50 EU / mL. The results are as follows: Figure 3 shown.

[0063] from Figure 3 It can be seen that the purity of the purified recombinant 2.1d subtype classical swine fever E2 protein is high, the purification efficiency of the above process is high, the operation is simple, and it is easy to scale up.

[0064] 1.4.2 Identification of recombinant 2.1d subtype classical swine fever E2 protein In order to verify the antigenicity of the prokaryotically expressed soluble recombinant 2.1d subtype classical swine fever E2 protein, it was identified using the classical swine fever standard positive serum from the China Veterinary Drug Administration. The specific operation process is as follows: 1) SDS-PAGE: SDS-PAGE electrophoresis was performed on the negative control protein, the purified recombinant 2.1d subtype CSFV E2 protein (0.5 μg loading amount), and the 2.1d subtype CSFV tissue abrasives. 2) Transfer and Block: Use a semi-dry transfer apparatus to transfer the proteins from the gel to a NC membrane. Block the membrane with 5% skim milk powder for 1 hour at 37°C. 3) Primary Antibody Incubation: Dilute the Classical Swine Fever positive serum 500-fold with blocking buffer and incubate overnight at 4°C. Wash the membrane three times with PBST solution, each time for 5 minutes. 4) Secondary antibody incubation: Dilute HPR-labeled goat anti-pig IgG antibody 20,000-fold in blocking buffer and incubate at 37°C for 2 h. Wash the membrane three times with PBST solution, each time for 5 min. 5) Development and imaging: Use ECL colorimetric solution for development, and use a Bio Rad gel imager for exposure and imaging.

[0065] Western Blot results are as follows Figure 4 As shown, from Figure 4 As can be seen in the results, the prokaryotically expressed recombinant 2.1d subtype CSFV E2 protein reacted specifically with positive serum at 45 kDa, and the 2.1d subtype CSFV sample also reacted specifically with positive serum at approximately 55 kDa (full-length glycosylated E2). These results indicate that the recombinant 2.1d subtype CSFV E2 protein has good antigenicity.

[0066] Example 2 Evaluation of immune challenge of recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine 2.1 Preparation of recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine 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%. The mixture was mixed and inactivated at 4°C for 72 hours. An equal amount of biphasic adjuvant ISA 201 was added, and the mixture was sheared at low speed at 31°C for 10 minutes to form a stable emulsion. Finally, a sterility test was performed.

[0067] After passing the sterility test, the emulsion is the recombinant 2.1d subtype swine fever E2 protein subunit vaccine with an antigen content of 300 μg / mL.

[0068] 2.2 Immunization and challenge with recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine 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; there were 6 experimental animals in each group.

[0069] The challenge group (Group A) received multiple subcutaneous injections of a recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine (1 mL, 300 μg of antigen) at the back. Two weeks after the initial vaccination, a booster dose of 1 mL was administered. The challenge and blank control groups were not vaccinated but received only 1 mL of saline. Continuous observation of the experimental animals after immunization revealed no significant adverse reactions and the animals were in good spirits. Necropsy at the injection site revealed no adverse reactions, including nodules, abscesses, or ulcers.

[0070] Two weeks after the second immunization, the animals in the immune challenge group and the challenge control group were challenged with 1×10 6 Copies of 2.1d subtype CSFV virus fluid were loaded. 5 days after challenge, the rabbits were euthanized and autopsied.

[0071] 2.3 Rectal temperature monitoring of experimental animals CSFV can induce a stereotypical fever in rabbits. Therefore, rectal temperature was measured every 12 hours after challenge. A rectal temperature ≥ 38.5°C was considered a fever. A fever at two or more consecutive time points was considered a stereotypical fever, indicating exposure to the pathogenicity of CSFV. The rectal temperature monitoring results are shown in Table 2 below.

[0072] Table 2 Results of rabbit rectal temperature monitoring and fever determination after challenge

[0073] As shown in Table 2, the six rabbits in the challenge control group developed fever 36 hours after challenge, which persisted until 96 hours. Rectal temperatures reached a peak at 60 hours (all exceeding 41°C), demonstrating typical fever and settling fever, indicating that the challenge induced illness in the experimental rabbits. In contrast, the six rabbits in the immunization group developed a transient fever only at 48 hours, but no settling fever. None of the rabbits in the blank control group developed fever.

[0074] 2.4 Viral load determination 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 tissue freezer grinder at 50 Hz, -30°C, for 90 s, followed by a 160 s pause, repeated three times. The homogenate was centrifuged at 4000 g for 3 min, and the supernatant was separated. Total RNA was extracted from the grinding solution using an RNA extraction kit, and first-strand cDNA synthesis was performed using a reverse transcription kit.

[0075] 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 to perform fluorescence quantitative PCR according to the system in Table 3 below.

[0076] Table 3 Fluorescence quantitative PCR amplification reaction system

[0077] A standard plasmid was constructed, and a standard curve of nucleic acid copy number versus Ct was established using the aforementioned fluorescence quantitative PCR method. The viral load of CSFV subtype 2.1d in rabbit spleens was calculated using this standard curve. The results of the viral load determination are shown in Table 4 below.

[0078] Table 4 Viral load analysis results after challenge

[0079] As can be seen from Table 4, all 6 rabbits in the challenge control group were infected with classical swine fever virus, while none of the immune challenge group and the blank control group were infected with classical swine fever virus.

[0080] 2.5 Specific antibody detection Before challenge, sera were collected from each group of rabbits and post-immunization specific antibody levels were measured using a Classical Swine Fever Blocking ELISA kit. In Classical Swine Fever antibody testing, the blocking rate directly correlates to the specific antibody titer; generally, a higher blocking rate indicates a higher antibody titer in the sample. A blocking rate below 30% indicates a negative result for antibodies to the recombinant 2.1d subtype Classical Swine Fever E2 protein; a blocking rate above 40% indicates a positive result for antibodies to the recombinant 2.1d subtype Classical Swine Fever E2 protein. The results are shown in Table 5 below.

[0081] Table 5 Specific antibody detection results

[0082] As shown in Table 5, among the three groups of experimental animals, only the challenge group had the highest blocking rate, exceeding 30%, indicating the highest titer of antibodies against the recombinant 2.1d subtype classical swine fever E2 protein. In contrast, the blocking rate in both the challenge and blank control groups was below 30%, with no detectable classical swine fever antibodies. These results demonstrate that the recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine induced high-titer antibodies and provided 100% protection against the challenge in the experimental animals.

[0083] In summary, the recombinant 2.1d subtype classical swine fever E2 protein provided by the present invention has a high soluble expression level. After the 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.

[0084] The above-described embodiments merely illustrate the implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A recombinant 2.1d subtype classical swine fever E2 protein, characterized in that: The recombinant 2.1d subtype classical swine fever E2 protein is selected from any one of the following: A1) has the amino acid sequence shown in SEQ ID NO: 3; A2) amino acid sequences having one or more amino acid substitutions, deletions or additions compared to the amino acid sequences defined in A1); A3) an amino acid sequence having a sequence identity of 80% or more to the amino acid sequence defined in A1) or A2); A4) An amino acid sequence obtained by linking a tag or a signal peptide to the N-terminus and / or C-terminus of the amino acid sequence defined in A1) or A2) or A3).

2. A nucleic acid molecule encoding the recombinant 2.1d subtype classical swine fever E2 protein according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleic acid molecule is selected from any one 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 recombinant 2.1d subtype classical swine fever E2 protein of claim 1; B3) A nucleic acid molecule that has a sequence identity of more than 90% with the nucleic acid molecule defined in B1) or B2) and encodes the recombinant 2.1d subtype classical swine fever E2 protein according to claim 1.

4. A recombinant vector, characterized in that Comprising the nucleic acid molecule according to claim 2 or 3.

5. A recombinant cell, characterized in that The method comprises the nucleic acid molecule according to claim 2 or 3 or the recombinant vector according to claim 4, and at least one of the pTf16 and pG-KJE8 vectors.

6. A method for preparing a recombinant 2.1d subtype classical swine fever E2 protein, characterized in that: The steps include: Cultivating the recombinant cell according to claim 5 and inducing expression to obtain a culture; The recombinant 2.1d subtype classical swine fever E2 protein according to claim 1 is isolated from the culture.

7. A recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine, characterized in that: It comprises the recombinant 2.1d subtype classical swine fever E2 protein according to claim 1 and an adjuvant.

8. A method for preparing the recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine according to claim 7, characterized in that: The steps include: adding an inactivating agent to the recombinant 2.1d subtype classical swine fever E2 protein for inactivation to obtain an inactivated recombinant 2.1d subtype classical swine fever E2 protein; The inactivated recombinant 2.1d subtype classical swine fever E2 protein is mixed with an adjuvant and emulsified to obtain a recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine.

9. The method for preparing the recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine according to claim 8, characterized in that: The inactivator includes formaldehyde, and the final concentration of the inactivator is 0.05-0.15%. The inactivation treatment includes: inactivating at a temperature of 0-4°C for 60-80 hours; the adjuvant includes a biphasic adjuvant ISA 201; The content of the recombinant 2.1d subtype classical swine fever E2 protein in the recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine is 200-400 μg / mL.

10. Use of the recombinant 2.1d subtype classical swine fever E2 protein according to claim 1, the nucleic acid molecule according to any one of claims 2-3, the recombinant vector according to claim 4, the recombinant cell according to claim 5, the recombinant 2.1d subtype classical swine fever E2 protein prepared by the preparation method according to claim 6, the recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine according to claim 7, and the recombinant 2.1d subtype classical swine fever E2 protein subunit vaccine prepared by the preparation method according to any one of claims 8-9 in the preparation of a medicament for preventing and / or treating 2.1d subtype classical swine fever virus infection.

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