Classical swine fever virus E2 protein mutant and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a hog cholera virus E2 protein mutant and application thereof. The hog cholera virus E2 protein mutant comprises the following amino acid mutation sequence (1) or (2): (1) the hog cholera virus E2 protein is obtained by mutating the 15th site and the 25th site of the hog cholera virus E2 protein amino acid sequence; (2) mutating the 172nd site and the 323rd site of the amino acid sequence of the hog cholera virus E2 protein; wherein the amino acid sequence of the hog cholera virus E2 protein is as shown in SEQ ID NO. 1. The expression quantity of the hog cholera virus E2 protein mutant disclosed by the invention can reach 117-134 mu g / mL; the immune effect of the E2 protein mutant is better than that of the original sequence, and the positive rate of the E2 protein mutant after mice are immunized for 28 days reaches 100%.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a mutant of the E2 protein of classical swine fever virus and its uses. Background Technology

[0002] Classical swine fever (CSF) is a highly contagious disease caused by the classical swine fever virus (CSFV). It is easily co-infected with other viral and bacterial diseases, increasing the difficulty of disease control in pig farms and seriously jeopardizing the development of the pig industry. It is listed as a Category A infectious disease by the World Health Organization (OIE) and as a Class I animal disease in China. Faced with the severe CSF epidemic, the United States and the United Kingdom pioneered research on attenuated CSF vaccines in the 1940s. In the 1950s, China successfully developed a highly safe and effective attenuated CSF vaccine (C strain), which played a crucial role in CSF control in China and worldwide. However, it still has limitations in terms of the durability of immune protection, the protective effect against variant strains, the optimization of production processes, and the ability to distinguish between vaccine immunization and natural infection. With the expansion of intensive farming and the continuous emergence of new diseases, existing conventional vaccines (inactivated vaccines and attenuated vaccines) cannot meet the CSF eradication standards of breeding pig farms. At the same time, genetically engineered vaccines, such as viral vector vaccines, subunit vaccines, nanoparticle vaccines, and plant expression vaccines, can avoid many of the shortcomings of live vaccines (poor safety, poor stability, inability to identify and diagnose, etc.), improving the immunogenicity and protective effect of vaccines while being suitable for large-scale production.

[0003] CSFV belongs to the genus *Pestivirus* of the family Flaviviruses. It is a linear, single-stranded, positive-sense RNA, approximately 12.3 kb in size, with only a single open reading frame (ORF). It can sequentially translate four structural proteins: capsid protein C, envelope glycoprotein E, etc. rnsThe vaccine contains E1, E2, and seven non-structural proteins. Among these, E2 protein is the primary protective antigen and is widely used in the development of novel classical swine fever (CSF) vaccines. CSFV E2 is a "moderately stable" antigen (Tm≈53℃), and protein stability is a key quality attribute of subunit vaccines. Changes in protein spatial structure can lead to decreased activity, thus affecting immunogenicity. The baculovirus expression system is the most commonly used system for producing CSFV E2 protein, but the yield of E2 protein in this system is generally low. E2 subunits typically require booster immunization, thus demanding a large amount of antigen. Efficient expression is also crucial for reducing vaccine production costs. Therefore, a novel CSFV E2 protein mutant is proposed, exhibiting strong immunogenicity, inducing high-titer neutralizing antibodies, increasing expression levels, and being suitable for long-term storage. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing a mutant of the E2 protein of classical swine fever virus and its uses.

[0005] To address the aforementioned technical problems, this invention discloses a novel E2 protein mutant of classical swine fever virus and its applications. The specific technical solution is as follows: In a first aspect, the present invention provides a classical swine fever virus E2 protein mutant, wherein the classical swine fever virus E2 protein mutant comprises the amino acid mutation sequence described in (1) or (2) below: (1) It was obtained by mutations at positions 15 and 25 of the amino acid sequence of the classical swine fever virus E2 protein; (2) It was obtained by mutations at positions 172 and 232 of the amino acid sequence of the classical swine fever virus E2 protein; The amino acid sequence of the classical swine fever virus E2 protein is shown in SEQ ID NO.1.

[0006] Furthermore, the mutated amino acids are all cysteine ​​(Cys,C), that is, the amino acids at positions 15, 25, 172 or 232 of the amino acid sequence of the classical swine fever virus E2 protein are all mutated to cysteine ​​(Cys,C).

[0007] Furthermore, the classical swine fever virus E2 protein mutant also includes a V5 tag, a 6×his tag sequence, and a signal peptide hybrid sequence; wherein the V5 tag and the 6×his tag sequence are sequentially linked to the C-terminus of the amino acid mutant sequence, and the signal peptide hybrid sequence is linked to the N-terminus of the amino acid mutant sequence.

[0008] Furthermore, the amino acid sequence of the signal peptide Hybrid is shown in SEQ ID NO.5.

[0009] In some technical solutions, the classical swine fever virus E2 protein mutant is characterized by mutating both Thr (position 15) and Gly (position 25) of the amino acid sequence shown in SEQ ID NO.1 to Cys, and sequentially attaching a V5 tag and a 6×his tag sequence to the C-terminus of the mutated amino acid sequence, and a signal peptide Hybrid sequence to the N-terminus. Furthermore, the amino acid sequence of the classical swine fever virus E2 protein mutant is shown in SEQ ID NO.6.

[0010] In some technical solutions, the classical swine fever virus E2 protein mutant is characterized by mutating Val at position 172 and Ser at position 232 of the amino acid sequence shown in SEQ ID NO.1 to Cys, and sequentially attaching a V5 tag and a 6×his tag sequence to the C-terminus of the mutated amino acid sequence, and a signal peptide Hybrid sequence to the N-terminus. Furthermore, the amino acid sequence of the classical swine fever virus E2 protein mutant is shown in SEQ ID NO.8.

[0011] Secondly, the present invention provides a gene encoding a mutant of the classical swine fever virus E2 protein described in the first aspect. In some embodiments of the present invention, Cys is encoded by TGC or TGT.

[0012] In some embodiments of the present invention, the 5' end of the nucleotide sequence of the gene is attached with the Kozak sequence GCCACC. Preferably, the nucleotide sequence of the gene is as shown in SEQ ID NO.7 or SEQ ID NO.9.

[0013] Thirdly, the present invention provides a method for preparing the E2 protein mutant of the classical swine fever virus described in the first aspect, comprising the following steps: (1) The gene encoding the E2 protein mutant of the classical swine fever virus is amplified, cloned into a baculovirus plasmid vector, and transfected into insect cells to obtain recombinant baculovirus; in some embodiments of the present invention, the baculovirus plasmid vector includes pFAST-Bac1.

[0014] (2) Infect insect cells with the recombinant baculovirus at an MOI of 0.25-16 for 72-96 h, harvest the cell suspension, centrifuge and collect the supernatant to obtain the E2 protein mutant of the classical swine fever virus; preferably, the MOI of the recombinant baculovirus is 2-8; the cell density of the insect cells is 4E6 cells / mL.

[0015] Preferably, in step (1) or step (2), the insect cells are SF9 cells or Hi5 cells.

[0016] Fourthly, the present invention provides a classical swine fever virus vaccine comprising the classical swine fever virus E2 protein mutant described in the first aspect.

[0017] The classical swine fever virus vaccine further includes an adjuvant, which includes any one of ISA201VG, ISA563VG, or ISA660VG. In some embodiments of the present invention, the adjuvant is ISA563VG. The volume ratio of the adjuvant to the classical swine fever virus E2 protein mutant is 1:0.5-1.5. In some embodiments of the present invention, the volume ratio of the adjuvant to the classical swine fever virus E2 protein mutant is 1:1.

[0018] Fifthly, the present invention provides the application of the classical swine fever virus E2 protein mutant described in the first aspect, or the classical swine fever virus E2 protein mutant prepared by the preparation method described in the third aspect, or the classical swine fever virus vaccine described in the fourth aspect, in the preparation of products for the prevention of classical swine fever.

[0019] Beneficial effects: The E2 protein mutants mE2-T15-G25 and mE2-V172-S232 provided by this invention effectively increase the expression level of E2 protein, with the protein expression level exceeding 100 μg / mL. The mutants exhibit good immunogenicity. When mice were immunized with vaccines prepared using the mutants mE2-T15-G25 and mE2-V172-S232, the rate of antibody production and the antibody level in the mice were higher than those prepared with the unmutated protein (E2-E1) and commercial vaccines. Attached Figure Description

[0020] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0021] Figure 1 Electrophoretic identification of E2 and E2 mutant proteins (non-reduction).

[0022] Figure 2 This study aims to detect the average blocking rate of antibodies after immunization in mice.

[0023] Figure 3 The content of purified E2 protein was detected after different storage times at 4℃. Detailed Implementation

[0024] The present invention will be further described below through specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0025] Example 1: Construction and Protein Expression Identification Using GenBank sequence number AAS20416.1 as a template, the E2 region (423 aa-793 aa) of classical swine fever strain 1.1 was searched. The C-terminal transmembrane region and extracellular region were deleted, and the amino acid sequence is shown in SEQ ID NO.1 (the nucleotide sequence of the coding gene is shown in SEQ ID NO.2). A V5 tag and a 6×his tag sequence were sequentially linked to its C-terminus, and a signal peptide Hybrid (1-23 aa, the amino acid sequence of the signal peptide is shown in SEQ ID NO.5) was added to its N-terminus. This sequence was named E2-E1, and its amino acid sequence is shown in SEQ ID NO.3. The nucleotide sequence of the gene encoding the amino acid sequence shown in SEQ ID NO.3 was obtained by linking the 5' end of the Kozak sequence (GCCACC) to the nucleotide sequence shown in SEQ ID NO.4.

[0026] Mutation screening was performed on the sequence shown in SEQ ID NO.1, and 32 pairs of mutagenic sites were obtained. Cysteine ​​(Cys) was replaced at the mutagenic sites (resulting in amino acid mutant sequences). Based on this, the thermostability of the mutated protein was screened (ΔG_stability<0.5kcal / mol). The screening results are shown in Table 1. A total of 11 pairs of E2 mutants with good thermostability were obtained, which were named mE2-XY (X and Y represent the position and name of the mutated amino acid) for subsequent verification.

[0027] Table 1. Prediction of disulfide bond stability and screening of protein stability in E2 protein.

[0028] The nucleotide sequences encoding the E2-E1 genes with the Kozak sequence (GCCACC) added to the 5' end, as well as the nucleotide sequences encoding the 11 mutants in Table 1, were modified by adding restriction endonucleases BamHI and HindII to the 5' and 3' ends, respectively. These were then synthesized by General Biotech (Anhui) Co., Ltd. The synthesized genes were ligated into the pFAST-Bac1 vector via enzyme digestion and ligation. Selected single clones were sequenced, and the sequencing results were consistent with the nucleic acid sequences, thus constructing the recombinant plasmids pFAST-Bac1-E2-E1 and pFAST-Bac1-mE2-XY.

[0029] Transform 5-10 ng / μL of pFAST-Bac1-E2-E1 or pFAST-Bac1-mE2-XY plasmids into DH10Bac competent cells, respectively. Transfer the cells to 800 μL of antibiotic-free 2YT medium and incubate at 37 °C with shaking at 200 rpm for 1 h. Transfer 100 μL of the culture to a selection dish (2YT medium containing 50 μg / mL Kan, 7 μg / mL gentamicin, 10 μg / mL tetracycline, 100 μg / mL Bluo-gal, and 40 μg / mL IPTG) and incubate at 37 °C. Incubate at ℃ in the dark for 2-3 days. Pick white spots and streak them again on a new screening culture dish. The next day, screen for white positive clones. Use M13-F (5'-TATTCCGGATTATTCATACC-3') and M13-R primers (5'-ACAAATGTGGTATGGCTGA-3') for bacterial PCR identification (reaction system is shown in Table 2, reaction procedure is shown in Table 3). Expand the culture of the single-band clones and extract plasmids to obtain recombinant E2-E1-rBacmid and mE2-XY-rBacmid plasmids.

[0030] Table 2 PCR reaction system

[0031] Table 3 PCR reaction procedure

[0032] The recombinant rod plasmid was used with ExpiFectamine Sf TM SF9 cells were transfected with the transfection reagent (Gibco, catalog number: A38915) and cultured at 27°C and 125 rpm until the cell viability dropped to 80%-60%. The cells were then centrifuged to collect the virus, and recombinant baculovirus E2-rBV strain P0 generation virus expressing classical swine fever E2 original protein or mutant protein was obtained.

[0033] P0 generation E2-rBV virus was used to infect Hi5 cells at 1% v / v (Hi5 cell density 2E6 cells / mL). Cells were harvested 72-96 h post-infection, centrifuged at 7400 rpm for 30 min, and the protein supernatant was obtained. The harvested protein supernatant was mixed with 6× Protein Loading Buffer (Beijing TransGen Biotech Co., Ltd., catalog number: DL101-02) and boiled. The mixture was then loaded onto 12% SurePAGE. TMProtein expression in precast gels (Nanjing Genscript Biotech Co., Ltd., catalog number: M00669) was detected by reducing SDS-PAGE electrophoresis. The protein expression of E2-E1 and the E2 mutant mE2-XY is shown in Table 4. Mutants mE2-T15-G25, mE2-L200-A228, mE2-V61-P89, mE2-V172-S232 and mE2-A117-P130 have the ability to secrete E2 protein. Among them, mutant mE2-T15-G25 has a better secretion ability than E2-E1. The secretion ability of mE2-V172-S232 is slightly worse than that of E2-E1. The secretion abilities of mE2-L200-A228 and mE2-A117-P130 are similar, but both are slightly worse than that of E2-E1. The secretion ability of mE2-V61-P89 is the worst. The amino acid sequence of mutant mE2-T15-G25 is shown in SEQ ID NO.6 (the C-terminus of this sequence is connected with V5 and 6×his tags, and the N-terminus of the signal peptide Hybrid is added), and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.7 (the 5' end of this encoding gene sequence is added with the Kozak sequence GCCACC); the amino acid sequence of mutant mE2-V172-S232 is shown in SEQ ID NO.8 (the C-terminus of this sequence is connected with V5 and 6×his tags, and the N-terminus of the signal peptide Hybrid is added), and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.9 (the 5' end of this encoding gene sequence is added with the Kozak sequence GCCACC).

[0034] Table 4. Expression of secreted proteins in E2-E1 and E2 mutants

[0035] The supernatant of proteins identified as expressed by reducing SDS-PAGE was mixed with 5× Protein Loading Buffer (NoReducing Buffer, Sangon Biotech (Shanghai) Co., Ltd., catalog number: C516031) and loaded directly onto 12% SurePAGE buffer without boiling. TM Non-reducing SDS-PAGE electrophoresis and Western blotting were performed on precast protein gels (e.g., for protein identification). Figure 1 As shown, the primary antibody used in WB was a mouse-derived E2 monoclonal antibody, and the secondary antibody was goat anti-mouse HRP. The dimer of the E2 mutant was 1-2 kDa larger than that of E2-E1, indicating that the construction was successful.

[0036] Example 2 Protein purification and animal experiments Proteins with a secretion supernatant concentration ≥40 μg / mL obtained in Example 1 (including E2 primitive protein and E2 mutant protein) were purified by nickel column chromatography. The equilibration buffer consisted of 50 mM Tris and 300 mM NaCl, pH 8.5, equilibrated for 5 CV. The washing buffer consisted of 50 mM Tris, 300 mM NaCl, and 10 mM imidazole, pH 8.5, and was twice the volume of the sample loaded. The elution buffer consisted of 50 mM Tris, 300 mM NaCl, and 500 mM imidazole, pH 8.5, eluted for 5 CV. This yielded purified E2-E1 protein solutions and purified E2 mutant protein solutions.

[0037] Protein purification buffer was replaced with a Hisrep 26 / 10 desalting column (Cytiva, catalog number: 17508701) buffer (50 mM Tris, 300 mM NaCl, pH 8.0) and then quantified by SDS-PAGE. The purified buffer was then mixed with adjuvant ISA563VG at a 1:1 volume ratio to prepare a classical swine fever virus (CSF) E2 recombinant baculovirus vaccine with a uniform antigen content of 35 μg / mL. Six-week-old Balb / c female mice were randomly divided into seven groups (n=8 per group: blank control group, commercial vaccine group, E2-E1 purified group, mE2-L200-A228 group, mE2-T15-G25 group, mE2-V172-S232 group, and mE2-A117-P130 group) for primary immunization and a second immunization 28 days later. Immunization was administered via intramuscular injection, with each mouse receiving 200 μL. The commercially available vaccine is a recombinant baculovirus inactivated vaccine against classical swine fever virus E2 protein (WH-09 strain) (Wuhan Keqian Biotechnology Co., Ltd.). The blank control group consisted of an injection of the same volume of sterile saline. Whole blood was collected and serum was separated at 7-day intervals. The serum was stored at -20℃ and antibody detection was performed using a classic classical swine fever virus E2 protein antibody detection kit (IDEXX CSFV E2 Ab, catalog number: 99-43220). The blocking rate (%) and positive rate (%) of each group were calculated. The blocking rate (%) was calculated as follows: [NC (mean OD of negative control) - S (OD of sample) / NC] × 100%, where the negative control was an internal control set within the kit; the positive rate (%) was calculated as: number of positive samples / total number of samples × 100%, with a blocking rate ≥ 40% considered positive.

[0038] The results are shown in Table 5 and Figure 2As shown, mE2-T15-G25 and mE2-V172-S232 preferentially produce antibodies compared to E2-E1 and commercial vaccines, and their titers are also higher than those of E2-E1 and commercial vaccines. Among them, the positivity rate of mE2-T15-G25 was 100% at 21 days after immunization and remained at 100% throughout the course of immunization, with no decline at 42 days, and the earliest and most stable peak. Although the positive reaction of mE2-V172-S232 started earliest at 7 days after immunization, the positivity rate was 66% at 21 days after immunization and reached 100% at 28 days after immunization, and the induction was both rapid and stable, the rapid rise phase of mE2-T15-G25 was significantly faster.

[0039] Table 5. Positive rates (%) after immunization with E2-E1 and E2 mutants

[0040] This embodiment also verified the stability of the immunogenic mE2-T15-G25 and mE2-V172-S232 proteins compared to E2-E1 by storing them at 4℃ for different periods. Samples were taken after storage at 4℃ for 7, 14, 30, 60, and 90 days. The obtained samples were directly mixed with 6× loading buffer and boiled for sample preparation. SDS-PAGE analysis showed that the purified mE2-T15-G25 and mE2-V172-S232 proteins had higher stability than E2-E1. After storage at 4℃ for at least 3 months, no protein precipitation occurred, and the protein content did not change significantly. In contrast, the protein concentration of E2-E1 began to decrease after 2 months of storage. Figure 3 As shown.

[0041] Example 3 Optimization of Protein Expression Conditions The P0 generation E2-E1-rBV, mE2-T15-G25-rBV, and mE2-V172-S232-rBV viruses prepared according to the method described in Example 1 were used to infect Hi5 cells (cell density 4E6 cells / mL) with MOIs of 0.25, 0.5, 1, 2, 4, 8, and 16, respectively. Cells were harvested 72-96 h post-infection, centrifuged at 7400 rpm for 30 min, and the protein supernatant was obtained. The supernatant was mixed with 6× Protein loading buffer (Beijing TransGen Biotech Co., Ltd., catalog number: DL101-2) and boiled for sample preparation. The protein secretion expression results of all culture supernatants were analyzed by SDS-PAGE, and are shown in Table 6.

[0042] The results showed that when the MOI was between 0.25 and 16, the secretion levels of the E2 mutants mE2-T15-G25 and mE2-V172-S232 were higher than those of the unmutated E2-E1. When the MOI was between 2 and 8, the protein expression levels of the E2 mutants mE2-T15-G25 and mE2-V172-S232 were both higher than 100 μg / mL. Among them, the E2 mutant mE2-T15-G25 had the highest protein expression level (134 μg / mL) under viral infection at MOI=8, while the E2 mutant mE2-V172-S232 had the highest protein expression level (117 μg / mL) under viral infection at MOI=2.

[0043] Table 6. Protein secretion levels after viral infection of cells with different MOIs

[0044] In summary, the E2 mutants mE2-T15-G25 and mE2-V172–S232 recombinant baculovirus of this invention can produce Hi5 cells with a protein secretion rate of 117-134 μg / mL, significantly improving the secretory expression capacity of E2 protein. The two E2 mutants also exhibit better stability, remaining stable at 4 °C for at least 3 months. Furthermore, in mouse immunization experiments, the rate of antibody production, duration of antibody retention, and antibody positivity rate of the two E2 mutants were significantly superior to those of the unmutated protein and commercially available vaccine protein.

[0045] The secretory expression capacity of the classical swine fever virus E2 mutant protein designed in this invention is significantly superior to that of existing technologies: for example, the E2 protein content secreted into the viral fluid by Chinese patent CN110747215A is approximately 75 μg / mL. Another example is the E2 protein secreted and expressed by Chinese patent CN115850510A at 50 μg / mL.

[0046] This invention provides a method and approach for identifying a mutant of the classical swine fever virus E2 protein and its uses. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A mutant of the E2 protein of the porcine pestivirus, characterized in that, Contains the amino acid mutant sequence described in (1) or (2) below: (1) It was obtained by mutations at positions 15 and 25 of the amino acid sequence of the classical swine fever virus E2 protein; (2) It was obtained by mutations at positions 172 and 232 of the amino acid sequence of the classical swine fever virus E2 protein; The amino acid sequence of the classical swine fever virus E2 protein is shown in SEQ ID NO.

1.

2. The CSFV E2 protein mutant according to claim 1, characterized in that, The mutated amino acids are all cysteine.

3. The CSFV E2 protein mutant of claim 1, wherein, The described classical swine fever virus E2 protein mutant also includes a V5 tag, a 6×his tag sequence, and a signal peptide hybrid sequence; Wherein, the V5 tag and 6×his tag sequences are sequentially linked to the C-terminus of the amino acid mutation sequence, and the signal peptide Hybrid sequence is linked to the N-terminus of the amino acid mutation sequence; preferably, the amino acid sequence of the signal peptide Hybrid is as shown in SEQ ID NO.

5.

4. The CSFV E2 protein mutant according to claim 3, characterized in that, The amino acid sequence of the classical swine fever virus E2 protein mutant is shown in SEQ ID NO.6 or SEQ ID NO.

8.

5. A gene encoding the E2 protein mutant of classical swine fever virus as described in any one of claims 1 to 4.

6. The gene of claim 5, wherein The nucleotide sequence of the gene is linked to the 5' end with the Kozak sequence GCCACC.

7. The gene of claim 6 wherein, The nucleotide sequence of the gene is shown in SEQ ID NO.7 or SEQ ID NO.

9.

8. A method for preparing the mutant of the E2 protein of the porcine pestivirus according to any one of claims 1 to 4, characterized in that, Includes the following steps: (1) The gene encoding the E2 protein mutant of the classical swine fever virus was amplified, cloned into a baculovirus plasmid vector, and transfected into insect cells to obtain recombinant baculovirus; (2) Infect insect cells with the recombinant baculovirus at an MOI of 0.25-16 for 72-96 h, harvest the cell suspension, centrifuge and collect the supernatant to obtain the classical swine fever virus E2 protein mutant.

9. A swine fever virus vaccine, characterized in that, It includes the classical swine fever virus E2 protein mutant according to any one of claims 1 to 4.

10. The classical swine fever virus E2 protein mutant according to any one of claims 1 to 4, or the classical swine fever virus E2 protein mutant prepared by the preparation method of claim 8, or the classical swine fever virus vaccine according to claim 9, in the preparation of products for the prevention of classical swine fever.

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