Swine erysipelas rSpaA-Fc fusion protein and application thereof in vaccine

Abstract

The present application relates to a kind of swine erysipelas rSpaA-Fc fusion protein and its application in vaccine.The protective antigen protein SpaA of swine erysipelas is designed, a fusion protein sequence is obtained, the protein sequence has swine erysipelas serum 1a type and swine erysipelas serum 2 type antigen protein, and Thelper and IgG Fc sequence are fused simultaneously.The sequence is optimized according to E. coli codon, and the rSpaA-Fc fusion protein sequence of swine erysipelas is obtained;Vector construction, transformation, induction expression, inclusion body purification obtain rSpaA-Fc fusion protein;The fusion protein is made into vaccine with adjuvant Gel-02 to immunize piglets, can earlier induce high level of humoral immunity and cellular immunity, improve the protection of swine erysipelas serum 1a and 2 type strong poison attack.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a porcine erysipelas rSpaA-Fc fusion protein and its application in vaccine preparation. Background Technology

[0002] *Erysipelothrix rhusiopathiae*, commonly known as swine erysipelas bacillus, is a facultative anaerobic, Gram-positive, slender bacillus belonging to the genus *Erysipelothrix* in the family Erysipelothrionaceae. It can cause erysipelas in pigs and other animals. Swine erysipelas is an acute, febrile infectious disease, characterized by high fever, acute septicemia, skin rashes, chronic verrucous endocarditis, skin necrosis, and polyarthritis (non-suppurative arthritis). *Erysipelothrix rhusiopathiae* has several serotypes, with most cases caused by serotypes 1a and 2.

[0003] Currently, this disease is distributed in most countries worldwide, occurring sporadically or in epidemics, causing significant economic losses to the pig farming industry and posing a threat to human health. Swine erysipelas, along with classical swine fever and swine pneumonia, is considered one of the three major infectious diseases in China's pig farming industry, causing severe economic losses. The most effective way to prevent swine erysipelas is through vaccination. Currently, the vaccines used on the market are mainly inactivated swine erysipelas vaccines and attenuated live vaccines. However, in these two types of vaccines, attenuated strains have the problem of virulence reversion, while inactivated vaccines have the problems of incomplete inactivation and low immunity. Therefore, developing novel subunit vaccines is the main direction of research on swine erysipelas vaccines. Although genetically engineered subunit vaccines have the advantages of high biosafety, simple operation, and low cost, their inability to induce cellular immunity is a limiting factor. At the same time, the current design of novel subunit vaccines for swine erysipelas mainly targets a specific genotype of SpaA protein (as shown in patent applications 201910442887.2 and 202011366261.7). Therefore, it is urgent to upgrade existing technologies to improve antibody levels, immune coverage, and protection rates. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention patent designs a porcine erysipelas rSpaA-Fc fusion protein and simultaneously prepares a subunit vaccine product containing this fusion protein, thus overcoming the shortcomings of the prior art.

[0005] First, this invention discloses a porcine erysipelas rSpaA-Fc fusion protein, the amino acid sequence of which is shown in SEQ ID NO:2.

[0006] The amino acid sequence of the porcine erysipelas rSpaA-Fc protein is formed by linking the signal peptide sequence as shown in SEQ ID NO:3, the T helper sequence as shown in SEQ ID NO:4, the first sequence of porcine erysipelas serum 1a SpaA protective antigen as shown in SEQ ID NO:5, the first sequence of linker1 as shown in SEQ ID NO:6, the first sequence of porcine erysipelas serum 2SpaA protective antigen as shown in SEQ ID NO:7, the second sequence of porcine erysipelas serum 1a SpaA protective antigen as shown in SEQ ID NO:8, the second sequence of linker1 as shown in SEQ ID NO:6, the second sequence of porcine erysipelas serum 2SpaA protective antigen as shown in SEQ ID NO:9, the third sequence of porcine erysipelas serum 1a SpaA protective antigen as shown in SEQ ID NO:10, the linker2 sequence as shown in SEQ ID NO:11, the Fc sequence as shown in SEQ ID NO:12, and the 6his tag sequence as shown in SEQ ID NO:13.

[0007] The base sequence encoding the rSpaA-Fc fusion protein of porcine erysipelas is shown in SEQ ID NO:1; the gene was optimized according to the codon preference of Escherichia coli.

[0008] The rSpaA-Fc fusion protein for porcine erysipelas of the present invention is used to prepare subunit vaccines and diagnostic reagents for porcine erysipelas.

[0009] Compared with existing technologies, the subunit vaccine prepared from the porcine erysipelas rSpaA-Fc fusion protein designed in this invention has the characteristics of being able to simultaneously induce humoral and cellular immunity, having high antibody levels, and high protection rates against porcine erysipelas gene types 1a and 2, thus laying a solid foundation for the production of porcine erysipelas subunit vaccines. Attached Figure Description

[0010] Figure 1 Example 4 uses ELISA to determine the detection results of γ-interferon in porcine serum. Detailed Implementation

[0011] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention.

[0012] All reagents used in the embodiments of this invention are commercially available products.

[0013] The list of reagents, strains, and plasmid sources for this invention is as follows:

[0014] The competent cells BL21 used in this invention were purchased from Beijing Qingke Biotechnology Co., Ltd.

[0015] The culture medium was purchased from Shanghai Yuanye Biotechnology Co., Ltd.;

[0016] Gel02 adjuvant was purchased from Sepico (Shanghai) Specialty Chemicals Co., Ltd.

[0017] The IFN-γ cytokine detection kit was purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.

[0018] The 96-well microplate was purchased from Corning Incorporated.

[0019] The pET28a vector was purchased from Genscript Biotech Inc.

[0020] Example 1

[0021] rSpaA-Fc sequence synthesis and recombinant plasmid construction

[0022] Based on the SpaA antigen sequences of porcine erysipelas serotype 2 strain C43311 (GenBank: EF635597.1) and porcine erysipelas serotype 1a strain LA150627 (GenBank: KU214211.1) reported by NCBI, sequences of SpaA-DC1 protein containing only serotype 1a antigen characteristics, SpaA-DC2 protein containing only serotype 2 antigen characteristics, and rSpaA-Fc fusion protein containing both porcine erysipelas serotype 1a and serotype 2 antigen proteins were synthesized (SEQ ID NO: 1). The SpaA-DC1 and SpaA-DC2 sequences are 1239 bases in size; the rSpaA-Fc sequence contains 2238 bases. The sequences were synthesized and ligated between the BamHI and EcoRI restriction sites of the pET28a vector to construct the prokaryotic expression vectors pET28a-SpaA-DC1, pET28a-SpaA-DC2, and pET28a-rSpaA-Fc.

[0023] Example 2

[0024] Construction of expression strains and induction of protein expression and purification

[0025] The method for preparing SpaA-DC1 protein, SpaA-DC2 protein, and rSpaA-Fc fusion protein includes the following steps: 1) BL21 competent cell transformation and identification of positive expression strains; 2) positive strain culture and protein induction expression; 3) inclusion body purification.

[0026] 1) Transformation of BL21 competent cells and identification of positive expression strains

[0027] The positive plasmid was transformed into BL21 competent cells. After transformation, single colonies were picked and cultured in LB liquid medium containing kanamycin at 37°C overnight on a shaker. After centrifugation at 8000 r / min for 5 min, the cells were collected, boiled in a water bath for 5 min, and centrifuged at 12000 r / min for 3 min. The supernatant was then collected for PCR identification. After correct identification, the culture was expanded.

[0028] 2) Cultivation of positive strains and protein induction expression

[0029] The PCR-positive strains were inoculated into 150 mL of TB medium and cultured in a shaker at 37 °C until OD600 = 0.6. IPTG was added to a final concentration of 1 mmol / L and induced for 4 h. After centrifugation, the cultures were resuspended in 1 / 10 volume of PBS, sonicated, and the supernatant and precipitate were collected by centrifugation for protein identification.

[0030] 3) Inclusion body purification

[0031] Add the precipitate to cold washing buffer (20 mM Tris, 2 mM EDTA, 1% Trixon 100, 1% Tween 20, 0.5 M Urea, pH 8.2) at a ratio of 1:10 (w:v) and stir for 20 minutes. Centrifuge at 12000 rpm for 25 minutes at 2–8 °C, discard the supernatant, and collect the precipitate. Repeat once (washing for 2–4 hours to remove membrane debris and membrane proteins). Wash the resulting precipitate once more with 50 mmol / L Tris-HCl under the same centrifugation conditions, discarding the supernatant. The resulting precipitate is the washed inclusion bodies.

[0032] The washed inclusion body precipitate was resuspended in inclusion body denaturation and lysis buffer (50 mM Tris, 8 M Urea, 5 mM GSH, pH 8.2) at a ratio of 1:10 (w:v). The mixture was stirred at room temperature for 30–60 minutes to ensure complete dissolution. The mixture was then centrifuged at 12,000 rpm for 15 minutes at 2–8 °C. The precipitate was discarded, and the supernatant was collected. The supernatant was slowly and evenly added to 7 volumes of PBS solution. The mixture was incubated overnight at 2–8 °C for renaturation. The supernatant was centrifuged and filtered through a 0.22 μm filter membrane for sterilization. This was the renatured protein antigen solution. The washed inclusion body precipitate was resuspended in inclusion body denaturation and dissolution buffer at a ratio of 1:10 (w:v). The mixture was stirred at room temperature for 30–60 minutes to ensure complete dissolution. The mixture was then centrifuged at 12,000 rpm for 15 minutes at 2–8°C. The precipitate was discarded and the supernatant was collected. The supernatant was slowly and evenly added to 7 times its volume of PBS solution and incubated overnight at 2–8°C for renaturation. The supernatant was centrifuged and filtered through a 0.22 μm filter membrane for sterilization. This is the renatured protein antigen solution.

[0033] Example 3

[0034] vaccine preparation

[0035] 1) Inactivation: Add the renatured protein antigen solution to 1 mol / L BEI solution to a final concentration of 0.005 mol / L while stirring continuously until homogeneous. Inactivate at 37°C for 24 hours. After inactivation, add a filtered and sterilized 2 mol / L sodium thiosulfate (Na₂S₂O₃) solution to a final concentration of 0.005 mol / L. Stir thoroughly and react at 37°C for 1 hour to terminate inactivation. Store at 2–8°C.

[0036] 2) Inspection of semi-finished products: Sterility test shall be conducted in accordance with the current appendix of the Chinese Veterinary Pharmacopoeia; Protein content shall be determined by SDS method; Inactivation test shall be judged as qualified if no colony growth is observed.

[0037] 3) Vaccine preparation: Dilute the above semi-finished product to a concentration of 22 mg / ml, mix it with adjuvant at a ratio of 9:1, and prepare the vaccine according to the standard procedure.

[0038] 4) The dosage inspection was carried out in accordance with the current appendix of the Chinese Veterinary Pharmacopoeia and met the requirements.

[0039] 5) The sterility test was conducted in accordance with the current appendix of the Chinese Veterinary Pharmacopoeia and met the requirements.

[0040] Example 4

[0041] 1) Immunoassay

[0042] Forty healthy Landrace pigs aged 3-4 weeks (negative for erysipelas antibody antigen, porcine circovirus antigen, African swine fever antigen, and porcine reproductive and respiratory syndrome antigen) were selected for an immunization test. A booster immunization was administered 3 weeks (21 days) after the initial immunization. The immunization was administered via intramuscular injection into the neck. The experiment was divided into four groups (G1 group: SpaA-DC1 antigen, 10 pigs; G2 group: SpaA-DC2 antigen, 10 pigs; G3 group: SpaA-Fc antigen, 10 pigs; G4 group: negative control, 10 pigs). Blood samples were collected 21 days after the initial immunization and 14 days after the booster immunization, and serum was separated. After blood collection, challenge was performed. Five pigs from each group were randomly selected to be challenged with a virulent erysipelas Ery-1a strain, and five pigs were challenged with a virulent erysipelas Ery-2 strain. The strains used were isolated and provided by Beijing Dingchi Biotechnology Co., Ltd., and the challenge dose was 2 × 10⁻⁶. 8 CFU / dose, the route of attack was intravenous injection, and the patient was observed for 14 consecutive days, with morbidity and mortality recorded.

[0043] (1) Whole blood was collected 21 days after the first immunization and 14 days after the second immunization, and serum was separated. The changes in swine erysipelas antibody levels were measured using the indirect ELISA method.

[0044] (2) The levels of swine erysipelas antibodies were measured 21 days after the initial immunization and 14 days after the second immunization (S / P ≥ 0.4 was considered positive for antibodies). The results are shown in Table 1.

[0045] Table 1

[0046]

[0047] The results recorded in Table 1 show that the antibody levels of the first immunization were positive for swine erysipelas antibodies in all G3 groups, and some antibodies in G1 and G2 groups. The antibody levels in G3 groups were significantly higher than those in G1 and G2 groups. The data from the second immunization 14 days later showed that all three immunization groups were positive for antibodies, with G3 groups having higher antibody levels.

[0048] (3) The highly virulent Ery-1a strain was used as the challenge strain for infection. The morbidity and mortality of piglets after challenge are shown in Table 2. Table 2

[0049]

[0050] The statistical data in Table 2 shows that 3 heads died and 2 heads developed the disease in Group G4, all heads in Groups G1 and G3 were protected, and 3 heads in Group G2 developed the disease. The data shows that the vaccines in Groups G1 and G3 were able to resist the attack of the Ery-1a strain, and 3 heads in Group G4 died and 2 heads developed the disease.

[0051] (4) Ery-2 was used as the challenge strain for virus challenge. The statistical results of morbidity and mortality in piglets after challenge are shown in Table 3. Table 3

[0052]

[0053] The statistical data in Table 3 shows that all animals in groups G2 and G3 were protected, 2 animals in group G1 developed the disease, and 2 animals in group G4 died while 3 animals developed the disease. The results indicate that groups G2 and G3 provided better protection against serotype 2 virus.

[0054] (5) The content of interferon-γ in porcine serum was determined by ELISA, and the results are as follows: Figure 1 As shown. Figure 1 The results showed that the G3 group was higher than the G1 and G2 groups, and the difference was significant, while the G1 and G2 groups were at a similar level.

[0055] In summary, this invention provides a porcine erysipelas rSpaA-Fc fusion protein encoded by the expression gene of the rSpaA-Fc fusion protein. When a subunit vaccine prepared using this protein as the main antigen component is used to immunize piglets, it can induce high antibody levels in piglets, and the antibody production time is early. At the same time, it provides protection against challenge to virulent porcine erysipelas serum strains 1a and 2, and can induce piglets to produce high levels of interferon-γ.

[0056] The above description is merely a preferred embodiment of the present invention and should not be construed as limiting the scope of the present invention. Any simple equivalent changes and modifications made in accordance with the claims and description of the present invention shall still fall within the scope of the present invention.

Claims

1. A porcine erysipelas rSpaA-Fc fusion protein, characterized in that, The amino acid sequence of the rSpaA-Fc fusion protein is shown in SEQ ID NO:

2.

2. A gene encoding the rSpaA-Fc fusion protein of porcine erysipelas as described in claim 1, characterized in that, Its base sequence is shown in SEQ ID NO:

1.

3. The gene according to claim 2, characterized in that, The gene was optimized according to the codon preference of E. coli.

4. A subunit vaccine, characterized in that, The subunit vaccine is prepared using the porcine erysipelas rSpaA-Fc fusion protein of claim 1 as an antigen.

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