A clostridium perfringens fusion protein, subunit vaccine composition and application thereof

Abstract

The present application relates to the field of biotechnology and animal epidemic prevention, and particularly relates to a Clostridium perfringens fusion protein, a subunit vaccine composition and application thereof. The present application constructs a new Clostridium perfringens fusion protein beta1-alpha1-F through molecular design, and the amino acid sequence of the fusion protein is shown as SEQ ID NO: 13. The fusion protein is used as an antigen, and can be efficiently and massively expressed in Staphylococcus epidermidis (concentration is greater than or equal to 580 μg / mL, and purity is greater than or equal to 86%). After inactivation by 0.2% formaldehyde, the fusion protein is emulsified with 26% v / v oil-in-water adjuvant to obtain a Clostridium perfringens subunit vaccine composition with good immunization effect, stable quality, high safety and simplified preparation process. The serum after immunization by the vaccine has a neutralization titer of A / C type toxin greater than or equal to 5 MLD / 0.1 mL; the protection rate of rabbits and cattle after challenge is 100%, and the immunization duration is greater than or equal to 180 days, which provides a better choice for the prevention and control of Clostridium perfringens disease.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and animal disease prevention, specifically to a fusion protein and subunit vaccine composition of Clostridium perfringens and its application. Background Technology

[0002] Clostridium perfringens, also known as Clostridium velutipes, is an important zoonotic pathogen. It can cause traumatic gas gangrene and food poisoning in humans, and is also the cause of many serious animal diseases, such as necrotic enteritis in cattle and sheep, sheep enterotoxemia, and lamb dysentery, thus posing a significant threat to animal husbandry.

[0003] Clostridium perfringens can be classified into five toxin types: A, B, C, D, and E, based on the types of exotoxins it secretes. All types secrete α-toxin, with type A secreting the largest amount. Types B and C also secrete β-toxin. Both α-toxin and β-toxin are the main immunogens of Clostridium perfringens.

[0004] Clostridium perfringens is characterized by rapid onset, short course, and high mortality. Humans and animals are prone to sudden death once infected. Therefore, vaccination is an effective means of controlling the disease it causes. Currently, traditional commercial vaccines are mainly inactivated vaccines. Their preparation method typically involves culturing type A or type C Clostridium perfringens in anaerobic liver broth for 24 hours, extracting and determining the virulence of α-toxin (for type A) or β-toxin (for type C) from the culture medium, then adding formaldehyde for inactivation, and finally mixing the inactivated culture medium with aluminum hydroxide adjuvant in a specific ratio to prepare the vaccine.

[0005] However, the preparation of this traditional inactivated vaccine has significant drawbacks: (1) the anaerobic liver broth has complex components and is difficult to control in terms of quality, resulting in large batch-to-batch differences in the vaccine; (2) the vaccine antigen is derived from the inactivated whole bacterial culture medium, which contains a large amount of bacterial miscellaneous proteins and other culture medium components in addition to the effective α or β toxins, further affecting the stability of vaccine quality between different batches; (3) due to the high virulence of Clostridium perfringens, the formaldehyde content used in the toxin inactivation process is high and the inactivation time is long, which increases the operational risks and potential safety hazards; (4) the toxin content secreted by Clostridium perfringens is low, and the culture medium usually needs to be concentrated 5 to 15 times to achieve the antigen content required in the vaccine, which increases the complexity and cost of the production process.

[0006] Therefore, it is both necessary and urgent to develop a novel Clostridium perfringens subunit vaccine that is effective, stable in quality, and safe, based on specific antigenic components rather than the whole bacterium. Summary of the Invention

[0007] The technical problem to be solved by the first aspect of the present invention is to address the shortcomings of existing Clostridium perfringens vaccines, such as large batch-to-batch variability, poor immunization effect, unstable vaccine quality, poor safety and short immune period, and to provide a new Clostridium perfringens fusion protein.

[0008] The technical problem to be solved by the second aspect of the present invention is to provide a nucleic acid molecule.

[0009] The technical problem to be solved by the third aspect of the present invention is to provide a recombinant expression vector.

[0010] The technical problem to be solved by the fourth aspect of the present invention is to provide a host cell.

[0011] The technical problem to be solved by the fifth aspect of the present invention is to provide the use of the fusion protein as an antigen in the preparation of subunit vaccine compositions for the prevention and / or treatment of Clostridium perfringens.

[0012] The technical problem to be solved by the sixth aspect of the present invention is to provide a subunit vaccine composition of Clostridium perfringens.

[0013] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0014] In a first aspect, the present invention provides a fusion protein of Clostridium perfringens, the fusion protein comprising Clostridium perfringens β-toxin protein fragment β1, Clostridium perfringens α-toxin protein fragment α1, and Salmonella typhimurium flagellar protein fragment F.

[0015] The amino acid sequence of the Clostridium perfringens β-toxin protein fragment β1 is shown in SEQ ID NO:1; the amino acid sequence of the Clostridium perfringens α-toxin protein fragment α1 is shown in SEQ ID NO:7; and the amino acid sequence of the Salmonella typhimurium flagellate protein fragment F is shown in SEQ ID NO:11.

[0016] The connection relationship between the Clostridium perfringens β-toxin protein fragment β1, the Clostridium perfringens α-toxin protein fragment α1, and the Salmonella typhimurium flagellar protein fragment F is: Clostridium perfringens β-toxin protein fragment β1 - Clostridium perfringens α-toxin protein fragment α1 - Salmonella typhimurium flagellar protein fragment F.

[0017] The Clostridium perfringens β toxin protein fragment β1, Clostridium perfringens α toxin protein fragment α1, and Salmonella typhimurium flagella protein fragment F are connected by a flexible linker peptide.

[0018] Specifically, the flexible linker peptide is n×(GGGGS), where n=1~10.

[0019] In some embodiments of the present invention, the Clostridium perfringens β toxin protein fragment β1, the Clostridium perfringens α toxin protein fragment α1, and the Salmonella typhimurium flagella protein fragment F are connected in pairs by 3×(GGGGS).

[0020] Specifically, the flagellin fragment of Salmonella typhimurium was fused into a fusion protein composed of β-toxin protein fragment and α-toxin protein fragment via 3×(GGGGS), ensuring that the toxin protein can stimulate the body to produce effective protective antibodies after entering the body.

[0021] The fusion protein, namely the fusion protein β1-α1-F of the present invention, has the amino acid sequence shown in SEQ ID NO:13.

[0022] Secondly, the present invention provides a nucleic acid molecule that encodes the fusion protein.

[0023] The nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:14.

[0024] Thirdly, the present invention provides a recombinant expression vector containing the aforementioned nucleic acid molecule.

[0025] Fourthly, the present invention provides a host cell containing the aforementioned nucleic acid molecule or the aforementioned recombinant expression vector.

[0026] Fifthly, the present invention provides the use of the fusion protein as an antigen in the preparation of subunit vaccine compositions for the prevention and / or treatment of Clostridium perfringens.

[0027] In a sixth aspect, the present invention provides a subunit vaccine composition for Clostridium perfringens, comprising the fusion protein and a pharmaceutically acceptable adjuvant.

[0028] The concentration of the fusion protein in the subunit vaccine composition is 35-100 μg / mL.

[0029] In some embodiments of the present invention, the concentration of the fusion protein in the subunit vaccine composition is 35 μg / mL, or 50 μg / mL, or 100 μg / mL.

[0030] The adjuvant may be an adjuvant known to those skilled in the art.

[0031] Preferably, the adjuvant is an oil-in-water adjuvant; more preferably, the adjuvant is ISA 35VG adjuvant.

[0032] In some embodiments of the invention, the adjuvant comprises 26% v / v of the total volume of the subunit vaccine composition.

[0033] Beneficial effects:

[0034] (1) The Clostridium perfringens fusion protein provided by the present invention has high expression level, high protein expression purity and good immunogenicity, and can effectively induce the body to produce corresponding antibodies, making it suitable for preparing products for the prevention of Clostridium perfringens.

[0035] (2) The method for preparing Clostridium perfringens fusion protein provided by the present invention is simple to operate, stable, efficient and quality controllable. The fusion protein is expressed in a Gram-positive bacterial expression system, which can achieve secretory expression of the target protein, and the protein content is not less than 580 μg / mL and the protein purity is not less than 86%.

[0036] (3) The Clostridium perfringens recombinant subunit vaccine provided by the present invention uses the above-mentioned fusion protein as the active substance. It has good safety, can effectively stimulate the body's immune response, has good immune effect, high neutralizing antibody titer, long duration of immunity, and few immune side effects. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0038] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0039] In the following embodiments, the pYL vector is constructed using a method disclosed in Chinese Patent Application No. 2020109079457. Specifically, the pYL plasmid vector is a recombinant pYL plasmid vector obtained by inserting the Xyl / tet fragment, the ori region, and the Erm resistance gene into the multiple cloning site and resistance region of the pRB373 plasmid. The Staphylococcus aureus strain RN4220 and Staphylococcus epidermidis (SE) were purchased from ATCC. The E. coli DH5α competent cells were purchased from Takara Bio Engineering (Dalian) Co., Ltd.

[0040] Example 1: Construction of fusion protein gene sequence and recombinant plasmid

[0041] The key pathogenic factors and major immunogens secreted by *Clostridium perfringens* (types A-E) are α-toxin (secreted by all types, with type A secreting the most) and β-toxin (secreted by types B and C). Since α-toxin and β-toxin are cytotoxic, their direct use in vaccine preparation is unsafe. Therefore, this embodiment designed different α-toxin and β-toxin protein fragments and introduced a *Salmonella typhimurium* flagellated protein fragment (F). These three protein fragments were linked together using a flexible linker peptide (GGGGS) to form different fusion proteins, further validating the expression levels, safety, and immunogenicity of each fusion protein.

[0042] Specifically, the designed β-toxin protein fragment (β1, β2, or β3) and α-toxin protein fragment (α1 or α2) are linked together using three flexible linkers (GGGGS) to ensure the fusion protein folds correctly and maintains its activity. The linking order is: β-toxin protein fragment (β1, β2, or β3) - 3 × flexible linker - α-toxin protein fragment (α1 or α2).

[0043] The Salmonella typhimurium flagellate protein fragment F was then linked to the C-terminus of the fusion protein via three flexible linkers (GGGGS) to construct a new fusion protein. The linking sequence was: β-toxin protein fragment (β1, or β2, or β3) - 3× flexible linker - α-toxin protein fragment (α1, or α2) - 3× flexible linker - Salmonella typhimurium flagellate protein fragment F.

[0044] Table 1 shows the names and corresponding sequence information of the β-toxin protein fragment (β1, β2, or β3), the α-toxin protein fragment (α1, α2), the Salmonella typhimurium flagellar protein fragment F, and the fusion protein.

[0045] Table 1. Sequence information of different protein fragments and fusion proteins

[0046]

[0047] 3. Gene synthesis and cloning

[0048] The nucleotide sequences corresponding to the amino acid sequences of the fusion proteins β1-α1-F, β2-α1-F, β3-α1-F, β1-α2-F, β2-α2-F, and β3-α2-F were entrusted to Sangon Biotech (Shanghai) Co., Ltd. for chemical synthesis.

[0049] Gene fragments of the synthesized fusion proteins β1-α1-F, β2-α1-F, β3-α1-F, β1-α2-F, β2-α2-F, and β3-α2-F were inserted into the pYL vector via molecular cloning and transformed into E. coli DH5α competent cells. Single colonies after transformation were picked and identified by colony PCR. Recombinant plasmids pYL-β1-α1-F, pYL-β2-α1-F, pYL-β3-α1-F, pYL-β1-α2-F, pYL-β2-α2-F, and pYL-β3-α2-F were then extracted from PCR-positive colonies for DNA sequencing. The recombinant plasmids pYL-β1-α1-F, pYL-β2-α1-F, pYL-β3-α1-F, pYL-β1-α2-F, pYL-β2-α2-F, and pYL-β3-α2-F were finally identified and correctly sequenced.

[0050] Example 2: Expression and identification of fusion proteins β1-α1-F, β2-α1-F, β3-α1-F, β1-α2-F, β2-α2-F, and β3-α2-F

[0051] 1. Construction of recombinant strains

[0052] The recombinant plasmids pYL-β1-α1-F, pYL-β2-α1-F, pYL-β3-α1-F, pYL-β1-α2-F, pYL-β2-α2-F, and pYL-β3-α2-F from Example 1 were transformed into Staphylococcus aureus strain RN4220, respectively. The transformed bacterial suspensions were plated on TSB solid medium containing 5 μg / mL erythromycin and incubated at 33–36°C for 20–24 h to form single colonies. Five single colonies were picked from each plate and transferred to 10 mL of TSB liquid medium containing 5 μg / mL erythromycin. The plates were then incubated at 33–36°C with shaking at 200 rpm for 16–20 h. The bacterial culture was processed according to the instructions of the nucleic acid extraction kit to extract the recombinant plasmid, which was then electroporated into competent cells of Staphylococcus epidermidis SE, ultimately obtaining recombinant strains SE / pYL-β1-α1-F, SE / pYL-β2-α1-F, SE / pYL-β3-α1-F, SE / pYL-β1-α2-F, SE / pYL-β2-α2-F, and SE / pYL-β3-α2-F for induction expression.

[0053] 2. Fusion protein induction, expression, and purification

[0054] The prepared seed cultures of recombinant strains SE / pYL-β1-α1-F, SE / pYL-β2-α1-F, SE / pYL-β3-α1-F, SE / pYL-β1-α2-F, SE / pYL-β2-α2-F, and SE / pYL-β3-α2-F were inoculated into TSB liquid medium containing 5 μg / mL erythromycin at a ratio of 1% v / v. ATC inducer was added to a final concentration of 300 ng / mL, and the cultures were incubated in a constant temperature shaking incubator at 36–37 °C with shaking at 200 r / min for 20 h. The cultures were then centrifuged at high speed, and the supernatant was collected. An appropriate amount of ammonium sulfate was added to the harvested supernatant, and after standing, the supernatant was clarified and filtered. The eluted target protein was collected by hydrophobic chromatography. The harvested target proteins were identified by SDS-PAGE, and the protein purity was determined using AlphaEaseFC software. The percentage content of all bands in the culture medium supernatant was calculated using the "AanlysisTools" function. The results are shown in Table 2.

[0055] Table 2. SDS-PAGE identification results of each fusion protein

[0056]

[0057] Example 3: Mouse toxicity test of fusion proteins β1-α1-F, β2-α1-F, β3-α1-F, β1-α2-F, β2-α2-F, and β3-α2-F

[0058] The toxicity test in mice was conducted according to the provisions of Part III of the *Pharmacopoeia of the People's Republic of China for Veterinary Medicine* (2020 edition). The experimental animals were 18±2 g SPF mice, with 5 mice per group, randomly divided into 22 groups. The experimental groups were as follows: 18 experimental groups, immunized with three injection doses of fusion protein β1-α1-F, β2-α1-F, β3-α1-F, β1-α2-F, β2-α2-F, and β3-α2-F, respectively; one negative control group containing culture medium; two positive control groups containing MLD α-toxin and MLD β-toxin, respectively; and one blank control group. Samples were diluted with gelatin buffer and injected via tail vein, with a total injection volume of 200 μL. Mice were observed for 1–3 days, and mortality was recorded.

[0059] Experimental Results: Mice in the blank control group, negative control group, three experimental groups of fusion protein β1-α1-F, three experimental groups of fusion protein β3-α1-F, and the 3 μg experimental groups of fusion proteins β2-α1-F, β1-α2-F, β2-α2-F, and β3-α2-F all survived. Mice in the two positive control groups, and the 30 μg and 300 μg experimental groups of fusion proteins β2-α1-F, β1-α2-F, β2-α2-F, and β3-α2-F all died. Therefore, in subsequent validation, fusion proteins β2-α1-F, β1-α2-F, β2-α2-F, and β3-α2-F were discarded, and only the safer fusion proteins β1-α1-F and β3-α1-F were retained.

[0060] Example 4: High-level expression of fusion proteins β1-α1-F and β3-α1-F

[0061] 1. Preparation of culture medium

[0062] (1) TSA solid medium: Weigh 40 g tryptic soy agar powder (TSA), add 940 mL of water for injection, dissolve it completely, autoclave at 121℃ for 15 minutes, cool to about 50℃, add erythromycin as needed to a final concentration of 5 μg / mL, pour into plates and set aside.

[0063] (2) TSB liquid culture medium: Weigh 30 g tryptic soy broth powder (TSB), add 940 mL of water for injection, dissolve thoroughly, and autoclave at 121°C for 15 minutes. Cool to about 50°C, and add erythromycin to a final concentration of 5 μg / mL.

[0064] 2. Preparation of Seed Liquid Production

[0065] (1) Primary production seed culture: After surface sterilization, recombinant strains SE / PYL-β1-α1-F and SE / PYL-β3-α1-F were taken out, and a small amount of TSB liquid medium was added. They were then streaked on TSB plates containing 5 μg / mL erythromycin and cultured at 36-38℃ for 12-16 h. Single colonies were picked and inoculated into TSB liquid medium with a final erythromycin concentration of 5 μg / mL and cultured at 36-38℃ and 200 r / min for 16-24 h to obtain the primary production seed culture.

[0066] (2) Secondary production seed liquid: The primary production seed liquid was inoculated into 200 mL of TSB liquid medium with a final concentration of 5 μg / mL of erythromycin at an inoculation rate of 0.2% v / v, and cultured at 36~38℃ and 200 r / min for 8~12 h to obtain the secondary production seed liquid.

[0067] 3. Large-scale expression in a 15 L fermenter

[0068] 5 L of TSB medium containing 5 μg / mL erythromycin and 0.01% antifoaming agent was injected into a 15 L fermenter using aseptic filtration. The parameters were set as follows: temperature 36–37 °C; pH 7.0 (pH was controlled by automatically adding 1 mol / L hydrochloric acid or 30% ammonia); dissolved oxygen 20% (dissolved oxygen was maintained by manually adjusting the stirring speed and aeration rate).

[0069] The two secondary production seed cultures were inoculated into fermenters at an inoculation rate of 1% v / v and fermented until OD. 600 At a concentration of 2.0, glycerol was used as the supplemental carbon source, and ATC was added to a final concentration of 500 ng / mL to induce expression for 24 h. After fermentation, the culture was centrifuged at high speed to collect the supernatant. The supernatant was used for SDS-PAGE analysis. Protein markers, supernatant, and BSA standards were subjected to SDS-PAGE gel electrophoresis. After electrophoresis, Coomassie Brilliant Blue R250 staining was performed for 30 minutes, and destaining was carried out until the bands were clear.

[0070] Use Launch Vision Works LS software to plot a standard curve when R... 2 When the concentration is ≥0.99, the sample can be tested. The protein concentration in the supernatant was analyzed using a standard curve as a reference. Results: The content of fusion protein β1-α1-F was determined to be 580 μg / mL, and the content of fusion protein β3-α1-F was determined to be 300 μg / mL.

[0071] Protein purity was determined using AlphaEaseFC software. The percentage content of all bands in the supernatant was calculated using the "Analysis Tools" function and recorded. Results: The purity of the fusion proteins β1-α1-F and β3-α1-F expressed at large scale was 86% and 85%, respectively.

[0072] Example 5: Preparation of recombinant subunit vaccine composition

[0073] The supernatants of the obtained fusion proteins β1-α1-F and β3-α1-F were added to 0.2% v / v formaldehyde solution and placed in a constant temperature shaking incubator at 36-37℃, shaking at 100 r / min for 24-48 h to inactivate residual bacteria. The qualified recombinant proteins β1-α1-F and β3-α1-F were added to the oil-in-water adjuvant ISA 35VG at different concentrations, continuously stirred with an emulsifier at 800 rpm for 12 min during the process, and stored at 4℃ to obtain different recombinant subunit vaccine compositions. The specific proportions of each component in the prepared vaccine are shown in Table 3 below.

[0074] Table 3. Composition ratio of recombinant subunit vaccine composition

[0075]

[0076] Example 6: Safety test of recombinant subunit vaccine composition

[0077] Eight guinea pigs weighing 350–450 g were randomly divided into four groups of two. Each group received an intramuscular injection of vaccine 1, vaccine 2, vaccine 3, and vaccine 4, respectively, 2 mL per pig. Survival and the presence of necrosis at the injection site were observed. Results showed that all vaccine-immunized guinea pigs survived without necrosis at the injection site. This indicates that the constructed fusion proteins β1-α1-F and β3-α1-F exhibited no toxicity, making the vaccines safer.

[0078] Example 7: Rabbit Immunization Test of Recombinant Subunit Vaccine Composition

[0079] 1. Rabbit serum neutralization test of β1-α1-F recombinant subunit vaccine composition and β3-α1-F recombinant subunit vaccine composition.

[0080] Thirty-six domestic rabbits weighing 1.5–2.0 kg were randomly divided into 10 groups. Groups 1–8 were the immunization groups (n=4 per group), and groups 9–10 were the control groups (n=2 per group), designated as control group A and control group C. Groups 1 and 2 received an intramuscular injection of vaccine 1; groups 3 and 4 received an intramuscular injection of vaccine 2; groups 5 and 6 received an intramuscular injection of vaccine 3; and groups 7 and 8 received an intramuscular injection of vaccine 4. Each rabbit received 0.5 mL of vaccine intramuscularly.

[0081] Twenty-one days after immunization, blood was collected and serum was separated. Equal volumes of serum from four rabbits in each group were mixed. For groups 1, 3, 5, and 7, 0.4 mL of the mixed serum was incubated with 0.8 mL of Clostridium perfringens type A toxin (C57-1) containing different toxin concentrations at 36–37°C for 40 min. Then, two mice (15–20 g each) were intravenously injected with 0.3 mL of the mixed serum. For groups 2, 4, 6, and 8, 0.4 mL of the mixed serum was incubated with 0.8 mL of Clostridium perfringens type C toxin (C59-2) containing different toxin concentrations at 36–37°C for 40 min. Then, two mice (15–20 g each) were intravenously injected with 0.3 mL of the mixed serum. Simultaneously, two control mice of each type were injected with 1 MLD of the same toxin as the serum-toxin mixture as a control. Mice were observed for one day. Based on the correspondence between serum neutralization titer and toxin content in Table 4, the results of the serum neutralization test in rabbits were determined.

[0082] The results are shown in Table 5. The neutralizing titers of rabbit serum against Clostridium perfringens types A and C in the three groups immunized with the β1-α1-F recombinant subunit vaccine all reached 5 or higher (neutralizing 5 MLD toxins in 0.1 mL of immunized animal serum), and increased with increasing antigen dose. However, the neutralizing titer of serum against Clostridium perfringens types A and C in the β3-α1-F recombinant subunit vaccine immunized group was 3 (neutralizing 3 MLD toxins in 0.1 mL of immunized animal serum), significantly lower than that of the β1-α1-F recombinant subunit vaccine immunized group. This indicates that the fusion protein β3-α1-F cannot effectively stimulate the body to produce sufficiently high levels of protective antibodies, resulting in insufficient vaccine immunogenicity.

[0083] 2. Challenge protection test of β1-α1-F recombinant subunit vaccine composition

[0084] Twenty-one days after immunization, rabbits in groups 1, 3, and 5, along with two control rabbits (group A) under the same conditions, were injected intravenously via the ear vein with one minimum lethal dose (MLD) of Clostridium perfringens type A toxin (C57-1). Rabbits in groups 2, 4, and 6, along with two control rabbits (group C) under the same conditions, were injected intravenously via the ear vein with one minimum lethal dose (MLD) of Clostridium perfringens type C toxin (C59-2). Observation was conducted for one day, and rabbit mortality was recorded.

[0085] The results are shown in Table 5. In the immunization group, 4 / 4 of the cells in the group that received intravenous injection of Clostridium perfringens type A and type C toxins into one rabbit MLD were protected, while all cells in the control group died. This indicates that the α-toxin and β-toxin protein fragments of this application are non-toxic, making the vaccine safer, and can also stimulate the body to produce high levels of neutralizing antibodies to withstand strong viral attacks.

[0086] Table 4. Correspondence between serum neutralization titer and toxin content

[0087]

[0088] Table 5. Results of neutralizing titer and challenge test in rabbit serum.

[0089]

[0090] Example 8: Bovine Immunization Test of Recombinant Subunit Vaccine Composition

[0091] 1. Calf serum neutralization test of β1-α1-F recombinant subunit vaccine composition

[0092] Twenty-four healthy calves aged 3-6 months were randomly divided into eight groups. Groups 1-6 were the immunization groups, with three calves in each group. The remaining six calves served as control groups, with three calves in each group (Control A and Control C). Groups 1 and 2 received an intramuscular injection of vaccine 1, groups 3 and 4 received an intramuscular injection of vaccine 2, and groups 5 and 6 received an intramuscular injection of vaccine 3. Each calf received 2 mL of vaccine intramuscularly.

[0093] Twenty-one days after immunization, blood was collected and serum was separated. Equal volumes of serum from three cattle in each group were mixed. For groups 1, 3, and 5, 0.4 mL of the mixed serum was incubated with 0.8 mL of Clostridium perfringens type A toxin (C57-1) containing different toxin concentrations at 36-37°C for 40 minutes. Then, two mice (15-20 g each) were intravenously injected with 0.3 mL of the mixed serum. For groups 2, 4, and 6, 0.4 mL of the mixed serum was incubated with 0.8 mL of Clostridium perfringens type C toxin (C59-2) containing different toxin concentrations at 36-37°C for 40 minutes. Then, two mice (15-20 g each) were intravenously injected with 0.3 mL of the mixed serum. Simultaneously, two control mice of each type were injected with 1 MLD of the same toxin as the serum-toxin mixture. Mice were observed for one day. The results of the calf serum neutralization test were determined according to the relationship between serum neutralization titer and toxin concentration as shown in Table 4.

[0094] The results are shown in Table 6. The neutralizing titer of calf serum immunized with the β1-α1-F recombinant subunit vaccine against type A or type C Clostridium perfringens toxin was above 5 (0.1 mL of immunized animal serum neutralized 5 MLD toxin), and increased with increasing antigen dose, indicating that the vaccine prepared by the fusion protein β1-α1-F has good safety and excellent immunogenicity.

[0095] 2. Challenge protection test of β1-α1-F recombinant subunit vaccine composition

[0096] Twenty-one days post-immunization, cattle in groups 1, 3, and 5, along with three cattle from control group A under the same conditions, were intravenously injected with one minimum lethal dose (MLD) of Clostridium perfringens type A toxin (C57-1). Calves in groups 2, 4, and 6, along with three cattle from control group C under the same conditions, were intravenously injected with one minimum lethal dose (MLD) of Clostridium perfringens type C toxin (C59-2). Calves were observed for three days, and mortality was recorded.

[0097] The results are shown in Table 6. When the immunized group was intravenously injected with one bovine minimum lethal dose (MLD) of Clostridium perfringens type A or C, 3 / 3 of the immunized group were protected, while all of the control group died. This indicates that the vaccine prepared by the fusion protein β1-α1-F can stimulate the body to produce a good immune response and effectively resist the attack of the virulent virus.

[0098] Table 6. Results of neutralizing titer and challenge test of calf serum

[0099]

[0100] Example 9: Duration of Immunity Study of Recombinant Subunit Vaccine Composition

[0101] Sixteen rabbits weighing 1.5–2.0 kg were randomly divided into four groups of four. Each group received an intramuscular injection of vaccine 1 (0.5 mL per rabbit). Eight rabbits were also divided into four control groups (A1, A2, C1, and C2), with two rabbits in each group receiving a subcutaneous injection of 0.5 mL of physiological saline as a challenge control. Twenty-one days after immunization, group 1, along with two rabbits in control group A1 under identical conditions, received an intravenous injection of one rabbit MLD of Clostridium perfringens type A (C57-1). Group 2, along with two rabbits in control group C1 under identical conditions, received an intravenous injection of one rabbit MLD of Clostridium perfringens type C (C59-2). Rabbit mortality was recorded after one day of observation.

[0102] 180 days after immunization, rabbits in group 3, along with two control rabbits (A2) under the same conditions, were each injected intravenously via the ear vein with one rabbit MLD of type A Clostridium perfringens toxin (C57-1); rabbits in group 4, along with two control rabbits (C2) under the same conditions, were each injected intravenously via the ear vein with one rabbit MLD of type C Clostridium perfringens toxin (C59-2). Rabbit mortality was recorded after one day of observation.

[0103] The results, as shown in Table 7, indicate that 21 days and 180 days after immunization, intravenous injection of type A or type C Clostridium perfringens toxin from rabbit MLD resulted in 4 / 4 protection in the immunized group, while all controls died. This demonstrates that the vaccine prepared using the fusion protein β1-α1-F has good safety, excellent immunogenicity, and a long duration of immunity, providing long-lasting protection.

[0104] Table 7 Results of the duration of immunity test

[0105]

[0106] This invention provides a fusion protein and subunit vaccine composition of Clostridium perfringens, and the ideas and methods for their application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. 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 fusion protein of Clostridium perfringens, characterized in that, The fusion protein includes Clostridium perfringens β toxin protein fragment β1, Clostridium perfringens α toxin protein fragment α1, and Salmonella typhimurium flagella protein fragment F. The amino acid sequence of the Clostridium perfringens β-toxin protein fragment β1 is shown in SEQ ID NO:1; the amino acid sequence of the Clostridium perfringens α-toxin protein fragment α1 is shown in SEQ ID NO:7; and the amino acid sequence of the Salmonella typhimurium flagellate protein fragment F is shown in SEQ ID NO:

11.

2. The fusion protein according to claim 1, characterized in that, The connection relationship between the Clostridium perfringens β-toxin protein fragment β1, the Clostridium perfringens α-toxin protein fragment α1, and the Salmonella typhimurium flagellar protein fragment F is: Clostridium perfringens β-toxin protein fragment β1 - Clostridium perfringens α-toxin protein fragment α1 - Salmonella typhimurium flagellar protein fragment F.

3. The fusion protein according to claim 2, characterized in that, The Clostridium perfringens β toxin protein fragment β1, Clostridium perfringens α toxin protein fragment α1, and Salmonella typhimurium flagella protein fragment F are linked by a flexible linker peptide.

4. The fusion protein according to any one of claims 1 to 3, characterized in that, The fusion protein has the amino acid sequence shown in SEQ ID NO:

13.

5. A nucleic acid molecule, characterized in that, Encodes the fusion protein according to any one of claims 1 to 4.

6. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid molecule as described in claim 5.

7. A host cell, characterized in that, Contains the nucleic acid molecule of claim 5 or the recombinant expression vector of claim 6.

8. The use of the fusion protein according to any one of claims 1 to 4 as an antigen in the preparation of a subunit vaccine composition for the prevention and / or treatment of Clostridium perfringens.

9. A subunit vaccine composition for Clostridium perfringens, characterized in that, It includes the fusion protein as described in any one of claims 1 to 4 and a pharmaceutically acceptable adjuvant.

10. The subunit vaccine composition according to claim 9, characterized in that, The concentration of the fusion protein in the subunit vaccine composition is 35-100 μg / mL.

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