Multi-epitope fusion protein for preventing infection of A-type to G-type clostridium perfringens and application

Abstract

The invention discloses a multi-epitope fusion protein for preventing infection of A-type to G-type clostridium perfringens and application, the multi-epitope fusion protein MVCP prepared by the invention is used for preventing infection of A-type to G-type clostridium perfringens of livestock and poultry, and the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 1. Mouse experiments verify that hyper-immune serum prepared by using the fusion protein as an antigen can prevent infection of any one of A-type and G-type clostridium perfringens, meanwhile, after MVCP immunizes a female mouse, the female mouse can generate high-level IgG antibodies, the antibodies are effectively transmitted to a newborn mouse through milk, the newborn mouse obtains resistance to infection of the A-type clostridium perfringens, and the newborn mouse can be prevented from suffering from infection of the A-type clostridium perfringens. Therefore, the effectiveness of the protein in preventing young animals from being infected with clostridium perfringens is verified.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, specifically to a fusion protein for preventing Clostridium perfringens infections of types A to G and its applications. Background Technology

[0002] *Clostridium perfringens* is a Gram-positive anaerobic short bacillus, an opportunistic pathogen. When the body's immunity is weakened, it proliferates rapidly, causing food poisoning, gastrointestinal diseases, and gas gangrene in humans and animals. The disease course is short, and the mortality rate is high. *Clostridium perfringens* primarily causes disease by secreting toxins. Based on different combinations of six toxins associated with its pathogenicity—α-toxin (CPA), β-toxin (CPB), ε-toxin (ETX), iota-toxin (ITX), *Clostridium perfringens* enterotoxin (CPE), and necrotic enteritis B-like toxin (NetB)—*Clostridium perfringens* is classified into seven toxin types (AG types), all of which can cause infection and disease in livestock and poultry. Young animals, due to their immature intestinal flora, are more susceptible to *Clostridium perfringens* infection, experiencing higher morbidity and mortality rates. Once infected, the disease progresses rapidly, treatment is ineffective, and large numbers of deaths occur within a short period.

[0003] One of the important means of preventing infection by this bacterium is immunization. Currently, the prevention of Clostridium perfringens infection is achieved through traditional inactivated vaccines, multivalent vaccines, or toxoid vaccines. Although these vaccines can induce animals to produce certain protective antibodies, they have drawbacks such as low safety, limited coverage, poor stability, and significant post-immunization side effects, which greatly limit their application. Developing a complete vaccine covering serotypes A to G is a key goal for preventing infection by this pathogen. Currently published inventions mainly focus on the modification of Clostridium perfringens α toxin, and a few inventions involve the fusion expression of multiple toxin proteins of Clostridium perfringens, such as CN105999252B (containing α, β, and ε fusion genes) and CN116444679A (containing α, β1, β2, and ε toxins). However, there is currently no commercially available complete vaccine invention that simultaneously contains all six toxin antigens of Clostridium perfringens.

[0004] This invention analyzes six important protective epitopes of toxins, arranges them in a specific order, and links them using a protein linker to ultimately construct a soluble multi-epitope fusion protein. The multi-epitope protein prepared by this method has the following advantages: (1) the co-expression of multiple protective epitopes provides comprehensive protection for inoculated animals; (2) the multi-epitope fusion protein contains only the effective immune components necessary for generating a protective immune response, removing components unrelated to immunity, thus increasing safety; (3) the physicochemical properties of the multi-epitope fusion protein are optimized during design, resulting in greater physicochemical stability and a higher probability of soluble expression. Summary of the Invention

[0005] Technical problem solved: In view of the shortcomings of existing vaccine design strategies, this invention provides a soluble multi-epitope fusion protein (MVCP) that can effectively prevent Clostridium perfringens infection of types A to G, its encoding gene and application.

[0006] Technical solution: A Clostridium perfringens multi-epitope fusion protein, the amino acid sequence of which is shown in SEQ ID NO:1.

[0007] A gene encoding the above-mentioned Clostridium perfringens multiepitope fusion protein, the nucleic acid sequence of which is shown in SEQ ID NO:2.

[0008] A recombinant expression plasmid containing the aforementioned gene, wherein the plasmid includes a promoter sequence and a terminator sequence to enable the expression of the gene in a host cell.

[0009] A recombinant host cell, wherein the recombinant expression plasmid described above is introduced into the host cell and is capable of expressing and producing the fusion protein described above.

[0010] The application of the above-mentioned fusion protein in the preparation of Clostridium perfringens vaccine, wherein the vaccine contains the fusion protein as an immunogenic component.

[0011] The above-mentioned fusion protein is used in the preparation of Clostridium perfringens-specific antibodies, wherein the fusion protein serves as an immunogen that stimulates the body to produce antibodies.

[0012] A Clostridium perfringens vaccine composition comprising the above-mentioned fusion protein and a pharmaceutically acceptable adjuvant selected from at least one of aluminum adjuvants, oil emulsion adjuvants, or CpG oligonucleotides.

[0013] A method for preparing the vaccine composition includes the following steps: mixing the above-mentioned fusion protein with an adjuvant and preparing an injectable suspension or lyophilized formulation.

[0014] A Clostridium perfringens diagnostic reagent comprising the above-mentioned fusion protein, used to detect the presence of Clostridium perfringens antibodies in a sample by immunological methods.

[0015] A drug delivery system comprising the aforementioned fusion protein and a nanoparticle carrier, wherein the nanoparticle carrier is a polylactic-co-glycolic acid copolymer (PLGA) or a liposome material.

[0016] Beneficial Effects: The multi-epitope fusion protein (MVCP) provided by this invention exhibits broad protective efficacy and safety compared to traditional vaccines, indicating its potential application in disease prevention strategies. Specifically, animal experiments have verified that hyperimmune serum containing the MVCP provides significant protection against lethal doses of Clostridium perfringens types A to G in mice, with a protection rate of up to 100%. After immunization of mother mice, the MVCP not only successfully elicited an immune response, producing high levels of IgG antibodies, but these antibodies were also effectively transferred to offspring through breast milk, resulting in significant immunoprotective effects against Clostridium perfringens type A infection in the offspring. This verifies the effectiveness of this vaccine in preventing infection in young animals. Attached Figure Description

[0017] Figure 1 This is a 3D structural prediction of the Clostridium perfringens multi-epitope fusion protein MVCP.

[0018] Figure 2 These are the SDS-PAGE and WB plots of MVCP. Figure 2 In -A, Line M is the protein marker; Line 1 is the pre-induction bacterial culture; Line 2 is the supernatant before purification; and Line 3 is the supernatant after purification. Figure 2 In line B, Line M is the protein marker; Line 1 is the bacterial culture before induction; Line 2 is the inclusion body precipitate after induction; and Line 3 is the supernatant after induction.

[0019] Figure 3 Passive protection results in mice with hyperimmune serum containing the multi-epitope fusion protein MVCP. Figure 3 Survival curve of -A mice challenged with Clostridium perfringens type A. Figure 3 Survival curve of -B mice challenged with type B bacteria. Figure 3 Survival curve of -C mice challenged with type C bacteria. Figure 3 Survival curve of -D mice challenged with type D bacteria. Figure 3 Survival curve of -E mice challenged with type E bacteria. Figure 3 Survival curve of -F mice challenged with type F bacteria. Figure 3 Survival curve of -G mice challenged with G-type bacteria.

[0020] Figure 4 This is the serum titer curve after MVCP immunization. Among them, Figure 4-A represents the serum IgG antibody titer at different time points after MVCP immunization of pregnant mice. Figure 4 -B represents the serum IgG antibody titer of baby mice at different time points after birth. Figure 4 -C represents the serum IgG antibody titer at different time points after MVCP immunization of rabbits.

[0021] Figure 5 These are the results of protective experiments conducted on 3-week-old pups challenged with MVCP and Clostridium perfringens type A whole-cell inactivated vaccines, respectively. Figure 5 -A represents the survival curves of pups in the Clostridium perfringens type A whole-cell inactivated vaccine and the PBS group. Figure 5 -B represents the survival curves of pups in the MVCP and PBS groups. Detailed Implementation

[0022] The following embodiments are provided to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. Modifications and substitutions made to the strategies, methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are all within the scope of the present invention.

[0023] Unless otherwise specified, all examples were performed under standard experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2001), or as recommended by the manufacturer’s instructions.

[0024] Example 1

[0025] According to the present invention, the above-mentioned Clostridium perfringens multi-epitope recombinant protein MVCP can be obtained by first artificially synthesizing its encoded gene (selecting Escherichia coli preferred codons) and then through biological expression.

[0026] 1) Synthesis of MVCP plasmid

[0027] Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the pCold II plasmid carrying the MVCP gene based on the MVCP amino acid sequence.

[0028] 2) Chemical transformation of the MVCP target plasmid

[0029] The plasmid carrying the target gene was introduced into DH5α and BL-21 strains and preserved. Finally, PCR detection and sequencing were performed using pCold II universal primers to determine whether the plasmid was successfully introduced into DH5α and BL-21 strains, as shown in Table 1.

[0030] Table 1

[0031]

[0032] 3) Expression and purification of MVCP

[0033] BL-21 bacterial culture successfully transfected with the target plasmid was cultured in 400 mL of ampicillin-resistant LB broth at a 1:100 ratio for 4 hours until the logarithmic growth phase. Then, IPTG inducer was added at a 1:1000 ratio, and the culture was induced at 16°C for 16 hours. The culture was centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in PBS. This process was repeated three times. The precipitate was resuspended in 25 mL of supernatant dissolving buffer and the cells were then sonicated. After sonication, the cells were centrifuged at 12000 rpm for 15 min to separate the supernatant from the inclusion body precipitate. SDS-PAGE and Western blotting confirmed that MVCP expression was present in the supernatant. MVCP extraction and purification were performed using a nickel column purification method. The final protein concentration was determined using a BCA assay kit.

[0034] 4) Preparation of hyperimmune serum for MVCP

[0035] Healthy, mature New Zealand white rabbits were selected. MVCP and adjuvant (MONTANIDE ISA 50V2) were mixed at a 1:1 volume ratio, and the rabbits were immunized intradermally in the back at 14-day intervals. The immunization dose was 500 μg per rabbit, and three immunizations were performed. Blood was collected from the ear vein before each immunization, and the serum was negative before immunization. The titer of the corresponding IgG antibody in the rabbit serum was detected using an indirect ELISA method. Ten days after the last immunization, blood was collected from the heart, and the serum was collected as MVCP hyperimmune serum. The serum was stored at -80℃ for later use.

[0036] Dilute MVCP to 5 μg / mL with ELISA coating buffer, add 100 μL to each well of the microplate, and incubate overnight at 4°C. Add 200 μL of PBST to each well and let stand for 1 min, then discard the liquid and wash. Repeat this step 3 times. Dilute the immune serum 1:200 with PBS, add 100 μL to each well, and incubate at 37°C for 2 h. Discard the liquid and wash 3 times with PBST. Add 200 μL of 1:2000 diluted horseradish peroxidase-labeled IgG antibody to each well and incubate at 37°C for 30 min. Discard the liquid and wash 3 times with PBST again. Add 100 μL of TMB chromogenic buffer to each well and develop for 15 min. Add stop solution to stop the development. Measure OD using a microplate reader. 450nm Absorbance value.

[0037] Example 2

[0038] Evaluation of MVCP as a candidate protein for the prevention of Clostridium perfringens infection (AG type).

[0039] (1) Safety test

[0040] Three 4-week-old ICR mice were selected and subcutaneously injected with 200 μL of MVCP multi-epitope vaccine and 200 μL of PBS, respectively. After 7 days of observation, there were no differences in diet, weight gain and activity between the mice subcutaneously injected with MVCP multi-epitope vaccine and the mice injected with PBS.

[0041] (2) Immunogenicity evaluation

[0042] Two 6-week-old female ICR mice (gestational age 19-20 days) were selected and subcutaneously immunized with 200 μL of MVCP vaccine (containing 20 μg antigen). After the initial immunization, booster immunizations were administered every 10 days for a total of three immunizations, with the antigen dose remaining constant for each immunization. Two identical pregnant mice were used as a control group, immunized with 200 μL of sterile PBS. Blood was collected from the submandibular vein before each immunization, and at 10-day intervals until 50 days post-immunization. Six pups were randomly selected from each group after birth, and at 20 days of age, blood was collected from the submandibular vein every 10 days until they reached 50 days of age. The corresponding IgG antibody titers in mouse serum were detected using an indirect ELISA method.

[0043] (3) Evaluation of the efficacy of passive immunotherapy

[0044] Mice were injected with MVCP hyperimmune serum followed by infection with a lethal dose of Clostridium perfringens AG to evaluate the efficacy of passive immunotherapy. The specific procedure was as follows: Three-week-old female ICR mice were selected and intraperitoneally injected with 200 μL of MVCP hyperimmune serum 24 h later, then infected with a lethal dose of Clostridium perfringens AG. A control group of 200 μL of rabbit negative serum was also included. When the Clostridium perfringens AG culture reached the logarithmic growth phase, it was centrifuged at 5000 rpm for 5 min, resuspended in PBS, and washed three times. The culture was then resuspended in 1 mL of PBS, and the OD value was measured. 600 The bacterial solution was diluted according to the required concentration for the experiment, and the diluted bacterial solution was counted using a dropper plate to control the concentration of the challenge bacterial solution to approximately 1 × 10⁻⁶. 9 CFU / mouse; 200 μL of bacterial solution was injected into each mouse intraperitoneally, and its survival was observed for 7 consecutive days.

[0045] The results showed that the hyperimmune serum prepared after immunization with the Clostridium perfringens multi-epitope protein MVCP obtained in this invention could protect mice from lethal doses of Clostridium perfringens AG infection, with a protection rate of 100%. Figure 3 ).

[0046] (4) Evaluation of the immune protection effect in pups

[0047] When the pups born to MVCP-immunized pregnant mice reached 3 weeks of age, 10 pups from each proteome were selected and intraperitoneally injected with a lethal dose of Clostridium perfringens type A. The specific procedure was as follows: Clostridium perfringens type A culture was cultured to the logarithmic growth phase, centrifuged at 5000 rpm for 5 min, resuspended in an appropriate amount of PBS, and the process was repeated three times. After resuspending the culture in 1 mL of PBS, the OD value was measured. 600 The bacterial solution was diluted according to the required concentration for the experiment, and the diluted bacterial solution was counted using a dropper plate to control the concentration of the challenge bacterial solution to approximately 1 × 10⁻⁶. 9 CFU / mouse; 200 μL of bacterial solution was injected into each mouse intraperitoneally, and its survival was observed for 7 consecutive days.

[0048] The results showed that MVCP immunization of pregnant mice produced high levels of IgG antibodies, which could be passed to pups through breast milk. Survival curves of pups infected with Clostridium perfringens type A indicated that MVCP provided 100% protection against 3-week-old ICR mice. Figure 4 , Figure 5 .

[0049] It should be noted that the above examples are only used to illustrate the technical solution of the present invention and not to limit it. They are not limited to preventing infection with a single type of Clostridium perfringens, but rather provide protection against infection with any 7, 6, 5, 4, 3, 2, or 1 of the 7 types of Clostridium perfringens (AG). The effectiveness of this vaccine in preventing infection with Clostridium perfringens type A in young animals when used to immunize mothers has also been verified. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions to the technical solution of the present invention as needed, without departing from the spirit and scope of the present invention.

Claims

1. A Clostridium perfringens multi-epitope fusion protein, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO:

1.

2. A gene encoding the Clostridium perfringens multi-epitope fusion protein of claim 1, characterized in that, The nucleic acid sequence of the gene is shown in SEQ ID NO:

2.

3. A recombinant expression plasmid, characterized in that, The plasmid contains the gene of claim 2, and the plasmid contains a promoter sequence and a terminator sequence to enable the expression of the gene in the host cell.

4. A recombinant host cell, characterized in that, The host cell is introduced with the recombinant expression plasmid of claim 3, and is able to express and produce the fusion protein of claim 1.

5. The use of the fusion protein according to claim 1 in the preparation of Clostridium perfringens vaccine, characterized in that, The vaccine contains the fusion protein as an immunogenic component.

6. The use of the fusion protein according to claim 1 in the preparation of Clostridium perfringens specific antibodies, characterized in that, The fusion protein serves as an immunogen that stimulates the body to produce antibodies.

7. A Clostridium perfringens vaccine composition, characterized in that, The invention comprises the fusion protein of claim 1 and a pharmaceutically acceptable adjuvant selected from at least one of aluminum adjuvants, oil emulsion adjuvants, or CpG oligonucleotides.

8. A method for preparing the vaccine composition of claim 7, characterized in that, The method includes the following steps: mixing the fusion protein of claim 1 with an adjuvant and preparing an injectable suspension or lyophilized formulation.

9. A diagnostic reagent for Clostridium perfringens, characterized in that, The fusion protein of claim 1 is included for use in detecting the presence of Clostridium perfringens antibodies in a sample by immunological methods.

10. A drug delivery system, characterized in that, It includes the fusion protein as described in claim 1 and the nanoparticle carrier.

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