EG95-PA fusion protein and application

Abstract

The invention relates to an EG95-PA fusion protein and application thereof, and belongs to the technical field of biology. The EG95-PA fusion protein is obtained by connecting the sequence-optimized EG95 protein with the sequence-optimized PA protein through the connecting peptide, and the vaccine prepared from the EG95-PA fusion protein can prevent echinococcosis and anthracnose at the same time, and is good in safety, better in immune effect and free of any side effect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an EG95-PA fusion protein and its applications. Background Technology

[0002] Echinococcosis is an important zoonotic parasitic disease caused by the larvae (hydatid larvae) of *Echinococcus granulosus*. It is widespread globally, especially in livestock-producing regions, posing a serious threat to human health and the livestock industry. The life cycle of *Echinococcus granulosus* is complex, involving canines (definitive host) and ruminants (intermediate host). Humans are usually infected by ingesting eggs found in the feces of infected dogs. The EG95 gene encodes an antigenic protein specifically expressed in the infective larval stage (protocercariae) of *Echinococcus granulosus*. The EG95 protein is highly immunogenic and can induce a strong immune response in the host.

[0003] Anthrax is a zoonotic infectious disease caused by Bacillus anthracis. Bacillus anthracis is a Gram-positive, aerobic spore-forming bacillus capable of forming durable spores. This bacterium primarily affects livestock (such as cattle, sheep, and horses), but humans can also become infected through contact with infected animals or their products. The pathogenicity of Bacillus anthracis relies mainly on three toxin proteins it secretes: protective antigen (PA), lethal factor (LF), and edema factor (EF). PA is a key component of the anthrax toxin system; it binds to receptors on the surface of host cells, forming an entry point for the toxin complex. Furthermore, after being cleaved by furin in the host cell, PA binds to LF or EF to form lethal toxin (LT) or edema toxin (ET), thereby entering the cell and exerting its toxic effects. PA possesses strong immunogenicity, inducing the host to produce protective antibodies, and is currently the main immunogen in anthrax vaccines. Echinococcosis and anthrax are common diseases in livestock, and their symptoms share certain similarities, making diagnosis and treatment challenging. Currently, there is no suitable vaccine that can simultaneously prevent livestock from contracting both diseases. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is that there is no effective vaccine for preventing echinococcosis and anthrax in the prior art.

[0005] To address the aforementioned technical problems, this invention provides an EG95-PA fusion protein and its applications.

[0006] The first objective of this invention is to provide an EG95-PA fusion protein comprising the EG95 protein encoded by the sequence shown in SEQ ID NO.1 and the PA protein encoded by the sequence shown in SEQ ID NO.2.

[0007] Furthermore, the EG95 protein and PA protein of the fusion protein are linked by a linker peptide; preferably, the amino acid sequence of the linker peptide is shown in SEQ ID NO.3.

[0008] Furthermore, the nucleotide sequence of the EG95-PA fusion protein is shown in SEQ ID NO.4.

[0009] Furthermore, the amino acid sequence encoding the above-mentioned EG95-PA fusion protein is shown in SEQ ID NO.5.

[0010] The second objective of this invention is to provide a method for preparing a recombinant antigen, comprising the following steps: preparing production cells containing the above-mentioned fusion protein, culturing the production cells to express the fusion protein, and obtaining the recombinant antigen.

[0011] Furthermore, the production cells are Escherichia coli.

[0012] A third objective of this invention is to provide a bivalent vaccine that simultaneously immunizes against echinococcosis and anthrax, the bivalent vaccine comprising the EG95-PA fusion protein as described above.

[0013] Furthermore, the bivalent vaccine also includes an immune adjuvant.

[0014] Furthermore, the immune adjuvant includes an aluminum adjuvant.

[0015] A fourth objective of this invention is to provide the application of the above-described EG95-PA fusion protein, the recombinant antigen prepared by the above-described preparation method, or the above-described vaccine in the preparation of products for the prevention of echinococcosis and / or anthrax.

[0016] The beneficial effects of this invention are:

[0017] The EG95-PA fusion protein provided by this invention exhibits superior immunogenicity and good safety. Vaccines derived from this fusion protein can simultaneously prevent echinococcosis and anthrax, with significantly higher immunization efficacy than existing echinococcosis and anthrax vaccines. It is safe, highly effective, and has no side effects, making it widely applicable for the simultaneous prevention and treatment of echinococcosis and anthrax. Attached Figure Description

[0018] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0019] Figure 1 This is an electrophoretic pattern of the EG95(A1)-linker-PA(B1) fusion protein, where M represents the protein marker; A represents the fusion protein at a concentration of 500 μg / mL; B represents the fusion protein at a concentration of 250 μg / mL; C represents the fusion protein at a concentration of 125 μg / mL; D represents the fusion protein at a concentration of 25 μg / mL; E represents the EG95(A1)-linker-PA(B1) fusion protein; and F represents the EG95(A)-linker-PA(B) fusion protein. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0021] Table 1. Sequences involved in the embodiments

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[0034] Example 1: Construction of Recombinant Vector

[0035] Based on the EG95 gene sequence (GenBank number: EU595964.1) and PA gene (GenBank number: M22589.1) indexed by NCBI, the EG95 gene and PA gene were optimized based on the original sequence A of the EG95 gene (as shown in SEQ ID NO.6) and the original sequence B of the PA gene (as shown in SEQ ID NO.7) to obtain the optimized EG95 genes A1 (as shown in SEQ ID NO.1) and A2 (as shown in SEQ ID NO.8), and the optimized PA genes B1 (as shown in SEQ ID NO.2) and B2 (as shown in SEQ ID NO.9). A His tag was added before the stop codon of the EG95 gene sequence. The gene sequence A1 of the EG95 gene and the gene sequence of the linker (as shown in SEQ ID NO.3) were inserted into the pET-28a vector to form the EG95-linker-pET-28a vector. The optimized gene sequence B1 of PA (as shown in SEQ ID NO.2) was then inserted into the EG95-linker-pET-28a vector using DNA ligase to construct a plasmid. The plasmid was then introduced into ampicillin-resistant host bacteria such as TOP10 or DH5α for plasmid cloning. The host bacteria carrying the plasmid were cultured overnight under resistant conditions, and the plasmid was extracted using a plasmid extraction kit to finally obtain the EG95(A1)-linker-PA(B1)-pET-28a plasmid. The following plasmids were constructed using the same method: EG95(A1)-linker-PA(B)-pET-28a, EG95(A)-linker-PA(B1)-pET-28a, EG95(A1)-linker-PA(B2)-pET-28a, EG95(A2)-linker-PA(B1)-pET-28a, EG95(A1)-pET-28a, PA(B1)-pET-28a, EG95(A2)-linker-PA(B2)-pET-28a, and EG95(A)-linker-PA(B)-pET-28a.

[0036] Example 2: Expression and Identification of Recombinant Proteins

[0037] The EG95(A1)-linker-PA(B1)-pET-28a plasmid from Example 1 was added to 200 µL of competent E. coli BL21 (ED3), incubated on ice for 30 min, heat-shocked at 42 °C for 90 s, and incubated on ice for another 5 min. Then, 700 µL of antibiotic-free medium was added, and the mixture was incubated at 37 °C with shaking for 1 h. 100 µL of the bacterial culture was plated onto LB agar plates containing kanamycin and incubated overnight. Recombinant Escherichia coli was obtained after antibiotic screening.

[0038] Single colonies of recombinant *E. coli* were incubated overnight at 37°C with continuous shaking (200 rpm) in 5 mL of LB broth containing kanamycin (100 μg / mL). After 12 hours, 500 μL of the culture was transferred to 200 mL of LB broth and incubated. The culture was then vigorously shaken (200 rpm) at 37°C until it reached an OD of 0.6 nm. 600 Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 1 mM, and the mixture was cultured at 37°C and 200 rpm for 4 hours with shaking. The protein yield was analyzed on 12% SDS-PAGE. After induction culture, the mixture was centrifuged at 8000 rpm for 20 minutes, the supernatant was discarded, and the precipitate was retained. The precipitate was resuspended in Na2HPO4 (1 M), and disrupted three times using a high-pressure cell disruptor: high pressure 1600, low pressure 25, centrifuged at 12000 rpm for 10 minutes, the supernatant was retained, and the precipitate was discarded. Bcsp31 was purified using an affinity chromatography Ni-NTA column, and protein folding was performed using the Pierce Protein Refolding Kit (Thermo Scientific, No. 89867). The purified EG95(A1)-linker-PA(B1) fusion protein was obtained.

[0039] The following fusion proteins were prepared using the same method: EG95(A1)-linker-PA(B) fusion protein, EG95(A)-linker-PA(B1) fusion protein, EG95(A1)-linker-PA(B2) fusion protein, EG95(A2)-linker-PA(B1) fusion protein, EG95(A1) protein, PA(B1) protein, EG95(A2)-linker-PA(B2) fusion protein, and EG95(A)-linker-PA(B) fusion protein.

[0040] Example 3: Preparation of a bivalent vaccine

[0041] The harvested EG95(A1)-linker-PA(B1) fusion protein was diluted to different concentrations using PBS solution. The diluted fusion protein solutions were then mixed with aluminum adjuvant at a 1:1 ratio of antigen component to adjuvant to prepare vaccines with different antigen gradients: 0.5 μg / dose, 5 μg / dose, 10 μg / dose, and 30 μg / dose. The mixture was stirred at 8000 rpm for 8-10 minutes until thoroughly mixed. After passing sterility, viscosity, and stability tests according to the appendix of the current edition of the Chinese Veterinary Pharmacopoeia, the vaccines were stored at 4°C for later use (A1+B1 group). The same method was used to prepare bivalent vaccines for EG95(A1)-linker-PA(B) fusion protein, EG95(A)-linker-PA(B1) fusion protein, EG95(A1)-linker-PA(B2) fusion protein, EG95(A2)-linker-PA(B1) fusion protein, EG95(A1) protein, PA(B1) protein, EG95(A2)-linker-PA(B2) fusion protein and EG95(A)-linker-PA(B) fusion protein, with a vaccine protein content of 150 μg / mL.

[0042] Example 4: Safety and Antibody Testing of Recombinant Bivalent Subunit Vaccine

[0043] 1. Safety Inspection

[0044] Healthy 4-week-old Balb / c mice were randomly divided into four groups of five each: a non-optimized group (using a bivalent vaccine constructed with the EG95(A)-liner-PA(B) fusion protein), an optimized group, anthrax live vaccine group, echinococcosis vaccine group, and a blank control group. After one week of acclimatization, the mice were first immunized subcutaneously, with a booster immunization 14 days after the first immunization. The non-optimized and optimized groups were injected with different bivalent vaccines prepared in Example 3, respectively. The anthrax vaccine group received commercially available anthrax spore vaccine No. II (Tiankang Biopharmaceutical Co., Ltd.), the echinococcosis vaccine group received commercially available echinococcosis (hydatid disease) genetically engineered subunit vaccine (Chongqing Aolong Biological Products Co., Ltd.), and the blank control group received no injection. Blood samples were collected 14 days after the booster immunization to detect EG95 and PA antibody levels, and the mice's weight changes during the immunization period were observed, along with any fever, anorexia, or other adverse reactions.

[0045] 2. Validity verification

[0046] 2.1 Animal Immunization

[0047] Use healthy, susceptible sheep aged 3 months or older, with at least 5 sheep in each group. In the immunization group, each sheep is injected with 1 mL of vaccine into the neck muscle, while the control group is not injected. A second immunization is performed 21 days after the first immunization, using the same route and the same dose.

[0048] 2.2 Serum antibody detection

[0049] Clinical serum from sheep immunized with the recombinant vaccine was collected and analyzed by indirect ELISA using recombinant EG95 / PA as the antigen. Specifically, purified recombinant EG95 / PA protein was coated onto an immunoassay plate (Corning 42592, USA) at a concentration of 1 μg / mL in carbonate coating buffer and incubated overnight at 4°C. The wells were cleaned, washed three times with phosphate-buffered saline-Tween 20 (PBST), and then blocked with 10% rabbit serum at 37°C for 2 hours. Plates were filled with 1 / 100 diluted serum and incubated at 37°C for 1 hour. After washing five times with PBST, the plates were incubated with HRP-conjugated buffer at 37°C for 1 hour. After washing with PBST, a substrate solution containing TMB (3,3′,5,5′-tetramethylbenzidine) was added to the wells of the plate and incubated in the dark at room temperature for 5 minutes. The reaction was then terminated by adding stop solution (SeraCare Life Sciences KPL TMB Microwell Peroxidase). In an ELISA reader, absorbance and OD are measured at 650 nm. 650nm <0.2 is considered negative, OD 650nm A value ≥0.2 is considered positive.

[0050] 3. Test Results

[0051] 3.1 Vaccine safety testing

[0052] The weight gain of mice in each experimental group after immunization was not significantly different from that in the control group, and there were no adverse reactions such as fever or anorexia. All mice survived, indicating that the vaccine of the present invention has good safety.

[0053] 3.2 ELISA results of immune serum titers

[0054] ELISA results showed that sheep in different groups produced antibodies, but the immunogenicity of the antibodies was poor. The optimized A1+B1 bivalent vaccine showed good immunogenicity against both Eg95 and PA, with an antibody efficacy rate (number of positive antibodies / total number of serum samples) of 80% against Eg95 and 80% against PA, which was significantly higher than that of other vaccines and existing anthrax and echinococcosis vaccines.

[0055] Table 2 Serum titer results

[0056]

[0057] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An EG95-PA fusion protein, characterized in that, The EG95-PA fusion protein comprises an EG95 protein encoded by the sequence shown in SEQ ID NO. 1 and a PA protein encoded by the sequence shown in SEQ ID NO.

2.

2. The EG95-PA fusion protein according to claim 1, characterized in that, The EG95 protein and the PA protein of the fusion protein are connected by a connecting peptide; preferably, the amino acid sequence of the connecting peptide is shown in SEQ ID NO.

3.

3. The EG95-PA fusion protein according to claim 1, wherein The nucleotide sequence encoding the EG95-PA fusion protein is shown in SEQ ID NO.

4.

4. The EG95-PA fusion protein according to claim 1, wherein The amino acid sequence of the EG95-PA fusion protein is shown in SEQ ID NO.

5.

5. A method of preparing a recombinant antigen, characterized by, comprising the following steps: producing cells containing the fusion protein according to any one of claims 1-4, culturing the production cells to express the fusion protein, and obtaining the recombinant antigen.

6. The production method according to claim 5, characterized by, The production cells are Escherichia coli.

7. A bivalent vaccine for simultaneous immunization against echinococcosis and anthrax, characterized in that, The bivalent vaccine comprises the EG95-PA fusion protein according to any one of claims 1-4.

8. The tetravalent vaccine of claim 7, characterized in that, The bivalent vaccine further comprises an immunological adjuvant.

9. The tetravalent vaccine of claim 8, characterized in that, The immunological adjuvant comprises an aluminum adjuvant.

10. Use of the EG95-PA fusion protein according to any one of claims 1-4, the recombinant antigen prepared by the method according to claim 5 or 6, or the vaccine according to any one of claims 7-9 in the preparation of a product for preventing echinococcosis and / or anthrax.

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