Subunit vaccine for preventing and treating canine echinococcosis and application

Abstract

The invention belongs to the technical field of genetic engineering, and relates to a subunit vaccine for preventing and treating canine echinococcosis and application. The vaccine is a recombinant fusion protein vaccine for preventing canine echinococcosis granulosus tapeworm infection, novel recombinant fusion protein based on canine echinococcosis EG95 antigen is constructed and expressed, and is prepared into a subunit vaccine for immunizing a dog, and the canine echinococcosis can be effectively prevented by stimulating cellular immunity and humoral immunity in the dog. Therefore, the life transmission cycle of echinococcus granulosus tapeworms is blocked, and a preventive vaccine is provided for echinococcosis terminal host prevention and control.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering and relates to a subunit vaccine for preventing and treating canine echinococcosis and application thereof. BACKGROUND

[0002] Echinococcosis is a chronic helminth disease caused by echinococcus infection in humans and animals. As the final host, dogs are the main source of infection and play a key role in the transmission of the disease. The eggs are excreted with the feces of the dog, and then the food such as water, vegetables and liver consumed by humans are contaminated with the eggs, which can easily cause echinococcosis infection in humans, causing serious infection and huge economic losses while endangering human health.

[0003] Although a lot of resources have been invested and many measures have been taken to prevent and control echinococcosis, due to the complex biological characteristics and transmission mechanism of the disease, the existing prevention and control measures are still difficult to achieve the goal of completely eradicating the disease. Echinococcosis is a typical dual-host parasitic disease, the adult stage mainly parasitizes the small intestinal mucosa of dogs (final host), and the larval stage (hydatid) mainly parasitizes the liver, lungs and other solid organs of sheep and other domestic animals (intermediate host), forming a characteristic cyst structure. This unique life cycle determines that the prevention and control of the disease needs to consider both the final host and the intermediate host.

[0004] In recent years, the research and application of recombinant vaccine antigen (EG95) for the prevention and control of intermediate host infection have made breakthrough progress. The EG95-based vaccine has shown good immune protection effect and can significantly reduce the echinococcus infection rate of intermediate hosts such as sheep, providing an effective technical means for blocking the intermediate link of echinococcosis. However, the immune prevention and control of the final host dog, which is a key link in the transmission chain of echinococcosis, has been neglected for a long time. Existing studies have confirmed that monthly praziquantel deworming treatment for dogs can effectively reduce the risk of infection of intermediate hosts. However, considering the frequent outdoor activities of dogs, the high probability of re-infection, and the limitations of this strategy such as high implementation cost and difficulty in continuous application, it is difficult to be widely applied as a long-term control strategy. Therefore, the development of new and sustainable control technologies has become a key requirement for long-term control of echinococcosis. At present, the development of canine echinococcosis vaccine as an active immunization control strategy is gradually becoming the research focus and technical bottleneck breakthrough direction in the field of echinococcosis prevention and control, with the advantages of inducing long-term immune protection and reducing the risk of transmission.

[0005] In the echinococcosis epidemic area, the number of dogs is much less than that of sheep, but the gravid segment of intestinal echinococcus tapeworm can produce thousands of eggs per day and excrete them to the external environment with feces, so it is more reasonable and economical to vaccinate dogs. SUMMARY

[0006] The present application aims to provide a recombinant fusion protein vaccine for preventing Echinococcus granulosus infection in dogs. A novel recombinant fusion protein based on the EG95 antigen of canine echinococcosis is constructed and expressed, and is prepared into a subunit vaccine for immunizing dogs, thereby blocking the life cycle of Echinococcus granulosus in the body of dogs by stimulating cellular immunity and humoral immunity, and providing a preventive vaccine for the prevention and control of the final host of echinococcosis.

[0007] The present application first proposes a recombinant fusion protein for preventing Echinococcus granulosus infection in dogs, which comprises two tandem EG95 antigen domains, a canine IgG antibody Fc fragment and a tag sequence composed of 6xHis from N-terminal to C-terminal; the two tandem EG95 antigen domains are connected by a flexible linker, and the amino acid sequence of the fusion protein is shown as SEQ ID NO: 2.

[0008] A polynucleotide encoding the above-mentioned fusion protein, wherein the polynucleotide sequence is optimized for the codon bias of CHO cells, and a Hind III site is introduced at the 5' end, and a EcoR I site is introduced at the 3' end; the polynucleotide sequence is shown as SEQ ID NO: 1.

[0009] A recombinant expression vector, which is obtained by double digestion of pEE12.4 with Hin d III and Eco R I, and then ligating with the polynucleotide which is also digested with Hin d III and Eco R I, to obtain the recombinant vector pEE12.4-EG95-Fc.

[0010] A cell line stably expressing the recombinant fusion protein, wherein the recombinant expression vector is integrated into the genome of the cell line, the cell is a CHO cell, and the cell line can continuously secrete the fusion protein of claim 1 in a selection medium without glutamine and containing 50 µM MSX, and the expression amount of the fusion protein is 5.869 g / L.

[0011] A veterinary subunit vaccine composition, comprising: an immunologically effective amount of the fusion protein, and an adjuvant ISA 201VG; the final concentration of the fusion protein in the vaccine is 50 µg / mL, and the vaccine is used for subcutaneous inoculation of dogs.

[0012] The use of a veterinary subunit vaccine composition in the preparation of a medicament for blocking the life cycle of Echinococcus granulosus in dogs, wherein the vaccine stimulates humoral immunity and cellular immunity in dogs.

[0013] A method for evaluating the immunization effect of a veterinary subunit vaccine composition, comprising: (a) immunize dogs with the vaccine of claim 6 at a dose of 1 mL per dog containing 50 µg of fusion protein, subcutaneously, with an immunization schedule of 0, 2, 4 weeks; (b) at least 42 weeks after the last immunization, the serum of the dogs is positive for anti-EG95 antibodies, and the serum IFN-γ and IL-10 levels are significantly higher than the baseline before immunization.

[0014] Compared with the prior art, the beneficial effects of the present application are that, 1. The EG95 protein double copy sequence of echinococcosis is first serially fused with the Fc functional domain of canine IgG, creating a new type of immunomolecule with high-density antigen epitopes and long-acting function in vivo. The Fc fragment of canine IgG is selected to ensure that the fusion protein can efficiently interact with canine FcRn in the canine body, thereby achieving the best half-life extension effect and avoiding the immune rejection problem that may be caused by heterologous proteins. The double copy EG95 truncated gene design significantly improves the transcription and protein expression level, overcoming the bottleneck of traditional single copy expression.

[0015] 2. The CHO (Chinese hamster ovary) cell eukaryotic expression system is used, compared with prokaryotic systems (such as E. coli), the CHO cell can complete complex post-translational modifications such as glycosylation, making the expressed protein closer to its natural conformation, having higher biological activity and immunoreactivity, and at the same time reducing the risk of immunogenicity abnormalities. The introduction of His tag simplifies the protein purification process, and the concentration of the purified protein is 5.869 mg / ml, which improves the recovery rate and purity and reduces the production cost.

[0016] 3. The EG95 vaccine antigen originally used for intermediate hosts (sheep) is developed for the end host (dog) through innovative modification. By blocking the key link of the parasite transmission chain, it shows important public health application value. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Enzyme digestion identification results of pEE12.4-EG95-Fc (1, 2, 3: pEE12.4-EG95-Fc enzyme digestion samples).

[0018] Figure 2 Eg95-Fc protein cell line cooling expression. A is the SDS-PAGE result, and B is the Western Blot result.

[0019] Figure 3 Vaccine immunized dogs at different times ELISA antibody detection results (serum OD450nm <0.30, determine antibody negative; serum OD450nm≥0.30, determine antibody positive).

[0020] Figure 4The levels of IFN-γ and IL-10 in the serum of the dogs were detected by ELISA (A, IFN-γ; B, IL-10). DETAILED DESCRIPTION

[0021] The purpose of the present application is achieved by the following technical solutions: Example 1 Construction of recombinant eukaryotic expression vector pEE12.4-EG95-Fc 1. Gene synthesis and sequence design The Fc fragment of canine IgG is added after the EG95 gene is linked, and the His tag sequence is designed in the C segment of the sequence. According to the optimization strategy of CHO cells, the above sequence is optimized, and Hin d III, sequence 3' end is added Eco R I (SEQ ID NO. 1), and the entire sequence (1230bp) is synthesized by Kingsun Biotech Co., Ltd. The sequence before optimization is shown in SEQ ID NO. 3.

[0022] 2. Construction of recombinant eukaryotic expression vector The above synthesized plasmid pUC57-EG95-Fc and pEE12.4 empty vector are both digested with Hind III and EcoR I double enzyme; after double enzyme digestion, the vector fragment is recovered, and the two are ligated, transformed E. coli DH5α, and coated on LB plate containing ampicillin to screen positive clones.

[0023] Among them, the enzyme digestion conditions are: 37℃ for 3h; the enzyme digestion reaction system is: pUC57-EG95-Fc 5μg; Hin d III and Eco R I each 5μl; 10×Buffer 4μl; supplement H2O to 40μl. The ligation conditions are: 22℃ for 3h; the ligation system is: EG95-Fc fragment 13μl, purified pEE12.4 4μl, T4 ligase 1μl, 10×Buffer 2μl.

[0024] Ligation product transformation E. coli DH5α competent (ice bath for 30min, 42℃ heat shock for 90s, ice bath for 5min), add 1ml LB medium to recover for 1h, then coat on LB plate containing ampicillin, and invert culture at 37℃ for 16h.

[0025] 3. Identification of positive clones Independent colonies are randomly picked from the LB plate containing ampicillin for plasmid extraction, and double enzyme digestion identification, and the enzyme digestion products are subjected to 1% agarose gel electrophoresis, as shown in Figure 1As shown, the positive clone includes a 7569bp vector fragment and a 1230bp target band.

[0026] The correctly digested clones were subjected to gene sequencing analysis. The obtained sequences were compared with the reference sequence (SEQ ID NO.1), requiring 100% coverage, no frameshift, and no mutations. The finally confirmed recombinant plasmid was named pEE12.4-EG95-Fc.

[0027] The correctly sequenced plasmid was selected for amplification to obtain the eukaryotic expression plasmid pEE12.4-EG95-Fc, which was used for subsequent CHO-GS cell transfection and expression evaluation.

[0028] Example 2: Stable transfection and expression of pEE12.4-EG95-Fc fusion protein plasmid into CHO cells. The pEE12.4-EG95-Fc fusion protein particle obtained in Example 1 was utilized Pvu Linearization was performed by single enzyme digestion with enzyme I. The digestion system contained 100 ng of plasmid. Pvu I 10 μl; 10×Buffer 20 μl; add H2O to 200 μl. Incubate at 37 °C for 3 hours. After purification and confirmation, the linearized plasmid pEE12.4-EG95-Fc was obtained.

[0029] CHO cells were washed with glutamine-free CD-CHO-AGT medium and then washed with electroporation buffer to adjust the cell density to 3 × 10⁶ cells / year. 7 cells / ml.

[0030] Take two sterile 1.5ml EP tubes, add 0.5ml of cell suspension resuspended in electroporation buffer to each tube, then add 5μg of plasmid to each tube, and electroporate the linearized plasmid pEE12.4-EG95-Fc into CHO cells according to the preset program, and then culture them in a shaker at 37℃.

[0031] After 48 hours of culture, the cell culture medium was replaced with CD-CHO-AGT medium containing 50 μM MSX, and cultured for another 48 hours. The cells were cultured until the cell density reached 1.0 × 10⁻⁶ cells / year. 6 After reaching a cell / ml density, cells were passaged to a density of approximately 1.0 × 10⁶ cells / ml using CD-CHO-AGT medium containing 50 μM MSX. 5 cells / ml, continue culturing. Repeat the pressurization process three times.

[0032] The cells were transferred to CHO-2 suspension culture for large-scale production, and after acclimatization, they were cryopreserved to obtain a stable proliferating cell line, which was named CHO-EG95-Fc.

[0033] Example 3 Identification of EG95-Fc fusion protein The cell line CHO-EG95-Fc from Example 2 was seeded in CHO-2 medium with 50 μM MSX and induced by cooling. The samples were then centrifuged at 12000 r / min for 1 min daily, and the supernatant was collected and stored at -80°C for later use.

[0034] First, the supernatant was analyzed by SDS-PAGE; subsequently, Western blot was performed using His mouse antibody as the primary antibody and HRP-labeled goat anti-mouse IgG (H+L) as the secondary antibody to specifically detect and analyze the target protein, such as... Figure 2 As shown in Figure A.

[0035] like Figure 2 The SDS-PAGE results shown in Figure A indicate the presence of a protein band of approximately 45 kDa in the supernatant from the cooling-induced expression. This band is close to the theoretical molecular weight (45 kDa) of the target protein EG95-Fc, suggesting that this band may represent the target protein. Figure 2 As shown in Figure B, the Western blot analysis revealed that this band exhibited a specific immune response to the His-tagged monoclonal antibody. This result not only further validated that its molecular weight was consistent with expectations but also confirmed that the band represented the target protein EG95-Fc from the perspective of target protein-antibody specific binding. Combining the SDS-PAGE and Western blot results, the band was determined to be the recombinant EG95-Fc protein expressed in CHO cells. This confirms that the band represents the EG95-Fc fusion protein expressed in CHO cells.

[0036] Example 4 Purification of EG95-Fc fusion protein After inoculating the CHO-EG95-Fc cell line from Example 2 into the CHO-2 culture product and centrifuging it, the culture supernatant containing the EG95-Fc fusion protein was filtered and purified using a 0.22 μm microporous membrane. The filtrate was then loaded onto a Ni-TED affinity chromatography column.

[0037] Impurities were removed using 50 mmol / L imidazole buffer, and the target protein was eluted with a high-concentration imidazole solution of 500 mmol / L. The eluent was added to a 40 kDa dialysis bag and dialyzed at 4°C for 48 hours.

[0038] The total protein content of the purified protein after dialysis was determined using a BCA protein concentration assay kit.

[0039] The results showed that a target band of approximately 45 KD was visible in the eluent. After purification, the total protein concentration of EG95-Fc was determined by the BCA protein kit to be 5.869 mg / ml. Compared with the concentration of 2.1 mg / ml of purified recombinant protein in the prior art CN202111681190 or the expression level of 2~3 g / L of purified protein in CN111675758A, this indicates that a high concentration of the target fusion protein was obtained through gene recombination and purification.

[0040] Example 5: Immunogenicity test of EG95-Fc genetically engineered vaccine in dogs Ten healthy beagle dogs aged 8-12 months, regardless of sex, were selected and tested negative for echinococcosis serum antibodies.

[0041] Immunization group: Five beagle dogs were randomly selected and each dog was subcutaneously vaccinated in the neck with the EG95-Fc vaccine obtained in Example 4. The dose was 1.0 ml / dog. A total of three immunization programs were implemented, with each immunization interval of 2 weeks.

[0042] Blank control group: Five beagle dogs were randomly selected and injected subcutaneously into the neck of each dog using the same injection method as the immunization group. 1.0 ml of PBS emulsion was injected into the neck of each dog.

[0043] Vaccine preparation: The final concentration of EG95-Fc was 5.869 mg / mL (Example 4). It was diluted to 100 µg / mL with sterile PBS and emulsified with ISA 201 VG at a 1:1 (v / v) ratio. The emulsification was carried out at 3000 r / min for 10 min until no stratification occurred, which constituted the vaccine (batch number: EG95-20230901). Each milliliter of vaccine contained 50 µg of EG95-Fc; the PBS emulsification process was the same for the control group.

[0044] Immunization schedule: 1.0 mL / animal subcutaneously injected into the dorsal side of the neck at weeks 0, 2, and 4.

[0045] Sampling time points: Blood samples were collected from dogs via the anterior vena cava before immunization (0d), 2 weeks (before the second immunization), 5 weeks, 8 weeks, 12 weeks, 14 weeks, 16 weeks, 19 weeks, 23 weeks, 27 weeks and 42 weeks. The collected blood samples were centrifuged to obtain serum, which was stored at -80℃ for later use in subsequent testing.

[0046] (1) Detection of antibody levels in canine serum by ELISA Antibody levels in canine serum were detected using an enzyme-linked immunosorbent assay (ELISA). Purified EG95-Fc protein was used as the coating antigen, diluted to a concentration of 5 μg / ml, and added to the ELISA plate at a volume of 100 μl / well. The plate was incubated overnight at 4°C to complete antigen coating. Canine serum was diluted 1:400 to serve as the primary antibody, and horseradish peroxidase (HRP)-labeled goat anti-canine IgG was diluted 1:7500 to serve as the secondary antibody.

[0047] The antibody levels in canine serum at different time points after immunization were detected using ELISA. Results showed that two weeks after the first immunization, serum antibody levels in the immunized dogs were positive, and antibody levels showed a sharp upward trend after the second and third immunizations. Five weeks after the first immunization, the antibody levels in the immunized dogs were significantly different from those in the control group, and this difference persisted until week 27. Figure 3 The results showed that the EG95-Fc genetically engineered subunit vaccine could effectively induce dogs to produce high levels of antibodies.

[0048] (2) Detection of lymphokine levels in canine serum by ELISA Following the instructions for use of the test kit, canine serum samples were diluted 1:2 before immunization and at 5, 27, and 42 weeks after the first immunization. The levels of lymphokines IFN-γ and IL-10 in the diluted canine serum were then measured. The results showed that the levels of both IFN-γ and IL-10 in canine serum increased after immunization. Figure 4 Five weeks after the first immunization, the mean level of the lymphokine IFN-γ in the immunized group reached 461 pg / mL. -1 (The control group had a concentration of only 109 pg·mL) -1 (P<0.001); at 42 weeks post-immunization, the lymphokine IFN-γ in the immunized group decreased slightly but remained significantly higher than that in the control group. At 5 weeks post-immunization, the lymphokine IL-10 and IFN-γ in the immunized group increased synchronously, reaching 147 pg·mL⁻¹. -1 (The control group was only 20 pg·mL) -1 (P<0.001), indicating a balanced Th1 / Th2 immune response. This suggests that the EG95-Fc genetically engineered subunit vaccine can elicit both humoral and cellular immune responses in dogs.

[0049] Therefore, the serum antibody test results showed that the EG95-Fc subunit vaccine (100 µg / dog, three doses, 201 VG adjuvant) can induce rapid, long-lasting and significant humoral and cellular immune responses in beagle dogs, with good safety profile, and is worthy of being included in challenge protection trials.

Claims

1. A recombinant fusion protein for preventing infection with *Echinococcus granulosus* in dogs, characterized in that, The fusion protein comprises, from N-terminus to C-terminus, two tandem EG95 antigen domains, a canine IgG antibody Fc fragment, and a tag sequence consisting of 6×His; the two tandem EG95 antigen domains are flexibly linked, and the amino acid sequence of the fusion protein is shown in SEQ ID NO:

2.

2. A polynucleotide encoding the fusion protein of claim 1, characterized in that, The polynucleotide sequence is shown in SEQ ID NO:

1.

3. A recombinant expression vector, characterized in that, The carrier is made of pEE12.4 via Hin d III and Eco After double digestion with RI, it was compared with the same enzyme digestion method. Hin d III and Eco The recombinant vector pEE12.4-EG95-Fc is obtained by linking the polynucleotides described in claim 2 through RI double digestion.

4. A cell line stably expressing the recombinant fusion protein of claim 1, characterized in that, The cell line genome integrates the recombinant expression vector of claim 3, the cell is a CHO cell, and the cell line can continuously secrete and express the fusion protein of claim 1 in a glutamine-free selective medium containing 50 µM MSX.

5. A veterinary subunit vaccine composition, characterized in that, The vaccine comprises: an immunogenic amount of the fusion protein of claim 1, and the adjuvant ISA 201 VG; the final concentration of the fusion protein in the vaccine is 50 µg / mL, for subcutaneous administration to dogs.

6. The use of the vaccine composition of claim 5 in the preparation of a medicament for blocking the life cycle of *Echinococcus granulosus* in dogs, characterized in that... The vaccine works by stimulating humoral and cellular immunity in dogs.

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