A virus-like particle vaccine for preventing avian metapneumovirus type B and a preparation method thereof

Abstract

The application discloses a virus-like particle vaccine for preventing avian metapneumovirus type B and a preparation method thereof, and belongs to the field of veterinary biological products. The virus-like particle vaccine comprises a fusion protein with a coding gene sequence shown as SEQ ID NO: 2, and the fusion protein can self-assemble to form a virus-like particle. The application further discloses a preparation method and application of the virus-like particle vaccine for preventing avian metapneumovirus type B. The virus-like particle vaccine for preventing avian metapneumovirus type B has good immunization effect, small immunization dose, and can effectively prevent diseases caused by avian metapneumovirus type B. The preparation method is simple, can prepare antigen proteins of avian metapneumovirus type B in a large amount, has short time consumption, high expression amount, greatly reduces production cost, and is favorable for large-scale production.

Description

Technical Field

[0001] This invention relates to a virus-like particle vaccine for the prevention of avian metapneumovirus type B and its preparation method, belonging to the field of veterinary biological products. Background Technology

[0002] Avian metapneumovirus (aMPV) belongs to the genus Metapneumovirus of the subfamily Pneumoviruse within the family Paramyxoviridae. It is a single-stranded negative-sense RNA virus that can infect various poultry species, including chickens and turkeys. This virus is a major pathogen causing turkey rhinotracheitis and Swollen Head Syndrome (SHS). Infection leads to respiratory symptoms, decreased egg production, and immunosuppression, and the virus is prone to secondary bacterial infections, causing severe economic losses to the global poultry industry.

[0003] Based on differences in their surface glycoprotein G gene, aMPV can be divided into four subtypes: A, B, C, and D. Among them, subtype B avian metapneumovirus (aMPV / B) is widespread globally. However, aMPV / B virus strains have not yet been successfully isolated and identified from clinical cases, resulting in a lack of in-depth, localized research on the molecular characteristics, epidemiological patterns, and pathogenic mechanisms of this pathogen. Related diagnostic and control technologies heavily rely on foreign products and standards.

[0004] This technological gap has brought about many problems: the protective efficacy of existing foreign commercial vaccines (mainly A and B subtype attenuated vaccines and inactivated vaccines) against the prevalent strains in China is uncertain; secondly, the difficulty in virus isolation has also restricted the development and standardization of precision diagnostic technology, resulting in a low clinical diagnosis rate and passive epidemic control; furthermore, due to insufficient understanding of the biological characteristics of the virus, it is difficult to formulate scientific and effective comprehensive prevention and control strategies.

[0005] In 2016, researchers successfully isolated a chicken-derived aMPV / B strain from chickens suffering from swollen head syndrome, named it LN16, and completed its whole-genome sequencing. Genome analysis showed high homology with a foreign turkey-derived reference strain, but with differences in intergenic regions. Animal experiments confirmed its pathogenicity in SPF chickens, commercial laying hens, broilers, and yellow-feathered broilers. However, transforming virus isolates into applicable disease control products still faces challenges. Traditional inactivated vaccine development requires optimized culture, inactivation, and adjuvant processes to ensure immunogenicity and safety; while the development of attenuated live vaccines requires artificial attenuation through in vitro passages and systematic evaluation of their genetic stability, safety, and immunogenicity. Therefore, developing a vaccine (whether inactivated or attenuated live) that can induce good immune protection, has high safety, and is suitable for chicken flocks, and establishing its quality standards and evaluation methods, has significant scientific value and application prospects for breaking technological dependence and achieving effective control of this disease. Summary of the Invention

[0006] To address the shortcomings of the existing technology, this invention provides a virus-like particle vaccine for the prevention of avian metapneumovirus type B and its preparation method, aiming to solve the technical problem of the lack of a vaccine with low production cost, high production efficiency and good immunization effect for the prevention of avian metapneumovirus type B in the existing technology.

[0007] The first technical solution provided by the present invention is an avian metapneumovirus type B aMPV F-ft protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] The second technical solution provided by the present invention is a gene encoding the protein described in the first technical solution.

[0009] In some embodiments, the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0010] The third technical solution provided by the present invention is a recombinant plasmid containing the gene described in the second technical solution.

[0011] In some embodiments, the recombinant plasmid uses pFastBac I as an expression vector.

[0012] The fourth technical solution provided by the present invention is to express the avian metapneumovirus type B aMPVF-ft protein described in the first technical solution, or to contain the gene described in the second technical solution, or to transform recombinant cells with the recombinant vector described in the third technical solution.

[0013] In some embodiments, the recombinant cells use Escherichia coli or insect cells as host cells.

[0014] The fifth technical solution provided by the present invention is a virus-like particle vaccine for the prevention of avian metapneumovirus type B, wherein the vaccine contains the avian metapneumovirus type B aMPV F-ft protein described in the first technical solution.

[0015] In some embodiments, the vaccine composition also includes an adjuvant.

[0016] In some embodiments, the adjuvant in the vaccine composition is a mineral oil adjuvant. In some embodiments, the content of aMPV F-ft protein in the vaccine is 5 to 10 μg / mL; preferably, the content of aMPV F-ft protein in the vaccine is 7.5 μg / mL.

[0017] The sixth technical solution provided by this invention is a method for preparing the virus-like particle vaccine for preventing avian metapneumovirus type B as described in the fifth technical solution, comprising the following steps: (1) The gene with the synthesized nucleotide sequence as shown in SEQ ID NO.2 was cloned into the pFastBac I vector to obtain the recombinant plasmid; (2) Transform the recombinant plasmid obtained in step (1) into Escherichia coli DH10 Bac competent cells and obtain recombinant rod granules by transposition; (3) Transfect insect cells with the recombinant baculovirus obtained in step (2); (4) The recombinant baculovirus from step (3) was inoculated into HF cells for large-scale culture, and the culture supernatant was collected by centrifugation to obtain aMPV F-ft recombinant protein. After purification, adjuvant was added, emulsified, and finally mixed.

[0018] The seventh technical solution provided by the present invention is the aMPV F-ft protein described in the first technical solution, or the gene described in the second technical solution, or the recombinant plasmid described in the third technical solution, or the recombinant cell described in the fourth technical solution, or the virus-like particle vaccine for preventing avian metapneumovirus type B described in the fifth technical solution, or the application of the method described in the sixth technical solution in the preparation of a drug for preventing and / or treating diseases related to avian metapneumovirus type B or diseases related to infection by avian metapneumovirus type B.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: The avian metapneumovirus type B virus-like particle vaccine of the present invention utilizes aMPV F-ft recombinant protein, exhibiting good immunogenicity, requiring a small immunization dose, and effectively preventing avian metapneumovirus type B-related diseases or diseases caused by avian metapneumovirus type B infection. The preparation method of the present invention is simple, enabling large-scale production of avian metapneumovirus type B antigen protein, with short processing time and high expression levels, significantly reducing production costs and facilitating large-scale production. Attached Figure Description

[0020] Figure 1 This is an SDS-PAGE assay for recombinant baculovirus expression products (M: protein marker; 1: sample; 2: control).

[0021] Figure 2 These are electron microscopy results of the aMPV F-ft recombinant protein. Detailed Implementation

[0022] refer to Figures 1-2 The preferred embodiments of the present invention will be described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0023] Materials used in the examples: 1. The plasmid pFastBac I was purchased from Thermo Fisher Scientific.

[0024] 2. Escherichia coli DH10 Bac was purchased from Thermo Biotech.

[0025] 3. sf9 cells were purchased from Thermo Fisher Scientific.

[0026] 4. SPF chickens were purchased from Merial.

[0027] Example 1: Construction of Recombinant Baculovirus 1. Transfer vector construction: The aMPV F-ft protein with the nucleotide sequence shown in SEQ ID NO.2 and the amino acid sequence shown in SEQ ID NO.1 was synthesized by Wuxi Cysofi Biotechnology. The gene with the nucleotide sequence shown in SEQ ID NO.2 was ligated with the pFastBac I vector to obtain the transfer vector, which was named aMPVBac.

[0028] 2. Construction of recombinant baculovirus: The synthesized intermediate plasmid aMPVBac was transformed into Escherichia coli DH10 Bac competent cells, and positive clones were selected for PCR identification using M13 primers.

[0029] M13-F:TGTAAAACGACGGCCAGT M13-R: CAGGAAACAGCTATGAC The PCR reaction system (total volume 25 μL) consisted of: 0.5 μL DNA template, 0.5 μL each of M13-F and M13-R, 12.5 μL DNA polymerase, and 11 μL sterile water. The PCR conditions were: 93℃ for 5 min; 94℃ for 30 s, 55℃ for 45 s, 72℃ for 5 min, 30 cycles; 72℃ for 10 min. 1% agarose gel electrophoresis showed successful amplification of a specific band of approximately 3000-5000 bp, consistent with the expected size. The positive recombinant rod-like particle was named rBacmid-aMPVBac and purified.

[0030] 3. Transfection of sf9 cells with recombinant baculovirus: sf9 cells were transfected with recombinant baculovirus rBacmid-aMPVBac using liposome transfection. The specific operation method was performed in accordance with the instructions of Thermo Fisher Scientific (China) Co., Ltd.’s Cellfectin transfection reagent to obtain F1 generation recombinant baculovirus raMPVBac.

[0031] Example 2: Preparation of recombinant protein 1. Amplification of recombinant baculovirus: The F1 generation recombinant baculovirus raMPVBac from Example 1 was inoculated into insect cells sf9 and cultured at 27°C for 4 days. The culture was collected, and the supernatant was obtained by centrifugation to obtain the F2 generation recombinant baculovirus. 2. Identification of expressed proteins: (1) The above-mentioned F2 generation recombinant baculovirus was inoculated into insect cells sf9 at an MOI of 5~10, cultured at 27℃ for 4 days, the culture was collected, and the supernatant was obtained by centrifugation to obtain the recombinant protein. (2) SDS-PAGE identification: The supernatant was subjected to SDS-PAGE electrophoresis; after electrophoresis, staining and destaining revealed that the aMPV F-ft sample band was 76 kDa. Figure 1 As shown, the band sizes in the electrophoresis results are consistent with the theoretical molecular weight of the target protein, proving that the protein expression was successful.

[0032] Example 3: Vaccine Preparation 1. Inactivation: The aMPV F-ft recombinant protein prepared in Example 2 was added to an inactivation vessel, and BEI inactivation agent with a final concentration of 0.2% to 0.5% was added. The mixture was then inactivated at 37°C for 24 h.

[0033] 2. Inspection of semi-finished products (1) Sterility test: Sterility test shall be conducted in accordance with the appendix of the current Chinese Pharmacopoeia.

[0034] (2) Protein content determination: Protein content was determined by the BCA method.

[0035] (3) Inactivation test: Take insect sf9 cells from the inactivated protein solution and incubate them at 27℃ for 72 hours. If no lesions appear, the inactivation test is deemed qualified.

[0036] 3. Vaccine preparation: After passing inspection, the semi-finished protein antigen is used to prepare the vaccine (the liquid components in the following formulation are by volume ratio).

[0037] (1) Preparation of oil phase: Take 95 parts of veterinary white oil and 1 part of aluminum stearate, place them in an oil phase preparation tank and heat to 80°C. Then add 5 parts of Span-80 and maintain the temperature until it reaches 115°C. After cooling, it is ready for use.

[0038] (2) Aqueous phase preparation: Dilute each recombinant protein with physiological saline to 100 μg / mL and mix them in an appropriate ratio. Take 5 parts of sterilized Tween-80 and add them to the mixing tank, along with 95 parts of the seedling protein solution. Stir for 20-30 minutes to completely dissolve the Tween-80.

[0039] (3) Emulsification: Take 2 parts of the oil phase and place them in a high-speed shear press. Start the motor and rotate it slowly while stirring. At the same time, slowly add 1 part of the water phase and emulsify at 10,000 rpm for 5 minutes. After emulsification, take 10 mL and centrifuge at 3,000 rpm for 15 minutes. The amount of water separated at the bottom of the tube should not exceed 0.5 mL.

[0040] Example 4: Inspection of Finished Vaccine Products 1. Characteristics Appearance: The vaccine should be a milky white emulsion, free of impurities, and the outer packaging should be qualified; Dosage form: Water-in-oil emulsion. Take a clean pipette, draw a small amount of vaccine, and drop it into cold water. Except for the first drop, it should not spread.

[0041] Stability: Add 10 mL of vaccine to a centrifuge tube and centrifuge at 3000 rpm for 15 min. The amount of water precipitated at the bottom of the tube should not exceed 0.5 mL.

[0042] Viscosity: As per the appendix of the current Chinese Veterinary Pharmacopoeia, it should comply with the regulations.

[0043] 2. Content inspection: Conducted according to the appendix of the current Chinese Veterinary Pharmacopoeia, and should comply with the regulations.

[0044] 3. Sterility test: Conducted according to the appendix of the current Chinese Veterinary Pharmacopoeia, and should meet the requirements.

[0045] 4. Safety Inspection: The avian metapneumovirus type B virus-like particle vaccine has three batch numbers: raMPV-001P (protein content of 5μg / ml), raMPV-002P (protein content of 7.5μg / ml), and raMPV-003P (protein content of 10μg / ml).

[0046] Thirty SPF chickens aged 3-4 weeks were selected, and each chicken was injected subcutaneously with 1.0 ml of vaccine in its neck. The chickens were observed for any obvious local or systemic adverse reactions within 14 days.

[0047] 5. Validity verification: 5.1 Grouping and Challenge: Eighty 3-4 week old SPF chickens were randomly divided into four groups (A-D), with 30 chickens in each group. Groups A, B, and C were the immunization groups, with each chicken receiving a subcutaneous injection of 0.5 ml of vaccine in the neck. Group D was the non-immunized control group. Chickens were challenged with a virulent strain on days 21 and 28 post-immunization, and the disease incidence was observed within 7 days.

[0048] 5.2 Efficacy Test Results: Eighty 3-4 week old SPF chickens were randomly divided into four groups (A-D), with 30 chickens in each group. Groups A, B, and C were the immunization groups, with each chicken receiving a subcutaneous injection of 0.5 ml of vaccine in the neck. Group D was the non-immunized control group. On days 21 and 28 post-immunization, chickens were challenged with a virulent strain via intranasal instillation (0.1 ml in each nostril, with a total dose of 10 ml). 4 TCID 50 / bird), and observe the disease incidence of experimental chickens within 7 days. See Table 1 for details. As can be seen from the table, 21 days after immunization with the three batches of vaccines, the protection rate of the immunized group was not less than 90%, and the protection rate was 100% 28 days after immunization. This data shows that the vaccine involved in this patent can effectively prevent infection with avian metapneumovirus type B.

[0049] Table 1. Results of efficacy testing for avian metapneumovirus type B virus-like particle vaccine.

[0050] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A type B avian metapneumovirus (aMPV) F-ft protein, characterized in that, The amino acid sequence of the avian metapneumovirus type B aMPV F-ft protein is shown in SEQ ID NO.

1.

2. The gene encoding the protein of claim 1.

3. A recombinant plasmid containing the gene described in claim 2.

4. The recombinant plasmid according to claim 3, characterized in that, The recombinant plasmid was expressed using plasmid pFastBac I as the expression vector.

5. A recombinant cell expressing the avian metapneumovirus type B aMPV F-ft protein of claim 1, or containing the gene of claim 2, or transformed with the recombinant vector of any one of claims 3 to 4.

6. The recombinant cell according to claim 5, characterized in that, The recombinant cells use Escherichia coli or insect cells as host cells.

7. A virus-like particle vaccine for the prevention of avian metapneumovirus type B, characterized in that, The vaccine contains the avian metapneumovirus type B aMPV F-ft protein as described in claim 1.

8. The virus-like particle vaccine according to claim 7, characterized in that, The vaccine contains 5–10 μg / mL of aMPV F-ft protein.

9. A method for preparing a virus-like particle vaccine against avian metapneumovirus type B as described in any one of claims 7-8, characterized in that, Includes the following steps: (1) The gene with the synthesized nucleotide sequence as shown in SEQ ID NO.2 was cloned into the pFastBac I vector to obtain the recombinant plasmid; (2) Transform the recombinant plasmid obtained in step (1) into Escherichia coli DH10 Bac competent cells and obtain recombinant rod granules by transposition; (3) Transfect insect cells with the recombinant baculovirus obtained in step (2); (4) The recombinant baculovirus from step (3) was inoculated into HF cells for large-scale culture, and the culture supernatant was collected by centrifugation to obtain aMPV F-ft recombinant protein. After purification, adjuvant was added, emulsified, and finally mixed.

10. The use of the aMPV F-ft protein of claim 1, or the gene of claim 2, or the recombinant plasmid of any one of claims 3-4, or the recombinant cell of any one of claims 5-6, or the virus-like particle vaccine for the prevention of avian metapneumovirus type B of any one of claims 7-8, or the method of claim 9 in the preparation of a medicament for the prevention and / or treatment of diseases related to avian metapneumovirus type B or diseases related to infection with avian metapneumovirus type B.

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