Faviadenovirus D11 type tandem multi-epitope vaccine

Abstract

The invention provides an avian adenovirus D11 type tandem multi-epitope vaccine, and belongs to the technical field of veterinary vaccines. According to the invention, an amino acid sequence of fiber protein of the fowl adenovirus D11 type is analyzed, 10 potential B cell epitopes are screened out, 10 antigen epitopes are artificially synthesized to prepare the vaccine, and six antigen epitopes with high neutralizing antibody titer are screened out by immunizing chicken. Flexible peptides are designed among the screened six antigen epitopes for connection, and a series multi-epitope antigen is designed. The oil emulsion vaccine is prepared by expressing tandem multi-epitope antigen protein through recombinant escherichia coli, purifying through a nickel column, and emulsifying with a mineral oil adjuvant. All test chickens immunized by the series multi-epitope vaccine in an over-dose manner have no abnormities in spirit, ingestion and water drinking, injection parts have no red and swollen reactions, and the vaccine is good in safety. The series multi-epitope vaccine has a good immune effect, can enable the immunized chicken to generate a neutralizing antibody to achieve challenge protection, and can block the detoxification of the challenge chicken.

Description

Technical Field

[0001] The invention belongs to the field of veterinary technology, and in particular relates to an avian adenovirus D11 type tandem multi-epitope vaccine. Background Art

[0002] Avian adenoviruses are divided into three groups: I, II, and III; Groups II and III can clearly cause hemorrhagic enteritis and egg drop syndrome in turkeys. There are five species in Group I, namely A, B, C, D, and E, and 12 serotypes. The pathogenicity of most Group I avian adenoviruses has not been fully clarified, but the pathogenicity of different serotypes varies greatly. Species A is FAdV-1, species B is FAdV-5, species C is FAdV-4 and FAdV-10, species D is FAdV-2, FAdV-3, FAdV-9, and FAdV-11, and species E includes FAdV-6, FAdV-7, FAdV-8a, and FAdV-8b.

[0003] Since 2015, hepatitis-pericardial effusion syndrome caused by group I C4 avian adenovirus has been widely prevalent throughout the country. It is a highly contagious and highly lethal infectious disease for broilers and laying hens. Subsequently, its host range has been expanded to poultry such as ducks and geese, causing huge economic losses. At the same time, inclusion body hepatitis caused by FAdV-8a, FAdV-8b and FAdV-D11 is often co-infected with hepatitis-pericardial effusion syndrome caused by C4 avian adenovirus. In recent years, inclusion body hepatitis caused by single infection with FAdV-8a, FAdV-8b and FAdV-D11 has often occurred in chickens and ducks, causing huge economic losses. Inclusion body hepatitis mainly affects broilers aged 3 to 6 weeks. The clinical manifestations of affected chickens are depression, reduced diet, and slow growth. The mortality rate can reach about 20%. Autopsy shows liver necrosis and the presence of eosinophilic or basophilic nuclear inclusions in hepatocytes as the main features.

[0004] Currently, commercial vaccines and egg yolk antibodies against FAdV-4 are widely used, effectively controlling the large-scale infection of FAdV-4 and reducing the losses caused by hepatitis-pericardial effusion syndrome. However, there is currently no commercial vaccine against FAdV-D11, and the spread of avian inclusion body hepatitis in poultry and the resulting losses are still expanding. Therefore, it is urgent to develop a vaccine against FAdV-D11 to control the harm of inclusion body hepatitis caused by FAdV-D11 to the poultry industry. Summary of the invention

[0005] The invention aims to provide an avian adenovirus D11 type tandem multi-epitope vaccine, belonging to the technical field of veterinary medicine.

[0006] To achieve the above object, the present invention provides the following technical solutions: Firstly, the present invention provides an avian adenovirus D11 type tandem multi-epitope vaccine, which is an oil emulsion vaccine prepared by emulsifying the avian adenovirus D11 type tandem multi-epitope antigen protein expressed by recombinant Escherichia coli and a mineral oil adjuvant.

[0007] Furthermore, the vaccine preparation method comprises the following steps: (1) The recombinant Escherichia coli expressing the tandem multi-epitope antigen protein of avian adenovirus D11 was transferred to 10 ml of LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured at 37°C and 250 r / min shaking for 12 h as the primary seed liquid; (2) According to the fermentation volume, the first-level seed liquid was inoculated with LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured at 37°C and 250 r / min shaking for 12 h to prepare the second-level seed liquid and multi-level seed liquid; (3) The seed liquid was inoculated with LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1% in a fermenter and fermented for 3 h until the OD600nm was 0.6-0.8; (4) Add 100 mM / L α-lactose to a final concentration of 0.4 mM / L, lower the culture temperature to 30 °C, and culture for another 5 h to terminate the fermentation; (5) The fermentation broth was centrifuged at 12000 r / min to collect the cells, and PBS (10 times the cell weight) was added to suspend the cells, and the cells were broken by ultrasonic wave and centrifuged at 12000 r / min. The supernatant was filtered through a 0.22 μm filter and the antigen protein was purified by Ni column. (6) Add 10% formaldehyde solution to the purified antigen protein solution and mix thoroughly. The final concentration of the formaldehyde solution is 0.1%. Inactivate at 2-8°C for 72 hours to prepare the antigen solution. (7) One part of antigen solution and two parts of mineral oil adjuvant are emulsified to prepare a tandem multi-epitope vaccine of avian adenovirus D11.

[0008] Furthermore, the amino acid sequence of the fowl adenovirus D11 type tandem multi-epitope antigen protein is SEQ ID NO.1.

[0009] Secondly, the present invention provides an avian adenovirus D11 type tandem multi-epitope antigen, wherein the multi-epitope antigen is an avian adenovirus D11 type tandem multi-epitope antigen protein expressed by recombinant Escherichia coli, and the amino acid sequence of the tandem multi-epitope antigen protein is SEQ ID NO.1.

[0010] Furthermore, the nucleotide sequence of the tandem multi-epitope antigen protein is SEQ ID NO.2.

[0011] Furthermore, the method for preparing the tandem multi-epitope antigen protein comprises the following steps: (1) The recombinant Escherichia coli expressing the tandem multi-epitope antigen protein of avian adenovirus D11 was transferred to 10 ml of LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured at 37°C and 250 r / min shaking for 12 h as the primary seed liquid; (2) According to the fermentation volume, the first-level seed liquid was inoculated with LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured at 37°C and 250 r / min shaking for 12 h to prepare the second-level seed liquid and multi-level seed liquid; (3) The seed liquid was inoculated with LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1% in a fermenter and fermented for 3 h until the OD600nm was 0.6-0.8; (4) Add 100 mM / L α-lactose to a final concentration of 0.5 mM / L, lower the culture temperature to 30 °C, and culture for another 5 h to terminate the fermentation; (5) The fermentation broth was centrifuged at 12,000 r / min to collect the bacteria, and PBS (10 times the volume of the bacteria) was added to suspend the bacteria. The bacteria were disrupted by ultrasonic wave and centrifuged at 12,000 r / min. The supernatant was filtered through a 0.22 μm filter and the antigen protein was purified by Ni column.

[0012] Beneficial effects: The present invention analyzes the amino acid sequence of the fiber protein of FAdV-D11, screens out 10 potential B cell epitopes, artificially synthesizes 10 antigen epitopes to prepare vaccines, and immunizes chickens to screen out 6 antigen epitopes with high neutralizing antibody titers. Flexible peptides are designed to connect the 6 selected antigen epitopes, and a tandem multi-epitope antigen is designed. The tandem multi-epitope antigen protein is expressed by recombinant Escherichia coli, and the oil emulsion vaccine is prepared by emulsification with a mineral oil adjuvant after nickel column purification. The prepared tandem multi-epitope vaccine is safe for overdose immunization of SPF chickens. The tandem multi-epitope vaccine has a good immune effect, can make the immunized chickens produce high levels of neutralizing antibodies, achieve a protective effect against the virus, and can block the detoxification of the virus-challenged chickens. The tandem multi-epitope vaccine of avian adenovirus D11 developed by the present invention is safe and effective, can reduce the damage caused to chickens by wild-type avian adenovirus D11 infection, and can effectively control the spread of the epidemic. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Detection of tandem multi-epitope antigen protein by SDS-PAGE electrophoresis. DETAILED DESCRIPTION

[0014] Example 1: Fiber protein antigen epitope screening Fiber protein is a spike protein on the surface of adenovirus particles, mediating the binding of the virus to host cell receptors and playing a key role in the process of virus adhesion and invasion of host cells. Therefore, fiber protein is the most important antigenic protein of adenovirus. The specific antibodies against fiber protein produced by the host after adenovirus invasion have the activity of neutralizing the virus and can neutralize the invasion of the virus into the host. Humoral immunity is dominant in the immune response of avian adenovirus. Inactivated adenovirus antigens can stimulate the body to produce high levels of neutralizing antibodies to protect the body from live virus attacks. Fiber protein immunization can produce neutralizing antibody levels close to those produced by inactivated antigens of whole virus particles. Neutralizing epitopes on fiber protein are the basis of immune protection. Mining the neutralizing epitopes of fiber protein can prepare highly specific and high-titer subunit vaccines.

[0015] With reference to the amino acid sequence of GenBank: WIM48614.1, the amino acid sequence of the fiber protein of FAdV-D11 was analyzed using the online epitope prediction tool of the Immune Epitope Database (IEDB), and 10 potential B cell epitopes were screened out. The epitope sequences and positions on the fiber protein are shown in Table 1. Sangon Biotech (Shanghai) Co., Ltd. was commissioned to synthesize the 10 epitope peptides and couple them to keyhole limpet hemocyanin (KLH) respectively.

[0016] The 10 synthesized epitope peptides were emulsified with 201 adjuvant to prepare subunit vaccines, so that the peptide content in the vaccine was 100 μg / mL. Five 21-day-old SPF chickens were immunized with each of the 10 prepared peptide vaccines, 0.5 ml / chicken, and 5 non-immunized SPF chickens were set as the control group. 21 days after immunization, all test chickens were bled and serum was separated for neutralizing antibody detection.

[0017] As shown in Table 1, the three amino-terminal epitopes of the fiber protein of FAdV-D11 have no virus neutralizing activity, and the neutralizing activity of one epitope is too low, only 1:7.35; the neutralizing activity of the six epitopes at the carboxyl terminus is greater than or equal to 1:16.38, which can be used for multi-epitope vaccine design.

[0018] Table 1 Prediction of fiber protein epitopes and determination of neutralization activity of FAdV-8b

[0019] Example 2: Construction of recombinant Escherichia coli expressing tandem multi-epitope antigens (1) Tandem multi-epitope vaccine sequence design: Select epitopes 5, 6, 7, 8, 9, and 10 in Table 1, connect epitope sequences 5 and 6 with GSG, epitope sequences 6 and 7 with GSG, epitope sequences 7 and 8 with SG, epitope sequences 8 and 9 with SG, and epitope sequences 9 and 10 with GSG. The amino acid sequence of the tandem multi-epitope protein is shown in SEQ ID NO.1.

[0020] (2) Construction of expression plasmid Based on the codon-optimized DNA sequence for expressing tandem multi-epitope proteins in E. coli, we commissioned Shanghai Sangon Biotechnology Co., Ltd. to synthesize SEQ ID NO.2 and cloned it between the BamHI and XhoI sites of the pET28a plasmid, named pET28a-6P.

[0021] (3) Construction of recombinant expression strain The pET28a-6P plasmid was used to transform E. coli BL21 competent cells, and a single colony was picked for PCR identification and the positive transformant was named E. coli BL21 / pET28a-6P. The single colony was cultured in 5 mL of LB medium containing 50 μg / mL kanamycin sulfate at 37°C and 250 r / min for 12 h to serve as the seed culture.

[0022] (4) Expression and identification of target protein A single colony of E. coli BL21 / pET28a-6P was selected and inoculated into 10 ml of LB culture medium containing 50 μg / mL kanamycin. The culture was shaken at 37°C until the OD600nm was 0.6-0.8. 100 mM / L α-lactose was added to a final concentration of 0.4 mM / L, and the culture temperature was lowered to 30°C and cultured for another 6 h. After removal, the cells were centrifuged at 12,000 r / min for 10 min to collect the cells. PBS with a volume of 10 times the weight of the cells was added to suspend the cells and ultrasonically disrupted. Centrifuge at 4°C, 12,000 r / min for 15 min. The supernatant and precipitate were treated with SDS-PAGE loading buffer, respectively, and analyzed by SDS-PAGE and Western blot.

[0023] (5) Purification of recombinant protein Before loading, use 10 column volumes of Binding Buffer to equilibrate the nickel column at a flow rate of 5 mL / min. After centrifugation and filtration, the protein solution is loaded onto the equilibrated Ni column through a constant flow pump at a flow rate of 1 mL / min, and the penetration liquid is collected. The sample can be loaded repeatedly to improve the column-hanging efficiency of the sample. The Ni column that has bound the target protein is re-equilibrated with 10 column volumes of Binding Buffer at a flow rate of 5 mL / min to remove unbound proteins. Elute the Ni column with an equal volume mixture of Elution Buffer and Binding Buffer at a flow rate of 5 mL / min, measure the OD280 value, and collect the eluate corresponding to the protein absorption peak. Take an appropriate amount of eluate for SDS-PAGE electrophoresis detection. The protein band size is about 14.41 kD. The electrophoresis results are as follows: Figure 1 .

[0024] Example 3: Preparation of tandem multi-epitope vaccines (1) 0.1 ml of seed solution was transferred to 10 ml of LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured at 37°C and 250 rpm for 12 h as the primary seed solution.

[0025] (2) 10 ml of the first-level seed solution was transferred to 1000 ml of LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured at 37°C and 250 r / min for 12 h as the second-level seed solution.

[0026] (3) Calibrate the pH electrode and dissolved oxygen electrode of the fermentation tank, and perform flow calibration of the peristaltic pump.

[0027] (4) Prepare 70 L of LB medium, add to a 100 L fermenter, and sterilize the medium, fermenter, and pipes at 121°C for 30 min.

[0028] (5) When the culture medium in the fermentation tank is cooled to 37°C, add kanamycin sulfate at a final concentration of 50 μg / mL, add 700 ml of secondary seed liquid to the fermentation tank, set the fermentation tank parameters to a stirring speed of 500-800 r / min, a tank pressure of 9 psi, a temperature of 37°C, and set the DO value (dissolved oxygen) to above 20%. Fermentation was carried out for 3 hours until the OD600nm was 0.6-0.8.

[0029] (6) Add 100 mM / L α-lactose to a final concentration of 0.4 mM / L, set the temperature to 30°C, and incubate for 5 h to terminate the fermentation.

[0030] (7) The fermentation broth was centrifuged at 12000 r / min to collect the cells. PBS with a volume of 10 times the weight of the cells was added to suspend the cells, and the cells were broken by ultrasonic wave and centrifuged at 12000 r / min. The supernatant was filtered through a 0.22μm filter and loaded onto the equilibrated Ni column at a flow rate of 1mL / min through a constant flow pump. The Ni column that had bound the target protein was re-equilibrated with 10 column volumes of Binding Buffer at a flow rate of 5mL / min to remove unbound proteins. The Ni column was eluted with an equal volume mixture of Elution Buffer and Binding Buffer at a flow rate of 5mL / min, and the OD280 value was measured. The eluate corresponding to the protein absorption peak was collected. The purified protein solution was stored at 2-8℃ for later use.

[0031] (8) Take the purified protein solution and add 10% formaldehyde solution, mix thoroughly, and the final concentration of the formaldehyde solution is 0.1%. Inactivate at 2-8℃ for 72 hours. Store at 2-8℃ for later use.

[0032] (9) Vaccine emulsification: Mix mineral oil, S-80 and aluminum stearate in a ratio of 95:5:1 to prepare the oil phase. Maintain the temperature at 115°C for 40 minutes. After cooling, the oil phase is obtained. 95 parts of inactivated antigen solution and 5 parts of sterile Tween-80 are mixed thoroughly to completely dissolve the Tween-80 to obtain the water phase. 2 parts of the oil phase are placed in an emulsification tank and slowly sheared and stirred at a speed of 2000 rpm / min. At the same time, 1 part of the water phase is slowly added and emulsified at a speed of 14000 rpm / min to prepare the finished vaccine.

[0033] Example 4: Tandem multi-epitope vaccine properties, safety, and efficacy testing (1) Property inspection Carefully observe the color of the vaccine and whether there are any impurities.

[0034] Use a 1 ml pipette to draw up a small amount of vaccine, first drop 1 drop of vaccine on the water surface 2 to 3 cm above the water surface, and then drop 2 to 3 drops of vaccine. Pay attention to the diffusion of the vaccine on the water surface and record the diffusion of the droplets. The first drop and subsequent drops should be recorded separately.

[0035] Stability: Pipette 10 ml of the vaccine into a centrifuge tube, centrifuge at 3000 r / min for 15 minutes, and observe whether there is water phase precipitation at the bottom of the tube.

[0036] The viscosity is measured at room temperature (20℃±0.1℃) using a rotary viscometer.

[0037] (2) For safety testing, 10 21-day-old SPF chickens were used. 1.0 ml of the vaccine was injected subcutaneously at the neck of each chicken. The chickens were observed for 14 days, and their clinical reactions such as eating and drinking were recorded. Vaccine absorption at the injection site was also observed by dissection.

[0038] (3) Efficacy test: 20 21-day-old SPF chickens were used, 10 of which were injected subcutaneously with 0.2 ml of vaccine in the neck, and the other 10 were used as controls. 21 days after vaccination, blood was collected from each chicken, and serum was separated. The neutralizing antibody titer was determined by virus neutralization test. At the same time, all immunized chickens and control chickens were intramuscularly injected with group I D11 avian adenovirus AdV11 strain virus liquid, 0.1 ml (containing 1×10 5.5 TCID 50 ). After 14 days of observation, the incidence of immunized chickens and control chickens was recorded. On the third day after the challenge, anal swabs were collected from all test chickens, viral DNA was extracted, and the excretion of group I D11 avian adenovirus was detected by fluorescent quantitative PCR. On the 14th day after the challenge, all test chickens were dissected to observe liver lesions.

[0039] As shown in Table 1, the properties of the prepared tandem multi-epitope vaccine are consistent with those of conventional oil-emulsion inactivated vaccines and have good stability.

[0040] Table 1 Test results of tandem multi-epitope vaccine characteristics

[0041] As shown in Table 2, all the chickens immunized with the prepared tandem multi-epitope vaccine in overdose showed no abnormality in spirit, feeding and drinking, and no redness or swelling reaction at the injection site. It can be seen that the tandem multi-epitope vaccine has good safety.

[0042] Table 2 Safety test results of tandem multi-epitope vaccine

[0043] As shown in Tables 3 and 4, 21 days after immunization with the tandem multi-epitope vaccine, the neutralizing antibody titer of the immunized chickens was ≥1:64, and the neutralizing antibody of the chickens in the control group was all negative; the challenge protection results showed that the challenge protection rate of the vaccine immunization group was 10 / 10, and 10 / 10 were negative for detoxification on the third day after the challenge. It can be seen that the tandem multi-epitope vaccine has a good immunization effect, which can make the immunized chickens produce neutralizing antibodies, achieve challenge protection, and block the detoxification of challenged chickens.

[0044] Table 3 Results of the efficacy test of tandem multi-epitope vaccines

[0045] Table 4 Statistical results of the efficacy test of tandem multi-epitope vaccines

Claims

1. A tandem multi-epitope vaccine of avian adenovirus D11, characterized in that: The vaccine is an oil emulsion vaccine prepared by emulsifying avian adenovirus D11 type tandem multi-epitope antigen protein expressed by recombinant Escherichia coli and a mineral oil adjuvant.

2. The vaccine according to claim 1, characterized in that The preparation method of the vaccine comprises the following steps: (1) The recombinant Escherichia coli expressing the tandem multi-epitope antigen protein of avian adenovirus D11 was transferred to 10 ml of LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured at 37°C and 250 r / min shaking for 12 h as the primary seed liquid; (2) According to the fermentation volume, the first-level seed liquid was inoculated with LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured at 37°C and 250 r / min shaking for 12 h to prepare the second-level seed liquid and multi-level seed liquid; (3) The seed liquid was inoculated with LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1% in a fermenter and fermented for 3 h until the OD600nm was 0.6-0.8; (4) Add 100 mM / L α-lactose to a final concentration of 0.4 mM / L, lower the culture temperature to 30 °C, and culture for another 5 h to terminate the fermentation; (5) The fermentation broth was centrifuged at 12000 r / min to collect the cells, and PBS (10 times the cell weight) was added to suspend the cells, and the cells were broken by ultrasonic wave and centrifuged at 12000 r / min. The supernatant was filtered through a 0.22 μm filter and the antigen protein was purified by Ni column. (6) Add 10% formaldehyde solution to the purified antigen protein solution and mix thoroughly. The final concentration of the formaldehyde solution is 0.1%. Inactivate at 2-8°C for 72 hours to prepare the antigen solution. (7) One part of antigen solution and two parts of mineral oil adjuvant are emulsified to prepare a tandem multi-epitope vaccine of avian adenovirus D11.

3. The vaccine according to claim 1, characterized in that The amino acid sequence of the fowl adenovirus D11 type tandem multi-epitope antigen protein is SEQ ID NO.

1.

4. A tandem multi-epitope antigen of avian adenovirus D11, characterized in that: The multi-epitope antigen is a tandem multi-epitope antigen protein of fowl adenovirus D11 type expressed by recombinant Escherichia coli, and the amino acid sequence of the tandem multi-epitope antigen protein is SEQ ID NO.

1.

5. The tandem multi-epitope antigen according to claim 4, characterized in that: The nucleotide sequence of the tandem multi-epitope antigen protein is SEQ ID NO.

2.

6. The tandem multi-epitope antigen according to claim 4, characterized in that: The method for preparing the tandem multi-epitope antigen protein comprises the following steps: (1) The recombinant Escherichia coli expressing the tandem multi-epitope antigen protein of avian adenovirus D11 was transferred to 10 ml of LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured at 37°C and 250 r / min shaking for 12 h as the primary seed liquid; (2) According to the fermentation volume, the first-level seed liquid was inoculated with LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured at 37°C and 250 r / min shaking for 12 h to prepare the second-level seed liquid and multi-level seed liquid; (3) The seed liquid was inoculated with LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1% in a fermenter and fermented for 3 h until the OD600nm was 0.6-0.8; (4) Add 100 mM / L α-lactose to a final concentration of 0.5 mM / L, lower the culture temperature to 30 °C, and culture for another 5 h to terminate the fermentation; (5) The fermentation broth was centrifuged at 12,000 r / min to collect the bacteria, and PBS (10 times the volume of the bacteria) was added to suspend the bacteria. The bacteria were disrupted by ultrasonic wave and centrifuged at 12,000 r / min. The supernatant was filtered through a 0.22 μm filter and the antigen protein was purified by Ni column.

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