A tandem multi-epitope vaccine against fowl adenovirus type D11

Abstract

The present invention provides a tandem multi-epitope vaccine against fowl adenovirus type D11, belonging to the technical field of veterinary vaccines. The present invention analyzes the amino acid sequence of the fiber protein of fowl adenovirus type D11, screens out 10 potential B-cell epitopes, artificially synthesizes 10 antigenic epitopes to prepare a vaccine and immunizes chickens to screen out 6 antigenic epitopes with relatively high neutralizing antibody titers. Flexible peptides are designed to be linked between the 6 screened antigenic epitopes to design a tandem multi-epitope antigen. The tandem multi-epitope antigen protein is expressed by recombinant Escherichia coli, emulsified with a mineral oil adjuvant after purification by a nickel column to prepare an oil emulsion vaccine. All the experimental chickens immunized with the tandem multi-epitope vaccine at an overdose showed no abnormalities in spirit, feeding and drinking, and there was no swelling reaction at the injection site, indicating good vaccine safety. The tandem multi-epitope vaccine has a good immunization effect, can enable the immunized chickens to produce neutralizing antibodies, achieve protection against challenge, and can block the excretion of challenged chickens.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary medicine, and particularly relates to a tandem multi-epitope vaccine against fowl adenovirus serotype D11. Background Art

[0002] Fowl adenoviruses are divided into three groups: Group I, Group II, and Group III; Group II and Group III are clearly known to cause hemorrhagic enteritis and egg drop syndrome in turkeys. There are five species (A, B, C, D, and E) and 12 serotypes in Group I. The pathogenicity of most Group I fowl adenoviruses has not been fully clarified, but there are significant differences in the pathogenicity of different serotypes. 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-hydropericardium syndrome caused by Group I serotype C4 fowl adenovirus is an infectious disease with high infectivity and high lethality to broiler chickens and laying hens, and subsequently its host range has been broadened to poultry such as ducks and geese. At the same time, inclusion body hepatitis caused by FAdV-8a, FAdV-8b, and FAdV-D11 is often co-infected with hepatitis-hydropericardium syndrome caused by serotype C4 fowl adenovirus. In recent years, inclusion body hepatitis caused by single infection of FAdV-8a, FAdV-8b, and FAdV-D11 has often occurred in chicken and duck flocks. Inclusion body hepatitis mainly affects broiler chickens at 3 to 6 weeks of age. The clinical manifestations of diseased chickens are listlessness, reduced food intake, slow growth, and the mortality rate can reach about 20%. The main pathological features found by autopsy are liver necrosis and the presence of eosinophilic or basophilic intranuclear inclusion bodies in hepatocytes.

[0004] At present, commercial vaccines and yolk antibodies against FAdV-4 have been widely applied, effectively controlling the large-scale infection of FAdV-4 and reducing the losses caused by hepatitis-hydropericardium syndrome. However, there is currently no commercial vaccine against FAdV-D11, and the spread of fowl inclusion body hepatitis in poultry and the resulting losses are still expanding. Therefore, there is an urgent need to develop a vaccine against FAdV-D11 to control the harm of inclusion body hepatitis caused by FAdV-D11 to the poultry farming industry. Summary of the Invention

[0005] The purpose of the present invention is to provide a tandem multi-epitope vaccine against fowl adenovirus serotype D11, belonging to the technical field of veterinary medicine.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] First, the present invention provides a tandem multi-epitope vaccine of fowl adenovirus type D11, and the vaccine is an oil emulsion vaccine prepared by emulsifying a tandem multi-epitope antigen protein of fowl adenovirus type D11 expressed by recombinant Escherichia coli with a mineral oil adjuvant.

[0008] Further, the preparation method of the vaccine comprises the following steps:

[0009] (1) The recombinant Escherichia coli expressing the tandem multi-epitope antigen protein of fowl adenovirus type D11 was transferred to 10 ml of LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured in a shaker at 37 °C and 250 r / min for 12 h as the primary seed liquid;

[0010] (2) According to the fermentation volume, the primary seed liquid was inoculated into the LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured in a shaker at 37 °C and 250 r / min for 12 h to prepare the secondary seed liquid and multi-stage seed liquid;

[0011] (3) The seed liquid was inoculated into the 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 reached 0.6 - 0.8;

[0012] (4) 100 mM / L of α-lactose was added to a final concentration of 0.4 mM / L, and the culture temperature was reduced to 30 °C and cultured for another 5 h to end the fermentation;

[0013] (5) The fermented broth was continuously centrifuged at 12000 r / min to collect the thalli, and the thalli were suspended with 10 times the volume of PBS based on the weight of the thalli, sonicated, continuously centrifuged at 12000 r / min, and the supernatant was filtered through a 0.22 μm filter, and the antigen protein was purified by an Ni column;

[0014] (6) The purified antigen protein solution was quantitatively added with a 10% formaldehyde solution, and mixed well. The final concentration of the formaldehyde solution was 0.1%, and inactivated at 2 - 8 °C for 72 h to prepare the antigen solution;

[0015] (7) 1 part of the antigen solution and 2 parts of the mineral oil adjuvant were emulsified to prepare the tandem multi-epitope vaccine of fowl adenovirus type D11.

[0016] Further, the amino acid sequence of the tandem multi-epitope antigen protein of fowl adenovirus type D11 is SEQ ID NO.1.

[0017] Second, the present invention provides a tandem multi-epitope antigen of fowl adenovirus type D11, and the multi-epitope antigen is a tandem multi-epitope antigen protein of fowl adenovirus type D11 expressed by recombinant Escherichia coli, and the amino acid sequence of the tandem multi-epitope antigen protein is SEQ ID NO.1.

[0018] Furthermore, the nucleotide sequence of the tandem multi-epitope antigen protein is SEQ ID NO.2.

[0019] Furthermore, the method for preparing the tandem multi-epitope antigen protein comprises the following steps:

[0020] (1) Recombinant Escherichia coli expressing the tandem multi-epitope antigen protein of fowl adenovirus type D11 was transferred to 10 ml of LB culture medium containing 50 μg / mL kanamycin sulfate at a ratio of 1% by volume, and cultured in a shaker at 37 °C and 250 r / min for 12 h as the primary seed solution;

[0021] (2) According to the fermentation volume, the primary seed solution was inoculated into LB culture medium containing 50 μg / mL kanamycin sulfate at a ratio of 1% by volume, and cultured in a shaker at 37 °C and 250 r / min for 12 h to prepare the secondary seed solution and multiple-stage seed solutions;

[0022] (3) The seed solution was inoculated into LB culture medium containing 50 μg / mL kanamycin sulfate at a ratio of 1% by volume in a fermenter, and fermented for 3 h until the OD600nm reached 0.6 - 0.8;

[0023] (4) 100 mM / L of α-lactose was added to a final concentration of 0.5 mM / L, and the culture temperature was reduced to 30 °C and cultured for another 5 h to end the fermentation;

[0024] (5) The fermented broth was continuously centrifuged at 12000 r / min to collect the bacteria, and the bacteria were suspended in PBS at 10 times the volume of the bacteria weight, sonicated, continuously centrifuged at 12000 r / min, and the supernatant was filtered through a 0.22 μm filter, and the antigen protein was purified by Ni column.

[0025] Beneficial effects: In the present invention, the amino acid sequence of the fiber protein of FAdV-D11 was analyzed, 10 potential B cell epitopes were screened out, 10 antigenic epitopes were artificially synthesized to prepare a vaccine, and chickens were immunized to screen out 6 antigenic epitopes with higher neutralizing antibody titers. Flexible peptides were designed to connect between the 6 screened antigenic epitopes to design a tandem multi-epitope antigen. The tandem multi-epitope antigen protein was expressed by recombinant Escherichia coli, emulsified with a mineral oil adjuvant after purification by Ni column to prepare an oil emulsion vaccine. The tandem multi-epitope vaccine prepared had good safety when super-dose immunized SPF chickens. The tandem multi-epitope vaccine had good immune effects, could enable the immunized chickens to produce high-level neutralizing antibodies, achieve the effect of protecting against challenge, and could block the excretion of challenged chickens. The tandem multi-epitope vaccine of fowl adenovirus type D11 developed in the present invention was safe and effective, could reduce the damage caused by the infection of wild fowl adenovirus type D11 to chicken flocks, and could effectively control the spread of the epidemic. Brief Description of the Drawings

[0026] Figure 1 SDS-PAGE electrophoresis detection of tandem multi-epitope antigen protein. Specific implementation mode

[0027] Example 1: Screening of fiber protein antigen epitopes

[0028] The fiber protein is the spike protein on the surface of adenovirus particles, which mediates the binding of the virus to the host cell receptor and plays a key role in the process of virus adhesion and invasion of host cells. Therefore, the fiber protein is the most important antigen protein of adenovirus. The specific antibodies against the 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 plays a dominant role in the immune response of avian adenovirus. Inactivating adenovirus antigen can stimulate the body to produce a high level of neutralizing antibodies, which can protect the body from live virus attack. Immunization with fiber protein can produce a neutralizing antibody level close to that produced by inactivated antigen of whole virus particles. The neutralizing epitopes on the fiber protein are the basis of immune protection. Mining the neutralizing epitopes of the fiber protein can prepare subunit vaccines with high specificity and high titer.

[0029] Referring 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 their positions on the fiber protein are shown in Table 1. Synthesize 10 epitope peptides and conjugate them with keyhole limpet hemocyanin (KLH) respectively by Shanghai Sangon Biotech Co., Ltd.

[0030] The 10 synthesized epitope peptides were emulsified with 201 adjuvant to prepare subunit vaccines, so that the peptide content in the vaccines was 100 μg / mL. Five 21-day-old SPF chickens were immunized with each of the 10 prepared peptide vaccines, 0.5 ml per chicken. At the same time, 5 non-immunized SPF chickens were set as the control group. Twenty-one days after immunization, all experimental chickens were bled and the sera were separated for neutralizing antibody detection.

[0031] As can be seen from Table 1, none of the 3 epitopes at the amino terminus of the fiber protein of FAdV-D11 have the activity of neutralizing the virus, and the neutralizing activity of 1 epitope is too low, only 1:7.35; the neutralizing virus activity of 6 epitopes at the carboxyl terminus is greater than or equal to 1:16.38, which can be used for the design of multi-epitope vaccines.

[0032] Table 1 Prediction and neutralizing activity determination of fiber protein epitopes of FAdV-8b

[0033]

[0034] Example 2: Construction of recombinant Escherichia coli expressing tandem multi-epitope antigen

[0035] (1)Design of tandem multi-epitope vaccine sequence: Select the epitopes No. 5, 6, 7, 8, 9, and 10 in Table 1. Connect the sequences of epitopes No. 5 and 6 with GSG, connect the sequences of epitopes No. 6 and 7 with GSG, connect the epitopes No. 7 and 8 with SG, connect the epitopes No. 8 and 9 with SG, and connect the epitopes No. 9 and 10 with GSG. The amino acid sequence of the tandem multi-epitope protein is shown in SEQ ID NO.1.

[0036] (2)Construction of expression plasmid: Optimize the DNA sequence for expressing the tandem multi-epitope protein according to the codon preference of Escherichia coli, entrust Sangon Biotech (Shanghai) Co., Ltd. to synthesize SEQ ID NO.2, and clone it between the BamHI and XhoI sites of the pET28a plasmid, named pET28a-6P.

[0037] (3)Construction of recombinant expression strain: Transform the pET28a-6P plasmid into Escherichia coli BL21 competent cells, pick a single colony for PCR identification of positive transformants, named E.coli BL21 / pET28a-6P. Incubate a single colony in 5 mL of LB culture medium containing 50 μg / mL kanamycin sulfate at 37°C and 250 r / min on a shaker for 12 h as the seed culture.

[0038] (4)Expression and identification of target protein: Pick a single colony of E.coli BL21 / pET28a-6P and inoculate it into 10 ml of LB culture medium containing 50 μg / mL kanamycin, and culture it at 37°C with shaking until the OD600nm reaches 0.6 - 0.8. Add 100 mM / L of α-lactose to a final concentration of 0.4 mM / L, and lower the culture temperature to 30°C and culture for another 6 h. After taking out, centrifuge at 12000 r / min for 10 min to collect the bacteria. Add PBS with a volume 10 times the weight of the bacteria to suspend the bacteria and break them by ultrasonic wave. Centrifuge at 4°C and 12000 r / min for 15 min, and treat the supernatant and precipitate with SDS-PAGE loading buffer respectively, and perform SDS-PAGE and Western blot analysis.

[0039] (5)Purification of the recombinant protein Before loading the sample, equilibrate the nickel column with 10 column volumes of Binding Buffer at a flow rate of 5 mL / min. After centrifugation and filtration, load the protein solution onto the equilibrated Ni column through a constant flow pump at a flow rate of 1 mL / min, and collect the flow-through. The sample can be loaded repeatedly to improve the binding efficiency of the sample to the column. Re-equilibrate the Ni column with the bound target protein 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 the eluate for SDS-PAGE electrophoresis detection. The size of the protein band is approximately 14.41 kD. The electrophoresis results are as Figure 1 。

[0040] Example 3: Preparation of tandem multi-epitope vaccine

[0041] (1) Transfer 0.1 ml of the seed solution to 10 ml of LB culture medium containing 50 μg / mL kanamycin sulfate at a ratio of 1% by volume, and culture it in a shaker at 37 °C and 250 r / min for 12 h as the primary seed solution.

[0042] (2) Transfer 10 ml of the primary seed solution to 1000 ml of LB culture medium containing 50 μg / mL kanamycin sulfate at a ratio of 1% by volume, and culture it in a shaker at 37 °C and 250 r / min for 12 h as the secondary seed solution.

[0043] (3) Calibrate the pH electrode and dissolved oxygen electrode of the fermenter, and calibrate the flow rate of the peristaltic pump.

[0044] (4) Prepare 70 L of LB medium, add it to a 100 L fermenter, and autoclave the medium, fermenter and pipeline at 121 °C for 30 min.

[0045] (5) When the culture solution in the fermenter cools to 37 °C, add kanamycin sulfate with a final concentration of 50 μg / mL, add 700 ml of the secondary seed solution to the fermenter, and set the parameters of the fermenter as follows: stirring speed 500 - 800 r / min, tank pressure 9 psi, temperature 37 °C, and set the DO value (dissolved oxygen) above 20%. Ferment and culture for 3 h until the OD600nm reaches 0.6 - 0.8.

[0046] (6) Add α-lactose at a final concentration of 0.4 mM / L to 100 mM / L, and set the temperature to 30 °C and then culture for another 5 h to end the fermentation.

[0047] (7) The fermentation broth was centrifuged continuously at 12,000 r / min to collect the thalli. PBS with a volume 10 times that of the thalli weight was added to suspend the thalli, followed by ultrasonic disruption. Then, it was centrifuged continuously at 12,000 r / min. The supernatant was filtered through a 0.22-μm filter and loaded onto the equilibrated Ni column at a flow rate of 1 mL / min using a constant flow pump. The Ni column bound with the target protein was re-equilibrated with 10 column volumes of Binding Buffer at a flow rate of 5 mL / 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 5 mL / 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°C for later use.

[0048] (8) A measured amount of the purified protein solution was added to 10% formaldehyde solution and mixed well. The final concentration of the formaldehyde solution was 0.1%, and it was inactivated at 2 - 8°C for 72 hours. It was stored at 2 - 8°C for later use.

[0049] (9) Vaccine emulsification: The oil phase was prepared by mixing mineral oil, Span - 80, and aluminum stearate in a ratio of 95:5:1 and heating to 115°C and maintaining for 40 minutes. After cooling, it was the oil phase. 95 parts of the inactivated antigen solution and 5 parts of sterile Tween - 80 were mixed well to completely dissolve Tween - 80, which was the aqueous phase. 2 parts of the oil phase were put into the emulsification tank, and the shear machine was set at 2000 rpm / min for slow shear stirring. While slowly adding 1 part of the aqueous phase, the shear machine was set at 14,000 rpm / min for emulsification for 5 - 10 minutes to prepare the finished vaccine.

[0050] Example 4: Character, safety, and potency tests of the tandem multi - epitope vaccine

[0051] (1) Character inspection

[0052] Appearance: Carefully observe the color of the vaccine and the presence of impurities.

[0053] Dosage form: Use a 1 - ml pipette to suck a small amount of the vaccine. First, drop 1 drop of the vaccine on the water surface 2 - 3 cm above the water surface, and then drop 2 - 3 more drops of the vaccine. Pay attention to observing the diffusion of the vaccine on the water surface and record the diffusion of the liquid drops. The first drop and each subsequent drop should be recorded separately.

[0054] Stability: Pipette 10 ml of the vaccine into a centrifuge tube and centrifuge at 3000 r / min for 15 minutes. Observe whether there is aqueous phase precipitation at the bottom of the tube.

[0055] Viscosity: It was detected using a rotational viscometer at room temperature (20°C ± 0.1°C).

[0056] (2)For safety inspection, 10 21-day-old SPF chickens were used. Each chicken was injected subcutaneously at multiple points in the neck with 1.0 ml of the vaccine. Observation was carried out for 14 days, and the clinical responses such as food intake and water drinking of the test chickens were recorded. Then, autopsy was performed to observe the absorption of the vaccine at the injection site.

[0057] (3)For potency inspection, 20 21-day-old SPF chickens were used. 10 chickens were each injected subcutaneously in the neck with 0.2 ml of the vaccine, and the other 10 chickens were used as controls. 21 days after inoculation, blood was collected from each chicken, serum was separated, and the neutralizing antibody titer was determined by virus neutralization test. At the same time, all immunized chickens and control chickens were intramuscularly injected with the virus solution of Group I, Type D11 avian adenovirus strain AdV11, 0.1 ml per chicken (containing 1×10 5.5 TCID 50 ). Observation was carried out for 14 days after challenge, and the morbidity of immunized chickens and control chickens was recorded. On the 3rd day after challenge, anal swabs were collected from all test chickens, virus DNA was extracted, and the excretion of Group I, Type D11 avian adenovirus was detected by fluorescence quantitative PCR. On the 14th day after challenge, all test chickens were autopsied to observe the liver lesions.

[0058] As shown in Table 1, the properties of the prepared tandem multi-epitope vaccine conform to those of conventional oil-emulsion inactivated vaccines, and it has good stability.

[0059] Table 1 Test results of the properties of the tandem multi-epitope vaccine

[0060]

[0061] As shown in Table 2, for all test chickens with super-dose immunization of the prepared tandem multi-epitope vaccine, there were no abnormalities in spirit, food intake, and water drinking, and there was no swelling reaction at the injection site. It can be seen that the tandem multi-epitope vaccine has good safety.

[0062] Table 2 Test results of the safety of the tandem multi-epitope vaccine

[0063]

[0064] As shown in Table 3 and Table 4, 21 days after immunization with the tandem multi-epitope vaccine, the neutralizing antibody titers of immunized chickens were all ≥1:64, and the neutralizing antibodies of chickens in the control group were all negative; the challenge protection results showed that the challenge protection rate of the vaccine immunization group was 10 / 10, and the excretion was negative for 10 / 10 on the 3rd day after challenge. It can be seen that the tandem multi-epitope vaccine has good immunization effect, can enable immunized chickens to produce neutralizing antibodies, achieve challenge protection, and block the excretion of challenged chickens.

[0065] Table 3 Test results of the potency of the tandem multi-epitope vaccine

[0066]

[0067] Table 4 Statistical results of the potency test of the tandem multi-epitope vaccine

[0068]

Claims

1. A tandem multi-epitope vaccine against fowl adenovirus type D11, characterized in that, The vaccine is an oil emulsion vaccine prepared by emulsifying a tandem multi-epitope antigen protein of fowl adenovirus type D11 expressed by recombinant Escherichia coli with a mineral oil adjuvant. The amino acid sequence of the tandem multi-epitope antigen protein of fowl adenovirus type D11 is SEQ ID NO.

1.

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 fowl adenovirus type D11 was transferred to 10 ml of LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured in a shaker at 37 °C and 250 r / min for 12 h as the primary seed solution; (2) According to the fermentation volume, the primary seed solution was inoculated into LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured in a shaker at 37 °C and 250 r / min for 12 h to prepare the secondary seed solution; (3) The secondary seed solution was inoculated into 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 reached 0.6 - 0.8; (4) 100 mM / L of α-lactose was added to a final concentration of 0.4 mM / L, and the culture temperature was reduced to 30 °C and cultured for another 5 h to end the fermentation; (5) The fermented broth was continuously centrifuged at 12,000 r / min to collect the bacteria. The bacteria were suspended in PBS at 10 times the volume of the bacteria weight, sonicated, centrifuged continuously at 12,000 r / min, and the supernatant was filtered through a 0.22 μm filter, and the antigen protein was purified by Ni column; (6) The purified antigen protein solution was measured and added with 10% formaldehyde solution, and mixed well. The final concentration of the formaldehyde solution was 0.1%, and inactivated at 2 - 8 °C for 72 h to prepare the antigen solution; (7) Mineral oil, Span-80, and aluminum stearate were mixed evenly at a ratio of 95:5:1 to prepare the oil phase. When the temperature reached 115 °C, it was maintained for 40 minutes, and after cooling, it was the oil phase; 95 parts of the inactivated antigen solution and 5 parts of sterile Tween-80 were mixed evenly to completely dissolve Tween-80, which was the water phase; 2 parts of the oil phase were put into an emulsifying tank, and the shear machine was at 2000 rpm / min for slow shear stirring. At the same time, 1 part of the water phase was slowly added, and then the shear machine was at 14,000 rpm / min for emulsification for 5 - 10 min to prepare the finished vaccine.

3. A tandem multi-epitope antigen of fowl adenovirus type D11, characterized in that, The multi-epitope antigen is a tandem multi-epitope antigen protein of fowl adenovirus type D11 expressed by recombinant Escherichia coli. The amino acid sequence of the tandem multi-epitope antigen protein is SEQID NO.

1.

4. The tandem multi-epitope antigen according to claim 3, wherein The nucleotide sequence of the tandem multi-epitope antigen protein is SEQ ID NO.

2.

5. The tandem multi-epitope antigen according to claim 3, wherein The preparation method of the tandem multi-epitope antigen protein comprises the following steps: (1) The recombinant Escherichia coli expressing the tandem multi-epitope antigen protein of fowl adenovirus type D11 was transferred to 10 ml of LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured in a shaker at 37 °C and 250 r / min for 12 h as the primary seed solution; (2)According to the fermentation volume, inoculate the primary seed liquid into the LB culture medium containing 50 μg / mL kanamycin sulfate at a ratio of 1% by volume, and culture it in a shaker at 37 °C and 250 r / min for 12 h to prepare the secondary seed liquid; (3)Inoculate the secondary seed liquid into the LB culture medium containing 50 μg / mL kanamycin sulfate at a ratio of 1% by volume in a fermenter, and ferment and culture for 3 h until the OD600nm reaches 0.6 - 0.8; (4)Add 100 mM / L of 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 end the fermentation; (5)Centrifuge the fermentation broth continuously at 12000 r / min to collect the bacteria, add PBS with a volume 10 times that of the bacteria weight for bacterial suspension, break the bacteria by ultrasonic wave, centrifuge continuously at 12000 r / min, filter the supernatant through a 0.22 μm filter, and purify the antigen protein through a Ni column.

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