Preparation method and immune efficacy evaluation method of foot-and-mouth disease subunit recombinant protein vaccine

Abstract

The invention discloses a method for preparing a foot-and-mouth disease subunit recombinant protein vaccine and evaluating immune efficacy of the foot-and-mouth disease subunit recombinant protein vaccine. By optimizing the prokaryotic expression process of the O-type FMDV VP1 protein, the obtained recombinant VP1 protein is mainly expressed in the form of an inclusion body; an adjuvant compatibility scheme adaptive to the recombinant protein is further designed, so that the immunogenicity of the vaccine is enhanced; meanwhile, a mouse-based immune efficacy evaluation method is established, and low-cost and short-period vaccine efficacy evaluation is realized by detecting the immunogenicity of the VP1 protein. The method effectively solves the problems that the prokaryotic expression VP1 subunit vaccine is insufficient in immune efficacy and high in evaluation cost, and provides efficient and practical technical support for research, development and industrialization of foot-and-mouth disease subunit recombinant protein vaccines.

Description

TECHNICAL FIELD

[0001] The application relates to a preparation method of a foot-and-mouth disease subunit recombinant protein vaccine and an immune efficacy evaluation method thereof, and belongs to the technical field of life science. BACKGROUND

[0002] Foot-and-mouth disease virus (FMDV) is a highly contagious animal pathogen that mainly infects bovine and porcine animals, and causes significant economic losses to the global livestock industry. At present, vaccination is the core means for preventing and controlling the spread of foot-and-mouth disease, among which the traditional inactivated vaccine is most widely used in the clinic due to its mature technology, rapid immune response and other characteristics. However, this type of vaccine needs to be prepared by culturing a large amount of live virus, which has the risk of biological safety leakage, and the immune duration is short, so it needs to be frequently boosted, and it may also cause local inflammatory reactions in the animal body, which is difficult to meet the long-term demand of modern livestock industry for high-efficiency and safe vaccines.

[0003] With the development of genetic engineering technology, foot-and-mouth disease subunit recombinant protein vaccines have become an important direction in the field of vaccine research and development due to the advantages of not needing to culture live viruses, high biological safety, and precise targeting of viral antigen sites. O-type foot-and-mouth disease virus is the most common serotype, and the VP1 protein as the main antigen site plays a key role in virus recognition and immune response. The immunogenicity of the VP1 recombinant protein alone is weak, and it needs to work with adjuvants to activate effective immune responses in the body. However, there is a lack of optimization research on the adjuvant compatibility of prokaryotic expression VP1 protein in the prior art, resulting in insufficient vaccine protection efficacy.

[0004] Immune efficacy evaluation is a key link in the development of VP1 subunit vaccines, and the current evaluation system for prokaryotic expression VP1 recombinant protein vaccines has significant defects, such as insufficient standardization of evaluation models. Existing researches mostly use target animals such as pigs and cattle for efficacy evaluation, which is costly and time-consuming. Mouse models are more suitable for early-stage research and development due to their convenient operation and good reproducibility. The evaluation of the synergistic effect of adjuvants and antigens is lacking. The existing evaluation does not design targeted detection schemes for the characteristics of prokaryotic expression recombinant VP1 protein combined with adjuvants, making it difficult to accurately determine the improvement of adjuvants on the immune efficacy of VP1 protein. These problems seriously hinder the development process and industrialization of prokaryotic expression VP1 subunit vaccines. Therefore, developing an efficient prokaryotic expression process of VP1 protein, optimizing the adjuvant compatibility scheme, and establishing a scientific mouse immune efficacy evaluation method have become key problems to be solved in the field of foot-and-mouth disease subunit vaccines. SUMMARY

[0005] In view of the existing technical deficiencies, the purpose of the present application is to construct a preparation method of a foot-and-mouth disease subunit recombinant protein vaccine and an immune efficacy evaluation method thereof, to help effectively prevent and control foot-and-mouth disease.

[0006] To achieve the above-mentioned purpose, the technical scheme of the present application is:

[0007] A preparation method and immune efficacy evaluation method of a foot-and-mouth disease subunit recombinant protein vaccine, comprising the following steps:

[0008] (1) Design and synthesis of primers

[0009] According to the O-type foot-and-mouth disease virus VP1 gene sequence (GenBank accession number: reference published sequence), a pCold-I-VP1 plasmid containing the VP1 gene was synthesized by Shanghai Shengong Bioengineering Co., Ltd. According to the FMDV genome sequence, the primers of VP1 and PCold-I genes were designed using Primer Premier 5 software. The upstream primer of the VP1 gene was inserted into the ECORV restriction endonuclease, and the downstream primer was inserted into the SAC I restriction endonuclease. The upstream primer of the PCold-I gene was inserted into the SAC I restriction endonuclease, and the downstream primer was inserted into the ECOR V restriction endonuclease, and was synthesized by Nanjing Qianke Biological Technology Co., Ltd. Among them, the primer pCold-I-VP1-F / R is used to amplify the 633bp VP1 gene fragment, and the primer PCold-I-F / R is used to amplify the 4407bp empty PCold-I gene fragment.

[0010] (2) PCR amplification of VP1 and PCold-I gene fragments

[0011] Based on the two enzyme digestion sites of ECOR V and SAC I, the pCold-I-VP1 plasmid containing the VP1 gene was used as the template, and the primers pCold-I-VP1-F / R and PCold-I-F / R were used to amplify the VP1 and PCold-I gene fragments, respectively. The reaction system and reaction conditions are shown in Table 1.1-1.2. After the amplification was completed, 10 μL of the PCR amplification product was subjected to nucleic acid electrophoresis in a 1.5% agarose gel (containing EB), and the voltage was 100V. After electrophoresis for 30 min, the results were observed on a gel imaging instrument. Table 1.1 PCR amplification system Table 1.2 PCR reaction conditions

[0012] (3) Recovery of VP1 and PCold-I gene PCR products

[0013] The gel band of the target DNA fragment was observed on the ultraviolet gel imaging instrument and cut into a clean EP tube. The target DNA was extracted according to the AXYGEN DNA gel recovery and purification kit instructions, and the recovered product was stored at -20℃ for standby.

[0014] (4) The connection of the target gene and the expression vector

[0015] The PCR recovery product VP1 was connected with PCold-I by using homologous recombination enzyme, and the reaction was carried out at 37°C for 30 min. After the reaction, the connection product was transformed into DH5α competent cells. Table 1.3 Connection system of PCR product

[0016] (5) Transformation of DH 5α competent cells

[0017] 100 μL of DH 5α competent cells stored in a -80°C refrigerator were thawed in an ice bath for about 5 min, 10 μL of the connection product was added, and the mixture was gently mixed and placed in an ice bath for 30 min. Then, the mixture was heated in a 42°C water bath for 90 s, quickly transferred to an ice bath for 2-3 min, 800 μL of LB medium was added to the centrifugal tube, and the mixture was evenly mixed and then placed in a 37°C water bath for 1 h at a speed of 200 r / min. Then, the mixture was centrifuged at 8000 r / min for 1 min, 800 μL of supernatant was discarded, and the remaining 100 μL of culture solution was evenly mixed and then plated on an LB plate containing kanamycin. The plate was then incubated in a 37°C incubator for 12-16 h.

[0018] (6) Identification of positive transformants

[0019] A single colony of suspected E. coli was picked from the LB solid agar medium plate in step (5) and streaked on an LB agar medium plate containing 100 μg / mL ampicillin. Then, the inoculation loop was placed in a PCR tube to which 10 μL of ddH2O had been added (10-fold dilution of the template). The mixture was then boiled in boiling water for 5 min, centrifuged at 10,000 rpm for 5 min, and the supernatant was used as the template for PCR amplification. The amplification system is shown in Table 1.4, and the reaction conditions are the same as those in Table 1.2. After the amplification was completed, 10 μL of the PCR amplification product was subjected to nucleic acid electrophoresis on a 1.5% agarose gel (containing EB). The voltage was 100 V, and the electrophoresis was carried out for 30 min. Then, the results were observed on a gel imaging instrument. Table 1.4 PCR amplification system After the plate containing 100 μg / mL ampicillin was incubated in a 37°C incubator for 12-16 h, the bacteria were inoculated into 10 mL of LB medium containing 100 μg / mL ampicillin for subculture. After the culture was incubated at 37°C in a 180 rpm shaker for 6 h, a small amount of plasmid extraction kit was used to extract the plasmid, which was sent to Genescript Biotech Co., Ltd. for sequencing, and the plasmid was also saved.

[0020] (7) Transformation and identification of BL21 competent cells

[0021] The recombinant plasmid extracted in step (6) was transformed into BL21 competent cells according to step (5), and the positive transformants were identified.

[0022] (8) Inducing expression of recombinant protein and identification

[0023] The prokaryotic expression bacteria BL21-pCold-I-VP1 were inoculated into LB liquid medium containing 100 μg / mL ampicillin and cultured overnight at 37°C with 180 rpm shaking. Then, the bacteria were inoculated into LB liquid medium containing 100 μg / mL ampicillin at a 1 / 100 inoculation amount, and cultured at 37°C with 180 rpm shaking for 2 h. Then, IPTG was added to a final concentration of 0.5 mM, and expression was induced at 16°C with 180 rpm shaking for 18-24 h. The bacteria were collected, 500 uL of the bacteria were resuspended with 1 x Binding Buffer (4 M NaCl, 20 mM Tris-HCl, 5 mM imidazole) buffer, and the bacteria were lysed by ultrasonic cell disruptor for 5 min. The supernatant was collected by centrifugation at 12000 rpm for 10 min at 4°C, and the precipitate was resuspended with an equal volume of 1 x Binding Buffer. Then, 30 mL of the supernatant and the precipitate were mixed with 10 uL of sample buffer (SDS-PAGE loading buffer), respectively, and boiled for 5 min. The mixture was centrifuged at 7000 rpm for 5 min, and the supernatant was used as the sample for SDS-PAGE electrophoresis. The specific operation steps of SDS-PAGE electrophoresis are as follows: ① Preparation of 12% protein separation gel: prepare the gel plate according to the product manual of Bio-Rad, add the separation gel (the height between the thin plate and the gel surface is about 2 cm), slowly add deionized water on the gel surface until it is full, and place it in a 37°C constant temperature box for about 20 min, and the separation gel can be fully solidified; ② Discard the water on the surface of the separation gel, and dry it with a water-absorbing paper, add the newly prepared stacking gel, and install the comb (the comb and the stacking gel cannot have air bubbles directly), and place it in a 37°C constant temperature box for about 20 min, and the stacking gel can be fully solidified; ③ Assemble the electrophoresis tank according to the product manual of Bio-Rad, fill the inner electrophoresis tank with electrophoresis liquid, and fill the outer electrophoresis tank with 2-4 times the volume of the inner electrophoresis liquid; ④ Pull out the comb, and load the sample: take 10 mL of the treated sample and add it to the loading hole of the stacking gel; ⑤ Electrophoresis: electrophorese at a voltage of 90 V for 20 min, then change the voltage to 120 V and continue electrophoresis until the bromophenol blue reaches the bottom of the separation gel, and cut off the power to terminate electrophoresis; ⑥ Staining: Remove the gel from the gel plate, discard the stacking gel portion, and place the separating gel into a container containing Coomassie Brilliant Blue R-250 staining solution. Boil in a water bath for 5-10 minutes to stain. ⑦ Decolorization: Place the stained separating gel into a container filled with water and boil it in a water bath to decolorize until clear protein bands are visible and the background color is relatively light, about 20-30 minutes. ⑧ Development: Imaging and photographing in a gel electrophoresis imaging system. The specific steps for Western blotting are as follows: ① The electrophoresis process is the same as the SDS-PAGE process described above; ② Transfer: On the negative electrode (black side) of the transfer apparatus, place the sponge, 3 layers of filter paper, gel, PVDF membrane, 3 layers of filter paper, and sponge in sequence. Remove air bubbles after each layer to ensure complete adhesion between the filter paper and gel, gel and membrane, and membrane and filter paper. After placement, fill the container with 4... ℃ Pre-cooled transfer solution, 100V, 70min; ③ Blocking: After the transfer is completed, the PVDF membrane is taken out, washed in TBST for 5 minutes, and then transferred to blocking solution (5% skim milk powder or BSA) and blocked at room temperature for 1 hour. ④ Primary antibody incubation: Refer to the WB dilution ratio in the antibody instructions, dilute His-TagAntibody (primary antibody) with antibody diluent, remove the membrane from the blocking solution, absorb excess liquid with filter paper, place it in the antibody incubation box, pour in the diluted primary antibody, and incubate overnight. ⑤ Secondary antibody incubation: Dilute Goat Anti-Mouse IgG HRP (secondary antibody) with antibody diluent according to the WB dilution ratio in the antibody instructions. Wash the PVDF membrane incubated with primary antibody three times with TBST for 5 minutes each time. Remove the membrane from the washing solution, blot off excess liquid with filter paper, place it in the antibody incubation box, pour in the diluted secondary antibody, and incubate at room temperature for 1 hour. ⑥ Wash the membrane: Wash the membrane 3 times with TBST, 5 minutes each time; ⑦ Exposure: Take equal volumes of ECL luminescent solution A and solution B, mix well, and store in the dark for later use. Place the PVDF film in a dark box containing the developing solution and incubate for 5 minutes, gently shaking the dark box continuously during incubation. Then place it in the instrument for exposure and photography.

[0024] (9) Preparation and refolding of inclusion bodies

[0025] Under the condition of 4℃ low temperature, centrifuge at 4000 rpm for 10 min, discard the supernatant, resuspend the bacterial slurry with 1x Binding Buffer, add Tween 2.0 at the amount of 1%, mix well, then perform ultrasonic lysis for 5 min in ice bath, centrifuge at 12000 rpm for 5 min, discard the supernatant, and finally collect the precipitate; dissolve the precipitate with 8M urea, mix well by pipetting, place in 4℃ overnight to achieve complete dissolution, centrifuge at 12000 rpm for 5 min under the condition of 4℃ low temperature, and collect the supernatant; place the supernatant in a dialysis bag with a molecular weight of 3KDa, and then place the dialysis bag in a large beaker filled with 6M urea, 4℃, 2h; replace the 6M urea in the beaker with 4M urea, 4℃, 4h; gradually replace with 3M, 2M, 1.5M, and observe until precipitate is formed, then stop dialysis, collect the protein, and store at -80℃.

[0026] (10) BCA assay of protein concentration

[0027] The BCA protein quantitative determination kit was used to determine the protein concentration, and the specific method was as follows: the collected protein was taken out from the refrigerator and placed at room temperature for 1h; the protein standard was added to the 96-well plate according to 0, 1, 2, 4, 6, 8, 10 μL, and double-distilled water (sterilized) was diluted to 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1 mg / mL according to a ratio of 1:10; 10 μL of the purified NP sample was added to the well, and two parallel samples were set; A liquid and B liquid were prepared into working solution according to a ratio of 50:1, mixed well, 200 μL / well, and incubated at 37℃ for 30 min; the OD562nm value was read by using a microplate reader, a standard curve was drawn by using the protein standard, and the protein sample concentration was calculated.

[0028] (11) Preparation of vaccine

[0029] The purified VP1 protein was concentrated to 1 mg / mL by using an ultrafiltration tube, and the VP1 protein was mixed with Freund's complete adjuvant / Freund's incomplete adjuvant at a volume ratio of 1:1 according to the antigen and adjuvant formula ratio in Table 1.5, and the vaccine was prepared by shaking and emulsifying for 3 min. Table 1.5 Antigen and adjuvant ratio

[0030] (12) Immunization grouping and procedure

[0031] Ten 5-week-old female BALB / c mice were randomly divided into two groups: a blank control group and a 100 μg VP1 antigen group, with five mice in each group. Mice were immunized subcutaneously in the back according to the immunization grouping and dosage specified in Table 1.6, with immunizations every two weeks for a total of three immunizations. The first immunization used Freund's complete adjuvanted vaccine, and the two booster immunizations used Freund's incomplete adjuvanted vaccine. Blood was collected from the eyeballs one day before immunization, incubated at 4°C for 2 hours, and centrifuged at 3000 rpm at 4°C for 15 minutes to separate the serum. A portion was aliquoted and stored at -20°C for later use, while the remainder was stored at -80°C for long-term preservation. Table 1.6 Antigen and Adjuvant Ratios Attached Figure Description

[0032] Figure 1 SDS-PAGE image of recombinantly expressed VP1 protein.

[0033] Figure 2 This is a graph showing the detection of specific antibodies in the serum of VP1-immunized mice. Detailed Implementation

[0034] The specific embodiments of the present invention will be further described in detail below with reference to examples.

[0035] pCold-I-VP1-F:5 ′ -CCATGGCTGATATCACCACCTCTACCGGT-3′, pCold-I-VP1-R: 5′-GCAAGCTTGTCGACGGAGCTCGGCTGTTTAACC-3′, PCold-IF: 5′-CCGAGCTCCGTCGACAAGCTTGC-3′, PCold-IR: 5′-GATATCAGCCATGGCCTTGTCGTCGTC-3′.

[0036] (1) Primer design and synthesis

[0037] The pCold-I-VP1 plasmid containing VP1 gene was synthesized by Shanghai Shengong Bioengineering Co., Ltd. according to the sequence of O-type foot-and-mouth disease virus VP1 gene (GenBank accession number: reference published sequence). The primers of VP1 and PCold-I genes were designed by Primer Premier 5 software according to the sequence of FMDV genome. The upstream primer of VP1 gene was inserted into ECORV restriction endonuclease, and the downstream primer was inserted into SAC I restriction endonuclease. The upstream primer of PCold-I gene was inserted into SAC I restriction endonuclease, and the downstream primer was inserted into ECOR V restriction endonuclease, and was synthesized by Nanjing Qianke Biological Technology Co., Ltd. Among them, the primer pCold-I-VP1-F / R was used to amplify the 633bp VP1 gene fragment, and the primer PCold-I-F / R was used to amplify the 4407bp empty PCold-I gene fragment.

[0038] (2) PCR amplification of VP1 and PCold-I gene fragments

[0039] Based on the two enzyme cutting sites of ECOR V and SAC I, the pCold-I-VP1 plasmid containing VP1 gene was used as a template, and the primers pCold-I-VP1-F / R and PCold-I-F / R were used to amplify the VP1 and PCold-I gene fragments by PCR, respectively. The reaction system and reaction conditions are shown in Table 1.1-1.2. After the amplification was completed, 10 μL of PCR amplification product was taken for nucleic acid electrophoresis in 1.5% agarose gel (containing EB), the voltage was 100V, and the electrophoresis was performed for 30 min. The results were observed on the gel imaging instrument. Table 1.1 PCR amplification system Table 1.2 PCR reaction conditions

[0040] (3) Recovery of VP1 and PCold-I gene PCR products

[0041] The gel band of the target DNA fragment was observed on the ultraviolet gel imaging instrument and cut into a clean EP tube. The target DNA was extracted according to the AXYGEN DNA gel recovery and purification kit instructions, and the recovered product was stored at -20℃ for standby.

[0042] (4) Connection of target gene and expression vector

[0043] The PCR product VP1 is connected with PCold-I by homologous recombination enzyme, and the reaction is carried out at 37℃ for 30 min. After the reaction is completed, the connection product is transformed into DH5α competent cells. Table 1.3 PCR product connection system

[0044] (5) Transformation of DH 5α competent cells

[0045] Take 100 μL of DH 5α competent cells stored in a-80℃ refrigerator, melt in an ice bath, and mix gently. Place in an ice bath for 30 min, heat shock at 42℃ water bath for 90 s, and quickly transfer to an ice bath for 2-3 min. Add 800 μL of LB medium to the centrifuge tube, mix well, and incubate at 37℃, 200 r / min for 1 h. Centrifuge at 8000 r / min for 1 min, discard 800 μL of supernatant, and mix the remaining 100 μL of culture solution evenly on a LB plate containing kanamycin, and incubate at 37℃ for 12-16 h.

[0046] (6) Identification of positive transformants

[0047] In step (5), the LB solid agar medium plate is inoculated with a sterile inoculation ring to pick up a single colony of suspected E. coli on a LB agar medium plate containing 100 μg / mL ampicillin. Then, the inoculation ring is placed in a PCR tube pre-added with 10 μL of ddH2O (10-fold dilution of the template), and then boiled in boiling water for 5 min. Centrifuge at 10000 rpm for 5 min, and use the supernatant as the template for PCR amplification. The amplification system is shown in Table 1.4, and the reaction conditions are the same as those in Table 1.2. After amplification, 10 μL of PCR amplification product is subjected to nucleic acid electrophoresis on a 1.5% agarose gel (containing EB), at a voltage of 100 V for 30 min. The results are observed on a gel imaging instrument. Table 1.4 PCR amplification system After incubating the streaked plate containing 100 μg / mL ampicillin at 37℃ for 12-16 h, the bacteria are inoculated into 10 mL of LB medium containing 100 μg / mL ampicillin for enrichment culture. After incubation at 37℃, 180 rpm on a shaker for 6 h, a small amount of plasmid extraction kit is used to extract the plasmid, which is sent to Genescript Biotech Co., Ltd. for sequencing, and the plasmid is also saved.

[0048] (7) Transformation and identification of BL21 competent cells

[0049] The recombinant plasmid extracted in step (6) was transformed into BL21 competent cells according to step (5), and positive transformants were identified.

[0050] (8) Inducing expression of recombinant protein and identification

[0051] The prokaryotic expression bacteria BL21-pCold-I-VP1 were inoculated into LB liquid medium containing 100 μg / mL ampicillin and cultured overnight at 37°C with 180 rpm shaking. Then, the bacteria were inoculated into LB liquid medium containing 100 μg / mL ampicillin at a ratio of 1 / 100, and cultured at 37°C with 180 rpm shaking for 2 h. Then, IPTG was added to a final concentration of 0.5 mM, and expression was induced at 16°C with 180 rpm shaking for 18-24 h. The bacteria were collected, 500 uL of which was resuspended with 1 x Binding Buffer (4 M NaCl, 20 mM Tris-HCl, 5 mM imidazole) buffer, and the bacteria were lysed by ultrasonic wave for 5 min. The supernatant was collected by centrifugation at 12000 rpm for 10 min at 4°C, and the precipitate was resuspended with an equal volume of 1 x Binding Buffer. Then, 30 mL of the supernatant and the precipitate were mixed with 10 uL of sample buffer (SDS-PAGE loading buffer), respectively, and boiled for 5 min. The mixture was centrifuged at 7000 rpm for 5 min, and the supernatant was used as the sample for SDS-PAGE electrophoresis. The specific operation steps of SDS-PAGE electrophoresis are as follows: ①Preparation of 12% protein separation gel: according to the product manual of Bio-Rad, the gel plate was prepared, the separation gel was added (the height between the thin plate and the gel surface was about 2 cm), and deionized water was slowly added until the gel surface was filled. The separation gel was placed in a 37°C incubator for 20 min or so until it was fully solidified. ②The water on the surface of the separation gel was discarded, and the newly prepared stacking gel was added. The comb was installed (there should be no air bubbles between the comb and the stacking gel), and the stacking gel was placed in a 37°C incubator for 20 min or so until it was fully solidified. ③According to the product manual of Bio-Rad, the electrophoresis tank was prepared, and the inner electrophoresis tank was filled with electrophoresis solution, and the outer electrophoresis tank was filled with 2-4 times the volume of the inner electrophoresis solution. ④Remove the comb and load the sample: 10 mL of the treated sample was added to the loading hole of the stacking gel. ⑤Electrophoresis: electrophoresis was performed at a voltage of 90 V for 20 min, and then the voltage was changed to 120 V for continuous electrophoresis until the bromophenol blue reached the bottom of the separation gel. The power was turned off, and the electrophoresis was terminated. ⑥Staining: the gel was taken out of the gel plate, the stacking gel was discarded, and the separation gel was placed in a container containing Coomassie Brilliant Blue R-250 staining solution. The container was placed in a water bath and boiled for 5-10 min. ⑦Decolorization: Put the separated gel into a container containing water, and boil in water bath until clear protein bands and light background color are observed, about 20-30 min; ⑧Development: Develop and take a photo in the gel electrophoresis imaging instrument. The specific operation steps of Western Blot are as follows: ①Electrophoresis process is the same as the above SDS-PAGE process; ②Membrane transfer: Place sponges, 3 layers of filter paper, gels, PVDF membranes, 3 layers of filter paper, and sponges in the negative electrode (black side) of the membrane transfer instrument in turn, and make sure that the filter paper is completely attached to the gel, the gel is completely attached to the membrane, and the membrane is completely attached to the filter paper. After placing, add 4℃ pre-cooled membrane transfer solution, 100V, 70min; ③Blocking: After membrane transfer is completed, take out the PVDF membrane, wash it in TBST for 5min, and then transfer it to the blocking solution (5% skimmed milk powder or BSA) for 1h at room temperature; ④Primary antibody incubation: According to the WB dilution ratio of the antibody instruction, dilute His-Tag Antibody (primary antibody) with antibody diluent, take out the membrane from the blocking solution, absorb the excess liquid with filter paper, place it in the antibody incubation box, pour in the diluted primary antibody, and incubate overnight; ⑤Secondary antibody incubation: According to the WB dilution ratio of the antibody instruction, dilute Goat Anti-Mouse IgG HRP (secondary antibody) with antibody diluent. Wash the primary antibody incubated PVDF membrane with TBST for 3 times, 5min each time, take out the membrane from the washing solution, absorb the excess liquid with filter paper, place it in the antibody incubation box, pour in the diluted secondary antibody, and incubate at room temperature for 1h; ⑥Washing membrane: Wash the membrane with TBST for 3 times, 5min each time; ⑦Exposure: Take equal volume of ECL luminescent liquid A and B, mix them, and store them in the dark for standby. Place the PVDF membrane in the developing liquid containing dark box and incubate for 5min. During the incubation, shake the dark box gently. Then, place it in the instrument for exposure and photography. The SDS-PAGE analysis result of recombinant VP1 protein expression is shown in Figure 1 M: 10-250kDa; 1: pCold-I empty load without induction of whole bacteria; 2: pCold-I empty load induction of whole bacteria; 3: pCold-I-VP1 without induction of whole bacteria; 4: pCold-I-VP1 induction of whole bacteria; 5: pCold-I-VP1 induction supernatant; 6: pCold-I-VP1 induction precipitate.

[0052] (9) Preparation and renaturation of inclusion bodies

[0053] Under the condition of 4℃ low temperature, centrifuge at 4000 rpm for 10 min, discard the supernatant, resuspend the bacterial slurry with 1x Binding Buffer, add Tween 2.0 at a rate of 1%, mix well, then perform ultrasonic lysis for 5 min in ice bath, centrifuge at 12000 rpm for 5 min, discard the supernatant, and finally collect the precipitate; dissolve the precipitate with 8M urea, mix well by pipetting, and place it at 4℃ overnight to ensure complete dissolution; centrifuge at 12000 rpm for 5 min under the condition of 4℃ low temperature, and collect the supernatant; place the supernatant in a 3KDa dialysis bag, and then place the dialysis bag in a large beaker filled with 6M urea, 4℃, 2h; replace the 6M urea in the beaker with 4M urea, 4℃, 4h; gradually replace it with 3M, 2M, 1.5M, and observe until precipitate is formed, then stop dialysis, collect the protein, and store it at -80℃.

[0054] (10) BCA assay of protein concentration

[0055] The BCA protein quantitative determination kit was used to determine the protein concentration, and the specific method was as follows: the collected protein was taken out of the refrigerator and placed at room temperature for 1h; protein standard was added to the 96-well plate at 0, 1, 2, 4, 6, 8, 10μL, and double-distilled water (sterilized) was added to dilute the various concentrations to 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1mg / mL at a ratio of 1:10; 10μL of purified NP sample was added to the well, with 2 replicates; A and B liquids were prepared into working solution at a ratio of 50:1, mixed well, 200μL / well, and incubated at 37℃ for 30min; the OD562nm value was read using a microplate reader, a standard curve was drawn by the protein standard, and the protein sample concentration was calculated.

[0056] (11) Vaccine preparation

[0057] The purified VP1 protein was concentrated to 1mg / mL using an ultrafiltration tube, and the VP1 protein was mixed with Freund's complete adjuvant / Freund's incomplete adjuvant at a volume ratio of 1:1 according to the antigen and adjuvant formulation ratio in Table 1.5, and the vaccine was prepared by shaking and emulsifying for 3min. Table 1.5 Antigen and adjuvant ratio

[0058] (12) Immunization grouping and procedure

[0059] 10 five-week-old female BALB / c mice were randomly divided into two groups, 5 mice in each group, namely the blank control group and the 100 μg VP1 antigen group. According to Table 1.6, the mice were injected subcutaneously on the back with the grouping and immunization dose, and immunized once every two weeks for a total of three times. The first immunization used Freund's complete adjuvant vaccine, and the two booster immunizations used Freund's incomplete adjuvant vaccine. One day before immunization, the mice were subjected to eyeball blood collection, and the serum was separated by centrifugation at 3000 r / s, 4°C for 15 min. A portion of the serum was stored at -20°C for later use, and the remaining serum was stored at -80°C. Table 1.6 Antigen and adjuvant ratio

[0060] (13) Chessboard method to optimize indirect ELISA detection method

[0061] The conditions of the chessboard method to optimize the indirect ELISA detection method of VP1 protein antibody were optimized, including the coating antigen concentration (0.5, 1, 2 μg / mL) and the dilution factor of the secondary antibody (1:30000, 1:50000, 1:80000). The OD450nm value was read by an enzyme label instrument, and the P / N (test value / negative value) was calculated. When P / N≥2.1, it was positive, and when P / N was the maximum value, it was the optimal condition of the method. The operation steps of indirect ELISA are as follows: dilute VP1 recombinant protein with protein coating solution, add 100 μL to each well of an ELISA high-binding force enzyme label plate, and coat in a 37°C constant temperature incubator for 2 h. Discard the coating solution, wash with PBST 5 times, 1 min each time, 200 μL per well. After the last discarding, pat dry the residual liquid in the well, add 200 μL of blocking solution to each well, and block at 4°C for 1 h. Discard the blocking solution, wash with PBST 5 times, 1 min each time, 200 μL per well. Dilute the test serum and negative mouse serum with the universal diluent, pat dry the residual liquid in the well after the last discarding, and then add 50 μL / well to the ELISA enzyme label plate, and incubate at 37°C in a constant temperature incubator for 25 min. Discard the primary antibody, wash with PBST 5 times, 1 min each time, 200 μL per well. Use HRP-labeled goat anti-mouse IgG as the secondary antibody, dilute with the universal diluent, pat dry the residual liquid in the well after the last discarding, add 50 μL / well to the ELISA enzyme label plate, and incubate at 37°C in a constant temperature incubator for 25 min. Discard the secondary antibody, wash with PBST 5 times, 1 min each time, 200 μL per well. After washing, add 500 μL of TMB developing solution to each well, and develop in the dark. React at room temperature for 3-5 min. Add 50 μL of stop solution to each well, and read the OD 450 nm value by an enzyme label instrument.

[0062] (14) Detection of the immunogenicity of VP1 protein

[0063] The VP1 positive sera were tested by the established optimal ELISA method (13) with the normal sera group as negative control. The test group A 450 / negative control group A 450 ≥ 2.1 was considered as positive result. The highest dilution fold with positive reaction was regarded as the titer of the test group. The results of VP1 immune mouse serum specific antibody detection were shown in Table 2. Figure 2 Table 2. VP1 immune mouse serum specific antibody detection results

Claims

1. This invention discloses a method for preparing a foot-and-mouth disease (FMD) subunit recombinant protein vaccine and evaluating its immunogenicity. By optimizing the prokaryotic expression process of type O FMDV VP1 protein, the recombinant VP1 protein is mainly expressed in the form of inclusion bodies. Furthermore, an adjuvant compatibility scheme adapted to this recombinant protein is designed to enhance vaccine immunogenicity. Simultaneously, a mouse-based immunogenicity evaluation method is established. By detecting the immunogenicity of the VP1 protein, a low-cost, short-cycle vaccine efficacy assessment is achieved. This invention effectively solves the problems of insufficient immunogenicity and high evaluation cost of prokaryotic VP1 subunit vaccines, providing efficient and practical technical support for the research and industrialization of FMD subunit recombinant protein vaccines.

2. The method for preparing a foot-and-mouth disease subunit recombinant protein vaccine and evaluating its immunogenicity according to claim 1, characterized in that... The primers described in the detection method have the following sequences: pCold-I-VP1-F: 5′-CCATGGCTGATATCACCACCTCTACCGGT-3′, pCold-I-VP1-R: 5′-GCAAGCTTGTCGACGGAGCTCGGCTGTTTAACC-3′, PCold-IF: 5′-CCGAGCTCCGTCGACAAGCTTGC-3′, PCold-IR: 5′-GATATCAGCCATGGCCTTTGTCGTCGTC-3′.

3. The method for preparing a foot-and-mouth disease subunit recombinant protein vaccine and evaluating its immunogenicity according to claim 1, wherein the primers are designed and synthesized as follows: Based on the O-type foot-and-mouth disease virus VP1 gene sequence (GenBank accession number: Reference Publication Sequence), Shanghai Sangon Biotech Co., Ltd. synthesized the pCold-I-VP1 plasmid containing the VP1 gene. Primers for the VP1 and PCold-I genes were designed using Primer Premier 5 software based on the FMDV genome sequence. An ECOR V restriction endonuclease was inserted into the upstream primer of the VP1 gene, and a SAC I restriction endonuclease was inserted into the downstream primer. Similarly, an SACI restriction endonuclease was inserted into the upstream primer of the PCold-I gene, and an ECOR V restriction endonuclease was inserted into the downstream primer. These primers were synthesized by Nanjing Qingke Biotechnology Co., Ltd. The primer pCold-I-VP1-F / R was used to amplify the 633bp VP1 gene fragment, and the primer PCold-IF / R was used to amplify the 4407bp empty PCold-I gene fragment.

4. The preparation method of the foot-and-mouth disease subunit recombinant protein vaccine and its immunogenicity evaluation method according to claim 1, wherein the PCR amplification steps of VP1 and PCold-I gene fragments are as follows: Based on the two restriction sites ECOR V and SAC I, using the pCold-I-VP1 plasmid containing the VP1 gene as a template, PCR amplification of VP1 and PCold-I gene fragments is performed using primers pCold-I-VP1-F / R and PCold-IF / R, respectively. The reaction system and reaction conditions are shown in Tables 1.1-1.

2. After amplification, 10 μL of PCR amplification product is taken and subjected to nucleic acid electrophoresis on a 1.5% agarose gel (containing EB) at a voltage of 100V for 30 min. The results are then observed on a gel imaging instrument. The gel band of the target DNA fragment is observed and cut off on a UV gel imaging instrument and placed in a clean EP tube. The target DNA is extracted according to the instructions of the AXYGEN DNA gel recovery and purification kit. The recovered product is stored at -20℃ for later use. Table 1.1 PCR amplification system Table 1.2 PCR reaction conditions 5. The method for preparing a foot-and-mouth disease subunit recombinant protein vaccine and evaluating its immunogenicity according to claim 1, wherein the ligation step of the target gene and the expression vector is as follows: The PCR product VP1 recovered by homologous recombinase is ligated to PCold-I, and the reaction is carried out at 37°C for 30 min. After the reaction, this ligation product is transformed into DH5α competent peptide cells (ligation system is shown in Table 1.3). 100 μL of DH5α competent cells stored at -80°C are thawed in an ice bath for about 5 min, 10 μL of ligation product is added, and the mixture is gently mixed. The mixture is placed in an ice bath for 30 min, then heat-shocked in a 42°C water bath for 90 s. The cells are then quickly transferred to an ice bath for 2-3 min, and 800 μL of the ligation product is added to a centrifuge tube. After mixing thoroughly with LB medium, incubate at 37°C with shaking at 200 rpm for 1 h, centrifuge at 8000 rpm for 1 min, discard 800 μL of supernatant, mix the remaining 100 μL of culture medium thoroughly and spread it onto LB plates containing kanamycin, and incubate at 37°C for 12-16 h. Table 1.3 PCR product ligation system 6. The method for preparing a foot-and-mouth disease subunit recombinant protein vaccine and evaluating its immunogenicity according to claim 1, wherein the identification of positive transformants is as follows: On the LB solid agar plate in step 7 of claim, a single colony suspected to be *Escherichia coli* is picked up with a sterile inoculating loop and streaked onto a plate containing 100 μg / mL ampicillin on an LB agar plate. Then, the inoculating loop is placed in a PCR tube pre-filled with 10 μL ddH2O (to dilute the template 10-fold), and then boiled in boiling water for 5 min, centrifuged at 10000 rpm for 5 min, and the supernatant is used as the template for PCR amplification. The amplification system is shown in Table 1.4, and the reaction conditions are the same as in Table 1.

2. After amplification, 10 μL of the supernatant is taken... PCR amplification products were subjected to nucleic acid electrophoresis on a 1.5% agarose gel (containing EB) at 100V for 30 min. The results were then observed on a gel imaging system. Petri dishes containing 100 μg / mL ampicillin were incubated at 37℃ for 12-16 h. Bacteria were then picked and inoculated into 10 mL of LB medium containing 100 μg / mL ampicillin for enrichment culture. The culture was carried out at 37℃ and 180 rpm for 6 h. Plasmids were extracted using a small-scale plasmid extraction kit and sent to Qingke Biotechnology Co., Ltd. for sequencing. The plasmids were also preserved at the same time. Table 1.4 PCR amplification system 7. The method for preparing a foot-and-mouth disease subunit recombinant protein vaccine according to claim 1 and evaluating its immunogenicity, wherein the transformation and identification steps of BL21 competent cells are as follows: the recombinant plasmid extracted in step 8 of claim 1 is transformed into BL21 competent cells according to step 7 of claim 1, and positive transformants are identified; the prokaryotic expression bacterium BL21-pCold-I-VP1 is inoculated into LB liquid medium containing 100 μg / mL ampicillin and cultured overnight at 37°C with shaking at 180 rpm; then, it is inoculated into LB liquid medium containing 100 μg / mL ampicillin at a 1 / 100 inoculation rate and cultured at 37°C with shaking at 180 rpm for 2 h; then, IPTG is added to a final concentration of 0.5 mM, and expression is induced at 16°C. After culturing at 180 rpm for 18-24 h, the bacterial cells are collected, and 500 μL of bacterial cells are treated with 1x Binding Buffer (4M NaCl, 20 mM... Resuspend the cells in Tris-HCl (5mM imidazole) buffer, sonicate for 5 min, centrifuge at 12000 rpm for 10 min at 4℃, collect the supernatant, and resuspend the precipitate in an equal volume of 1x Binding Bufer buffer. Take 30 mL of supernatant and precipitate respectively, add 10 μL of sample buffer (SDS-PAGE loading buffer), mix well, boil for about 5 min, and centrifuge at 7000 rpm for 5 min to obtain the sample for SDS-PAGE electrophoresis.

8. The method for preparing a foot-and-mouth disease subunit recombinant protein vaccine and evaluating its immunogenicity according to claim 1, wherein the steps for preparing and refolding inclusion bodies are as follows: centrifuging at 4000 rpm for 10 min at 4℃, discarding the supernatant, resuspending the bacterial sludge with 1x Binding Buster, and adding Twen at 1% of the amount... 2.0 After mixing, sonicate on ice for 5 min, centrifuge at 12000 rpm for 5 min, discard the supernatant, and finally collect the precipitate; dissolve the precipitate with 8M urea, mix thoroughly by pipetting, and incubate overnight at 4℃ to allow it to dissolve completely. Centrifuge at 12000 rpm for 5 min at 4℃ and collect the supernatant; place the supernatant in a 3 kDa dialysis bag, and then place the dialysis bag in a large beaker filled with 6M urea, incubate at 4℃ for 2 h; replace the 6M urea in the beaker with 4M urea, incubate at 4℃ for 4 h; gradually replace with 3M, 2M, and 1.5M, observing carefully until a precipitate forms, then immediately stop dialysis, collect the protein, and store at -80℃.

9. The method for preparing a foot-and-mouth disease subunit recombinant protein vaccine and evaluating its immunogenicity according to claim 1, wherein the steps for determining the protein concentration using BCA are as follows: The protein concentration is determined using a commercially available BCA protein quantification kit, specifically as follows: The collected protein is removed from the refrigerator and placed at room temperature for 1 hour; protein standards are added to 96-well plates at concentrations of 0, 1, 2, 4, 6, 8, and 10 μL, and then diluted with sterile double-distilled water at a ratio of 1:10 to obtain concentrations of 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1 mg / mL; 10 μL of purified NP sample is added to each well, with two replicates; solution A and solution B are mixed at a ratio of 50:1 to prepare a working solution, 200 μL / well, and incubated at 37°C for 30 minutes; the OD562nm value is read using an ELISA reader, a standard curve is plotted using the protein standards, and the protein sample concentration is calculated.

10. The method for preparing a foot-and-mouth disease subunit recombinant protein vaccine and evaluating its immunogenicity according to claim 1, wherein the steps of vaccine preparation and animal experiment are as follows: the purified VP1 protein is concentrated to 1 mg / mL using an ultrafiltration tube, and the VP1 protein is mixed with Freund's complete adjuvant / Freund's incomplete adjuvant in a 1:1 volume ratio according to the antigen and adjuvant formulation ratio in Table 1.5, and the mixture is shaken and emulsified for 3 min to prepare the vaccine; 10 five-week-old female BALB / c mice are randomly divided into two groups, namely a blank control group and a 100 μg VP1 antigen group, with 5 mice in each group. The mice are subcutaneously injected on the back according to the immunization grouping and immunization dose in Table 1.6, and immunized once every 2 weeks for a total of 3 immunizations. The first immunization uses Freund's complete adjuvant vaccine, and the two booster immunizations use Freund's incomplete adjuvant vaccine. Blood is collected from the eyeballs one day before immunization, and the serum is separated by centrifugation at 3000 r / s, 4°C for 15 min after standing at 4°C for 2 h. A portion is aliquoted and stored at -20°C for later use, and the remainder is stored in a -80°C refrigerator for long-term storage. Table 1.5 Antigen and Adjuvant Ratios

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