A PCV2 subunit vaccine of PCV2d type coupled with COS

Abstract

The present invention belongs to the technical field of veterinary medicines, and provides a PCV2d type PCV2 subunit vaccine coupled with COS. The antigen of the vaccine is a COS-coupled PCV2d Cap protein, which is obtained by reacting a thiol-modified Cap protein with a maleimidized COS. Compared with the uncoupled protein, the COS-coupled PCV2d Cap protein has a different degree of improvement in the proportion of α-helix, antiparallel β-fold, parallel β-fold and β-turn in its secondary structure, while the proportion of irregular curling is greatly reduced; the thermal stability and structural stability are improved, and the level of antigen epitope display is also improved. The subunit vaccine prepared by the coupled protein can more efficiently stimulate the immune system to produce a strong and lasting immune response, and has commercial prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary medicines, and in particular relates to a PCV2 subunit vaccine. Background Art

[0002] Porcine circovirus ( porcine circovirus Porcine circovirus type 2 (PCV) was first discovered as a contaminant in the porcine kidney cell line PK-15 in 1974 and was named PCV1. It is a non-pathogenic virus. Since 1991, the clinical and pathological range of PCV2 infection has continued to expand. Porcine circovirus type 2 , PCV2) is the main pathogen of porcine circovirus disease (PCVAD), which can cause a variety of diseases such as postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis nephropathy syndrome (PDNS). PCV2 is also believed to affect the reproductive system, causing the so-called porcine respiratory disease complex (PRDC), enteritis, and proliferative and necrotizing pneumonia (PNP). PMWS was first described in Saskatchewan, Canada in 1991 as a sporadic disease characterized by emaciation and jaundice. The disease can reduce the immunity of infected pigs, cause growth and reproduction disorders, increase pig mortality, low feed return rate, and significantly reduce economic benefits. PCV2 is a DNA virus without a capsule covering its surface. PCV2 infection is widespread in almost all pig producing countries and is recognized as a global infectious disease of pigs.

[0003] PCV2 is a small single-stranded circular DNA virus and an important member of the Circoviridae family and the genus Circovirus. The diameter of PCV2 particles is about 17 nm, and there is no envelope on the surface of the virus. PCV2 virus particles are composed of icosahedral capsid proteins (Capsid, Cap protein) and genetic genes. The icosahedral structure is composed of 60 copies of Cap protein, arranged in 12 pentamer units. Its genome is about 1768 nt long and mainly contains three open reading frames (ORF): ORF1, ORF2 and ORF3. The Cap protein encoded by PCV2 ORF2 is a structural antigen protein of the virus, which is closely related to the pathogenicity of the virus and has a molecular weight of about 27.8 kDa. As a structural protein of PCV2, the Cap protein has antigenic epitopes at amino acids 25-39, 69-83, 117-131, 113-127, 169-183 and 193-207, which plays a key role in stimulating the host to produce specific antibodies and is essential for stimulating an effective immune response. PCV2 Cap protein can self-assemble into virus-like particles (VLPs) under appropriate conditions in vitro. Currently, PCV2 Cap protein has been widely used to produce PCV2 subunit vaccines and has good immune effects. However, the N-terminus of PCV2 Cap contains a large number of nuclear localization signal peptide (NLS) sequences, a large number of arginine residues and some rare codons, which hinder the expression of PCV2 Cap in Escherichia coli expression systems or baculovirus expression systems.

[0004] At present, PCV2 vaccines are mainly divided into two categories, PCV2 inactivated vaccines and PCV2 subunit vaccines. Among them, inactivated vaccines are vaccines made with inactivated PCV2 as antigens. PCV2 is inactivated by physical or chemical methods. PCV2 loses its ability to infect while maintaining good immunogenicity. The inactivated antigen is mixed with an adjuvant for emulsification to prepare the PCV2 inactivated vaccine. Subunit vaccines are vaccines made with PCV2 Cap protein or VLPs produced by genetic engineering as antigens. At present, PCV2 Cap protein is mainly produced by expressing PCV2 ORF2 gene through Escherichia coli expression system and baculovirus expression system. Subunit vaccines have higher safety than attenuated or inactivated vaccines, but lack the ability to induce humoral immunity and cellular immunity for a long time.

[0005] In the design and production of vaccines, in addition to antigens, adjuvants are also important factors that determine the effectiveness of vaccines. Adjuvants can enhance the body's immune response to antigens, increase antibody levels, and produce more effective protective immunity. PCV2 vaccine adjuvants are mainly divided into two categories: chemical adjuvants and molecular adjuvants. Most of the adjuvants used in commercial vaccines on the market are oil adjuvants. In addition to traditional oil adjuvants and water adjuvants, some cytokines, nanomaterials, macromolecular proteins, and gene fragments have excellent potential as adjuvants. At present, the design and production logic of almost all vaccines is the physical mixing of antigens and adjuvants. It is difficult to achieve a breakthrough in immune efficiency when adjuvants and antigens work alone. Studies by Bram Slütter and others have shown that direct coupling of antigens with adjuvants is a feasible and effective strategy that can greatly increase the immune reactivity of subunit vaccines. Combining antigens and adjuvants into a covalent compound may be a promising vaccine development strategy.

[0006] There are currently 8 genotypes of PCV2, of which PCV2a, PCV2b and PCV2d are the main genotypes, while PCV2c, PCV2e, PCV2f, PCV2g and PCV2h are less common genotypes. With the continuous mutation and evolution of PCV2 genes, the current main epidemic strain has changed from PCV2b to PCV2d. The independence of PCV2d has become stronger over time, and it has appeared on a large scale in pigs immunized with PCV2 vaccines. It is speculated that it may have broken through the protective effect of current commercial vaccines and caused more serious economic losses. Most of the commercial vaccines on the market were developed between 1999 and 2005 when PCV2 began to spread on a large scale. PCV2a was the main strain at that time, and most of the current commercial vaccines were developed based on the PCV2a subtype. Studies have shown that in recent years, the PCV2 positive rate in some pig farms in my country has remained high, and there is a phenomenon of mixed infection of multiple subtypes, with the more pathogenic PCV2d occupying the main position. Therefore, the prevention and control of PCV2d subtype is particularly important to ensure the production efficiency of live pigs. The development of effective PCV2d vaccine and its combination with clinical treatment methods are of great significance to improving the current status of disease prevention and control in pig farms, which can promote the purification of PCV2 in my country and ensure the healthy development of my country's pig industry. Summary of the invention

[0007] In response to the problems that the protective effect of commercial vaccines decreases or disappears due to changes in PCV2 epidemic strains, and that existing subunit vaccines lack the ability to induce humoral immunity and cellular immunity for a long time, the present invention provides a subunit vaccine against PCV2d epidemic strains. By coupling the Cap protein of the PCV2d subtype with chitosan oligosaccharide (COS), the proportion of α-helix, antiparallel β-fold, parallel β-fold and β-turn in the secondary structure of the protein can be increased, and the vaccine has a significant sustained-release effect and can maintain a high level of IgG for a long time.

[0008] To achieve the above purpose, the present invention adopts the following technical solution.

[0009] A method for preparing a COS-coupled PCV2d Cap protein comprises reacting a thiol-modified Cap protein with a maleimidized COS to obtain the COS-coupled PCV2d Cap protein.

[0010] Specifically, the above preparation method comprises the following steps:

[0011] (1) Obtaining PCV2d Cap protein solution;

[0012] (2) PCV2d Cap was cross-linked with 2-iminothiolane, and the thiolated Cap protein was obtained after separation and purification;

[0013] (3) Sodium periodate reacts with COS and then separates and purifies COS to obtain aldehyde-modified COS;

[0014] (4) The aldehyde-modified COS is reacted with N-(2-aminoethyl)maleimide and sodium cyanoborohydride, and then separated and purified to obtain maleimidized COS;

[0015] (5) The thiol-modified Cap protein reacts with the maleimidized COS to obtain the COS-coupled PCV2d Cap protein.

[0016] In step (1), the PCV2d Cap protein is a full-length protein, and its amino acid sequence is shown in SEQ ID NO: 1. The PCV2 Cap protein can be obtained by methods known in the art, such as expression in Escherichia coli, insect bacillus system, and yeast system. In order to adapt to the different codon preferences of various expression systems and thus improve the expression yield and solubility of the expression product, codon preference modification can be performed. In some embodiments, in order to ensure that PCV2 ORF2 can be efficiently expressed in Escherichia coli, ORF2 (SEQ ID NO: 1) of the PCV2d SD strain is optimized for Escherichia coli codons, and the optimized nucleic acid (SEQ ID NO: 2) is seamlessly cloned and connected with pET30a.

[0017] In step (2), in order to ensure the efficiency and coupling amount of COS coupling, 2-iminothiolane can be used in excess; using 1.2 times excess 2-iminothiolane is sufficient to ensure effective modification. Therefore, the mass ratio of PCV2d Cap to 2-iminothiolane is 1:1-2; the preferred mass ratio is 1:1.2-1.5.

[0018] The mass ratio of COS to sodium periodate is 1:1-1.2; the mass ratio of N-(2-aminoethyl)maleimide, sodium cyanoborohydride and COS is 5:5:2.

[0019] The mass ratio of thiolated Cap protein to maleimidized COS is 1:1-1.5.

[0020] In steps (2) and (5), separation and purification can be carried out by methods known in the art for purifying proteins. Generally, proteins and impurities can be separated by dialysis or by adsorption-desorption and exclusion methods of chromatographic columns, wherein the impurities are mainly salts and unreacted raw materials. The molecular weight cutoff of dialysis can be selected according to the specific relative molecular weights of impurities and proteins. For adsorption-desorption, impurities can be adsorbed or proteins can be adsorbed. In some embodiments, separation is performed using a polypropylene chromatographic column with a molecular weight cutoff of 7000, and the effluent is collected by centrifugation at about 1000g.

[0021] In steps (2), (4) and (5), the reaction temperature is 0-10° C. In step (3), the reaction condition is to avoid light.

[0022] In steps (3) and (4), the separation and purification step is performed by dialyzing in a PBS buffer at pH 7.4 using a dialysis bag with a molecular weight cutoff of 100Da-500Da.

[0023] For the above method, COS can be first aldehyded and maleimidized in sequence, and then the Cap protein can be thiolated. The order of derivatization of the two parts does not affect the structure of COS-coupled Cap protein. After coupling, the proportion of each component in the secondary structure of the protein has changed, and the proportions of α-helix, antiparallel β-fold, parallel β-fold and β-turn have increased to varying degrees, while the proportion of irregular curling has decreased significantly. These structural changes will have a positive impact on the various biological properties of PCV2d Cap protein, such as a certain level of improvement in thermal stability, structural stability, and antigen epitope display level, thereby potentially optimizing its underlying ability as a vaccine antigen. In addition, it can also produce a significant sustained-release effect of the vaccine. In some reports, the inactivated virus is first maleimidized, COS is thiolated, and then the coupling reaction is carried out. This method of derivatizing the two parts did not result in significant changes in the secondary structure of PCV2 after coupling. Since the connecting arm between PCV2 and COS is short, it is impossible to construct sufficient random conformation space at the amino acid level, which affects the coating of COS on the antigen and results in no obvious changes in the sustained release of the vaccine.

[0024] The present invention also provides a COS-coupled PCV2d Cap protein obtained by the above preparation method. The COS-coupled PCV2d Cap protein can be used to prepare a vaccine against the PCV2d subtype. The vaccine may also contain an adjuvant. In some embodiments, the adjuvant is JLC-3 carbomer adjuvant.

[0025] The present invention has the following advantages:

[0026] The COS-coupled PCV2d Cap protein provided by the present invention has a different degree of improvement in the proportion of α-helix, antiparallel β-fold, parallel β-fold and β-turn in its secondary structure compared to the uncoupled protein, while the proportion of irregular curling is greatly reduced; it has a positive impact on various biological properties of the PCV2d Cap protein, improves thermal stability and structural stability, and at the same time improves the level of antigen epitope display. Animal experiments have shown that the vaccine prepared by the coupled protein can stimulate higher antibody levels than ordinary subunit vaccines; and has an obvious sustained release effect, and the high level of IgG antibodies is maintained for a long time. The subunit vaccine can more efficiently stimulate the immune system to produce a strong and lasting immune response. The vaccine provided by the present invention has commercial prospects and lays a foundation for the current prevention and control and purification of PCV2d and the research and development of new PCV2 vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is the endogenous fluorescence detection of PCV2d Cap-COS and PCV2d Cap;

[0028] Figure 2 SDS-PAGE (left) and Western blot (right) analysis of PCV2d Cap-COS;

[0029] Figure 3 is the size exclusion chromatography fitting diagram of PCV2d Cap-COS and PCV2d Cap;

[0030] Figure 4 Transmission electron microscope images of PCV2d Cap-COS (left) and PCV2d Cap (right);

[0031] Figure 5 Comparison of antibody and cytokine levels and ADWG in different treatment groups;

[0032] Figure 6 This is a pathological section of a mouse organ (HE staining, 200×); the arrow indicates the lesion. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with the examples and drawings, but the present invention is not limited by the following examples. Unless otherwise specified, the reagents used in the examples are all purchased commercially.

[0034] Example 1 Preparation of COS-coupled PCV2d Cap protein

[0035] 1. Preparation of PCV2d Cap Protein

[0036] According to the sequence of PCV2d SD strain (GenBank: JQ653449.1), the ORF2 amino acid sequence (SEQ ID NO: 1) of PCV2dSD strain was modified according to the preference of Escherichia coli, and the nucleotide sequence was shown in SEQ ID NO: 2, and commercial synthesis was performed. The PCV2d ORF2 gene fragment was inserted into the pET30a linearized vector using Takara In-Fusion Snap Assembly cloning kits and connected to the pET30a-PCV2d ORF2 plasmid. The above plasmid was transformed into BL21 (DE3) competent cells to obtain engineered bacteria. The engineered bacteria were cultured in liquid LB to an OD600 of 0.8-1.2, IPTG was added to 1mM and induced at 28℃ for 7h, the bacterial cells were separated and resuspended in PBS with pH 7.4, and the supernatant was obtained by centrifugation after ultrasonic disruption. The supernatant was adsorbed to Ni-NTA resin and loaded onto a column for gradient elution with 50 mM-200 mM imidazole to obtain a purified PCV2d Cap protein solution.

[0037] 2. Oxidation reaction of COS

[0038] Take it to a clean beaker. Weigh 100 mg COS and add it to 50 mL acetic acid-sodium acetate buffer (pH 5.5). Use a magnetic stirrer to stir thoroughly for 15 min to ensure that COS is completely dissolved. After the dissolution is completed, add 100 mg sodium periodate to the COS solution system and perform the oxidation reaction for 1 h under light-proof conditions. After the reaction is terminated, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 100 Da-500 Da (Solarbio, Cat. YA1069) and dialyzed with PBS buffer for 24 h. During this period, the dialysate was changed 3 times to completely remove unreacted reagents and small molecule products. In this reaction, the hydroxyl group in COS will be oxidized to an aldehyde structure to obtain aldehyde-modified COS.

[0039] 3. Reductive amination of COS

[0040] Take 10 mL of aldehyded COS solution (20 mg) for later use, weigh 50 mg of N-(2-aminoethyl)maleimide and dissolve it in 150 μL of dimethyl sulfoxide solution, and weigh 50 mg of sodium cyanoborohydride and dissolve it in 200 μL of PBS buffer at pH 7.4. Mix the three solutions and react them at 4°C for 3 h. After the reaction, dialyze in PBS buffer for 12 h using a dialysis bag with a molecular weight cutoff of 100 Da-500 Da (Solarbio, Cat. YA1069) to remove the unreacted N-(2-aminoethyl)maleimide and sodium cyanoborohydride. In this reaction, the aldehyde group on the aldehyded COS undergoes a preliminary coupling reaction with the primary amine in N-(2-aminoethyl)maleimide to generate a reversible Schiff base intermediate. Then, under the reducing conditions of sodium cyanoborohydride, the intermediate will be further transformed into a stable and irreversible secondary amine bond to generate maleimidized COS.

[0041] 4. Thiolation reaction of PCV2d Cap

[0042] Take 20 mg of PCV2d Cap protein and use a 10 kDa ultrafiltration tube to replace the solution and system with a non-amine buffer (PBS buffer containing 2 mM EDTA and 10% Glycerol, pH 8.0). Depending on the volume of the reaction, the optional concentration of EDTA is 2mM-5mM to chelate the divalent metal ions in the solution to prevent sulfhydryl oxidation. Then add 25 mg of 2-iminothiolane hydrochloride to the solution and react at 4°C for 2 h; this reaction can introduce sulfhydryl groups into the Cap protein. After the reaction, use Zeba TM A desalting centrifugal column (7K MWCO) was used to separate the thiolated Cap protein from excess 2-iminothiolane hydrochloride and small molecule products. After loading, the sample was centrifuged at 1000 g for 2 min, and the effluent was collected, i.e., the thiolated PCV2d Cap protein solution.

[0043] 5. Bioconjugation of maleimidized COS and thiolated PCV2d Cap

[0044] The maleimidized COS solution was mixed with the thiolated PCV2d Cap protein solution and allowed to react in a 4°C refrigerator for 12 h. After the reaction, the mixture was dialyzed in a conjugation buffer (PBS, EDTA 2 mM, Glycerol 10%, pH 7.2) for 24 h using a 10 kDa dialysis bag to remove unbound COS. The solution was changed three times during the period. After dialysis, the PCV2d Cap-COS complex was obtained and stored at 4°C.

[0045] In natural proteins, there are a certain proportion of tryptophan, tyrosine and phenylalanine residues. When these three amino acids are excited by ultraviolet light with a wavelength of 280 nm, they emit fluorescence of a certain wavelength, usually between 300-400 nm. When PCV2d Cap is modified with COS, random binding occurs. Due to the shielding effect of COS on the fluorescence of 300-400 nm wavelength, the fluorescence intensity of the PCV2d Cap-COS complex excited is lower than that of the unmodified PCV2d Cap. Therefore, a high-precision microplate reader can be used to perform endogenous fluorescence detection on PCV2d Cap-COS and PCV2d Cap, and the bioconjugation effect can be judged by analyzing the fluorescence intensity of the two: take PCV2d Cap-COS complex sample and PCV2d Cap sample, adjust the sample concentration to 0.1 mg / mL, add to a black opaque microplate plate, 200 mL per well, use a high-precision microplate reader for fluorescence detection, set the excitation wavelength to 280 nm, and set the emission wavelength detection to 300-410 nm.

[0046] Test results such as Figure 1 As shown: between the wavelength of 300-400 nm, the fluorescence intensity of the PCV2d Cap-COS complex is lower than that of PCV2d Cap, which proves that the coupling reaction between PCV2d Cap and COS has been successfully carried out.

[0047] According to the method in the manual, the reducing sugar content detection kit (Beijing Solebow Technology Co., Ltd.) was used to detect the COS content in the PCV2d Cap-COS complex. At the same time, the BCA protein quantification kit (ThermoFisher, USA) was used to accurately detect the protein content in the sample, and the glycoprotein ratio of the sample was calculated based on the test results to evaluate the coupling efficiency. After testing, the sugar concentration in the sample was 0.582 mg / mL and the protein concentration was 0.862 mg / mL. The sugar concentration of the sample was divided by the protein concentration to obtain a glycoprotein ratio of 67.51% for the PCV2d Cap-COS complex, which means that on average every 100 units of protein can be coupled with 67.51 units of COS as a covalent compound, and COS has a high level of coupling efficiency with the PCV2d Cap protein.

[0048] 6. Physicochemical properties of COS-coupled PCV2d Cap protein

[0049] The PCV2d Cap-COS complex was subjected to SDS-PAGE electrophoresis to detect the protein molecular weight distribution, and its immunogenicity was analyzed by Western blot, using purified PCV2d Cap solution as a control.

[0050] The results of SDS-PAGE and Western blot analysis of PCV2d Cap-COS are shown in Figure 2 As shown in the figure, M is protein marker; 1 is PCV2d Cap-COS; 2 is PCV2d Cap. The results show that after PCV2d Cap is bioconjugated with COS, it can no longer cross the boundary between the upper and lower layers of gel, proving that its molecular weight has increased significantly. Western blot results show that the molecular weight of the PCV2dCap-COS complex is significantly larger than that of PCV2d Cap, while it can still bind to PCV2-specific antibodies and has immunogenicity.

[0051] The samples were analyzed by size exclusion chromatography to obtain the molecular weight distribution data of the samples and to observe whether the molecular weight of the PCV2d Cap-COS complex antigen was increased compared with the purified PCV2d Cap. Figure 3 As shown in the figure: PCV2d Cap-COS (1) began to peak at 16 min and ended at 18 min, while the peak time of PCV2d Cap (2) without COS modification was 18-21 min. The peak time of PCV2d Cap modified with COS was significantly shifted to the left, indicating that the molecular weight of PCV2d Cap-COS was significantly larger than that of PCV2d Cap.

[0052] TEM observation of PCV2d Cap before and after coupling with COS, as shown in the following pictures: Figure 4 As shown in the figure, the diameter of the PCV2d Cap-COS complex (indicated by the arrow in the left figure) can reach 100 nm. The coating effect of the random coupling of PCV2d Cap protein and COS makes the volume of the PCV2d Cap-COS complex significantly larger than that of the simple PCV2d Cap protein, which is also consistent with the molecular weight test results.

[0053] PCV2d Cap-COS complex samples and PCV2d Cap samples were scanned by far-ultraviolet circular dichroism (CD) at a wavelength of 190-260 nm. The collected wavelengths were analyzed by CDNN software to determine and deduce their secondary structures and analyze the structural ratio changes after PCV2d Cap coupling.

[0054] Table 1 Calculation of protein secondary structure ratios

[0055]

[0056] The results are shown in Table 1: After PCV2d Cap was coupled with COS, the proportions of various components in its secondary structure changed, and the proportions of α-helix, antiparallel β-fold, parallel β-fold and β-turn increased to varying degrees, while the proportion of irregular coil decreased significantly. The change in structural proportions can be used to determine that PCV2d Cap and COS were successfully coupled, and these structural changes will have a positive impact on the various biological properties of PCV2d Cap protein, such as a certain level of improvement in thermal stability, structural stability, and antigen epitope display, thereby potentially optimizing its underlying ability as a vaccine antigen.

[0057] Example 2 Preparation of PCV2d subunit vaccine

[0058] The vaccine was prepared using JLC-3 carbomer adjuvant. The adjuvant and antigen were mixed in a ratio of 1:1 (w / w) and oscillated in a 4°C refrigerator for 30 min. The specific groups were as follows:

[0059] (1) Bioconjugate vaccine: The PCV2d Cap-COS complex prepared in Example 1 was used as an antigen and mixed with JLC-3 carbomer adjuvant at a ratio of 1:1 (w / w), and the mixture was shaken and mixed in a refrigerator at 4°C for 30 min. This vaccine was named PCV2d Cap-COS / JLC-3. The antigen concentration of the vaccine was 100 μg / mL, that is, each mL of the vaccine contained 100 μg of PCV2d Cap-COS complex.

[0060] (2) Physical mixed vaccine: Take an appropriate amount of COS solution and mix it with PCV2d Cap solution, and control the mass ratio of protein to COS at 100:67.5. Then, mix the solution as an antigen solution with JLC-3 carbomer adjuvant at a ratio of 1:1 (w / w), shake and mix in a refrigerator at 4°C for 30 min, and control the antigen concentration at 100 μg / mL. This vaccine is named PCV2dCap / COS / JLC-3.

[0061] (3) PCV2d Cap-COS complex was directly used as a vaccine and diluted with PBS to a protein concentration of 100 μg / mL, named PCV2d Cap-COS.

[0062] (4) PCV2d Cap vaccine: The purified PCV2d Cap prepared in Example 1 was mixed as an antigen with JLC-3 carbomer adjuvant at a ratio of 1:1 (w / w), and the mixture was shaken and mixed in a refrigerator at 4°C for 30 min. This vaccine was named PCV2d Cap / JLC-3, and the antigen concentration of the vaccine was 100 μg / mL.

[0063] The prepared vaccines were stored in a 4°C refrigerator for 30 days for stability observation, and the four vaccines showed good stability.

[0064] Application Example 1 Animal Immunization and Virus Challenge Test

[0065] The vaccines used for animal experiment immunization were the four vaccines prepared in Example 2: PCV2d Cap-COS / JLC-3 vaccine, PCV2d Cap / COS / JLC-3 vaccine, PCV2d Cap-COS vaccine, and PCV2d Cap / JLC-3 vaccine. A PBS control group (pH7.4, immunization dose 0.25 mL) was also set up. The specific steps were as follows: 40 healthy 6-week-old BALB / c mice were used as experimental animals and divided into 5 groups, with 8 mice in each group. The experimental animals were immunized with a single subcutaneous injection of 0.25 mL of vaccine, and blood was collected from the orbital venous plexus every 7 days after immunization. The collected blood samples were placed in a 4°C refrigerator for 3 h, then centrifuged at low temperature and low speed to separate the serum and stored in a -20°C refrigerator. Mice were injected with 0.2 mL of PCV2d SD strain (GenBank: JQ653449.1) virus solution (titer of 10 5.5 TCID 50 / mL) were challenged with the virus and the animals were dissected 28 days after the challenge.

[0066] The levels of IgG antibodies, IFN-γ and TNF-α in serum were detected by ELISA kits, and the immune effects of vaccines in each group were compared. The body weight before and after the challenge was recorded and the average daily weight gain (ADWG) after the challenge was calculated: the difference between the body weight before dissection and the body weight before the challenge was divided by 28. After the challenge test, mice in each group were dissected and the lungs and kidneys were taken for pathological observation, and pathological tissue sections were prepared for pathological evaluation and histological analysis.

[0067] The levels of IgG antibodies, IFN-γ and TNF-α in the serum of mice in different experimental groups and the ADWG results can be used to preliminarily evaluate the immunogenicity and immune protection of each group of vaccines. Figure 5As shown: All vaccines can effectively induce specific antibody production and increase IFN-γ and TNF-α levels after vaccination. Among them, PCV2d Cap-COS / JLC-3 vaccine showed the highest antibody peak. Although its peak time was later than other control groups, its high-level antibody lasted the longest, which was consistent with the trend of IFN-γ and TNF-α. At the same time, the data of PCV2d Cap-COS group and PCV2d Cap-COS / JLC-3 group showed similar trends. The antibody levels of the four vaccination groups showed a regular change of first rising and then falling, and compared with the PBS control group, the antibody levels of each vaccine group were significantly increased, confirming that these vaccines have good immune activation effects. In addition, in the vaccinated mouse group, their ADWG after the virus attack was better than that of the PBS control group, showing better growth performance after resistance to PCV2 infection, especially the mice vaccinated with PCV2d Cap-COS / JLC-3 vaccine, whose daily weight gain increased most significantly.

[0068] Comprehensive analysis of various data shows that the PCV2d Cap-COS / JLC-3 vaccine not only showed better effects in overall immune response, but also showed certain advantages in improving the growth performance of the test mice and demonstrated a unique sustained-release effect.

[0069] Pathological tissue section results Figure 6 As shown: After PCV2d challenge, the lung interstitium of mice in the PBS group showed lymphocyte infiltration accompanied by interstitial congestion, and kidney sections showed that the glomeruli in the glomeruli were enlarged, resulting in narrowing of the renal capsule cavity. The mice in the PCV2dCap / JLC-3 group had the same kidney lesions as the mice in the PBS group, but no obvious pathological damage was found in the lungs. The mice immunized with PCV2d Cap-COS / JLC-3, PCV2d Cap / COS / JLC-3, and PCV2d Cap-COS vaccines did not have pathological damage in the lungs and kidneys.

Claims

1. A method for preparing COS-coupled PCV2d Cap protein, characterized in that: The following steps are involved: (1) Obtaining PCV2d Cap protein solution; (2) PCV2d Cap was cross-linked with 2-iminothiolane, and the thiolated Cap protein was obtained after separation and purification; (3) Sodium periodate reacts with COS and then separates and purifies COS to obtain aldehyde-modified COS; (4) The aldehyde-modified COS is reacted with N-(2-aminoethyl)maleimide and sodium cyanoborohydride, and then separated and purified to obtain maleimidized COS; (5) The thiol-modified Cap protein was reacted with the maleimidized COS to obtain the COS-coupled PCV2d Cap protein after separation and purification.

2. The preparation method according to claim 1, characterized in that: In step (1), the PCV2 Cap protein is a full-length protein; the PCV2 Cap protein is expressed by recombinant Escherichia coli, recombinant insect bacillus system or recombinant yeast system.

3. The preparation method according to claim 1, characterized in that: In step (1), the amino acid sequence of the PCV2 Cap protein is shown in SEQ ID NO:

1.

4. The preparation method according to claim 1, characterized in that: In step (1), the encoding nucleotide sequence of the PCV2 Cap protein is shown in SEQ ID NO:

2.

5. The preparation method according to claim 1, characterized in that: The mass ratio of PCV2d Cap to 2-iminothiolane is 1:1-2; The mass ratio of COS to sodium periodate is 1:1-1.2; The mass ratio of N-(2-aminoethyl)maleimide, sodium cyanoborohydride, and COS was 5:5:2; The mass ratio of thiolated Cap protein to maleimidized COS was 1:1-1.5; In steps (2) and (5), separation and purification are performed by dialysis or chromatographic column separation; In steps (2), (4) and (5), the reaction temperature is 0-10°C; In step (3), the reaction conditions are light-proof conditions; In steps (3) and (4), the separation and purification step is performed by dialyzing in a PBS buffer at pH 7.4 using a dialysis bag with a molecular weight cutoff of 100Da-500Da.

6. The preparation method according to claim 5, characterized in that: The mass ratio of PCV2d Cap to 2-iminothiolane is 1:1.2-1.5; In steps (2) and (5), separation and purification are performed using a polypropylene chromatography column with a molecular weight cutoff of 7000.

7. A COS-coupled PCV2d Cap protein obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the COS-coupled PCV2d Cap protein as claimed in claim 7 in the preparation of PCV2d subtype vaccine.

9. A vaccine prepared by coupling COS with PCV2d Cap protein as claimed in claim 7.

10. The vaccine according to claim 9, characterized in that The invention also comprises an adjuvant; the adjuvant is JLC-3 carbomer adjuvant.

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