A method for differential diagnosis of swine fever based on swine fever E2 subunit vaccine

Abstract

The application provides a method for diagnosing and differentiating classical swine fever based on a classical swine fever E2 subunit vaccine, which comprises the following steps: immunizing pigs in a pig farm with the classical swine fever E2 subunit vaccine, detecting E0 antibodies and E2 antibodies after one year of immunization, and eliminating the pigs with E0 antibodies until both the E0 antibodies and the CSFV nucleic acid detection are negative, which indicates that the classical swine fever is purified. The classical swine fever E2 subunit vaccine comprises a nucleic acid fragment encoding an antigen protein with the amino acid sequence of SEQ ID NO: 5 inserted into a recombinant expression vector. The classical swine fever E2 subunit vaccine provided by the application has good immunoprotective efficacy on pigs, and a classical swine fever epidemic disease purification method is established by using the subunit vaccine. The application effect of the method in a pig breeding company for two years shows that the method can effectively purify the classical swine fever, which indicates that the purification method provided by the application is feasible and can be popularized and applied.

Description

Technical Field

[0001] This invention belongs to the field of veterinary technology, and in particular relates to a purification method for identifying and diagnosing classical swine fever based on a classical swine fever E2 subunit vaccine. Background Technology

[0002] Classical swine fever (CSF), belonging to the genus *Vibrio* of the family Flaviviridae, is a highly acute, febrile, and contagious disease of pigs caused by the classical swine fever virus (CSFV). It is classified into peracute, acute, subacute, chronic, and mild forms. The disease is characterized by rapid onset, persistent high fever, degeneration of small blood vessel walls, widespread petechial hemorrhages, and splenic infarction. CSF is widespread, with high morbidity and mortality rates. The main routes of transmission are the digestive and respiratory tracts; infected sows can transmit the disease vertically to their fetuses via the placenta. It is prevalent year-round and can have a devastating impact on the pig industry, making it one of the most serious infectious diseases affecting pig farming.

[0003] In recent years, with the rapid development of large-scale and intensive pig farming in my country, the high-intensity immunization with C-strain live vaccines has reduced the probability of swine fever outbreaks. However, latent infections or immunized individuals carrying the virus remain widespread, and persistent and latent infections frequently occur. Clinical manifestations tend to be mild and atypical, posing challenges to the comprehensive prevention and control of swine diseases. Traditional swine fever C-strain live vaccines cannot distinguish whether the antibodies in pigs are produced by vaccine immunization or wild-type virus infection. In current farming practices, piglets vaccinated with swine fever C-strain live vaccines after farrowing require a separate initial immunization with swine fever vaccine two weeks after weaning, which poses certain biosecurity risks. Furthermore, traditional swine fever eradication methods require large-scale live tonsil sampling, which causes significant stress to the pig herd, severely impacting production and posing biosecurity risks. Summary of the Invention

[0004] The purpose of this invention is to provide a method for identifying and diagnosing classical swine fever based on a classical swine fever E2 subunit vaccine, thereby overcoming the shortcomings of the prior art.

[0005] The present invention first provides a classical swine fever E2 antigen protein, wherein the antigen protein is a fusion protein prepared by connecting an immune protein with the amino acid sequence SEQ ID NO:1 and an immune protein with the amino acid sequence SEQ ID NO:3 in series via a flexible linker;

[0006] The immune protein with the amino acid sequence SEQ ID NO:1 has a sequence encoding a nucleic acid fragment that is SEQ ID NO:2.

[0007] The immune protein with the amino acid sequence SEQ ID NO:3 has a sequence encoding a nucleic acid fragment that is SEQ ID NO:4;

[0008] As a specific example, the antigen protein has the amino acid sequence SEQ ID NO:5;

[0009] Furthermore, the nucleotide sequence of one of the encoding genes of the antigen protein is SEQ ID NO:6;

[0010] The present invention provides a classical swine fever E2 subunit vaccine, wherein the classical swine fever E2 subunit vaccine contains a nucleic acid fragment encoding the above-mentioned antigen protein inserted into a recombinant expression vector.

[0011] Another aspect of the present invention provides a method for purging swine fever in a breeding farm, which involves immunizing the pig herd with the aforementioned swine fever E2 subunit vaccine, detecting E0 antibodies and E2 antibodies one year after immunization, culling pigs with E0 antibodies, and continuing this process until both E0 antibody and CSFV nucleic acid tests are negative, indicating that swine fever purification is complete.

[0012] The classical swine fever E2 subunit vaccine provided by this invention has good immune protection efficacy against swine herds. Furthermore, a classical swine fever eradication method has been established using this subunit vaccine. After two years of application in a breeding pig company, the results show that the method of this invention can effectively achieve the eradication of classical swine fever, indicating that the eradication method provided by this invention is feasible and can be widely applied. Attached Figure Description

[0013] Figure 1 Electrophoretic detection image of recombinant expression of fusion antigen;

[0014] Figure 2 Immunogenicity detection diagram of recombinant protein;

[0015] Figure 3 Flowchart for the purification of classical swine fever based on E2 subunit vaccine testing;

[0016] Figure 4 : A chart showing the positive rates of CSFV and CSF antibodies for a certain company from 2020 to 2022. Detailed Implementation

[0017] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0018] Example 1: Preparation of classical swine fever E2 subunit vaccine

[0019] 1. Preparation of antigen fusion protein

[0020] Classical swine fever virus (CSFV) E2 protein (GenBank accession number: AF092448) was selected as the target antigen. Its antigenic epitopes, hydrophilicity and flexibility were analyzed using the DNASTAR Protean module. Two highly immunogenic regions, E2-T1 and E2-T5, were screened out to prepare antigen fusion proteins.

[0021] The E2-T1 amino acid sequence is as follows:

[0022] NNLPRKGDCRSGNHLGPVSGIYVKPGPVFYQDYMGPVY (SEQ ID NO:1);

[0023] The nucleotide sequence encoding this protein is as follows:

[0024] AATAACCTACCTAGGAAAGGAGACTGCAGGAGTGGCAACCATCTAGGCCCGGTTAGCGGGATATATGTAAAGCCCGGCCCTGTCTTTTATCAGGACTACATGGGCCCGGTCTAC (SEQ ID NO: 2);

[0025] The E2-T5 amino acid sequence is as follows: VTGSDGKLYHIYVCIDGCILLKLAKRGEPRT (SEQ ID NO:3);

[0026] The nucleotide sequence encoding this protein is as follows:

[0027] GTGACAGGTAGTGACGGAAAGCTTTACCATATATATGTGTGCATCGATGGTTGCATACTGCTGAAGCTAGCCAAGAGAGGCGAGCCAAGAACC (SEQ ID NO: 4);

[0028] E2-T1 and E2-T5 were linked together to form the fusion antigen E2-T1 / T5 using a flexible linker (GGGGSGGGGS). The nucleotide sequence of the antigen was artificially synthesized and cloned into the prokaryotic expression vector pET-28a(+), which was then transformed into E. coli BL21(DE3) and expressed by IPTG.

[0029] The amino acid sequence of the obtained fusion protein is as follows:

[0030] NNLPRKGDCRSGNHLGPVSGIYVKPGPVFYQDYMGPVYGGGGSGGGGSVTGSDGKLYHIYVCIDGCILLKLAKRGEPRT (SEQ ID NO:5);

[0031] The sequence of the corresponding nucleotide fragment encoding the gene is as follows:

[0032] AATAACCTACCTAGGAAAGGAGACTGCAGGAGTGGCAACCATCTAGGCCCGGTTAGCGGGATATATGTAAAGCCCGGCCCTGTCTTTTATCAGGACTACATGGGCCCGGTCTACGGTGGTGGTGGTAGCGGTGGTGGTGGTAGCGTGACAGGTAGTGACGGAAAGCTTTACCATATATATGTGTGCATCGATGGTTGCATACTGCTGAAGCTAGCCAAGAGAGGCGAGCCAAGAACC (SEQ ID NO:6).

[0033] SDS-PAGE showed that the fusion protein had a molecular weight of approximately 8.5 kDa and a purity >90%. Figure 1 ELISA assays showed that the reactivity of the fusion antigen E2-T1 / T5 with CSFV-positive porcine serum was significantly higher than that of the individual fragments E2-T1, E2-T5, and E2(T1 / T5). Figure 2 ).

[0034] 2. Constructing subunit vaccines

[0035] The E2-T1 / T5 coding gene fragment was cloned into the EcoRI / XhoI site of the eukaryotic expression vector pVAX1 to construct the recombinant plasmid pVAX1-E2-T1 / T5. The plasmid was verified to be correct by sequencing, with an endotoxin level <0.1 EU / mg (LAL method) and a concentration of 1.2 mg / mL (A260 / A280=1.88), meeting vaccine standards.

[0036] 3. Swine immune experiment

[0037] Thirty-five healthy 6-week-old piglets were purchased and randomly divided into three groups: an experimental group, a control group, and a blank control group (15 piglets in each group). The experimental group was injected intramuscularly with pVAX1-E2-T1 / T5 (100 μg / pig), the control group was injected with the same dose of pVAX1-E2-Full, and the blank control group was injected with an equal volume of PBS buffer. The piglets were immunized three times on days 0, 14, and 28, and a booster immunization was given on day 56.

[0038] Table 1: Serum-specific IgG antibody titers (indirect ELISA)

[0039]

[0040] As shown in Table 1, the experimental group increased the IgG level in pigs, indicating that the prepared subunit vaccine can more effectively activate B cells, induce high-titer neutralizing antibodies, and the effect is much higher than that of the original E2 protein.

[0041] Example 2: Identification and Diagnosis of Classical Swine Fever E2 Subunit Vaccine for the Purification Effect

[0042] This invention establishes a method for purging classical swine fever (CSF) in farms based on a subunit vaccine for CSF E2. The technical route is described below. Figure 2 .

[0043] 1. Investigation and control of wild-type swine fever virus infection

[0044] Spleen, tonsils, and umbilical cord blood can be collected from pigs that abort during pregnancy, have weak, stillborn, or deformed piglets during farrowing, or exhibit nerve tremors in suckling piglets. These samples are then tested for classical swine fever virus (CSF) nucleic acid using qRT-PCR. If the CSF nucleic acid test is positive, the corresponding sow is considered a carrier. Blood samples are then collected to test for E2 antibodies. If the E2 antibody test is negative, the corresponding breeding pig is culled. If the E2 antibody test is positive, enhanced immunization is administered, and close monitoring is maintained.

[0045] 2. Basic antibody monitoring for classical swine fever

[0046] Simultaneously with the first phase, blood samples were randomly collected from breeding sows and replacement gilts based on statistical principles to understand the status of E0 and E2 antibodies against classical swine fever in pig farms, providing guidance for customizing appropriate immunization programs and eradication plans.

[0047] The E0 antibody is an antibody prepared by immunizing pigs with the E0 antigen protein (also known as Erns, Envelope glycoprotein Erns). Naturally infected pigs will only produce antibodies against the E0 protein. However, the E2 subunit vaccine prepared in this invention does not contain the E0 protein and therefore will not induce an E0 antibody response.

[0048] 3. Vaccine selection and immunization

[0049] The classical swine fever (CSF) E2 subunit vaccine prepared using this invention is administered as follows: Breeding pigs are vaccinated twice annually with 2 mL of the CSF E2 subunit vaccine per pig each time; finishing pigs are vaccinated once at 70 days of age with 2 mL of the CSF vaccine per pig each time; and replacement gilts are vaccinated twice at 140 days and 210 days of age with 2 mL of the CSF E2 subunit vaccine per pig each time.

[0050] 4. Screening and culling of the breeding sow herd

[0051] Continuous vaccine monitoring should be conducted on pig farms that have received the E2 subunit vaccine. Quarterly sampling by parity is recommended, and E0 and E2 antibodies against classical swine fever (CSF) should be measured using ELISA. Data on sow reproductive performance, such as litter size and abortion rates, should also be collected. Pigs with negative E0 antibodies and an E2 antibody blocking rate of at least 70% are considered to have no history of CSF infection and are fully resistant to wild-type virus; they can remain in the herd. Pigs with an E2 antibody blocking rate of less than 70% only require a booster immunization, and post-immunization antibody changes should be monitored. Pigs with positive E0 antibodies but no history of exposure to live CSF vaccine may have a history of wild-type virus infection. Regardless of the E2 antibody blocking rate, tonsils should be collected for CSF antigen testing to determine if they are carrying the virus. If the antigen test is positive and the E2 antibody test is negative, the pig should be culled. If the E2 antibody test is positive, the pig should be closely monitored. If the antigen test is negative, it is highly likely to be a false positive for E0 and close monitoring is required. For sows that test positive for pathogens, their piglets will be tested for E0 and E2 antibodies. Piglets that test positive for pathogens or are negative for E2 antibodies will not be used for breeding.

[0052] Through several rounds of screening and culling, pigs that tested positive for classical swine fever (CSF) pathogen and those with immune tolerance were continuously eliminated. Simultaneously, replacement pigs were introduced with pathogen-negative, E2 antibody-qualified replacements to address any remaining issues. This process gradually brought the herd to a state of complete purification, with a CSF E0 antibody positivity rate of ≤10% and a CSF E2 antibody positivity rate of ≥90% in random sampling.

[0053] 5. The stage of complete eradication of swine fever in the entire herd

[0054] Every six months, random sampling of the breeding pig herd is conducted according to a 95% confidence interval, by parity, until the positive rate of classical swine fever pathogen equals 0 (serum E0 antibody detection combined with tonsil qRT-PCR detection), and the positive rate of E2 antibody in the pig herd is ≥95%. If the positive rate of classical swine fever pathogen equals 0 (serum E0 antibody detection combined with tonsil qRT-PCR detection) in two consecutive sampling tests, the eradication is considered successful, and the herd enters the eradication maintenance phase.

[0055] 6. Maintaining the eradication of African swine fever

[0056] Every 6 months, 10% of the pig herd will be randomly inspected to confirm the purification effect and monitor for the occurrence of swine fever. If the swine fever pathogen is detected or the disease occurs, the herd will be re-entered into the basic herd screening and culling stage.

[0057] Example 3: Achieving classical swine fever eradication through immunization with classical swine fever E2 subunit vaccine

[0058] A company began using a classical swine fever E2 subunit vaccine in April 2020, and the results are as follows: Figure 3 From the fourth quarter of 2020 to the fourth quarter of 2022, the CSFV sample positivity rate showed an overall downward trend. In the third and fourth quarters of 2022, the CSFV nucleic acid detection rate was 0% in two separate blood sample tests of pig herds.

[0059] From 2020 to 2022, the overall E2 and E0 antibody positivity rates of the company's sow herds showed that the E2 antibody positivity rate was above 95% for three consecutive years, and the E0 antibody positivity rate decreased from 49% in 2020 to 15% in 2022. Combined with the CSFV nucleic acid positivity rate in 2022, it can be concluded that the company basically achieved swine fever eradication by the end of 2022 and entered the swine fever eradication maintenance phase.

Claims

1. A classical swine fever E2 antigen protein, characterized in that, The amino acid sequence of the antigen protein is SEQ ID NO:5.

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