A method for preparing avian infectious bursal disease VP2 nanoparticle oral vaccine by using k. marxianus and application thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses a method for preparing an avian infectious bursal disease VP2 nanoparticle oral vaccine by using Kluyveromyces marxianus and application thereof. The applicant assembles and prepares a double replicon expression vector suitable for Kluyveromyces marxianus through screening, and the vector achieves the effect of high conversion rate and high expression efficiency in Kluyveromyces marxianus. The double replicon expression vector pGKD32 is shown in SEQ ID NO. 2. The applicant successfully expresses the avian infectious bursal disease VP2 in the form of nanoparticles by using the Kluyveromyces marxianus expression system provided in the application, and provides a new idea for the research and development of the avian infectious bursal disease virus oral immunization strategy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method and application of preparing an oral vaccine for avian infectious bursal disease VP2 nanoparticles using Kluyveromyces martensii. Background Technology

[0002] Infectious bursal disease (IBD) is one of the diseases required to be reported by the World Organisation for Animal Health (OIE). IBD has a high incidence rate, short course, and significant harm, seriously threatening the development of the poultry industry. Therefore, the prevention and treatment of IBD are of great importance to poultry farming. IBD is characterized by highly swollen or atrophied bursa of Fabricius, highly swollen kidneys with urate deposition, and hemorrhage at the junction of the bursa of Fabricius, leg muscles, pectoral muscles, proventriculus, and gizzard. The bursa of Fabricius is a central immune organ unique to poultry, the site of B cell maturation. Induced and mature B cells, upon stimulation by antigens, can transform into plasma cells to produce antibodies, participating in specific humoral immunity. IBD mainly affects chicks and young chickens aged 3-12 weeks, leading to necrosis and atrophy of the bursa of Fabricius. This results in the destruction of a large number of immature B lymphocytes in the bursa of Fabricius in young chickens, causing severe immunosuppression, making them prone to secondary infections and immune failure, leading to huge economic losses. Therefore, this disease has received high attention from the poultry industry and has also become a hot topic of research in the academic community. VP2 protein is the major structural protein of IBDV, encoding 441 amino acids with a molecular weight of approximately 37-40 kDa, accounting for 51% of the total viral protein. Located on the outer surface of the viral particle, it forms the nucleocapsid backbone together with VP3. VP2 contains the virus's major neutralizing epitopes, which can induce the body to produce serologically specific protective neutralizing antibodies, protecting the host from IBDV infection. It also possesses the ability to self-assemble into viral particles, making it a target protein for research on IBDV subunit vaccines.

[0003] Kluyveromyces marxianus (KM) is an unconventional yeast belonging to the Saccharomycetaceae family, the same family as Saccharomyces cerevisiae. It is a safe yeast with GRAS (Generally Recognized as Safe) certification from the US Food and Drug Administration (FDA) and QPS (Qualified Presumption of Safety) certification from the European Food Safety Authority (EFSA). It has also been approved as a new food ingredient by the National Health and Family Planning Commission of China. The applicant successfully isolated a strain KM-C2 (CN103952324A) from a natural pig farm in Huangpi District, Hubei Province in 2013, and deposited it at the China Center for Type Culture Collection (CCTCC) of Wuhan University, Wuhan, Hubei Province, China on March 3, 2014 (CCTCC NO: M2014059). This strain exhibits a biodegradation rate of over 80% for aflatoxin B1, zearalenone, and ochratoxin A, and can also be used for oral detoxification in animals. Therefore, this strain is highly suitable as a starting strain and as an oral carrier for IBDV nanoparticles in feed additives.

[0004] Currently, there are no nutritional auxotroph screening markers for this strain that can be used in food-safe yeast expression systems, nor are there knockout vectors suitable for genetic modification of this strain. There are also no suitable expression vectors for this strain. Therefore, this invention establishes a suitable genetic manipulation system for this strain, enabling genetic manipulation on the yeast genome. Different promoter-terminator and replicon combinations are matched to this strain to provide novel candidate expression elements. Furthermore, this invention is applied to IBDV-VP2 nanoparticle genetically engineered vaccines, laying the foundation for further IBDV vaccine development and providing a reference for the preparation of genetically engineered vaccines for other animal pathogens.

[0005] To address the aforementioned issues, the applicant has provided a double-replicon expression vector suitable for *Kluyveromyces martensii* and filed a patent application (application number 2024104862538, not published prior to this application). In this application, the applicant novelly constructed a double-replicon expression vector suitable for *Kluyveromyces martensii*, which exhibits advantages such as good stability, high exogenous protein expression levels, and high conversion efficiency. In this invention, the applicant further utilizes this replicon to prepare an oral vaccine for avian infectious bursal disease VP2 nanoparticles, laying the foundation for oral vaccines against avian infectious bursal disease. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing an oral vaccine for avian infectious bursal disease VP2 nanoparticles using Kluyveromyces martensii. The method is simple, easy to implement, and suitable for commercial production.

[0007] Another object of the present invention is to provide the application of the nanoparticles prepared by the above method in the preparation of avian infectious bursal disease subunit vaccines.

[0008] To achieve the above objectives, the present invention adopts the following technical measures:

[0009] A method for preparing an oral vaccine for avian infectious bursal disease VP2 nanoparticles using Kluyveromyces martensii includes the following steps:

[0010] The polynucleotide shown in SEQ ID NO.3 was homologously recombined with the pGKD32 vector linearized with EcoRI and HindIII and then transformed into auxotrophic Kluyveromyces martensii.

[0011] The polynucleotide sequence of the pGKD32 vector is shown in SEQ ID NO.2.

[0012] Preferably, the auxotrophic Kluyveromyces martensii is obtained by introducing the URA3-targeting knockout vector KM-KO-URA3 (shown in SEQ ID No. 1) into Kluyveromyces martensii.

[0013] Preferably, the Kluyveromyces martensii strain described above has the preservation number CCTCC NO: M2014059.

[0014] The scope of protection of this invention also includes:

[0015] The application of nanoparticles prepared by the above method in the preparation of avian infectious bursal disease subunit oral vaccine.

[0016] The application of the auxotrophic Kluyveromyces martensii containing nanoparticles prepared by the above method in the preparation of an oral vaccine for avian infectious bursal disease subunit.

[0017] The application of the auxotrophic Kluyveromyces martensii containing nanoparticles prepared by the above method in the preparation of feed additives. The application of the auxotrophic Kluyveromyces martensii containing nanoparticles prepared by the above method in increasing animal weight. Compared with the prior art, the present invention has the following advantages:

[0018] The applicant has constructed a novel double-replicon expression vector suitable for *Kluyveromyces martensii*, which exhibits advantages such as good stability, high expression level of exogenous protein, and high conversion rate. In this invention, the applicant further utilized this replicon to prepare VP2 nanoparticles for avian infectious bursal disease. Whether administered via injection or orally using *Kluyveromyces martensii* expressing the nanoparticles, the nanoparticles effectively protected chickens after challenge. Furthermore, the applicant found that chickens immunized orally with feed showed a significant increase in body weight, indicating that the feed additive prepared from the auxotrophic *Kluyveromyces martensii* containing nanoparticles not only possesses antiviral properties against infectious bursal disease but also promotes growth. Attached Figure Description

[0019] Figure 1 The fluorescence intensity represents the fluorescence intensity of the pGKD1, pGKD2, pGKD3, and pGKD4 vectors carrying the EGFP protein.

[0020] Figure 2 The transformation efficiency of pG1, pG2, pG3, and pGKD3 vectors carrying EGFP protein.

[0021] Figure 3 The fluorescence intensity represents the values ​​of the pG1, pG2, pG3, and pGKD3 vectors carrying the EGFP protein.

[0022] Figure 4 To assess the stability of pG1, pG2, pG3, and pGKD3 vector transformants carrying the EGFP protein.

[0023] Figure 5 The transformation efficiency of pGKD31, pGKD32, pGKD33, pGKD3, and pG2 vectors carrying EGFP protein.

[0024] Figure 6 The fluorescence intensity represents the fluorescence intensity of the pGKD31, pGKD32, pGKD33, pGKD3, and pG2 vectors carrying the EGFP protein.

[0025] Figure 7 To assess the stability of transformants from pGKD31, pGKD32, pGKD33, pGKD3, and pG2 vectors carrying the EGFP protein.

[0026] Figure 8 The results of KM-G / IBDV-VP2 expression purification;

[0027] Among them: lane 1 is the marker, lane 2 is the empty control, and lanes 3, 4 and 5 are the whole bacteria, crude pure and refined pure samples of KM-G / IBDV-VP2, respectively.

[0028] Figure 9 The electron microscopy results are for the purified KM-G / IBDV-VP2 nanoparticles after negative staining.

[0029] Figure 10 This is a schematic diagram of serum IBDV-VP2 IgG levels after immunization.

[0030] Figure 11 This is a diagram showing the final weight after the experiment.

[0031] Figure 12 This diagram illustrates the survival rates of different groups after IBDV infection.

[0032] Figure 13 This is a schematic diagram of bursal lesions in different chickens after necropsy following the completion of the experiment. Detailed Implementation

[0033] The starting strain used in this embodiment is Kluyveromyces marxianus C2 (patent number: ZL103952324A), which was deposited at the China Center for Type Culture Collection on March 3, 2014. The strain is classified and named as Kluyveromyces marxianus C2, with accession number CCTCC NO: M2014059, and the address is Wuhan University, Wuhan, Hubei Province, China.

[0034] Example 1:

[0035] URA3 auxotrophic host strain G was constructed using a CRISPR-Cas9-based traceless genome editing method.

[0036] 1.1 Construction of the KM-KO genome knockout vector without scarring and screening for antibiotic resistance

[0037] The Kluyveromyces martensii (KM) scarless genome knockout vector KM-KO uses three expression cassettes: TEF1-BleoR-CYC1, Cas9, and GAP-gRNA scaffold, two replicons: Pichia pastoris (panARS) and Escherichia coli (ori), and incorporates two BsaI point mutations. The preparation process is as follows:

[0038] Using the Pichia pastoris expression vector plasmid pPICZaA as a template, the first 192 bases of the TEF1 promoter in pPICZaA were amplified. Simultaneously, the C-to-T mutation at position 182 of the TEF1 promoter eliminated the Bsa I restriction site. The fragments starting at position 171 of TEF1, the BleoR gene, and up to position 18 after the CYC1 terminator were amplified. Simultaneously, the G-to-A mutation at position 9 after the CYC1 terminator eliminated the Bsa I restriction site. The ori replicon of the pPICZaA plasmid was also amplified. These three fragments were recovered by gel electrophoresis and fused with PCR to obtain a 1967 bp fragment containing TEF1, BleoR, the CYC1 terminator, and the *E. coli* ori replicon, which was identified by sequencing by Qingke Biotechnology. The Cas9 expression cassette and the gRNA expression cassette panARS sequences were synthesized by GenScript into the PUC57 vector and amplified separately. Fragments of 4600 bp and 2097 bp were recovered by gel electrophoresis. Homologous recombination was performed on the above three fragments, and the cells were transformed into DH5α competent cells. Single clones were selected and expanded for culture to verify the correctness of the gRNA expression cassette sequence and the Cas expression cassette. Double-positive plasmids were selected for activation, and after plasmid extraction, they were digested with Pme I. A band size of 1919bp + 6700bp was found to be the correct KM-KO genome knockout vector, named KM-KO.

[0039] KM-C2 plasmid (approximately 2 μg) was electroporated with different concentrations of Zeocin antibiotic for sensitivity screening (100 μg / mL, 200 μg / mL) to verify plasmid presence. Results showed that uniformly sized KM-C2 monoclonal antibodies grew on YPD plates with different antibiotic concentrations, indicating a normal screening system. To improve the positive screening rate, an antibiotic concentration of 200 μg / mL was ultimately selected as the screening concentration.

[0040] 1.2 Construction of URA3 auxotrophic host strain KM-G

[0041] URA3 was selected as a screening marker for nutritional deficiencies. The URA3 gene sequence information of KM was obtained by searching the NCBI database, and the following primers were designed and constructed: URA-F1 (5`-CAAGGATGCTCATCACAATACG-3`), URA-R1 (5`-GCAAGCATTAACAACCCTCTCTACATGTGTCTTCAATAGACAG-3`), URA-F2 (5`-TCTATTGAAGACACATGTAG-AGAGGGTTGTTAATGCTTG-3`), and URA-R2 (5`-GTATACAATGTGACGCAATGC-3`). The upper homologous arm URA3U of URA3 (629 bp) was amplified using URA-F1 and URA-R1, and the lower homologous arm URA3D of URA3 (492 bp) was amplified using URA-F2 and URA-R2. The URA3U and URA3D fragments were then fused by PCR to obtain the 1192 bp fragment URA3HX, which has the core region of the URA3 gene removed from positions 187-432.

[0042] URA3 knockout gRNA was designed using the online gRNA design platform (http: / / www.rgenome.net / cas-designer / ), specifically gRNA-△URA3 (5`-AGGTTCTTTCGTAACTTCCT-3`). Qingke Biotechnology synthesized gRNA-△URA3-F (5`-CGTC-AGGTTCTTTCGTAACTTCCT-3`) and gRNA-△URA3-R (5`-AAACAGGAAGTT ACGAAAGAACCT-3`). The gRNA fragments were obtained by annealing the upstream and downstream primers using PCR. KM-KO was digested with Bsa I, and the digestion products were recovered via gel electrophoresis. The gRNA fragments were ligated into a linearized KM-KO vector using Solution I, and the resulting vectors were transformed into DH5α, GPD-F, and CYCT-R for identification of positive single clones. Positive single clones were expanded and plasmids were extracted to obtain the URA3-targeting knockout vector KM-KO-URA3 (SEQ ID No. 1). 2 μg of KM-KO-URA3 and 8 μg of URA3HX fragments were electroporated into Km-C2 plates, plated on Zeocin-YPD solid plates, and 8 transformants were picked for colony PCR identification. Positive colonies were expanded and passaged continuously to verify growth stability. They were simultaneously plated on SC and YPD plates. The strain that could not grow on SC plates but grew normally on YPD plates was the URA3 auxotrophic strain. Because the Pichia pastoris panARS replicon on the KM-KO-URA3 vector has poor replication stability, continuous passage in antibiotic-free YPD medium resulted in plasmid loss. The strain could not grow when plated on YPD plates containing antibiotics, indicating that the introduced KM-KO-URA3 plasmid had been eliminated. The strain was allowed to be modified multiple times. The URA3 auxotrophic strain that successfully knocked out the URA3 core region, had good growth stability, and eliminated the KM-KO-URA3 plasmid was named strain KM-G.

[0043] Example 2:

[0044] Construction, screening, and optimization of expression elements for expression vectors of auxotrophic host strain KM-G:

[0045] 2.1 Screening of promoter and terminator combinations for KM-G strain

[0046] To achieve green, antibiotic-free production, the following strategy was used to design expression vectors for the KM-G strain. Four promoter-terminator combinations were designed and synthesized (refer to DOI:10.3389 / fbioe.2019.00097), with the KanR expression cassette and the *E. coli* *ori* multiple cloning site region between the promoter and terminator. The four promoter-terminator combinations are: Puc57-INU: INU(P)-KanR-ori-INU(T), Puc57-NC1: NC1(P)-KanR-ori-NC1(T), Puc57-PGK: PGK(P)-KanR-ori-PGK(T), and Puc57-TDH3: TDH3(P)-KanR-ori-TDH3(T).

[0047] PUC57 is the same as PUC57 plasmid.

[0048] The URA3 gene sequence information of KM was obtained by searching the NCBI database, and a URA3 expression cassette PUC57-URA3 with a truncated promoter sequence was designed and synthesized, with SalI and KpnI restriction sites at both ends. A plasmid PUC57-PKD1 containing the PKD1 sequence (PKD1 sequence reference: DOI:10.1002 / biot.202100382) was designed and synthesized, with KpnI and SacII restriction sites at both ends. Puc57-INU, Puc57-NC1, Puc57-PGK, and Puc57-TDH3 were double-digested with SacII and SalI. PUC57-URA3 was double-digested with SalI and KpnI. PUC57-PKD1 was double-digested with KpnI and SacII. Linearized PUC57-PKD1 and PUC57-URA3 were ligated to linearized Puc57-INU, Puc57-NC1, Puc57-PGK, and Puc57-TDH3, respectively, to obtain pGKD1, pGKD2, pGKD3, and pGKD4 vectors. EGFP was used as a marker gene to screen the expression intensity of different promoter-terminator combinations. The EGFP gene was optimized according to the KM codon table and synthesized into the PUC57 vector to obtain PUC57-EGFP. The pGKD1, pGKD2, pGKD3, and pGKD4 vectors were double-digested using EcoRI and HindIII, respectively. EGFP fragments were amplified using primers with homologous arms containing different promoter and terminator arms. Subsequently, the EGFP gene sequences with homologous arms were homologously recombinated with the linearized vectors pGKD1, pGKD2, pGKD3, and pGKD4, respectively. The recombination products were transformed into KM-G competent cells and plated on SD plates. Fluorescent transformants were selected using a blue light analyzer, and the EGFP gene was amplified using a colony PCR kit. After sequencing verification, the cells were expanded, and the fluorescence intensity at 400 nm was measured using a 0.1 OD seed culture with a fluorescence microplate reader.

[0049] The fluorescence intensity results are as follows: KM-G / pGKD3>KM-G / pGKD2>KM-G / pGKD1≈KM-G / pGKD4 Figure 1 Therefore, for the KM-G strain, the expression intensity is: PGK combination > NC1 combination > INU combination ≈ TDH3 combination. Because the resistance gene KanR and the E. coli element ori are removed during double digestion of the pGKD vector using EcoRⅠ and HindⅢ, the goal of green, antibiotic-free production is achieved.

[0050] 2.2 Screening of KM-G strain replicons

[0051] The replicon genes PUC57-C1, PUC57-C2, and PUC57-C3 containing C1 / CenD, C2 / CEN5, and C3 / CEN6 (refer to DOI:10.3389 / fbioe.2019.00097) were synthesized. The vectors pGKD3, PUC57-C1, PUC57-C2, and PUC57-C3 were digested with KpnI and SacII. The linearized C1, C2, and C3 were constructed into the linearized pGKD3, replacing the PKD1 replicon in pGKD3. The vectors carrying the C1, C2, and C3 replicons were named pG1, pG2, and pG3, respectively. pG1, pG2, pG3, and pGKD3 were double-digested using EcoRI and HindIII, respectively. The linearized vectors were then homologously recombinated with EGFP fragments containing a PGK promoter-terminator combination homologous arm. The recombination products were transformed into KM-G competent cells and plated on SD plates for 48 h. Each group was performed in triplicate, and the transformation efficiency was calculated (transformation efficiency = number of transformants / vector DNA mass). Subsequently, the three largest single clones from each group were selected and cultured overnight in 5 ml of SD medium. They were then transferred to 5 ml of SD medium at an OD of 600 nm (0.05) and cultured for 48 h. The 0.1 OD bacterial culture was used to measure the fluorescence intensity at 400 nm using a fluorescence microplate reader. Simultaneously, the three largest single clones from each group were selected and grown overnight in 5 ml of YPD liquid medium. The cultures were diluted and plated on YPD or SD plates, and the plasmid stability was measured (plasmid stability = number of colonies formed on SD plate / number of colonies formed on YPD plate).

[0052] The results showed that pG2 had the highest conversion rate. The average conversion rates of different plasmids were: pG2 > pG1 > pG3 > pGKD3. Figure 2 Although pGKD3 has the lowest conversion rate, it has the highest average fluorescence intensity. Average fluorescence intensity: pGKD3 > pG2 > pG1 > pG3 Figure 3 Furthermore, pGKD3 exhibits a stability of 72.09%, significantly higher than pG1, pG2, and pG3. Average plasmid stability: pGKD3 > pG1 ≈ pG2 ≈ pG3 Figure 4pKD1 is an endogenous multicopy plasmid identified in Kluyveromyces lactis, and pKD1-based plasmids are the only multicopy plasmids successfully applied to KM. pKD1 contains three main open reading frames, namely A, B, and C. Previously, Professor Lü Hong's team at Fudan University demonstrated the role of gene A in maintaining a high copy number of pKD1-based plasmids in KM. Deletion of genes B or C affects the stable replication of pKD1-based plasmids in KM, and this deficiency cannot be resolved by trans-expression of genes B and C. Therefore, pKD1 is essential for high-level and stable expression of exogenous proteins in KM. However, the transformation efficiency of pKD1 replicons is significantly lower than that of the other replicons screened in this example. Here, we attempt to combine the remaining replicons (C1, C2, C3) screened in this example with pKD1 to construct a dual replicon system, hoping to create a dual replicon system that can combine the desirable phenotypes of both replicons.

[0053] 2.3KM-G Dual Replication Subsystem Screening

[0054] The C1, C2, and C3 replicon sequences of pG1, pG2, and pG3 plasmids, containing partial homologous arms of the PGK terminator and partial homologous arms of the URA3 promoter, were amplified, respectively. pGKD3 was digested with SalI, and the C1, C2, and C3 replicon sequences with homologous arms were recombinated with SalI-linearized pGKD3 vectors. After transformation and confirmation, the double replicon vectors containing C1, C2, and C3-PKD1 were named pGKD31, pGKD32 (SEQ ID No. 2), and pGKD33, respectively. Their transformation rate, fluorescence intensity, and stability were determined using the method described in Example 2.2.

[0055] like Figure 5 , 6As shown in Figures 7 and 8, compared with the single-replicon vector pGKD3, the double-replicon vector pGKD31 failed to improve in transformation efficiency, fluorescence intensity, and replication stability. In fact, its transformation efficiency and fluorescence intensity were significantly lower than pG1, and its replication stability was also low. Similarly, pGKD33 also failed to improve in these areas, with its transformation efficiency significantly lower than pG3, and its fluorescence intensity and replication stability remaining low. Therefore, when double replicons are used in the same expression host, their function may be affected by various unknown factors, leading the expression host to not only fail to inherit the superior characteristics of both replicons but also potentially inherit inferior phenotypes. However, the pGKD32 vector not only inherited the superior phenotype of the pGKD3 vector in terms of fluorescence intensity and stability but also inherited the superior phenotype of pG2 in terms of replication stability. Furthermore, compared with pGKD3, it even significantly improved fluorescence intensity and stability. In summary, in the expression host KM-G, the dual-replication system does not necessarily inherit the superior replication phenotypes of both replicons; instead, it may inherit the inferior phenotypes. Only pGKD32 (a combination of C2 and PKD1) can inherit the superior phenotypes of both, achieving transformation efficiency comparable to the superior C2 replicon, and exhibiting significantly improved fluorescence intensity and stability compared to the superior PKD1 phenotype. Therefore, in subsequent examples, we will use the pGKD32 expression vector as the preferred vector for the KM-G strain.

[0056] Example 3:

[0057] Construction and Evaluation of the Protective Effect of KM-G / IBDV-VP2 Nanoparticle Oral Vaccine

[0058] 3.1 Construction and Identification of KM-G / IBDV-VP2 Nanoparticles

[0059] The IBDV-VP2 gene was referenced from GenBank Sequence ID: UWL63285.1. A His tag and flexible linker were introduced at the N-terminus, and the gene was optimized using the KM codon table and synthesized into the PUC57 vector. The His-IBDV-VP2 sequence (SEQ ID NO.3) with homologous arms of the PGK promoter and terminator was amplified, the fragment was recovered by gel electrophoresis, and homologously recombined with the EcoRI and HindIII linearized pGKD32 vector. pGKD32-IBDV-VP2 was obtained, and the recombinant product was transformed into KM-G, plated on solid SD plates, and incubated at 30°C for 36-48 h. Positive transformants were identified by colony PCR. Positive transformants were picked into 5 ml SD bacterial bottles, incubated at 220 rpm / 30°C for 24 h, and then transferred to 400 ml SD Erlenmeyer flasks for further incubation for 72 h. Bacterial cells were collected, washed twice with PBS, and then subjected to high-pressure disruption. KM-G / IBDV-VP2 nanoparticles were purified by crude purification with 40% ammonium sulfate and refined with molecular sieves. SDS-PAGE analysis and negative-stained transmission electron microscopy identification were performed.

[0060] The results showed that, compared with the control group, KM-G / IBDV-VP2 had an additional protein band at 42.1 KD. Figure 8 The band represents the IBDV-VP2 protein. Electron microscopy results show that IBDV-VP2 can self-assemble into nanoparticles. Figure 9 ).

[0061] Immunogenicity and protective efficacy against viral challenge of 3.2KM-G / IBDV-VP2 nanoparticle oral vaccine

[0062] Thirty-two SPF White Leghorn laying hens were selected and randomly divided into four groups.

[0063] Group 1 was the PBS control group, with a volume of 500 uL;

[0064] Group 2 was the VP2 injection immunization group (purified VP2 nanoparticles were fully emulsified with white oil adjuvant at a volume ratio of 1:2, and the dose of VP2 nanoparticles was 100ug / animal);

[0065] Group 3 was the oral immunization group (2 mL of 500OD bacterial lysate per animal by gavage, and 1 mg of VP2 nanoparticles per animal).

[0066] Group 4 was the feed-mixed immunization group (mixed with the same dose as Group 3).

[0067] Immunization was performed on days 7 and 21, oral challenge was performed on day 35, and euthanasia was performed on day 39. Blood samples were collected on days 14, 21, and 28 to detect antigen-specific IgG levels. At the end of the trial, survival rate, final weight, bursal disease lesions were observed and photographed.

[0068] like Figure 10 As shown, after oral immunization, compared with the control group, all immunized groups produced serum IgG against the IBDV-VP2 antigen. With increasing immunization frequency, the antigen-specific IgG level gradually increased. Antibody levels: injection immunization group > oral immunization group > dipped immunization group. This indicates that the IBDV-VP2 nanoparticles prepared in this invention can enhance humoral immune responses through both oral and injection immunization. Figure 11 As shown, compared with the control group and the injected immunization group, oral immunization significantly increased the final weight of chickens, indicating that the vaccine, as a feed additive, also has a growth-promoting effect. Figure 12 As shown, the challenged control group experienced mortality on the second day after challenge, with only one chicken in extremely poor condition surviving by the fourth day, while all chickens in the immunized group survived. Figure 13 As shown, after necropsy, the bursa of Fabricius in the challenge control group was severely atrophied and hemorrhagic; the injection immunization group showed slight atrophy and hemorrhagic symptoms; the feed-mixed immunization group showed slight swelling; and the bursa of Fabricius in the gavage group was in a relatively normal state.

[0069] Conventional IBDV challenge protection experiments typically do not use high challenge doses, and generally do not cause chicken mortality after challenge. The assessment is usually based on the bursal index and lesion score. However, in this example, to compare the protective effects of different immunization methods as thoroughly as possible, a highly virulent tissue virus (i.e., clinical isolate) was selected for challenge (infectious dose of 10 MID), a dose significantly higher than that used in conventional challenge protection experiments.

[0070] Four days after the viral challenge, only one chicken in the control group, in extremely poor condition, survived. In contrast, all chickens in the immunized group survived, but their bursae showed varying degrees of lesions. The reproducibility between the different groups was good, indicating that this embodiment can effectively evaluate the immunoprotective effect of different immunization methods and has important reference value for the development of oral vaccines and the prevention and control of IBDV.

[0071] The specific embodiments of the present invention have been described in detail above, but they are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A method for preparing an oral vaccine for avian infectious bursal disease VP2 nanoparticles using Kluyveromyces martensii, comprising the following steps: After homologous recombination of the polynucleotide shown in SEQ ID NO.3 with the pGKD32 vector linearized with EcoR Ⅰ and Hind Ⅲ, it was transformed into auxotrophic Kluyveromyces martensii to obtain auxotrophic Kluyveromyces martensii containing VP2 nanoparticles of avian infectious bursal disease. After the bacteria were broken up, separated and purified, VP2 nanoparticles of avian infectious bursal disease were obtained. The polynucleotide sequence of the pGKD32 vector is shown in SEQ ID NO.2; the auxotrophic Kluyveromyces martensii is obtained by introducing the URA3-targeting knockout vector KM-KO-URA3 into Kluyveromyces martensii; the polynucleotide sequence of the knockout vector KM-KO-URA3 is shown in SEQ ID NO.1; the preservation number of the Kluyveromyces martensii is CCTCC NO: M2014059.

2. The use of the auxotrophic Kluyveromyces martensii yeast containing VP2 nanoparticles of avian infectious bursal disease prepared by the method of claim 1 in the preparation of an oral vaccine for avian infectious bursal disease subunits.

3. The application of the auxotrophic Kluyveromyces martensii containing VP2 nanoparticles of avian infectious bursal disease prepared by the method of claim 1 in the preparation of chicken feed additives.

4. The use of the auxotrophic Kluyveromyces martensii containing VP2 nanoparticles of avian infectious bursal disease prepared by the method of claim 1 in the preparation of a chicken weight-promoting drug.

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