A method for efficient and stable expression of canine parvovirus VP2 nanoparticles in *Kluyveromyces martensii*

Abstract

This invention belongs to the field of biomedical technology and discloses a method for efficiently and stably expressing canine parvovirus VP2 nanoparticles in *Kluyveromyces martensii*. The applicant prepared a double-replicant expression vector suitable for *Kluyveromyces martensii* through screening, assembly, and analysis. This vector achieved high conversion rate and high expression efficiency in *Kluyveromyces martensii*. The double-replicant expression vector pGKD32 is shown in SEQ ID NO.2. Using the *Kluyveromyces martensii* expression system provided by this invention, the applicant successfully expressed canine parvovirus VP2 in nanoparticle form, providing a new approach for the development of oral immunization strategies for canine parvovirus.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for the efficient and stable expression of canine parvovirus VP2 nanoparticles by Kluyveromyces martensii. Background Technology

[0002] Canine parvovirus (CPV) originated from feline panleukopenia virus (FPV), which evolved from a mutation of feline parvovirus. Canine and feline parvoviruses share a 99% genetic sequence similarity, but they differ in antigenic characteristics, host range, and hemagglutination properties. Since its discovery in the United States in 1978, canine parvovirus has spread rapidly worldwide, seriously threatening the development of the dog breeding industry. High viral mutation rates and delayed vaccination of dogs lead to high morbidity and mortality rates in infected animals. Kluyveromyces marxianus (KM) is an unconventional yeast belonging to the family Saccharomycetaceae, 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 (Quacularized 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 of CPV antigens in feed additives.

[0003] 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, applying this invention to CPV nanoparticle genetically engineered vaccines lays the foundation for further CPV vaccine development and provides a reference for the preparation of genetically engineered vaccines for other animal pathogens.

[0004] 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 canine parvovirus nanoparticles, preparing for a canine parvovirus subunit vaccine. Summary of the Invention

[0005] The purpose of this invention is to provide a method for the efficient and stable expression of canine parvovirus VP2 nanoparticles by Kluyveromyces martensii. The method is simple, easy to implement, and suitable for commercial production.

[0006] Another object of the present invention is to provide the application of the nanoparticles prepared by the above method in the preparation of canine parvovirus subunit vaccines.

[0007] To achieve the above objectives, the present invention adopts the following technical measures:

[0008] A method for efficient and stable expression of canine parvovirus VP2 nanoparticles using Kluyveromyces martensii KM-C2 includes the following steps:

[0009] 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.

[0010] The polynucleotide sequence of the pGKD32 vector is shown in SEQ ID NO.2.

[0011] 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.

[0012] Preferably, the Kluyveromyces martensii strain described above has the preservation number CCTCC NO: M2014059.

[0013] The scope of protection of this invention also includes:

[0014] The application of nanoparticles prepared by the above method in the preparation of canine parvovirus subunit vaccines.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] The applicant has constructed a novel double-replicon expression vector suitable for Kluyveromyces martensii, which exhibits advantages such as good stability, high expression levels of exogenous proteins, and high conversion efficiency. In this invention, the applicant further utilizes this replicon to prepare canine parvovirus nanoparticles, preparing for a canine parvovirus subunit vaccine. Attached Figure Description

[0017] Figure 1 The fluorescence intensity represents the fluorescence intensity of the pGKD1, pGKD2, pGKD3, and pGKD4 vectors carrying the EGFP protein.

[0018] Figure 2 The transformation efficiency of pG1, pG2, pG3, and pGKD3 vectors carrying EGFP protein.

[0019] Figure 3 The fluorescence intensity represents the values ​​of the pG1, pG2, pG3, and pGKD3 vectors carrying the EGFP protein.

[0020] Figure 4 To assess the stability of pG1, pG2, pG3, and pGKD3 vector transformants carrying the EGFP protein.

[0021] Figure 5 The transformation efficiency of pGKD31, pGKD32, pGKD33, pGKD3, and pG2 vectors carrying EGFP protein.

[0022] Figure 6 The fluorescence intensity represents the fluorescence intensity of the pGKD31, pGKD32, pGKD33, pGKD3, and pG2 vectors carrying the EGFP protein.

[0023] Figure 7 To assess the stability of transformants from pGKD31, pGKD32, pGKD33, pGKD3, and pG2 vectors carrying the EGFP protein.

[0024] Figure 8The purification results for KM-G / CPV-VP2 expression are as follows: 1: Empty vector lysis supernatant. 2: Whole cell lysis. 3: Lysis supernatant. 4: Lysis precipitate. 5: Supernatant after 35% ammonium sulfate precipitation. 6: Redissolution of 35% ammonium sulfate precipitate. 7: Supernatant after redissolution of precipitate. 8: Precipitate after redissolution. 9 / 10: Molecular sieve purification.

[0025] Figure 9 The electron microscopy results are for the purified KM-G / CPV-VP2 nanoparticles after negative staining. Detailed Implementation

[0026] 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.

[0027] Example 1:

[0028] URA3 auxotrophic host strain G was constructed using a CRISPR-Cas9-based traceless genome editing method.

[0029] 1.1 Construction of the KM-KO genome knockout vector without scarring and screening for antibiotic resistance

[0030] The Kluyveromyces martensii (KM) scarless genome knockout vector KM-KO uses three expression cassettes: TEF1-BleoR-CYC1, Cas9, and GAP-gRNAscaffold, two replicons: Pichia pastoris (panARS) and Escherichia coli (ori), and incorporates two BsaI point mutations. The preparation process is as follows:

[0031] 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.

[0032] 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.

[0033] 1.2 Construction of URA3 auxotrophic host strain KM-G

[0034] URA3 was selected as a marker for nutritional malnutrition screening. 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`-TCT ATTGAAGACACATGTAG-AGAGGGTTGTTAATGCTTG-3`), and URA-R2 (5`-GTATACAATG TGACGCAATGC-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.

[0035] 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.

[0036] Example 2:

[0037] Construction, screening, and optimization of expression elements for expression vectors of auxotrophic host strain KM-G:

[0038] 2.1 Screening of promoter and terminator combinations for KM-G strain

[0039] 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).

[0040] PUC57 is the same as PUC57 plasmid.

[0041] 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.

[0042] 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.

[0043] 2.2KM-G strain replicon screening

[0044] 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).

[0045] 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.

[0046] 2.3KM-G Dual Replication Subsystem Screening

[0047] 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.

[0048] 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.

[0049] Example 3:

[0050] Construction and identification of KM-G / CPV-VP2 nanoparticles

[0051] The CPV-VP2 gene sequence was optimized using the KM codon table and synthesized into the PUC57 vector, referencing GenBank Sequence ID: WOC23116.1. The CPV-VP2 sequence (SEQ ID NO. 3) with homologous arms of the PGK promoter and terminator was amplified, the fragment was recovered via gel electrophoresis, and homologously recombined with the EcoRI and HindIII linearized pGKD32 vector. pGKD32-CPV-VP2 was obtained, and the recombinant product was transformed into KM-G, plated onto 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 subjected to high-pressure disruption. KM-G / CPV-VP2 nanoparticles were purified by crude purification with 35% ammonium sulfate and further purified by molecular sieve. SDS-PAGE analysis and negative staining transmission electron microscopy identification were performed. Results showed that, compared to the control group, KM-G / CPV-VP2 had an additional protein band at 68KD. Figure 8The band represents the CPV-VP2 protein. Electron microscopy results show that CPV-VP2 can self-assemble into nanoparticles. Figure 9 ).

[0052] These nanoparticles have the potential to be used to prepare canine parvovirus subunit vaccines.

[0053] 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 efficient and stable expression of canine parvovirus VP2 nanoparticles in Kluyveromyces martensii, comprising the following steps: The polynucleotide shown in SEQ ID NO.3 was homologously recombined with the pGKD32 vector linearized with EcoR I and Hind III and then transformed into auxotrophic Kluyveromyces martensii. The polynucleotide sequence of the pGKD32 vector is shown in SEQ ID NO.2; The aforementioned auxotrophic Kluyveromyces martensii was 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, and the preservation number of the Kluyveromyces martensii is CCTCC NO: M2014059.

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