Method for efficiently and stably expressing canine parvovirus VP2 nanoparticles by kluyveromyces marxianus

Abstract

The invention belongs to the technical field of biological medicines, and discloses a method for efficiently and stably expressing canine parvovirus VP2 nanoparticles by kluyveromyces marxianus. The dual-replicon expression vector applicable to the kluyveromyces marxianus is prepared by the applicant through screening and assembling, the vector realizes the effects of high conversion rate and high expression efficiency in the kluyveromyces marxianus, and the dual-replicon expression vector pGKD32 is shown as SEQ ID NO.2. An applicant successfully expresses the canine parvovirus VP2 in a nanoparticle form by using the kluyveromyces marxianus expression system provided by the invention, and a new thought is provided for research and development of canine parvovirus oral immunization strategies.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a method for Kluyveromyces marxianus to efficiently and stably express canine parvovirus VP2 nanoparticles. Background Art

[0002] Canine parvovirus (CPV) is originally derived from feline panleukopenia virus (FPV), which is derived from the mutation of feline parvovirus gene. The gene sequence similarity between canine parvovirus and feline parvovirus is as high as 99%, but the two have different antigenic characteristics, host range and hemagglutination characteristics. Since the discovery of canine parvovirus in the United States in 1978, the virus has spread rapidly around the world and seriously threatened the development of the dog breeding industry. Due to the high mutation rate of the virus and the failure to vaccinate dogs in time, the morbidity and mortality of infected animals are high. Kluyveromyces marxianus (KM) is an unconventional yeast belonging to the same family as Saccharomyces cerevisiae, and is a safe yeast with GRAS (Generally Recognized as Safe) certification by the U.S. Food and Drug Administration (FDA) and QPS (Qualified Presumption of Safety) certification by 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 KM-C2 strain (CN103952324A) in a natural pig farm in Huangpi, Hubei Province in 2013, and sent it to the China Center for Type Culture Collection of Wuhan University, Wuhan, Hubei Province, China for preservation on March 3, 2014 (CCTCC NO: M2014059). The biodegradation rate of the strain for aflatoxin B1, zearalenone and ochratoxin A is more than 80%, and it can also be used for oral detoxification of animals. Therefore, the strain is very suitable as a starting strain and can be used as an oral carrier carrying CPV antigen in feed additives.

[0003] At present, the strain does not have nutritional deficiency screening markers that can be used for edible safety level yeast expression system and knockout vectors that can be genetically modified to the strain. There is also no expression vector suitable for the strain. In view of this, the present invention establishes a suitable genetic manipulation system for the strain to realize genetic manipulation on the yeast genome. Different promoter-terminator combinations and replicon combinations are matched to the strain to provide novel candidate expression elements. In addition, the present invention is applied to CPV nanoparticle genetic engineering vaccines, which lays the foundation for further developing CPV vaccines and also provides reference for the preparation of other animal pathogenic genetic engineering vaccines.

[0004] In response to the above problems, the applicant provided a double replicon expression vector suitable for Kluyveromyces marxianus and filed a patent application with application number 2024104862538 (not disclosed before the submission of this application). In this application, the applicant newly constructed a double replicon expression vector suitable for Kluyveromyces marxianus, which has the advantages of good stability, large exogenous protein expression, and high conversion rate. In the present invention, the applicant further used the replicon to prepare canine parvovirus nanoparticles in preparation for canine parvovirus subunit vaccine. Summary of the invention

[0005] The purpose of the present invention is to provide a method for efficiently and stably expressing canine parvovirus VP2 nanoparticles by Kluyveromyces marxianus. The method is simple, easy to implement and suitable for commercial production.

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

[0007] In order to achieve the above object, the present invention adopts the following technical measures:

[0008] A method for efficiently and stably expressing canine parvovirus VP2 nanoparticles by Kluyveromyces marxianus KM-C2 comprises the following steps:

[0009] The polynucleotide shown in SEQ ID NO.3 was homologously recombined with the pGKD32 vector linearized by EcoRⅠ and HindⅢ, and then transformed into the auxotrophic Kluyveromyces marxianus;

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

[0011] In the above method, preferably, the auxotrophic Kluyveromyces marxianus is obtained by introducing the targeted URA3 knockout vector KM-KO-URA3 (shown in SEQ ID No. 1) into Kluyveromyces marxianus;

[0012] In the method described above, preferably, the deposit number of the Kluyveromyces marxianus is CCTCC NO: M2014059.

[0013] The protection scope of the present invention also includes:

[0014] Application of the nanoparticles prepared by the above method in the preparation of canine parvovirus subunit vaccine.

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

[0016] The applicant has newly constructed a double replicon expression vector suitable for Kluyveromyces marxianus, which has the advantages of good stability, large exogenous protein expression and high conversion rate. In the present invention, the applicant further uses the replicon to prepare canine parvovirus nanoparticles to prepare for canine parvovirus subunit vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

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

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

[0024] Figure 8The expression and purification results of KM-G / CPV-VP2. 1: Supernatant after empty cell crushing. 2: Whole cell crushing. 3: Supernatant after crushing. 4: Precipitate after crushing. 5: Supernatant after 35% ammonium sulfate precipitation. 6: Precipitate re-dissolved in 35% ammonium sulfate. 7: Supernatant after re-dissolving the precipitate. 8: Precipitate after re-dissolving the precipitate. 9 / 10: Molecular sieve purification.

[0025] Fig. 9 This is the electron microscopic identification result of the purified KM-G / CPV-VP2 nanoparticles after negative staining. DETAILED DESCRIPTION

[0026] The starting strain used in this example is Kluyeromyces marxianus C2 (authorized patent number: ZL103952324A), which was sent to the China Center for Type Culture Collection for preservation on March 3, 2014, and is classified and named: Kluyeromyces marxianus C2, with a preservation number of CCTCC NO: M2014059, and is located at Wuhan University, Wuhan, Hubei Province, China.

[0027] Embodiment 1:

[0028] Construction of URA3 auxotrophic host strain G based on CRISPR Cas9-based scarless genome modification

[0029] 1.1 Construction of KM scarless genome knockout vector KM-KO and screening of antibiotic tolerance

[0030] The Kluyveromyces marxianus (KM) scarless genome knockout vector KM-KO uses three expression frames, TEF1-BleoR-CYC1, Cas9, and GAP-gRNA scaffold, two replicons, Pichia pastoris panARS and Escherichia coli ori, and designs 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, and the 182nd base C of the TEF1 promoter was mutated to T to eliminate the Bsa I restriction site; the 171st base of TEF1, the BleoR gene, and the 18th base after the CYC1 terminator were amplified, and the 9th base G after the CYC1 terminator was mutated to A to eliminate the Bsa I restriction site; the ori replicon of the pPICZaA plasmid was amplified. The above three fragments were recovered by gel and fusion PCR was performed to obtain a 1967bp fragment containing TEF1, BleoR, CYC1 terminator and Escherichia coli ori replicon, which was sequenced and identified by Qingke Bio. The Cas9 expression frame and gRNA expression frame panARS sequences were synthesized by GenScript into the PUC57 vector and amplified separately, and 4600bp and 2097bp fragments were recovered by gel. The above three fragments were homologously recombined and transformed into DH5α competent cells. After selecting a single clone for expansion culture, verify that the gRNA expression frame sequence is correct; verify that the Cas expression frame is correct. Pick the double-positive plasmid for activation, extract the plasmid, and use Pme I to cut it. The band size of 1919bp+6700bp is the correct KM scarless genome knockout vector, named KM-KO.

[0032] About 2ug of KM-KO plasmid was used to electroporate KM-C2, and different concentrations of Zeocin antibiotics were applied for sensitivity screening (100ug / mL, 200ug / mL) to verify the presence of the plasmid. The results showed that KM-C2 monoclones of equal size could be grown on YPD plates with different antibiotic concentrations, indicating that the screening system was normal. In order to increase the screening positive rate, 200ug / ml antibiotic concentration was finally selected as the screening concentration.

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

[0034] URA3 was selected as the nutritional deficiency screening marker, and the URA3 gene sequence information of KM was obtained by retrieving the NCBI database. The following primers were designed: 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 with a length of 629 bp was amplified using URA-F1 and URA-R1, and the lower homologous arm URA3D of URA3 with a length of 492 bp was amplified using URA-F2 and URA-R2. The URA3U and URA3D fragments were subjected to fusion PCR to obtain the 1192 bp fragment URA3HX without the core region of the URA3 gene at positions 187-432.

[0035] The gRNA online design platform (http: / / www.rgenome.net / cas-designer / ) was used to design the URA3 knockout gRNA, gRNA-△URA3 (5`-AGGTTCTTTCGTAACTTCCT-3`). Qingke Biosynthesized gRNA-△URA3-F (5`-CGTC-AGGTTCTTTCGTAACTTCCT-3`) and gRNA-△URA3-R (5`-AAACAGGAAGTT ACGAAAGAACCT-3`). The gRNA fragment was obtained by annealing the upstream and downstream primers of gRNA using PCR. KM-KO was digested with Bsa I, and the digestion product was recovered by gel. The gRNA fragment was connected to the linearized KM-KO vector using solution I, and transformed into DH5α, GPD-F and CYCT-R to identify positive single clones. The positive single clone was expanded and cultured to extract the plasmid, and the targeted URA3 knockout vector KM-KO-URA3 (shown in SEQ ID No. 1) was obtained. 2ug KM-KO-UR A3 and 8ug URA3HX fragments were electrotransferred to Km-C2, and Zeocin-YPD solid plates were coated. 8 transformants were picked, and the colony PCR was used for identification. The positive colonies were expanded and cultured and continuously subcultured to verify the growth stability. They were simultaneously coated on SC plates and YPD plates. The strains that could not grow on SC plates and grew normally on YPD plates were URA3 auxotrophic strains. Since the replication stability of the Pichia pastoris panARS replicon on the KM-KO-URA3 vector was poor, the plasmid would be lost if it was continuously subcultured in YPD medium without antibiotics. It could not grow when it was coated on YPD plates containing antibiotics, indicating that the transferred KM-KO-URA3 plasmid had been eliminated. The strain was allowed to be transformed multiple times, and the URA3 auxotrophic strain with successful knockout of the URA3 core region, good growth stability and elimination of the KM-KO-URA3 plasmid was named KM-G strain.

[0036] Embodiment 2:

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

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

[0039] In order to achieve green and antibiotic-free production, the following strategy was used to design the expression vector of the KM-G strain. Four promoter-terminator combinations were designed and synthesized (reference DOI: 10.3389 / fbioe.2019.00097), the KanR expression cassette and the Escherichia coli ori in the multiple cloning site region between the promoter-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), Puc57-TDH3: TDH3 (P) -KanR-ori-TDH3 (T).

[0040] PUC57 is the PUC57 plasmid.

[0041] The URA3 gene sequence information of KM was retrieved by retrieving the NCBI database, and the URA3 expression cassette PUC57-URA3 with a truncated promoter sequence was designed and synthesized, with SalⅠ and KpnⅠ restriction sites at both ends. The plasmid PUC57-PKD1 containing the PKD 1 sequence gene (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 connected to linearized Puc57-INU, Puc57-NC1, Puc57-PGK, and Puc57-TDH3, respectively. pGKD1, pGKD2, pGK D3, and pGKD4 vectors were obtained. 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. EcoRⅠ and HindⅢ were used to double-digest the pGKD1, pGKD2, pGKD3, and pGKD4 vectors, and primers with different promoter and terminator homology arms were used to amplify the EGFP fragment. Then, the EGFP gene sequence with homology arms was homologously recombined with the linearized vectors pGKD1, pGKD2, pGKD3, and pGKD4, and the recombinant products were transformed into KM-G competent cells and coated on SD plates. Transformants with fluorescence were selected by a blue light instrument, and the EGFP gene was amplified using a colony PCR kit. After sequencing verification, the culture was expanded, and 0.1OD seed solution was taken and the fluorescence intensity at 400nm was measured using 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 KM-G strain, the expression intensity is: PGK combination > NC1 combination > INU combination ≈ TDH3 combination. When the pGKD vector is double-digested with EcoRⅠ and HindⅢ, the resistance gene KanR and the E. coli element ori are removed to achieve the purpose of green and non-antibiotic production.

[0043] 2.2 KM-G strain replicon screening

[0044] The replicon genes PUC57-C1, PUC57-C2, and PUC57-C3 containing C1 / CenD, C2 / CEN5, and C3 / CEN6 (reference DOI:10.3389 / fbioe.2019.00097) were synthesized, and the pGKD3, PUC57-C1, PUC57-C2, and PUC57-C3 vectors were digested with KpnⅠ and SacⅡ. The linearized C1, C2, and C3 were constructed into the linearized pGKD3, respectively, 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 with EcoRⅠ and HindⅢ, and the linearized vectors were homologously recombined with the EGFP fragments with the PGK promoter terminator combination homology arms. The recombinant products were transformed into KM-G competent cells and spread on SD plates for 48 hours. Three replicates were made for each group, and the transformation rate was calculated (transformation rate = number of transformants / vector DNA mass). Subsequently, the three largest single clones were picked out and cultured in 5ml SD medium overnight, and transferred to 5ml SD medium with an OD600nm of 0.05 for 48 hours. 0.1OD bacterial solution was taken and the fluorescence intensity at 400nm was measured using a fluorescence microplate reader. In addition, the three largest single clones in each group were picked out and grown in 5ml YPD liquid medium overnight. The culture was diluted and spread on YPD or SD plates to determine the stability of the plasmid (determination of 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 transformation rate. The average transformation rates of different plasmids were: pG2>pG1>pG3>pGKD3( Figure 2 ). Although pGKD3 has the lowest conversion rate, its average fluorescence intensity is the highest. Average fluorescence intensity: pGKD3>pG2>pG1>pG3( Figure 3 In addition, the stability of pGKD3 can reach 72.09%, which is much higher than pG1, pG2, and pG3. Average plasmid stability: pGKD3>pG1≈pG2≈pG3( Figure 4). pKD1 is an endogenous multicopy plasmid identified in Kluyveromyces lactis, and the plasmid based on pKD1 is the only multicopy plasmid successfully applied to KM. pKD1 contains three major open reading frames, namely A, B and C. Previously, the team of Professor Lv Hong of Fudan University demonstrated the role of gene A in maintaining the high copy number of pKD1-based plasmids in KM. The absence of B or C genes affects the stable replication of pKD1-based plasmids in KM, and this defect cannot be solved by trans-expression of B and C genes. Therefore, PKD1 is necessary for KM to express foreign proteins at high levels and stably. However, the conversion rate of PKD1 replicon is significantly lower than that of the other replicons screened in this example. Here, we try to combine the other replicons (C1, C2, C3) screened in this example with PK D1 to construct a dual replicon system, in the hope that a dual replicon system that can combine the excellent phenotypes of the two replicons can emerge.

[0046] 2.3 Screening of KM-G dual replicon system

[0047] The C1, C2, and C3 replicon sequences with the PGK terminator homology arms and the URA3 promoter homology arms of the pG1, pG2, and pG3 plasmids were amplified respectively. The pGKD3 was single-digested with SalⅠ, and the C1, C2, and C3 replicon sequences with homology arms were recombined with the SalⅠ linearized pGKD3 vector respectively. After transformation and identification, the double replicon vectors with C1, C2, and C3-PKD1 were named pGKD31, pGKD32 (SEQ ID No. 2), and pGKD33 respectively. The conversion rate, fluorescence intensity, and stability were determined using the method 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 terms of transformation efficiency, fluorescence intensity and replication stability, among which the transformation efficiency and fluorescence intensity were even significantly lower than pG1, and the replication stability was also at a low level; pGKD33 also failed to improve in terms of transformation efficiency, fluorescence intensity and replication stability, among which the transformation efficiency was even significantly lower than pG3, and the fluorescence intensity and replication stability were also at a low level. Therefore, when the double replicon is used in the same expression host, its function may be affected by a variety of unknown factors, resulting in the expression host not only failing to inherit the excellent properties of the two replicons, but may inherit the inferior phenotype. However, the pGKD32 vector not only inherited the excellent phenotype of the pGKD3 vector in terms of fluorescence intensity and stability, but also inherited the excellent phenotype of pG2 in terms of replication stability. In addition, compared with pGKD3, it even significantly improved the 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 the two replicons, but may inherit the inferior phenotypes. Only pGKD32 (C2 and PKD1 combination) can inherit the superior phenotypes of both, and the transformation efficiency is comparable to that of the superior phenotype C2 replicon, and the fluorescence intensity and stability are significantly improved compared to the superior phenotype PKD1. Therefore, in the subsequent examples, we use the pGKD32 expression vector as the preferred vector for the KM-G strain.

[0049] Embodiment 3:

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

[0051] The CPV-VP2 gene was optimized with reference to GenBank Sequence ID: WOC23116.1 and synthesized into the PUC57 vector using the KM codon table. The CPV-VP2 sequence with the PGK promoter and terminator homology arms was amplified (shown in SEQ ID NO.3), and the fragment was recovered by gel and homologously recombined with the pGKD32 vector linearized with EcoRⅠ and HindⅢ. pGKD32-CPV-VP2 was obtained, and the recombinant product was transformed into KM-G, coated on a solid SD plate and cultured at 30℃ for 36-48h, and positive transformants were identified by colony PCR. Positive transformants were picked into 5ml SD bacterial bottles, cultured at 220rpm / 30℃ for 24h, and transferred to 400ml SD conical bottles for further culture for 72h. The bacteria were collected and washed twice with PBS for high-pressure crushing, and the KM-G / CPV-VP2 nanoparticles were crudely purified by 35% ammonium sulfate and refined by molecular sieves. SDS-PAGE analysis and negative staining transmission electron microscopy were performed. The results showed that compared with the control group, KM-G / CPV-VP2 had an additional protein band at 68KD ( Figure 8), this band is CPV-VP2 protein. Electron microscopy results show that CPV-VP2 can self-assemble into nanoparticles ( Fig. 9 ).

[0052] The nanoparticles have the potential to be prepared as canine parvovirus subunit vaccines.

[0053] The specific embodiments of the present invention are described in detail above, but they are only 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 made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A method for efficiently and stably expressing canine parvovirus VP2 nanoparticles by Kluyveromyces marxianus, comprising the following steps: The polynucleotide shown in SEQ ID NO.3 was homologously recombined with the pGKD32 vector linearized by EcoR Ⅰ and Hind Ⅲ, and then transformed into the auxotrophic Kluyveromyces marxianus; The polynucleotide sequence of the pGKD32 vector is shown in SEQ ID NO.

2.

2. The method according to claim 1, wherein the auxotrophic Kluyveromyces marxianus is obtained by introducing a knockout vector KM-KO-URA3 targeting URA3 into Kluyveromyces marxianus; The polynucleotide sequence of the knockout vector KM-KO-URA3 is shown in SEQ ID NO.

1.

3. The method according to claim 2, wherein the Kluyveromyces marxianus has a deposit number of CCTCC NO: M2014059.

4. Use of the nanoparticles prepared by the method of claim 1 in preparing canine parvovirus oral vaccine.

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