Dual-replicon expression vector suitable for kluyveromyces marxianus and application of dual-replicon expression vector in preparation of ASFV-P54 nanoparticles

Abstract

The invention belongs to the technical field of biological medicines, and discloses a dual replicon expression vector suitable for kluyveromyces marxianus and application of the dual replicon expression vector in preparation of ASFV-P54 nanoparticles. An applicant prepares a dual-replicon expression vector through screening and assembling, the vector achieves the effects of high conversion rate and high expression efficiency in kluyveromyces marxianus, and the dual-replicon expression vector pGKD32 is shown as SEQ ID NO.2. The invention also discloses a preparation method of the kluyveromyces marxianus expression vector pGKD32. The Kluyveromyces marxianus expression system provided by the invention is utilized by the applicant to successfully express the ASFV-P54 protein in a nanoparticle form, and a new thought is provided for research and development of an ASFV oral immunization strategy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a double replicon expression vector suitable for Kluyveromyces marxianus and its application in preparing ASFV-P54 nanoparticles. Background Art

[0002] African swine fever (ASF) is an acute, febrile, highly contagious disease of pigs caused by infection with African swine fever virus (ASFV). The main symptoms are high fever, cyanosis of the skin, severe bleeding of lymph nodes and internal organs, and a mortality rate of up to 100%, which is a huge threat to the pig farming industry. The World Organization for Animal Health (OIE) lists it as a notifiable animal disease, and my country lists it as a Class I animal disease.

[0003] Kluyveromyces marxianus (KM) is an unconventional yeast that belongs to the Saccharomycetaceae family together with Saccharomyces cerevisiae. It is a safe yeast that has been certified GRAS (Generally recognized as safe) by the U.S. Food and Drug Administration (FDA) and QPS (Qualified Presumption of Safety) 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.

[0004] The applicant successfully isolated a KM-C2 strain (patent number: ZL 103952324A) 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 City, Hubei Province, China for preservation on March 3, 2014 (CCTCC NO: M2014059). The strain has a biodegradation rate of more than 80% for aflatoxin B1, zearalenone, and ochratoxin A, and can also be used for oral detoxification of animals. Therefore, the strain is very suitable as a starting strain and is used in feed additives as an oral carrier carrying ASFV antigens.

[0005] At present, the strain has no nutritional deficiency screening markers that can be used for the edible safety level yeast expression system and no knockout vectors that can be used for genetic modification of 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 achieve genetic manipulation on the yeast genome. Different promoter-terminator combinations and replicon combinations are matched to the strain to provide new candidate expression elements. Summary of the invention

[0006] The object of the present invention is to provide a double replicon expression vector pGKD32 suitable for Kluyveromyces marxianus, which is shown in SEQ ID NO.2.

[0007] Another object of the present invention is to provide an application of a double replicon expression vector pGKD32 in the expression of heterologous proteins in Kluyveromyces marxianus, which is particularly suitable for the preparation of African swine fever subunit vaccines.

[0008] In order to achieve the above object, the present invention adopts the following technical measures:

[0009] The applicant prepared a double replicon expression vector by screening, assembling, and the vector achieved high conversion rate and high expression efficiency in Kluyveromyces marxianus, as follows:

[0010] A double replicon expression vector pGKD32 suitable for Kluyveromyces marxianus is shown in SEQ ID NO.2.

[0011] Double replicon expression vectors containing target genes also fall within the protection scope of the present invention.

[0012] A recombinant auxotrophic Kluyveromyces marxianus is obtained by introducing the above-mentioned double replicon expression vector into auxotrophic Kluyveromyces marxianus, wherein the auxotrophic Kluyveromyces marxianus is obtained by introducing KM-KO-URA3 (shown in SEQID NO.1) into Kluyveromyces marxianus;

[0013] Application of the dual-replicon expression vector pGKD32 in the expression of heterologous proteins in Kluyveromyces marxianus;

[0014] Use of the double replicon expression vector pGKD32 or recombinant auxotrophic Kluyveromyces marxianus in the preparation of subunit vaccines;

[0015] Among the applications described above, the preferred method is to prepare African swine fever subunit vaccine.

[0016] In the application described above, preferably, an ASFV-P54 protein oral vaccine in the form of nanoparticles is prepared, specifically, the Cap-linker-P54 sequence (shown in SEQ ID NO.3) is homologously recombined with the double replicon expression vector pGKD32, and then introduced into the auxotrophic Kluyveromyces marxianus for expression.

[0017] The deposit number of the Kluyveromyces marxianus described above is CCTCC NO: M2014059.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] The dual replication system has been successfully used in the construction of plasmids. However, the existing dual replication system has only been successfully applied in shuttle plasmids, that is, the two replications belong to different hosts. Its purpose is to allow plasmids to replicate themselves in different expression hosts. For example, Escherichia coli and yeast, Escherichia coli and mammalian cells, etc., are generally propagated in Escherichia coli and then transformed into expression hosts to express foreign genes. This shuttle vector constructed by the dual replication system has only one replication function in different hosts, so the two replications generally do not interact with each other, and it is impossible to combine the replication characteristics of the two replications in the same host.

[0020] In the present invention, the applicant creatively connected several preferred replicons in series, resulting in a dual replicon system that can combine the excellent phenotypes of two replicons. Furthermore, the applicant used the Kluyveromyces marxianus expression system provided by the present invention to successfully express the ASFV-P54 protein in the form of nanoparticles, providing new ideas for the development of ASFV oral immunization strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the fluorescence intensity of pGKD1, pGKD2, pGKD3, and pGKD4 vectors carrying EGFP protein.

[0022] Figure 2 is the transformation efficiency of pG1, pG2, pG3, and pGKD3 vectors carrying EGFP protein.

[0023] Figure 3 is the fluorescence intensity of pG1, pG2, pG3, and pGKD3 vectors carrying EGFP protein.

[0024] Figure 4 It is the stability of pG1, pG2, pG3, and pGKD3 vector transformants carrying EGFP protein.

[0025] Figure 5is the transformation efficiency of pGKD31, pGKD32, pGKD33, pGKD3, and pG2 vectors carrying EGFP protein.

[0026] Figure 6 is the fluorescence intensity of pGKD31, pGKD32, pGKD33, pGKD3, and pG2 vectors carrying EGFP protein.

[0027] Figure 7 It is the stability of transformants of pGKD31, pGKD32, pGKD33, pGKD3, and pG2 vectors carrying EGFP protein.

[0028] Figure 8 The results of KM-G / cap-P54 expression and purification are shown in Figure 1. Lane 1 is the marker, lane 2 is the empty sample, lane 3 is KM-G / cap-P54, lane 4 is the crude KM-G / cap-P54, and lane 5 is the refined KM-G / cap-P54.

[0029] Fig. 9 This is the electron microscopic identification result of the purified KM-G / cap-P54 nanoparticles after negative staining.

[0030] Fig.10 is the serum P54 IgG level after immunization.

[0031] Fig.11 is the fecal P54 IgA level after immunization. DETAILED DESCRIPTION

[0032] 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.

[0033] Embodiment 1:

[0034] Construction of URA3 auxotrophic host strain G using a CRISPR Cas9-based scarless genome modification method

[0035] 1.1 Construction of KM scarless genome knockout vector KM-KO and screening of antibiotic tolerance

[0036] 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:

[0037] 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.

[0038] 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.

[0039] 1.2 Construction of URA3 auxotrophic host strain KM-G

[0040] 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.

[0041] 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`-AAACAGGAAGTTACGAAAGAACCT-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 SEQID No.1) was obtained. 2ug KM-KO-URA3 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.

[0042] Embodiment 2:

[0043] Construction of expression vector, screening and optimization of expression elements for the auxotrophic host strain KM-G:

[0044] 2.1 Screening of promoter and terminator combinations of KM-G strain

[0045] 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).

[0046] PUC57 is the PUC57 plasmid.

[0047] The URA3 gene sequence information of KM was obtained 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 S acⅡ. Linearized PUC57-PKD1 and PUC57-URA3 were connected with linearized P uc57-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 E GFP 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 pGKD1, pGKD2, pGKD3, and pGKD4 vectors, respectively, and primers with different promoter and terminator homology arms were used to amplify the EGFP fragment. Subsequently, 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 E GFP 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.

[0048] 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.

[0049] 2.2 KM-G strain replicon screening

[0050] 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, 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).

[0051] 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.

[0052] 2.3 Screening of KM-G dual replicon system

[0053] 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.

[0054] 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.

[0055] Embodiment 3:

[0056] Construction and oral immunogenicity of KM-G / cap-P54 nanoparticles

[0057] 3.1 Construction and identification of KM-G / cap-P54 nanoparticles

[0058] Cap-P54 was designed as follows: the Cap gene was based on the GenBank PCV2d KX808467 sequence; the ASFV P54 gene was based on the GenBank ASFV MK333180, and only the antigen extracellular region was retained; the flexible linker 4×GGS was used to connect the two, and a V5 tag was added to the C-terminus, i.e., Cap-linker-P54-V5. The KM codon table was used for optimization and synthesis into the PUC57 vector. The Cap-linker-P54 sequence with PGK promoter and terminator homology arms (shown in SEQ ID NO.3) was amplified, the fragment was recovered by gel and homologously recombined with the pGKD32 vector linearized with EcoRⅠ and HindⅢ. pGKD32-Cap-P54 was obtained, and the recombinant product was transformed into KM-G, spread onto a solid SD plate and cultured at 30℃ for 36-48h, and positive transformants were identified by colony PCR. Positive transformants were transferred to 5 ml SD bacterial bottles, cultured at 220 rpm / 30 °C for 24 h, and then transferred to 400 ml SD conical bottles for further culture for 72 h. The bacteria were collected and washed twice with PBS, and then crushed under high pressure. KM-G / cap-P54 nanoparticles were purified by 40% ammonium sulfate and molecular sieves. SDS-PAGE analysis was performed, and negative staining transmission electron microscopy was performed for identification.

[0059] The results showed that compared with the control group, there was an additional protein band at 44KD in KM-G / cap-P54 ( Figure 8 ), this band is cap-P54 protein. Electron microscopy results show that cap-P54 can self-assemble into nanoparticles ( Fig. 9 ).

[0060] 3.2 Immunogenicity of oral immunization with KM-G / cap-P54 nanoparticles

[0061] Fifteen 6-8 week old female SPF Balb / c mice were randomly divided into groups, with 5 mice in each group. Group 1 was the monomer P54 immunization group; Group 2 was the cap-P54 nanoparticle immunization group; Group 3 was the control group. The immunization group was orally administered with 200ug protein each time; the control group was orally administered with an equal volume of PBS. Oral immunization was performed on days 0, 14, and 28, respectively. Orbital blood was collected on days 28 and 42, respectively, to detect P54 IgG levels and fecal P54 IgA levels.

[0062] like Fig.10 , 11As shown in the results, after oral immunization, the immunized groups can produce serum IgG and mucosal IgA against P54 antigen compared with the control group (P < 0.05). Compared with P54 monomer antigen, P54 nanoparticles can produce higher levels of serum IgG and mucosal IgA. With the increase in the number of immunizations, the serum IgG and mucosal IgA produced by P54 nanoparticles are significantly higher than those of P54 monomer antigen (P < 0.05). This shows that oral immunization of ASFV P54 nanoparticles prepared by the present invention can significantly improve humoral immunity and mucosal immunity.

[0063] 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 double replicon expression vector pGKD32 suitable for Kluyveromyces marxianus, as shown in SEQ ID NO.

2.

2. The double replicon expression vector according to claim 1 containing a target gene.

3. A recombinant auxotrophic Kluyveromyces marxianus, wherein the recombinant auxotrophic Kluyveromyces marxianus is obtained by introducing the expression vector of claim 2 into the auxotrophic Kluyveromyces marxianus, and the auxotrophic Kluyveromyces marxianus is obtained by introducing the knockout vector shown in SEQ ID NO.1 into Kluyveromyces marxianus.

4. Use of the double replicon expression vector pGKD32 according to claim 1 in expressing heterologous proteins in Kluyveromyces marxianus.

5. Use of the double replicon expression vector pGKD32 according to claim 1 or the recombinant auxotrophic Kluyveromyces marxianus according to claim 3 in the preparation of a subunit vaccine.

6. The use according to claim 5, wherein the subunit vaccine is an African swine fever subunit vaccine.

7. According to the use of claim 6, the African swine fever subunit vaccine is an ASFV-P54 protein oral vaccine in the form of nanoparticles, specifically, the Cap-linker-P54 sequence shown in SEQ ID NO.3 is homologously recombined with the double replicon expression vector pGKD32 according to claim 1, and then introduced into the auxotrophic Kluyveromyces marxianus for expression.

Images