A dual-replicon expression vector suitable for Kluyveromyces marxianus and its application in the preparation of ASFV-P54 nanoparticles

Abstract

The present invention belongs to the field of biomedical technology, and discloses a dual-replicon expression vector applicable to Kluyveromyces marxianus and its application in the preparation of ASFV-P54 nanoparticles. Through screening, assembly and preparation, the applicant prepared a dual-replicon expression vector, which achieved high conversion rate and high expression efficiency in Kluyveromyces marxianus. The dual-replicon expression vector pGKD32 is shown as SEQ ID NO.2. By using the Kluyveromyces marxianus expression system provided by the present invention, the applicant successfully expressed the ASFV-P54 protein in the form of nanoparticles, providing a new idea for the research and development of ASFV oral immunization strategy.

Description

Technical Field

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

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

[0003] Kluyveromyces marxianus (KM) is an unconventional yeast, belonging to the same Saccharomycetaceae family as Saccharomyces cerevisiae. It is a safe yeast certified by the GRAS (Generally Recognized as Safe, food additive safety evaluation) of the US Food and Drug Administration (FDA) and the QPS (Qualified Presumption of Safety, safety qualification certification) of the European Food Safety Authority (EFSA), and has also been approved as a new food raw material by the Chinese National Health and Family Planning Commission.

[0004] The applicant successfully isolated a strain KM-C2 (authorized patent number: ZL 103952324A) from a natural breeding local pig farm in Huangpi, Hubei in 2013, and sent it to the China Center for Type Culture Collection, Wuhan University, Wuhan, Hubei, China for preservation on March 3, 2014 (CCTCC NO: M2014059). The biodegradation rate of this strain to aflatoxin B1, zearalenone, and ochratoxin A is over 80%, and it can also be used for oral detoxification of animals. Therefore, this strain is very suitable as a starting strain and as an oral carrier carrying ASFV antigen for application in feed additives.

[0005] Currently, there are no auxotrophic selection markers for the yeast expression system at the edible safety level and knockout vectors for genetic modification of this strain. Nor is there an expression vector suitable for this strain. In view of this, the present invention establishes a suitable genetic operation system for this strain to achieve genetic operations on the yeast genome. Different promoter-terminator combinations and replicon combinations are matched for this strain to provide novel candidate expression elements. Summary of the Invention

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

[0007] Another object of the present invention is to provide the application of the dual-replicon expression vector pGKD32 in the heterologous protein expression of Kluyveromyces marxianus, and this application is particularly suitable for the preparation of African swine fever subunit vaccines.

[0008] To achieve the above object, the present invention takes the following technical measures:

[0009] The applicant screened, assembled and prepared a dual-replicon expression vector, which achieved high transformation efficiency and high expression efficiency in Kluyveromyces marxianus, specifically as follows:

[0010] A dual-replicon expression vector pGKD32 suitable for Kluyveromyces marxianus, as shown in SEQ ID NO.2.

[0011] The dual-replicon expression vector containing the target gene also belongs to the protection scope of the present invention.

[0012] A recombinant auxotrophic Kluyveromyces marxianus is obtained by introducing the above dual-replicon expression vector into auxotrophic Kluyveromyces marxianus, and the auxotrophic Kluyveromyces marxianus is obtained by introducing KM-KO-URA3 (shown in SEQI D NO.1) into Kluyveromyces marxianus;

[0013] The application of the dual-replicon expression vector pGKD32 in the heterologous protein expression of Kluyveromyces marxianus;

[0014] The application of the dual-replicon expression vector pGKD32 or recombinant auxotrophic Kluyveromyces marxianus in the preparation of subunit vaccines;

[0015] In the above applications, preferably, it is for the preparation of African swine fever subunit vaccines.

[0016] In the above-mentioned application, preferably, the prepared oral vaccine of ASFV-P54 protein in the form of nanoparticles is specifically obtained by homologous recombination of the Cap-linker-P54 sequence (shown in SEQ ID NO.3) with the dual replicon expression vector pGKD32 and then introducing it into auxotrophic Kluyveromyces marxianus for expression.

[0017] The preservation number of the above-mentioned Kluyveromyces marxianus 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 for plasmid construction. However, the existing dual replicon systems have only been successfully applied in shuttle plasmids, that is, the two replicons belong to different hosts respectively. The purpose is to allow the plasmid to self-replicate in different expression hosts. Such as Escherichia coli and yeast, Escherichia coli and mammalian cells, etc. Generally, it is propagated in Escherichia coli and then transformed into the expression host to express foreign genes. The shuttle vector constructed by this dual replicon system has only one replicon functioning in different hosts. Therefore, the two replicons generally do not interact with each other and cannot combine the replication characteristics of the two replicons in the same host.

[0020] In the present invention, the applicant creatively concatenates several preferred replicons to obtain a dual replicon system that can combine the excellent phenotypes of the two replicons. Further, the applicant uses the Kluyveromyces marxianus expression system provided by the present invention to successfully express the ASFV-P54 protein in the form of nanoparticles, providing a new idea for the research and development of the ASFV oral immunization strategy. Brief Description of the Drawings

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

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

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

[0024] Figure 4 The stabilities of the transformants of the pG1, pG2, pG3, and pGKD3 vectors carrying the EGFP protein.

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

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

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

[0028] Figure 8 The expression and purification results of KM-G / cap-P54. Lane 1 is Marker, lane 2 is the empty vector sample, lane 3 is KM-G / cap-P54, lane 4 is the crudely purified KM-G / cap-P54, and lane 5 is the highly purified KM-G / cap-P54.

[0029] Figure 9 The electron microscopy identification results of negatively stained highly purified KM-G / cap-P54 nanoparticles.

[0030] Figure 10 The level of P54 IgG in serum after immunization.

[0031] Figure 11 The level of P54 IgA in feces after immunization. Detailed implementation methods

[0032] The starting strain used in this example is Kluyveromyces marxianus C2 (authorized patent number: ZL103952324A). This strain was sent to the China Center for Type Culture Collection for preservation on March 3, 2014. The taxonomic name is Kluyveromyces marxianus C2, and the preservation number is CCTCC NO: M2014059. The address is Wuhan University, Wuhan, Hubei Province, China.

[0033] Example 1:

[0034] Construction of URA3 auxotrophic host strain G strain based on the CRISPR Cas9-based scarless genome editing method

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

[0036] The markerless genomic knockout vector KM-KO of Kluyveromyces marxianus (KM) adopts three expression cassettes of TEF1-BleoR-CYC1, Cas9, and GAP-gRNA scaffold, two replicons of 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, amplify the first 192 bases before the TEF1 promoter in pPICZaA, and at the same time mutate the 182nd base C of the TEF1 promoter to T to eliminate the Bsa I restriction site; amplify from the 171st base of TEF1, the BleoR gene, to the 18th base after the CYC1 terminator, and at the same time mutate the 9th base G after the CYC1 terminator to A to eliminate the Bsa I restriction site; amplify the ori replicon of the pPICZaA plasmid. Gel extraction and recovery of the above three fragments and perform fusion PCR to obtain a 1967bp fragment containing TEF1, BleoR, CYC1 terminator, and Escherichia coli ori replicon, which is identified by Tsingke Biological Sequencing. The Cas9 expression cassette and the gRNA expression cassette panARS sequence are synthesized by GenScript into the PUC57 vector and amplified separately. Gel extraction and recovery of the 4600bp and 2097bp fragments. Homologous recombination of the above three fragments and transformation into DH5α competent cells. Select monoclonal colonies for expanded culture and then verify that the gRNA expression cassette sequence is correct; verify that the Cas expression cassette is correct. Pick double-positive plasmids for activation. After plasmid extraction, use Pme I digestion. The band sizes of 1919bp + 6700bp are the correct KM markerless genomic knockout vector, named KM-KO.

[0038] Electrotransform KM-C2 with about 2μg of the KM-KO plasmid, coat different concentrations of Zeocin antibiotics for sensitivity screening (100μg / mL, 200μg / mL), and verify the presence of the plasmid. The results show that KM-C2 monoclonal colonies of equal size can grow on YPD plates with different antibiotic concentrations, indicating that the screening system is normal. To improve the screening positive rate, a 200μg / ml antibiotic concentration is finally selected as the screening concentration.

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

[0040] URA3 was selected as the auxotrophic selection marker. The URA3 gene sequence information of KM was retrieved through the NCBI database, and the following primers were designed and synthesized: URA-F1 (5`-CAAGGATGCTCATCACAATACG-3`), URA-R1 (5`-GCAAGCATTAACAACCCTCTCTACATGTGTCTTCAATAGACAG-3`), URA-F2 (5`-TCT ATTGAAGACACATGTAG-AGAGGGTTGTTAATGCTTG-3`), and URA-R2 (5`-GTATACAATG TGACGCAATGC-3`). URA-F1 and URA-R1 were used to amplify the upstream homologous arm URA 3U of URA3 with a length of 629 bp, and URA-F2 and URA-R2 were used to amplify the downstream homologous arm URA3D of URA3 with a length of 492 bp. The URA3U and URA3D fragments were subjected to fusion PCR to obtain a fragment URA3HX with a length of 1192 bp, which removed the core region at positions 187-432 of the URA3 gene.

[0041] Design the URA3 knockout gRNA using the online gRNA design platform (http: / / www.rgenome.net / cas-designer / ), gRNA-△URA3 (5`-AGGTTCTTTCGTAACTTCCT-3`). Tsingke Biological synthesized gRNA-△URA3-F (5`-CGTC-AGGTTCTTTCGTAACTTCCT-3`) and gRNA-△URA3-R (5`-AAACAGGAAGTTACGAAAGAACCT-3`). Use PCR to anneal the upstream and downstream primers of the gRNA to obtain the gRNA fragment. Digest KM-KO with Bsa I and recover the digested product by gel extraction. Use solutionⅠ to ligate the gRNA fragment with the linearized KM-KO vector, transform it into DH5α, and identify positive monoclonal colonies with GPD-F and CYCT-R. Expand the culture of positive monoclonal colonies and extract plasmids to obtain the targeting URA3 knockout vector KM-KO-URA3 (shown in SEQ ID No.1). Electroporate 2 μg of KM-KO-URA3 and 8 μg of URA3HX fragment into Km-C2, coat on Zeocin-YPD solid plates, pick 8 transformants, and identify them by colony PCR. Expand the culture of positive colonies and continuously passage to verify growth stability. Synchronously coat on SC plates and YPD plates. The strain that cannot grow on SC plates but can grow normally on YPD plates is the URA3 auxotrophic strain. Since the replication stability of the Pichia pastoris panARS replicon on the KM-KO-URA3 vector is poor, the plasmid will be lost after continuous passage in YPD medium without antibiotics. Coating it on YPD plates containing antibiotics cannot grow, indicating that the transferred KM-KO-URA3 plasmid has been eliminated, allowing the strain to be modified multiple times. Name the URA3 auxotrophic strain that has successfully knocked out the core region of URA3, has good growth stability, and has eliminated the KM-KO-URA3 plasmid as the KM-G strain.

[0042] Example 2:

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

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

[0045] To achieve green and antibiotic-free production, the following strategies were used to design the expression vector of strain KM-G. Four promoter-terminator combinations (refer to DOI: 10.3389 / fbioe.2019.00097), the KanR expression cassette, and the Escherichia coli ori were designed and synthesized in the multiple cloning site region between the promoter and the 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).

[0046] PUC57 is the PUC57 plasmid.

[0047] Retrieve the URA3 gene sequence information of KM through the NCBI database, and design and synthesize the URA3 expression cassette PUC57-URA3 with the truncated promoter partial sequence, with SalⅠ and KpnⅠ restriction enzyme sites at both ends. Design and synthesize the plasmid PUC57-PKD1 containing the PKD 1 sequence gene (sequence reference of PKD1: DOI: 10.1002 / biot.202100382), with KpnⅠ and SacⅡ restriction enzyme sites at both ends. Use SacⅡ and SalⅠ to double-digest Puc57-INU, Puc57-NC1, Puc57-PGK, and Puc57-TDH3. Use SalⅠ and KpnⅠ to double-digest PUC57-URA3. Use KpnⅠ and SacⅡ to double-digest PUC57-PKD1. Connect the linearized PUC57-PKD1 and PUC57-URA3 with the linearized Puc57-INU, Puc57-NC1, Puc57-PGK, and Puc57-TDH3 respectively. Obtain the pGKD1, pGKD2, pGK D3, and pGKD4 vectors. Use EGFP as the marker gene to screen the expression intensity of different promoter-terminator combinations. The EGFP gene is optimized according to the KM codon table and synthesized into the PUC57 vector to obtain PUC57-EGFP. Use EcoRⅠ and HindⅢ to double-digest the pGKD1, pGKD2, pGKD3, and pGKD4 vectors respectively, amplify the EGFP fragment using primers with different promoter-terminator homologous arms, and then homologous recombine the EGFP gene sequence with the homologous arms into the linearized vectors pGKD1, pGKD2, pGKD3, and pGKD4 respectively. The recombinant products are transformed into KM-G competent cells and spread on the SD plate. Select the transformants with fluorescence through a blue light instrument, amplify the EGFP gene using a colony PCR kit, and after sequencing verification is correct, perform enlarged culture. Take 0.1OD of the seed solution and measure the fluorescence intensity at 400nm 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 the KM-G strain, the expression intensity: PGK combination > NC1 combination > INU combination ≈ TDH3 combination. Since the resistance gene KanR and the E. coli element ori are removed when using EcoRⅠ and HindⅢ to double-digest the pGKD vector, the purpose of green antibiotic-free production is achieved.

[0049] 2.2 Replicon screening of the KM-G strain

[0050] Synthesize 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). Use KpnⅠ and SacⅡ to digest the vectors pGKD3, PUC57-C1, PUC57-C2, and PUC57-C3. Construct linearized C1, C2, and C3 into linearized pGKD3 respectively, replacing the PKD1 replicon in pGKD3. The vectors carrying C1, C2, and C3 replicons are named pG1, pG2, and pG3 respectively. Use EcoRⅠ and HindⅢ to perform double digestion on pG1, pG2, pG3, and pGKD3 respectively. Homologously recombine the linearized vectors with the EGFP fragment with the PGK promoter terminator combination homologous arms. Transform the recombinant products into KM-G competent cells and spread them on SD plates for 48 h of culture. Do 3 replicates for each group and calculate the transformation efficiency (transformation efficiency = number of transformants / mass of vector DNA). Subsequently, pick out 3 of the largest monoclonal colonies and culture them overnight in 5 ml of SD medium. Transfer them to 5 ml of SD medium at OD600nm of 0.05 and culture for 48 h. Take 0.1 OD of the bacterial solution and measure the fluorescence intensity at 400 nm using a fluorescence microplate reader. Additionally, simultaneously pick the 3 largest monoclonal colonies from each group into 5 ml of YPD liquid medium and grow them overnight. Dilute the culture and spread it on YPD or SD plates respectively to determine the plasmid stability (determination of plasmid stability = number of colonies formed on SD plates / number of colonies formed on YPD plates).

[0051] The results showed that pG2 had the highest transformation efficiency. Average transformation efficiencies of different plasmids: pG2 > pG1 > pG3 > pGKD3( Figure 2 ). Although pGKD3 had the lowest transformation efficiency, its average fluorescence intensity was the highest. Average fluorescence intensity: pGKD3 > pG2 > pG1 > pG3( Figure 3 ). Additionally, the stability of pGKD3 could reach 72.09%, which was much higher than that of pG1, pG2, and pG3. Average plasmid stability: pGKD3 > pG1 ≈ pG2 ≈ pG3( Figure 4) pKD1 is an endogenous multicopy plasmid identified in Kluyveromyces marxianus. The plasmid based on pKD1 is the only multicopy plasmid that has been successfully applied to KM. pKD1 contains three major open reading frames, namely A, B, and C. Previously, the team of Professor Lü Hong from Fudan University demonstrated the role of the A gene in maintaining the high copy number of the plasmid based on pKD1 in KM. The deletion of the B or C gene affects the stable replication of the plasmid based on pKD1 in KM, and this defect cannot be resolved by trans-expressing the B and C genes. Therefore, PKD1 is essential for the high-level and stable expression of foreign proteins in KM. However, the transformation efficiency of the PKD1 replicon is significantly lower than that of the other replicons screened in this example. Here, we attempted to combine the other replicons (C1, C2, C3) screened in this example with PK D1 to construct a dual replicon system, hoping to obtain a dual replicon system that can combine the excellent phenotypes of the two replicons.

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

[0053] Amplify the C1, C2, and C3 replicon sequences with partial homologous arms of the PGK terminator and partial homologous arms of the URA3 promoter in the pG1, pG2, and pG3 plasmids, respectively. Single-digest pGKD3 with SalⅠ, and recombine the C1, C2, and C3 replicon sequences with homologous arms with the SalⅠ-linearized pGKD3 vector. After transformation and correct identification, the dual replicon vectors with C1, C2, and C3-P KD1 are named pGKD31, pGKD32 (SEQ ID No.2), and pGKD33, respectively. Use the method in Example 2.2 of this example to measure their transformation efficiency, fluorescence intensity, and stability.

[0054] As Figure 5 、 6As shown in Figures 7, compared with the single replicon vector pGKD3, the dual replicon vector pGKD31 did not show any improvement in transformation efficiency, fluorescence intensity, and replication stability. Among them, the transformation efficiency and fluorescence intensity were even significantly lower than those of pG1, and the replication stability was also at a low level. pGKD33 also did not show any improvement in transformation efficiency, fluorescence intensity, and replication stability. Among them, the transformation efficiency was even significantly lower than that of pG3, and the fluorescence intensity and replication stability were also at a low level. Therefore, when the dual replicon is applied to the same expression host, its function may be affected by various unknown factors, resulting in the expression host not only being unable to inherit the excellent characteristics of the two replicons, but instead may inherit the inferior phenotypes. However, the pGKD32 vector not only inherited the excellent phenotypes of the pGKD3 vector in fluorescence intensity and stability, but also inherited the excellent phenotypes of pG2 in replication stability. In addition, compared with pGKD3, the fluorescence intensity and stability were even significantly improved. In summary, in the expression host KM-G, the dual replication system does not necessarily inherit the excellent replication phenotypes of the two replicons, but instead may inherit the inferior phenotypes. Only pGKD32 (the combination of C2 and PKD1) can inherit the excellent phenotypes of both. The transformation efficiency can be comparable to that of the excellent phenotype C2 replicon, and the fluorescence intensity and stability are significantly improved compared with the excellent phenotype PKD1. Therefore, in the subsequent examples, we used the pGKD32 expression vector as the preferred vector for the KM-G strain.

[0055] Example 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] The Cap-P54 is designed as follows: The Cap gene refers to the GenBank PCV2d KX808467 sequence; the ASFV P54 gene refers to GenBank ASFV MK333180, and only the extracellular antigen region is retained; they are connected by a flexible linker 4×GGS, and a V5 tag is added to the C-terminus, namely Cap-linker-P54-V5, which is optimized using the KM codon table and synthesized into the PUC57 vector. Amplify the Cap-linker-P54 sequence with homologous arms of the PGK promoter and terminator (shown in SEQ ID NO.3), recover the gel fragment and perform homologous recombination with the pGKD32 vector linearized by EcoRⅠ and HindⅢ. Obtain pGKD32-Cap-P54, transform the recombinant product into KM-G, coat it on a solid SD plate and culture at 30 °C for 36 - 48 h, and identify positive transformants by colony PCR. Pick the positive transformants into a 5 ml SD bacterium bottle, culture at 220 rpm / 30 °C for 24 h, transfer them to a 400 ml SD conical flask and continue to culture for 72 h. Collect the bacteria and wash them twice with PBS for high-pressure crushing, and perform crude purification with 40% ammonium sulfate and purification with molecular sieve for KM-G / cap-P54 nanoparticles. Analyze by SDS-PAGE and identify by negative staining transmission electron microscopy.

[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 ), and this band was the cap-P54 protein. The electron microscopy results showed that cap-P54 could self-assemble into nanoparticles ( Figure 9 ).

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

[0061] Select 15 female SPF-grade Balb / c mice at 6 - 8 weeks old, randomly divide them into groups, with 5 mice in each group. Group 1 is the monomer P54 immunization group; Group 2 is the cap-P54 nanoparticle immunization group; Group 3 is the control group. Each immunization group orally administers 200 μg of protein; the control group orally administers an equal volume of PBS. Oral immunization is performed on days 0, 14, and 28 respectively. Blood is collected from the orbital cavity on days 28 and 42 to detect the P54 IgG level and the fecal P54 IgA level.

[0062] As Figure 10 、 11As shown, after oral immunization, compared with the control group, the immunized groups could all produce serum IgG and mucosal IgA against the P54 antigen (P < 0.05). Compared with the P54 monomer antigen, the P54 nanoparticles could 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 the P54 nanoparticles were significantly higher than those of the P54 monomer antigen (P < 0.05). It was indicated that the oral immunization with the ASFV P54 nanoparticles prepared in the present invention could significantly improve the humoral immunity and mucosal immunity.

[0063] The specific embodiments of the present invention have been 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 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 dual-replicon expression vector pGKD32 suitable for Kluyveromyces marxianus, as shown in SEQ ID NO.

2. The Kluyveromyces marxianus has a deposit number of CCTCC NO: M2014059.

2. The double replicon expression vector according to claim 1, comprising a target gene, wherein the double replicon expression vector is suitable for Kluyveromyces marxianus with a deposit number of CCTCC NO: M2014059.

3. A recombinant auxotrophic Kluyveromyces marxianus, wherein the recombinant auxotrophic Kluyveromyces marxianus is obtained by introducing the expression vector of claim 2 into auxotrophic Kluyveromyces marxianus, and the auxotrophic Kluyveromyces marxianus is obtained by introducing the knockout vector represented by SEQ ID NO. 1 into Kluyveromyces marxianus. The deposit number of the Kluyveromyces marxianus is CCTCC NO: M2014059.

4. Use of the double-replicon expression vector pGKD32 according to claim 1 in expressing heterologous proteins in Kluyveromyces marxianus, wherein the Kluyveromyces marxianus has a deposit number of CCTCC NO: M2014059.

5. Use of the double-replicon expression vector pGKD32 according to claim 1 in preparing a subunit vaccine using a protein expressed by Kluyveromyces marxianus, the deposit number of the Kluyveromyces marxianus being CCTCC NO: M2014059.

6. Use of the recombinant auxotrophic Kluyveromyces marxianus according to claim 3 in the preparation of subunit vaccines.

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

8. The use according to claim 7, wherein 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.

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