Method for preparing porcine epidemic diarrhea virus epitope nanoparticle by using k. marxianus and application thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses a method for preparing porcine epidemic diarrhea virus (PEDV) epitope nanoparticles by using Kluyveromyces marxianus and application thereof. The applicant assembles and prepares a double replicon expression vector suitable for the Kluyveromyces marxianus through screening, and the vector achieves the effect of high conversion rate and high expression efficiency in the Kluyveromyces marxianus. The double replicon expression vector pGKD32 is shown as SEQ ID NO. 2. The Kluyveromyces marxianus expression system provided by the application is used to successfully express the PEDV epitope in the form of nanoparticles, thereby providing a new idea for the research and development of the PED oral immunization strategy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a method for preparing porcine epidemic diarrhea virus epitope nanoparticles by using Kluyveromyces marxianus and application thereof. BACKGROUND

[0002] Porcine epidemic diarrhea (PED) is an infectious intestinal disease caused by porcine epidemic diarrhea virus (PEDV). The impact of PEDV on piglets is greater than that on adult pigs, and generally shows symptoms such as vomiting, watery diarrhea, dehydration, anorexia, weight loss, etc. PEDV has a very high mortality rate on newborn piglets, and poses a great threat to the global pig industry. Kluyveromyces marxianus (KM) is a non-conventional yeast, and belongs to the family Saccharomycetaceae together with Saccharomyces cerevisiae. It is a safe yeast certified by the Food and Drug Administration (FDA) of the United States (Generally recognized as safe, food additive safety evaluation) and the European Food Safety Authority (EFSA) (Qualified Presumption of Safety, safety qualification certification), and is also approved by the Chinese National Health and Family Planning Commission as a new food raw material. The applicant successfully isolated a KM-C2 strain (CN103952324A) in a natural breeding soil pig farm in Huangpi, Hubei in 2013, and sent it to the China Typical Culture Collection Center of Wuhan University in Wuhan, Hubei 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 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 PEDV antigen in feed additives.

[0003] At present, there is no nutritional deficiency screening marker for edible safety level yeast expression system and no knockout vector for genetic modification of the strain. There is also no expression vector suitable for the strain. In view of this, the present application establishes a suitable genetic operation system for the strain to realize genetic operation on the yeast genome. Different promoter-terminator combinations and replicon combinations are matched for the strain to provide new candidate expression elements. In addition, the application is applied to PEDV nanoparticle genetic engineering vaccine to lay a foundation for further development of PEDV vaccine and provide a reference for preparation of other animal pathogen genetic engineering vaccines.

[0004] In view of the above problems, the applicant provides a double replicon expression vector suitable for Kluyveromyces marxianus and makes a patent application, the application number of which is 2024104862538 (not published before the filing of the present application), in which the applicant newly constructs a double replicon expression vector suitable for Kluyveromyces marxianus. The expression vector has the advantages of good stability, large amount of expressed foreign protein and high transformation rate. In the present application, the applicant further utilizes the replicon to prepare porcine epidemic diarrhea virus nanoparticles to prepare porcine epidemic diarrhea subunit vaccine. SUMMARY

[0005] The present application aims to provide a method for preparing porcine epidemic diarrhea virus epitope nanoparticles by using Kluyveromyces marxianus. The method is simple, easy to operate and suitable for commercial production.

[0006] Another object of the present application is to provide the application of the nanoparticles prepared by the above method in the preparation of porcine epidemic diarrhea subunit vaccine.

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

[0008] A method for preparing porcine epidemic diarrhea virus epitope nanoparticles by using Kluyveromyces marxianus, comprising the following steps:

[0009] The polynucleotide shown in SEQ ID NO. 3 is homologously recombined with the linearized pGKD32 vector after EcoR I and Hind III, and then transformed into the auxotrophic Kluyveromyces marxianus;

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

[0011] Preferably, the auxotrophic Kluyveromyces marxianus is obtained by introducing the target URA3 knockout vector KM-KO-URA3 (shown in SEQ ID No. 1) into Kluyveromyces marxianus.

[0012] Preferably, the preservation number of the Kluyveromyces marxianus is CCTCC NO: M2014059.

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

[0014] The nanoparticles prepared by the above method are applied to the preparation of a porcine epidemic diarrhea subunit oral vaccine.

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

[0016] The applicant newly constructs a double replicon expression vector suitable for Kluyveromyces marxianus, which has the advantages of good stability, large amount of expressed exogenous protein and high transformation rate. In the present application, the applicant further utilizes the replicon to prepare porcine epidemic diarrhea virus nanoparticles, which prepares for a porcine epidemic diarrhea subunit vaccine. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The fluorescence intensity of pG1, pG2, pG3, pGKD3 vectors carrying EGFP protein.

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

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

[0020] Figure 4 The stability of pG1, pG2, pG3, pGKD3 vectors carrying EGFP protein.

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

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

[0023] Figure 7 The stability of pGKD31, pGKD32, pGKD33, pGKD3, pG2 vectors carrying EGFP protein.

[0024] Figure 8Figure 6. SDS-PAGE analysis of KM-G / cap-PEDV-AN expression and purification. Lane 1 is Marker, Lane 2 is empty sample, Lane 3 is whole cell sample of KM-G / cap-PEDV-AN after disruption, Lane 4 is supernatant sample of KM-G / cap-PEDV-AN after disruption, Lane 5 is precipitate sample of KM-G / cap-PEDV-AN after disruption.

[0025] Figure 9 Figure 7. TEM analysis of KM-G / cap-PEDV-AN after purification.

[0026] Figure 10 Figure 8. Serum PEDV-AN IgG level after immunization.

[0027] Figure 11 Figure 9. Fecal PEDV-AN IgA level after immunization. DETAILED DESCRIPTION

[0028] The starting strain used in this example is Kluyeromyces marxianus C2 (Patent No. ZL103952324A), which was sent to China Center for Type Culture Collection on March 3, 2014, and classified as Kluyeromyces marxianus C2 (CCTCC NO: M2014059), Wuhan, Hubei, China.

[0029] Example 1

[0030] Construction of URA3 auxotrophic host strain G based on CRISPR Cas9 scarless genome engineering method

[0031] 1.1 Construction of KM scarless genome knockout vector KM-KO and screening of antibiotic resistance

[0032] The Kluyeromyces marxianus (KM) scarless genome knockout vector KM-KO has three expression frames of TEF1-BleoR-CYC1, Cas9, and GAP-gRNA scaffold, two replicons of Pichia panARS and Escherichia coli ori, and two BsaI point mutations, and was prepared as follows:

[0033] The 192 bases before the TEF1 promoter in the Pichia pastoris expression vector plasmid pPICZaA were amplified as a template, and the 182nd base C of the TEF1 promoter was mutated to T to eliminate the Bsa I enzyme cutting site; the 171st base of the TEF1 promoter, 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 enzyme cutting site; the pPICZaA plasmid ori replicon was amplified. The above three fragments were fused by gel recovery PCR to obtain a 1967 bp fragment containing TEF1, BleoR, CYC1 terminator and E. coli ori replicon, which was identified by sequencing by Genescript. The Cas9 expression frame and gRNA expression frame panARS sequence were synthesized by Kingsriver into the PUC57 vector and amplified, respectively, and the 4600 bp and 2097 bp fragments were recovered by gel recovery. The above three fragments were subjected to homologous recombination and transformed into DH5α competent cells. After selecting single colonies for expansion culture, the gRNA expression frame sequence was verified to be correct; the Cas expression frame was verified to be correct. Double positive plasmids were selected for activation, and after extracting the plasmid, Pme I enzyme digestion was performed, and the band size was 1919 bp+6700 bp, which was the correct KM scarless genome knockout vector, named KM-KO.

[0034] About 2ug of KM-KO plasmid was used to electrotransform KM-C2, and different concentrations of Zeocin antibiotic were used for sensitivity screening (100ug / mL, 200ug / mL) to verify the presence of the plasmid. The results showed that YPD plates with different antibiotic concentrations could grow KM-C2 monoclones of equal size, indicating that the screening system was normal. To improve the positive rate of screening, 200ug / ml of antibiotic concentration was finally selected as the screening concentration.

[0035] 1.2 Construction of URA3 auxotrophic host strain KM-G strain

[0036] URA3 as an auxotrophic screening marker, the sequence information of URA3 gene of KM was obtained by searching NCBI database, and the following primers URA-F1 (5'-CAAGGATGCTCATCACAATACG-3'), URA-R1 (5'-GCAAGCATTAACAACCCTCTCTACATGTGTCTTCAATAGACAG-3'), URA-F2 (5'-TCTATTGAAGACACATGTAG-AGAGGGTTGTTAATGCTTG-3'), URA-R2 (5'-GTATACAATGTGACGCAATGC-3') were designed and synthesized. URA-F1 and URA-R1 were used to amplify the homologous arm URA3U of 629 bp on URA3, URA-F2 and URA-R2 were used to amplify the homologous arm URA3D of 492 bp below URA3, and the fragments of URA3U and URA3D were subjected to fusion PCR to obtain the fragment URA3HX of 1192 bp removing the core region of 187-432 of URA3 gene.

[0037] URA3 knockout gRNA, gRNA-△URA3 (5'-AGGTTCTTTCGTAACTTCCT-3'), was designed using gRNA online design platform (http: / / www.rgenome.net / cas-designer / ). Picosynthetic gRNA-△URA3-F (5'-CGTC-AGGTTCTTTCGTAACTTCCT-3') and gRNA-△URA3-R (5'-AAACAGGAAGTTACGAAAGAACCT-3'). gRNA fragment was obtained by annealing gRNA upstream and downstream primers using PCR. KM-KO was digested using Bsa I, and the digested product was recovered by gel. The gRNA fragment was ligated with linearized KM-KO vector using solution I, and transformed into DH5α. GPD-F and CYCT-R were used to identify positive monoclonal. The positive monoclonal was cultured to extract plasmid, and the target URA3 knockout vector KM-KO-URA3 (shown in SEQ ID No. 1) was obtained. 2ug KM-KO-URA3 and 8ug URA3HX fragment were electroporated into Km-C2, and plated on Zeocin-YPD solid plates. Eight transformants were picked, and colony PCR was used for identification. The positive colonies were cultured and subcultured to verify growth stability. They were simultaneously plated on SC plates and YPD plates. The strain that could not grow on SC plates but grew normally on YPD plates was a URA3 auxotrophic strain. Since the Pichia panARS replicon on the KM-KO-URA3 vector had poor replication stability, it would be lost during continuous subculture in YPD medium without antibiotics. It could not grow 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, and the URA3 core region was successfully knocked out. The URA3 auxotrophic strain with good growth stability and eliminated KM-KO-URA3 plasmid was named KM-G strain.

[0038] Example 2:

[0039] Construction of expression vector for auxotrophic host strain KM-G strain, screening and optimization of expression elements:

[0040] 2.1 Screening of promoter and terminator combinations for KM-G strain

[0041] To achieve green antibiotic-free production, the following strategy was used to design the expression vector for KM-G strain. Four promoter-terminator combinations (referring to DOI: 10.3389 / fbioe.2019.00097) were designed and synthesized, with KanR expression cassette and E. coli ori in the region of the multiple cloning site between the promoter and terminator. The four promoter-terminator combinations were 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).

[0042] PUC57 is a PUC57 plasmid.

[0043] The URA3 gene sequence information of KM was obtained by searching the NCBI database, and the URA3 expression frame PUC57-URA3 with a truncated promoter partial sequence was designed and synthesized, with Sal I and Kpn I enzyme digestion sites at both ends. The plasmid PUC57-PKD1 containing the PKD1 sequence gene (the sequence reference of PKD1: DOI: 10.1002 / biot.202100382) was designed and synthesized, with Kpn I and Sac II enzyme digestion sites at both ends. Puc57-INU, Puc57-NC1, Puc57-PGK and Puc57-TDH3 were double-digested with Sac II and Sal I. PUC57-URA3 was double-digested with Sal I and Kpn I. PUC57-PKD1 was double-digested with Kpn I and Sac II. Linearized PUC57-PKD1 and PUC57-URA3 were respectively connected with linearized Puc57-INU, Puc57-NC1, Puc57-PGK and Puc57-TDH3. The pGKD1, pGKD2, pGKD3 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 a PUC57 vector to obtain PUC57-EGFP. pGKD1, pGKD2, pGKD3 and pGKD4 vectors were double-digested with EcoR I and Hind III, respectively, EGFP fragments were amplified using primers with different promoter terminator homologous arms, and then the EGFP gene sequence with homologous arms was respectively homologously recombined with linearized vectors pGKD1, pGKD2, pGKD3 and pGKD4. The recombination products were transformed into KM-G competent cells and plated on SD plates. The 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 correct one was expanded and cultured, and the fluorescence intensity at 400 nm was measured using a fluorescence microplate reader.

[0044] 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. Since the resistance gene KanR and the E. coli element ori were removed when pGKD vectors were double-digested with EcoR I and Hind III, the purpose of green antibiotic-free production was achieved.

[0045] 2.2 Replicon screening of KM-G strain

[0046] Synthetic plasmid containing C1 / CenD, C2 / CEN5, C3 / CEN6 (reference DOI: 10.3389 / fbioe.2019.00097) replicon gene PUC57-C1, PUC57-C2, PUC57-C3, using Kpn I and Sac II enzyme pGKD3, PUC57-C1, PUC57-C2, PUC57-C3 vector, linearized C1, C2, C3 respectively constructed into linearized pGKD3, replace PKD1 replicon in pGKD3, the vector carrying C1, C2, C3 replicon is named as pG1, pG2 and pG3 respectively. Using EcoR I and Hind III respectively double enzyme digestion of pG1, pG2, pG3 and pGKD3, linearized vector is respectively homologous recombination with EGFP fragment with PGK promoter terminator set with homologous arm, the recombinant product is transformed into KM-G competent cells, and is coated on SD plate and cultured for 48 h. Each group is repeated for 3 times, and the transformation rate (transformation rate = number of transformants / mass of vector DNA) is calculated. Subsequently, 3 largest single colonies are respectively picked in 5 ml SD medium and cultured overnight, and are transferred to 5 ml SD medium at OD600nm of 0.05 and cultured for 48 h. 0.1 OD bacterial liquid is used to measure 400 nm fluorescence intensity by using a fluorescence enzyme label instrument. In addition, the above-mentioned largest 3 single colonies of each group are simultaneously picked into 5 ml YPD liquid medium and grown overnight. The culture is diluted and coated on YPD or SD plate respectively, and the stability of the plasmid is measured (the stability of the plasmid = the number of bacteria formed on the SD plate / the number of bacteria formed on the YPD plate).

[0047] The results show that the transformation rate of pG2 is the highest. The average transformation rate of different plasmids: pG2 > pG1 > pG3 > pGKD3 ( Figure 2 ). Although the transformation rate of pGKD3 is the lowest, the average fluorescence intensity is the highest. The average fluorescence intensity: pGKD3 > pG2 > pG1 > pG3 ( Figure 3 ). In addition, the stability of pGKD3 can reach 72.09%, which is much higher than that of pG1, pG2 and pG3. The average plasmid stability: pGKD3 > pG1 ≈ pG2 ≈ pG3 ( Figure 4). pKD1 is an endogenous multicopy plasmid identified in K. marxianus, and pKD1 -based plasmids are the only multicopy plasmids successfully applied to KM. pKD1 contains three major open reading frames, A, B, and C. Previously, Professor Lu Hong's team at Fudan University demonstrated the role of the A gene in maintaining high copy numbers of pKD1 -based plasmids in KM. Deletion of the B or C gene affects the stable replication of pKD1 -based plasmids in KM, and this defect cannot be resolved by expressing the B and C genes in trans. Therefore, PKD1 is essential for KM to express exogenous proteins at a high and stable level. However, the transformation efficiency of the PKD1 replicon is significantly lower than the rest of the replicons screened in this example. Here, we attempt to combine the rest of the replicons (C1, C2, C3) screened in this example with PKD1 to construct a dual replicon system, in the hope that a dual replicon system that combines the excellent phenotypes of the two replicons can be obtained.

[0048] 2.3 Screening of KM-G dual replicon system

[0049] The C1, C2, C3 replicon sequences with the PGK terminator partial homology arm and the URA3 promoter partial homology arm were amplified from the pG1, pG2, pG3 plasmids, respectively. pGKD3 was single- digested with Sal I, and the C1, C2, C3 replicon sequences with homology arms were recombined with linearized pGKD3 vectors, respectively. After transformation and correct identification, the dual replicon vectors with C1, C2, C3-PKD1 were named pGKD31, pGKD32 (SEQ ID No. 2), pGKD33, respectively. The transformation efficiency, fluorescence intensity, and stability were determined using the method in Example 2.2.

[0050] As Figure 5 , 6, 7, the transformation efficiency, fluorescence intensity and replication stability of the double replicon vector pGKD31 were not improved compared with the single replicon vector pGKD3, and the transformation efficiency and fluorescence intensity were even significantly lower than pG1, and the replication stability was also at a low level; the transformation efficiency, fluorescence intensity and replication stability of pGKD33 were also not improved, and 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 applied in the same expression host, its function may be affected by various unknown factors, resulting in that the expression host not only cannot inherit the excellent characteristics of the two replicons, but also may inherit the inferior phenotype. However, the pGKD32 vector not only inherits the excellent phenotype of the pGKD3 vector in fluorescence intensity and stability, but also inherits the excellent phenotype of the pG2 vector in replication stability. In addition, compared with pGKD3, the fluorescence intensity and stability are even significantly improved. In summary, in the expression host KM-G, the double replicon system may not necessarily inherit the excellent replication phenotype of the two replicons, but may inherit the inferior phenotype, only pGKD32 (C2 and PKD1 combination) can inherit the excellent phenotype of the two, the transformation efficiency can be comparable to 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 will use the pGKD32 expression vector as the preferred vector of the KM-G strain.

[0051] Example 3:

[0052] Construction and oral immunogenicity of KM-G / PEDV-AN nanoparticles

[0053] 3.1 Construction and identification of KM-G / PEDV-AN nanoparticles

[0054] PEDV-AN is composed of PEDV-SD epitope (NCTEPVLVYSNIGVCKSGSIGYVPSQSGQVK), PEDV-NTD epitope (GTNFSFVCSNSSDPHLATFAIPLGAIQVPYYCFLKVD), PEDV-HR2 epitope (TG EIADLEQRSESLRNTTEELQ) connected by a flexible linker GGS. Cap-PEDV-AN is designed as follows: Cap gene is referenced to GenBank PCV2d KX808467 sequence; PEDV-AN is added to the C-terminal of Cap, i.e. Cap-linker-PEDV-AN, which is optimized using KM codon table and synthesized into PUC57 vector. Cap-linker-PEDV-AN sequence (shown in SEQ ID NO. 3) with PGK promoter and terminator homologous arms is amplified, the recovered fragment is ligated with pGKD32 vector linearized by EcoR I and Hind III for homologous recombination. pGKD32-Cap-PEDV-AN is obtained, and the recombination product is transformed into KM-G, which is spread on solid SD plates and cultured at 30°C for 36-48 h, and positive transformants are identified by colony PCR. The positive transformants are picked into 5 ml of SD bacterial bottles, which are cultured at 220 rpm / 30°C for 24 h, and then transferred into 400 ml of SD conical flasks for further culture for 72 h. The bacterial bodies are collected and washed twice with PBS for high-pressure disruption, and KM-G / cap-PEDV-AN nanoparticles are obtained by 40% ammonium sulfate crude purification and molecular sieve fine purification. SDS-PAGE analysis is performed, and negative staining transmission electron microscopy is used for identification.

[0055] The results show that there is an additional protein band at 39.8 KD in KM-G / cap-PEDV-AN compared with the control group ( Figure 8 ), which is the cap-PEDV-AN protein. The electron microscopy results show that Cap-PEDV-AN can self-assemble into nanoparticles ( Figure 9 ).

[0056] 3.2 Immunogenicity of KM-G / cap-PEDV-AN nanoparticle oral immunization

[0057] Fifteen 6-8 week old female SPF Balb / c mice are selected and randomly divided into groups, with 5 mice in each group. Group 1 is the PBS control group, group 2 is the monomer PEDV-AN immunization group, and group 3 is the PEDV-AN nanoparticle immunization group. The immunization groups are orally administered with 200 ug of protein each time, and the control group is orally administered with the same volume of PBS. Oral immunization is performed on days 0, 14 and 28, respectively. The orbital blood is collected on days 14, 28 and 42 for detection of PEDV-AN IgG level, and the feces are collected for detection of PEDV-AN IgA level.

[0058] As Figure 10、 11 As shown in Figure 6, after oral immunization, the immune groups can produce serum IgG and mucosal IgA against PEDV-AN antigen compared with the control group (P<0.05). Compared with PEDV-AN monomer antigen, PEDV-AN nanoparticles can produce higher levels of serum IgG and mucosal IgA. With the increase of the number of immunization, the serum IgG and mucosal IgA produced by PEDV-AN nanoparticles are significantly higher than that of PEDV-AN monomer antigen (P<0.05). It is shown that the oral immunization of PEDV-AN nanoparticles prepared by the present application can significantly improve the humoral immunity and mucosal immunity.

[0059] The above detailed description of the specific embodiments of the present application is only as an example, and the present application is not limited to the above described specific embodiments. Any equivalent modifications and substitutions of the present application made by those skilled in the art are also within the scope of the present application. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present application should be covered within the scope of the present application.

Claims

1.A method for preparing porcine epidemic diarrhea virus (PEDV) epitope nanoparticle by using Kluyveromyces marxianus, comprising the following steps: homologous recombination of a polynucleotide shown in SEQ ID NO. 3 with a linearized pGKD32 vector after EcoR I and Hind III, and then transforming into an auxotrophic Kluyveromyces marxianus; the polynucleotide sequence of the pGKD32 vector is shown in SEQ ID NO. 2; the auxotrophic Kluyveromyces marxianus is obtained by introducing a knock-out vector KM-KO-URA3 targeting URA3 into Kluyveromyces marxianus, and the polynucleotide sequence of the knock-out vector KM-KO-URA3 is shown in SEQ ID NO. 1; the accession number of the Kluyveromyces marxianus is CCTCC NO: M2014059. 2.Use of the method of claim 1 in preparing a porcine epidemic diarrhea subunit oral vaccine. ​ ​

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