KR20070045817A - Transgenic plant to express antigen for preventing porcine epidemic diarrhea and method for producing the same - Google Patents

Abstract

The present invention relates to a transgenic plant producing a swine epidemic diarrheal virus antigen protein and a method for producing the same, and specifically, a spike protein of the swine epidemic diarrheal virus using a promoter expressed in a plant and a promoter expressed in a sweet potato storage root. The present invention relates to a transformed plant that produces an antigen protein by expressing a gene encoding a gene in a plant, and the transformed plant that produces the antigenic protein of the swine epidemic diarrheal virus of the present invention is useful for the use of feed for preventing diarrheal disease of pigs. It can be used to prevent infection of the swine epidemic diarrheal virus.

Swine Pandemic Diarrheal Virus, Transgenic Sweet Potato

Description

Transgenic plant to express antigen for preventing swine epidemic diarrheal disease and method for producing same {transgenic plant to express antigen for preventing porcine epidemic diarrhea and method for producing the same}

Figure 1 A depicts the schematic diagram of the transformation vector 35Sp :: PEDV-sp1 / pCAMBIA2300 for expressing a porcine epidemic diarrhea preventing protein antigen developed in this invention in plants [35Sp, CaMV 35S promoter; PEDV-sp1, a gene encoding a PEDV spike protein fragment; NosT, nopaline synthase terminator; LB, left border; RB, right border], B of FIG. 1 shows a schematic diagram of the transformation vector Spop :: PEDV-sp1 / pCAMBIA2300 for expressing the pig epidemic diarrheal disease preventing antigen protein developed in the present invention in plants [Spop, sweet potato storage root] High expressing sporamin promoter; PEDV-sp1, a gene encoding a PEDV spike protein fragment; NosT, nopaline synthase terminator; LB, left border; RB, right border],

FIG 35Sp :: is developed in this invention, A of the two-sp1 PEDV / pCAMBIA2300 vector and the transformed potato plants, introducing the picture is regenerated into plants from embryogenic callus in a kanamycin selection medium, and Fig. 2 of the present invention B is Transformed sweet potato plants into which the developed Spop :: PEDV-sp1 / pCAMBIA2300 vector were re-differentiated from the embryogenic callus to plants in kanamycin selection medium,

Figure 3 A shows that the introduction of the gene encoding the swine epidemic diarrheal virus spike protein in a transformed sweet potato plant to which the 35Sp :: PEDV-sp1 / pCAMBIA2300 vector developed in the present invention was introduced [M, size] Markers, 1 to 14 are kanamycin resistant sweet potato plants, N is a non-transformed sweet potato plant, P is a transformed vector DNA], and B of FIG. 3 is a Spop :: PEDV-sp1 / pCAMBIA2300 vector developed in the present invention. Introduced by PCR the introduction of the gene encoding the swine epidemic diarrheal virus spike protein in the transformed sweet potato plants introduced [M, size marker, 1 to 14 are kanamycin resistant sweet potato plants, N is non-transforming sweet potato plants And P is the transformation vector DNA],

Figure 4 shows the results of analyzing the Southern blot (Sourthern blot) that the gene encoding the swine epidemic diarrheal virus spike protein in the transformed sweet potato plant of the present invention [NT is a non-transforming sweet potato plant; 1, 2, 3, 4 were transgenic sweet potatoes into which 35Sp :: PEDV-sp1 / pCAMBIA2300 was introduced; 5, 6, and 7 are transgenic sweet potatoes into which the Spop :: PEDV-sp1 / pCAMBIA2300 vector is introduced.

Figure 5 A is 35Sp of the invention :: PEDV-sp1 / pCAMBIA2300 is introduced transgenic potato plants in the porcine epidemic diarrhea virus spike protein will to be the gene encoding the expression of a portion showing a result of analysis by RT-PCR [ NT is a non-transforming sweet potato plant; 1 from 7 35Sp :: PEDV-sp1 / pCAMBIA2300 is introduced transgenic potato], B of Figure 5 of the present invention Spop :: PEDV-sp1 / pCAMBIA2300 vector is introduced transformant and porcine epidemic diarrhea virus in Kumamoto Plant RT-PCR analysis of the expression of a gene encoding a portion of the spike protein is shown as [NT is a non-transforming sweet potato plant; 1 to 8 are transgenic sweet potatoes introduced with Spop :: PEDV-sp1 / pCAMBIA2300].

The present invention is a transgenic plant expressing a protein by introducing a gene expressing an antigen among genes encoding a spike protein of porcine epidemic diarrhea virus (PEDV), a method for producing the same and the transgenic plant It relates to the use as a preventive oral vaccine against diarrheal virus infection.

The most economically damaging disease of the hog industry in recent years is the pig epidemic diarrhea (PED). The causative agent of swine epidemic diarrhea is the porcine epidemic diarrhea virus (PEDV) belonging to the coronaviridae family (Pensaert and de Bouck, Arch. Viol., 58, 243-247, 1978). PEDV belongs to the family coronaviridae family, such as the transmissible gastroenteritis virus (TGEV), and the clinical symptoms are also very similar but antigenically distinct. PEDV particles range in diameter from 120-160 nm and have a variety of shapes with envelopes.

PEDV is composed of S (spike protein), M (membrane glycoprotein), E (small envelope protein), HE (hemagglutinin-esterase protein), N (nucleocapsid phosphoprotein) ICS (internal core-shell composed of M glcoprotein), NC (nucleocapsid) and the like (Egberink et al., Am. J. Vet. Res., 49, 1320-1324, 1988). The antigenicity of PEDV is determined by the coat protein and the spike protein, in particular the spike glycoprotein is involved in the formation of neutralizing antibodies and also binds to cellular receptors and causes fusion between cells.

PEDV proliferates in the villus epithelial cells of the swine intestine, causes diarrhea, anorexia and vomiting in infected pigs, severe dehydration in newborn pigs within 1 week of age, and dies within 3-4 days of infection (de Bouck and Pensaert, Am. J. Vet. Res., 41, 219-223, 1980). Decreased milk secretion from infected sows or the absence of milk causes severe mortality of maternal neonatal pigs, leading to increased mortality.

Swine pandemic diarrhea was first reported in the UK in 1972. In Korea, PEDV was confirmed by separating PEDV in 1993, and it is estimated that pig diarrhea caused by PEDV has been developed in pig farm since 1980s. In fact, the most damaging diseases to the domestic pig industry since 1990 are PED and TGE. However, since there are no statistically analyzed reports on the damage caused by PEDV, it is not possible to calculate the exact economic loss, but given that mortality rates of PEDV infected piglets range from at least 30% to up to 80%, The damage to pig farmers can be enormous.

Preventive methods of swine pandemic diarrheal disease include the purchase of live attenuated virus vaccine in addition to the prevention of spreading between farms when the pigs are purchased from uninfected farms, but have not been shown to be fully effective. The reason is that the current vaccine is used for intramuscular injection, so the formation of IgG in the blood is good, but the mucosal immunity (sIgA), which is important for the prevention of PED, is not well formed.

Expressing the antigenic protein from the plant and using it as an oral vaccine can reduce costs, facilitate purification, and cultivate the plant in the area of use, making it easy to move and store, as well as mass production. In 1992, masons expressed the surface antigen gene (HBdAg) of hepatitis B virus (HBV) in tobacco, and the vaccine was developed from plants, starting with the production of immunogenic antigen proteins (Mason et al., Proc). Natl.Acad.Sci. USA, 89, 11745-11749, 1992). Various plants have been developed since then, but considering the characteristics of antigenic proteins, it is important to use plants that can reproduce like sweet potatoes.

Reports of antigenic protein production for PEDV in transgenic plants have been reported for transgenic tobacco producing PEDV neutralizing antibody epitopes (Bae et al., Vaccine, 21: 4052-4058, 2003; Kang et al. , Protein Express.Purif., 38, 129-135, 2004; Kang et al., Vaccine, 23, 2294-2297, 2005). Korean Patent Application, Patent Application No. 10-2002-0024424, which describes a transformant producing an epitope protein of swine pandemic diarrheal virus, discloses a transgenic tobacco comprising an epitope gene of swine pandemic diarrheal virus. . However, cigarettes contain toxic alkaloids, which are not suitable for oral immunity and have environmental disadvantages as LMOs (Living modified organisms) in packaging. In contrast, the transgenic plant of the present invention introduced only a part of the gene of the spike protein antigenic to PEDV into the pCAMBIA2300 vector, in particular by transforming the sweet potato using a sporamin promoter specifically expressed in the sweet potato storage roots. Not only is easy for oral vaccines, but also shows the difference from the above patents due to its nutritional propagation properties and the absence of environmental risks.

  In addition, Korean Patent Application, Application No. 2003-0012280 reported a transgenic potato expressing a gene encoding a PEDV spike protein subunit and a gene encoding a membrane protein using a CaMV 35S promoter. However, in the above case, there is a disadvantage that the protein content is relatively low in tubers of potato and is not suitable for reproduction, and the antigenicity is low due to protein denaturation during processing, and the gene encoding the PEDV spike protein subunit and the membrane protein are encoded. In the case of the transformed potato expressing the gene, there is a disadvantage of producing a spike protein-producing potato and a membrane-producing transgenic potato, respectively. In contrast, the present invention introduces only a portion of genes that are antigenic in spike proteins relative to PEDV, thereby making transfection much easier than transforming the genes encoding the PEDV spike protein subunits and the genes encoding the membrane proteins, respectively. There is an advantage to switch.

Sweet potato has no environmental risks of living modified organism (LMO) because it does not bloom under domestic cultivation conditions and is maintained as a nutrient propagation, and it is a plant that can use all parts of plants such as root, leaf, and leaves. It is possible to minimize denaturation of antigenic protein when used as an oral vaccine. In addition, it is easy to cultivate as a propagation crop, short time to commercialization, it can grow well in poor soil, it is not only easy to cultivate but also has the highest productivity per unit area.

As described above, although sweet potatoes have many advantages, they are not easy to transform, and thus industrially useful sweet potatoes have not been developed, but the present inventors have established a sweet potato regeneration and transformation system, and a disaster-resistant transformation. Sweet potato plants have been developed.

Thus, the present inventors produced a transformed sweet potato to produce an antigenic protein by expressing the gene encoding the spike protein of the swine epidemic diarrheal virus in the sweet potato using a promoter that is highly expressed in sweet potato storage roots, the transformation of the present invention The present invention was completed by confirming that sweet potatoes produce the antigenic protein of the swine epidemic diarrheal virus.

The present invention provides a transgenic plant that produces an antigenic protein against swine pandemic diarrheal virus (PEDV). In addition, another object of the present invention to provide a vaccine composition for oral administration for preventing swine epidemic diarrheal disease containing the transformed plant as an active ingredient.

In order to achieve the above object,

The present invention provides a transgenic plant cell comprising a gene encoding a PEDV (porcine epidemic diarrhea virus) antigen protein of SEQ ID NO: 1 .

The present invention also provides a transgenic plant prepared by re-differentiation from the transgenic plant cells.

The present invention also provides a recombinant vector comprising a promoter for plant expression, the PEDV gene described in SEQ ID NO: 2 , and a transcription terminator, and a transformant comprising the same.

The present invention also provides a recombinant PEDV protein isolated from the transformed plant.

In addition, the present invention

1) preparing an expression vector comprising a gene encoding a PEDV antigen protein of SEQ ID NO: 1 ;

2) introducing the expression vector of step 1) into agrobacteria; And

3) It provides a method for producing a transformed plant comprising the step of transforming the plant cells by co-culturing the agrobacteria and plant cells of step 2).

The present invention also provides a vaccine composition for oral administration comprising the transformed plant, the protein extract of the transformed plant or the recombinant PEDV protein isolated from the transformed plant.

In addition, the present invention provides a feed composition for preventing PEDV comprising the transformed plant, the protein extract of the transformed plant or the recombinant PEDV antigen protein isolated from the transformed plant.

Hereinafter, the present invention will be described in more detail.

The present invention provides a transgenic plant cell comprising a gene encoding a PEDV (porcine epidemic diarrhea virus) antigen protein of SEQ ID NO: 1 . The present invention also provides a transgenic plant prepared by re-differentiation from the transgenic plant cells.

The PEDV protein of the present invention may include a PEDV protein or "variants" described by SEQ ID NO: 1 , and the PEDV gene encoding the same may be a gene described by SEQ ID NO: 2 , a "homologous gene thereof ( homologs ”and“ varients ”thereof. Thus, the category of homologous genes includes all kinds of genes capable of producing the PEDV protein described in SEQ ID NO: 1 when expressed.

The "variant protein" of the PEDV protein means a protein in which one or more amino acid residues of the PEDV protein represented by SEQ ID NO: 1 are deleted, added, inserted or substituted, and the "varients" of the PEDV gene are By PEDV protein is meant any gene encoding a variant protein. Preferably, the gene of the PEDV protein has a nucleotide sequence of SEQ ID NO: 2 , and the protein preferably has an amino acid sequence of SEQ ID NO: 1 .

The PEDV antigen protein of SEQ ID NO: 1 is an active site showing antigenicity in the PEDV spike protein, and the present inventors obtained a gene encoding a PEDV spike protein that acts as a PEDV antigen, and is represented by SEQ ID NO: 2 .

After the recombinant vector containing the gene was prepared, it was introduced into Agrobacterium, and co-cultured with sweet potato cells to prepare transformed sweet potato cells expressing the PEDV antigen.

Expression vectors can be introduced into plant cells by known methods such as polyethylene glycol method, electroporation, Agrobacterium mediated delivery and particle bombardment methods. have.

The plant cell into which the vector of the present invention is introduced is not particularly limited to a specific form as long as the cell can be regenerated into a plant. These cells include, for example, cultured cell suspensions, protoplasts, leaf sections, and callus.

In the present invention, an expression vector for plant transformation, agrobacterium strain into which the expression vector is introduced, and a transformed plant cell comprising a gene encoding a PEDV spike protein serving as a PEDV antigen for producing the transformed plant cell, All are included in the scope of the present invention, the expression vector and Agrobacterium strains can be applied to a variety of crops other than sweet potatoes shown in the examples. That is, there is no particular limitation on the plant cells expressing the PEDV antigen, and it is preferable that the crop is edible directly to pigs. In particular, sweet potatoes cultivated throughout the country are not cultivated under domestic cultivation conditions. It is more preferable because there is no environmental risk as LMO.

The present inventors transformed plant cells containing the PEDV gene encoding the PEDV (porcine epidemic diarrhea virus) protein of SEQ ID NO: 1 to KCTC 10857BP and KCTC 10858BP as of October 5, 2005, respectively, to the Genetic Bank of Korea Biotechnology Research Institute. Deposited.

The present invention also provides a recombinant vector comprising the promoter for plant expression, the PEDV gene described in SEQ ID NO: 2 , and a transcription terminator, and a transformant comprising the same.

The inventors isolated a gene (sp1) encoding a portion of the PEDV spike protein using PCR from the swine epidemic diarrheal virus and constructed a recombinant vector. Recombinant vector produced in the present invention was used as a sporamin promoter high expression in the CaMV 35S promoter and sweet potato storage roots based on the pCAMBIA2300 vector. As shown in FIG. 1 CaMV 35S promoter (35Sp), Spokane lamin promoter to (Spop), SEQ ID NO: 3 and SEQ ID NO: 4 gene (sp1) encoding the PEDV spike protein portion synthesized by PCR using primers described in, and no Nopaline synthase terminator (NosT). In the present invention, the 35Sp :: PEDV-sp1 using the CaMV 35S promoter and the Spop :: PEDV-sp1 gene construct using the sporamin promoter were introduced into the plant expression vector pCAMBIA2300, and these were respectively incorporated into the 35Sp :: PEDV-sp1 gene. / pCAMBIA2300 and Spop :: PEDV-sp1 / pCAMBIA2300, which were introduced into Agrobacterium to prepare transformed Agrobacterium, and were respectively assigned to KCTC 10855BP and the Korean Biotechnology Research Institute on October 5, 2005. Deposited with KCTC 10856BP.

In addition, the present invention

1) preparing an expression vector comprising a gene encoding a PEDV antigen protein of SEQ ID NO: 1 ;

2) introducing the expression vector of step 1) into agrobacteria; And

3) It provides a method for producing a transformed plant comprising the step of transforming the plant cells by co-culturing the agrobacteria and plant cells of step 2). In the method, the expression vector of step 1 is a gene encoding the PEDV antigen protein of SEQ ID NO: 1 is located in the CaMV 35S promoter or sporamin promoter forward, preferably comprises a kanamycin resistance gene as a selection marker, More preferably, 35Sp :: PEDV-sp1 / pCAMBIA2300 and Spop :: PEDV-sp1 / pCAMBIA2300 are used, but are not limited thereto. In addition, the expression vector of step 2 may be introduced into Agrobacterium according to a conventional method, and the transformed cells prepared by step 3 may prepare a transformed plant through a regeneration process.

In an embodiment of the present invention, somatic embryos were induced by culturing for about 1 month in MS basal medium (BMCK) to induce somatic embryos and plants from embryogenic callus transformed with PEDV antigen gene. Roots were induced and redivided into news bodies.

As a result of analyzing the transformed sweet potato prepared as described above by PCR and Southern blot, it was confirmed that the PEDV antigen gene of the present invention was introduced into the plant and preserved, and the reverse transcriptase-PCR (RT-PCR) method of the present invention. It was confirmed that the antigen gene is expressed.

The present invention also provides a recombinant PEDV protein isolated from the transgenic plant. In addition, the present invention provides a transgenic vaccine composition comprising a transgenic plant, a protein extract of the transgenic plant, or a recombinant PEDV protein isolated from the transgenic plant. In addition, the present invention provides a feed composition for preventing PEDV comprising the transformed plant, the protein extract of the transformed plant or the recombinant PEDV protein isolated from the transformed plant.

The effective amount of the transformed sweet potato blend of the present invention as an active ingredient can be suitably determined according to its purpose of use (prophylactic or therapeutic treatment), and the active sweet potato, which is an active ingredient, is normally used without any specific maximum tolerance because there is no problem in terms of safety. It is certain.

The recombinant PEDV protein separation method may be suitably used according to a conventional method, and the composition may be used in the form of a powder after use of the transformed plant or dried, and may also be used with other feed or feed additives. It can be suitably used according to a conventional method. The transformed sweet potato prepared in the present invention can be fed to pigs by feeding it, thereby inducing antibody formation against swine PEDV in pigs that have been ingested, thereby inducing an immune effect against PEDV. In addition, since the transformed sweet potato is used as a feed, the sweet potato is not only easily grown by the general public, but also has been used as a feed for farmers in the past, so there is no objection, so that the public can easily use it to induce immunization of pigs. You can.

Meanwhile, the protein was expressed using an expression vector prepared by inserting only 444 bps fragments of known antigenicity in the base sequence of the spike protein, and exhibited the effect of continuously administering the vaccine by ingesting the transformed sweet potato of the present invention in pigs. It is also safer than conventional attached virus vaccines. Thus, oral administration of the transformed sweet potato may induce mucosal immunity in swine, and ultimately, the oral vaccine effect on diarrheal disease in pigs can be expected.

Hereinafter, preferred examples are provided to aid in understanding the present invention.

However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

< Example  1> Preparation of Recombinant Vector

For the production of recombinant vector expressing PEDV spike protein, PEDV provided by Professor Hyun-Soo Kim of Chungnam National University was converted into plasmid using RT-PCR method. First, Hin dIII (Roche) and Xba I (Roche) sequences were attached before and after the CaMV 35S promoter and inserted into the same restriction enzyme site of the pCAMBIA2300 vector (cambia.org) (35Sp / pCAMBIA). The 444 bps fragment described by SEQ ID NO: 2 (Chang et al., Mol. Cells, 14, 295-299, 2002), known antigenic in the PEDV spike protein sequence, contains the Bam HI (Roche) sequence (GGATCC). The primers were synthesized by PCR (sp1) and inserted into the TOPO vector (Invitrogen). Primer (primer) is used, 5 ' and described in SEQ ID NO: 4 GGA TCC AAA AGA AAC GTC TGT GAT ACC-3 '5'- GGA TCC ATG GAA CAG CCA ATT TCT TTT GTT-3 described in SEQ ID NO: 3 to be. In addition, the primer described in SEQ ID NO: 3 includes an initiation codon sequence (ATG) for protein expression of the present invention.

PCR fragments were digested with Eco RV (Roche) and Sac I (Roche), and then 35S :: PEDV-sp1 / pCAMBIA2300 recombinant by inserting 35Sp / pCAMBIA2300 into Sma I (Roche) and Sac I. The vector was completed ( A in FIG. 1 ).

In addition, in addition to the CaMV 35S promoter, sporamin promoters were used as Hin dIII and Xba I to produce a vector using a sweet potato storage root high expressing sporamin promoter (Hattori and Nakamura, Plant Mol. Biol., 11, 417-426, 1988). After the cleavage, the previously prepared 35S :: PEDV-sp1 / pCAMBIA2300 vector was treated with the same enzyme to remove the CaMV 35S promoter, and a sporamin promoter was inserted into the Spop :: PEDV-sp1 / pCAMBIA2300 recombinant vector. 1 B ).

The two recombinant vectors were introduced into Agrobacterium tumefaciens EHA105 and used for sweet potato transformation. In the introduction method, recombinant vector DNA and agrobacteria were mixed and quenched in liquid nitrogen according to An, G, etc., and then dissolved at 37 ° C. for 5 minutes, and then incubated at 28 ° C. for 1 hour with the addition of YEP medium. YEP medium containing 100 mg / L rifampicin and 50 mg / L kanamycin was plated and incubated at 28 ° C. for 2 days (An et al., In Plant Molecular Biology Mannual Part A3: 1-19 Kluwer Academic Publisher, 1988). Agrobacterium into which the recombinant vector was introduced was deposited as KCTC 10855BP and KCTC 10856BP as of October 5, 2005, at the Korea Biotechnology Research Institute Gene Bank.

< Example  2> Agrobacterium Mediated Transformation and Plant Regeneration

Embryonic callus derived from apical meristem from sweet potato plants was extracted from MS (Murashige and Skoog, Physiol.Plant., 15, 473-497, 1962) base medium mineral salt 100 mg / L myo-inositol, 0.4 Mg / L thiamine-HCl, 30 g / L sucrose, 1 mg / L 2,4-D and 4 g / L Gelrite added medium (MS1D) at 4 week intervals Maintained by subculture (Kwon et al., Korean J. Plant Biotechnol., 29, 189-192, 2002). Embryonic callus 1 week after passage in MS1D solid medium was used as a transformation material.

After culturing the Agrobacterium prepared in Example 1 for 1 day, the Agrobacterium was collected by centrifugation, suspended in 10 mL of MS1D liquid medium, mixed with embryogenic callus, left for 30 minutes, and then sterilized filter paper ( filter paper) was used to remove the agrobacteria and the medium on the callus and co-cultured in MS1D medium for 2 days. Callus cultured with sterile water containing 400 mg / L claforan was washed three times to remove agrobacteria, and then in a selection medium containing 100 mg / L kanamycin and 400 mg / L claporan (MS1DCK). Kanamycin resistant callus was selected with subculture at three week intervals for about four months. Most callus died as passage was progressed in MS1DCK selection medium, but some callus proliferated while maintaining embryogenic capacity.

Regenerating plant cells, wherein the 35S :: PEDV-sp1 / pCAMBIA2300 vector was introduced and selected were named 35S-PEDV (sweet potato callus), and the spop :: PEDV-sp1 / pCAMBIA2300 vector was introduced and reselected. Available plant cells were named spop-PEDV (sweet potato callus), and the transformed sweet potato plant cells were deposited as KCTC 10857BP and KCTC 10858BP as of October 5, 2005, to the Korea Biotechnology Research Institute Gene Bank.

Transferred to MS base medium (100 mg / L kanamycin, containing 400 mg / L claforan: BMCK) free of 2,4-D to induce somatic embryos and plants from embryonic callus surviving for 4 months in selection medium. It was incubated at 16 ° C. under cool white fluorescence at a temperature of about 40 μmol · m ⁻² · sec ⁻¹ and a photoperiod. Somatic embryos were induced after 1 month of culturing in BMCK medium, and stems and roots were induced as a result of 1-2 months of incubation into news bodies ( FIG. 2 ).

Example 3 Transformed Plant Analysis

Chromosome DNA was isolated from plants re-differentiated into kanamycin-containing medium and analyzed by PCR. The primers used for PCR used the primers of Example 1 ( SEQ ID NO: 3 and SEQ ID NO: 4 ). As a result, bands of 444 bps were observed in more than 50% of the regenerated plants ( FIG. 3 ).

Southern blot analysis was performed on sweet potato plants identified by the introduction of PEDV gene by PCR. Genomic DNA was isolated using DNeasy Plant Maxi Kit (Qiagen, Germany) from the leaves of sweet potato plants growing in the incubator. The separated DNA (30 μg) was digested with XbaI and electrophoresed on 0.8% agarose gel, and then transferred to Zeta Probe membrane (Bio-Rad). A probe was used by synthesizing by PCR using the primers ( SEQ ID NO: 3 and SEQ ID NO: 4 ) prepared from specific portions of the PEDV spike protein (444 bps). This DNA was labeled with isotope [α- ³² P] dCTP and added to a prehybridization solution (for zeta probe membrane) and hybridized at 60 ° C. for 24 hours. After completion of the reaction, the membrane was washed and exposed to an X-ray film to identify a band. As a result, it was found that 1-2 copies of the PEDV gene were introduced into the transformed sweet potato plant ( FIG. 4 ).

In order to determine whether the PEDV gene introduced from the transformed sweet potato is normally expressed, RNA was extracted and RT-PCR was performed. In order to extract RNA, 1 g of the regenerated sweet potato leaves were ground finely in a mortar with liquid nitrogen. Here, 5 mL of TRI reagent (Molecular Research Center, MRC) was used to isolate RNA by routine methods. 1 μg of the isolated RNA was mixed with oligo dT and reacted at 70 ° C. for 5 minutes, and then mixed with the reaction solution of the primary cDNA synthesis kit (ImProm-II ^™ Reverse Transcription System, Promega) for 5 minutes at 25 ° C. The primary cDNA was synthesized by reacting for 60 minutes at 42 ° C. for 15 minutes at 70 ° C. PCR was performed using the above primers by taking 1-2 μL of the synthesized cDNA. PCR conditions were performed for 30 minutes at 1 minute at 94 ° C, 1 minute at 56 ° C, 1 minute at 72 ° C, and then at 72 ° C for 7 minutes. As a result, DNA fragments of 444 bps were synthesized in all of the transformed sweet potatoes.

It was expressed at similar levels of the genes introduced in the transgenic potato using a CaMV 35S promoter (A in Fig. 5). On the other hand, in the transformant using a sports lamin promoter was introduced genes are expressed at different levels for each plant (B in Fig. 5), this phenomenon appeared because Sporting lamin promoters and high expression in potato storage root, in other tissues weakly expressed Is grasped.

This experiment demonstrated that the products obtained by RT-PCR were derived from the expressed mRNA of the gene encoding the introduced PEDV spike protein, which means that the gene encoding the PEDV spike protein introduced from sweet potato is normally expressed. do.

As we have seen above,

The transformed sweet potato expressing the PEDV antigen protein of the present invention is easy to cultivate in farms and is a high-yielding crop and is already used as a feed, so it is possible to mass-produce transgenic sweet potatoes to easily produce PEDV antigen protein in piglet farms. When the transgenic sweet potato is used as a feed vaccine, it is not necessary to administer the vaccine periodically like the existing inactivated virus vaccine, and it can bring incomes to pig farmers such as cost reduction.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Transgenic plant to express antigen for preventing porcine          epidemic diarrhea and method for producing the same <130> 5p-10-18 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 148 <212> PRT <213> PEDV antigen amino acid sequence <400> 1 Glu Gln Pro Ile Ser Phe Val Thr Leu Pro Ser Phe Asn Asp His Ser   1 5 10 15 Phe Val Asn Ile Thr Val Ser Ala Ala Phe Gly Gly Leu Ser Ser Ala              20 25 30 Asn Leu Val Ala Ser Asp Thr Thr Ile Asn Gly Phe Ser Ser Phe Cys          35 40 45 Val Asp Thr Arg Gln Phe Thr Ile Thr Leu Phe Tyr Asn Val Thr Asn      50 55 60 Ser Tyr Gly Tyr Val Ser Lys Ser Gln Asp Ser Asn Cys Pro Phe Thr  65 70 75 80 Leu Gln Ser Val Asn Asp Tyr Leu Ser Phe Ser Lys Phe Cys Val Ser                  85 90 95 Thr Ser Leu Leu Ala Gly Ala Cys Thr Ile Asp Leu Phe Gly Tyr Pro             100 105 110 Ala Phe Gly Ser Gly Val Lys Leu Thr Ser Leu Tyr Phe Gln Phe Thr         115 120 125 Lys Gly Glu Leu Ile Thr Gly Thr Pro Lys Pro Leu Glu Gly Ile Thr     130 135 140 Asp Val Ser Phe 145         <210> 2 <211> 444 <212> DNA PEDV antigen DNA sequence <400> 2 gaacagccaa tttcttttgt tactttgcca tcatttaatg atcattcttt tgttaatatt 60 actgtctctg cggcttttgg tggtcttagt agtgccaatc tcgttgcatc tgacactact 120 atcaatgggt ttagttcttt ctgtgttgac actagacaat ttaccattac actgttttat 180 aatgttacaa acagttatgg ttatgtgtct aaatcacagg atagtaattg tcctttcacc 240 ttgcaatctg ttaatgatta cctgtctttt agcaaatttt gtgtttcaac cagccttttg 300 gctggtgctt gtaccataga tctttttggt taccctgcgt tcggtagtgg tgttaagttg 360 acgtcccttt attttcaatt cacaaaaggt gagttgatta ctggcacgcc taaaccactt 420 gaaggtatca cagacgtttc tttt <210> 3 <211> 30 <212> DNA <213> PEDV primer 1 <400> 3 ggatccatgg aacagccaat ttcttttgtt 30 <210> 4 <211> 27 <212> DNA <213> PEDV primer 2 <400> 4 ggatccaaaa gaaacgtctg tgatacc 27

Claims (16)

A transgenic plant cell comprising a gene encoding a PEDV (porcine epidemic diarrhea virus) antigen protein of SEQ ID NO: 1 . The transformed plant cell of claim 1, wherein the gene has a sequence represented by SEQ ID NO: 2. 6 . The transformed plant cell of claim 1, wherein the plant cell is a sweet potato cell. The transgenic plant cell according to claim 1, which is described by accession number KCTC 10857BP. The transgenic plant cell according to claim 1, which is described by accession number KCTC 10858BP. Transgenic plant prepared by re-differentiation from the plant cell of any one of claims 1 to 5. The transgenic plant of claim 6, wherein the transgenic plant is sweet potato. A recombinant vector comprising a promoter for plant expression, the PEDV gene set forth in SEQ ID NO: 2, and a transcription terminator. The recombinant vector 35Sp :: PEDV-sp1 / pCAMBIA2300 of claim 8, wherein the recombinant vector 35Sp :: PEDV-sp1 / pCAMBIA2300 is introduced into Agrobacterium tumefaciens EHA105 (Accession No. KCTC 10855BP). The recombinant vector Spop :: PEDV-sp1 / pCAMBIA2300 of claim 8, wherein the recombinant vector Spop :: PEDV-sp1 / pCAMBIA2300 is introduced into Agrobacterium tumefaciens EHA105 (Accession No. KCTC 10856BP). Agrobacterium tumefaciens EHA105 (Accession No. KCTC 10855BP) transformed with the recombinant vector of claim 9. Agrobacterium tumefaciens EHA105 (Accession No. KCTC 10856BP) transformed with the recombinant vector of claim 10. Recombinant PEDV protein isolated from the transgenic plant of claims 6-7. 1) preparing an expression vector comprising a gene encoding the PEDV antigen protein of SEQ ID NO: 1 ; 2) introducing the expression vector of step 1) into agrobacteria; And 3) transforming the plant cells by co-culturing the agrobacteria and plant cells of step 2). 8. A vaccine composition for oral administration comprising the transformed plant of claim 6 or 7, a protein extract of the transformed plant or a recombinant PEDV protein isolated from the transformant. A feed composition for preventing PEDV, comprising the transformed plant of claim 6 or 7, a protein extract of the transformed plant, or a recombinant PEDV protein isolated from the transformant.

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