KR100492820B1 - Neutralizing epitope for porcine epidemic diarrhea virus and use the same - Google Patents

Abstract

The present invention relates to swine pandemic diarrheal virus neutralizing epitopes and their use. In particular, the present invention provides a PEDV (porcine epidemic diarrhea virus) epitope comprising the polypeptide of SEQ ID NO: 1, which can effectively prevent infection of the virus by using it as a target antigen against the swine epidemic diarrheal virus.

Description

Swine pandemic diarrheal virus neutralizing epitope and its use {NEUTRALIZING EPITOPE FOR PORCINE EPIDEMIC DIARRHEA VIRUS AND USE THE SAME}

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a swine epidemic diarrheal virus neutralizing epitope and its use, and more particularly, to a method for preventing swine epidemic diarrheal virus infection by cloning the epitope gene acting on the neutralization of swine epidemic diarrheal virus.

[Private Technology]

Porcine epidemic diarrhea virus (PEDV) is a virus belonging to the coronaviridae system that causes epidemic diarrhea in pigs (Pensaert, MB and de Bouck, P. (1978) Arch. Virol. 58, 243-247). As a result, weight loss and death of weaning pigs are caused, leading to many economic losses. PEDV was first reported in 1978 in Belgium and the United Kingdom, and has been identified as a causative agent of enteropathogenic diarrhea in pigs (Debouck, P. and Pensaert, M. (1980) Am. J. Vet. Res. 41, 219-223). Since then, several outbreaks have been reported in pig breeding countries, particularly in Europe and Asia (Debouck, P., Callebaut, P., and Pensaert, M. (1982) Proc. Int. Vet. Soc. Cong. 7, 53; Kweon, C.-H., Kwon, B.-J., Jung, T.-S., Kee, Y.-J., Hur, D.-H., Hwang, E.-K., Rhee, J.-C., and An, S.-H. (1993) Kor. J. Vet. Res. 33, 249-254). PEDV causes damage and atrophy of chorionic cells in the jejunum and ileum, and not only causes swine enteritis, but also fatally acts on newborn pigs (Ducatelle, R., Coussement, W., Charlier, G., Debouck, P., and Hoorens, J. (1981) J. Vet. Med. 28, 483-493). In addition, the swine epidemic diarrheal disease caused by PEDV is more damaging in Asia than in the United States or Europe, which is thought to be due to poor pig environment.

Currently, vaccines are used to prevent PEDV infection, but the entire virus strains are used as vaccines without analysis of exact antigens such as live vaccines, live vaccines, and attenuated vaccines, and research on the primary target antigens of PEDV is ongoing. Is in. Among them, Spike protein is recognized as a target antigen for effective vaccine development against coronavirus because it recognizes the target cell and mediates essential biological functions such as fusion between virus and cell membrane (Spaan, W., Cavanagh, D). , and Horzinek, MC (1988) Coronavirus: structure and genome expression.J. Gen. Virol. 69, 2939-2952).

In order to solve the problems of the prior art, an object of the present invention is to provide an epitope polypeptide and gene in a spike protein that is used as a PEDV target antigen.

It is another object of the present invention to provide a PEDV antibody prepared using the epitope region as an antigen.

It is another object of the present invention to provide a vaccine composition using the epitope site as an antigen.

In order to achieve the above object, the present invention provides a PEDV (porcine epidemic diarrhea virus) epitope comprising the polypeptide of SEQ ID NO: 1.

The present invention also provides a PEDV (porcine epidemic diarrhea virus) epitope gene encoding the polypeptide of SEQ ID NO: 1.

In another aspect, the present invention (a) inserting the PEDV epitope gene in the expression vector to produce a recombinant vector, (b) transforming the recombinant vector to a host cell to prepare a transformant, (c) the transformation It provides a recombinant PEDV epitope protein prepared by expressing and isolating recombinant PEDV epitope protein from the body.

The present invention also provides an antiserum against PEDV obtained from an immunized animal by injecting the PEDV epitope protein in the animal.

The present invention also provides anti-serum from an immunized animal by injecting the PEDV epitope protein into the animal, and provides an antibody against PEDV isolated from the anti-serum.

The present invention also provides a vaccine composition comprising the PEDV epitope.

Hereinafter, the present invention will be described in detail.

The inventors have identified whether the PEDV spike protein can induce PEDV neutralizing antibodies and which site of the spike protein plays a critical role in inducing neutralizing antibodies.

Transmissible gastroenteritis virus (TIGV), which causes diarrheal disease in pigs, exhibits feline infectious peritonitis virus (FIPV) and cross-neutralization, which causes feline disease. 503-715 (Genebank AAA47109), which contains neutralizing epitopes of TGEV, 92% homology with the corresponding site of FIPV. However, in PEDV, which does not exhibit cross-neutralization with TGEV, the corresponding site is 28% homologous to TGEV. Especially, the 522-607 site of TGEV, which is the core site, is 503-568 (Genebank CAA80971) and the corresponding site of PEDV. Only 9% homology is seen. Therefore, the low homology seen in the core part was assumed to be due to no cross-neutralizing force between TGEV and PEDV. Therefore, the present inventors estimated that the core region of the PEDV spike protein may contain an epitope capable of neutralizing PEDV, and as a result, cloned an epitope capable of neutralizing PEDV.

PEDV neutralizing epitopes of the invention are polypeptides comprising the amino acid sequence of SEQ ID NO: 1. "X" described in the amino acid of SEQ ID NO: 1 represents a variety of amino acids by subtype of virus, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, Amino acids selected from the group consisting of phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine can be used. The PEDV epitope corresponds to the core portion of the PEDV spike protein.

In the present invention, the amino acid of SEQ ID NO: 1 in X 22 is X22 for convenience, 23 X is X23, 24 X is X24, 25 X is X25, 51 X is X51, 58 X is X58, 114 Burn X is represented by X114 and X137 as X137.

PEDV epitopes of the present invention

X22 is Asp or Gly,

X23 is Ser or Leu,

X24 is Ser or Gly.

X25 is Ser or Gly,

X51 is Thr or Arg,

X58 is Asn or Ser,

X114 is Leu or Phe, and

X137 is preferably Ile or Val.

More preferably X22 to X25 is "DSGG" or "GLSS", X51 is "T" or "R", X58 is "N" or "S", X114 is "L" or "F", X137 is It is preferable to be "I" or "V".

In addition, the PEDV epitope of the present invention may further include a sequence representing a spike protein other than the amino acid sequence of SEQ ID NO: 1, PEDV (GeneBank No. Z25483) spike protein may be used as a PEDV epitope.

Antibodies formed by the PEDV epitope proteins of the present invention not only recognize and bind PEDV, but also exhibit activity that can neutralize PEDV.

In the present invention, as an example of the nucleotide sequence encoding the PEDV epitope protein is shown in SEQ ID NO: 2.

In addition, the present invention can mass-express PEDV epitopes in recombinant protein form using genetic engineering methods to obtain large amounts of PEDV epitopes. This inserts a gene encoding a PEDV epitope into an expression vector to produce a recombinant vector, expresses the recombinant vector in a host cell, and isolates it. The expression vector is preferably designed to be expressed in prokaryotes or eukaryotes, more preferably is designed to be expressed in E. coli, yeast, fungi, plant cells or animal cells.

The present invention also provides antisera or antibodies against PEDV. The antibody is prepared by using a PEDV epitope or a recombinant PEDV epitope protein as an antigen, and can be prepared by injecting into a common laboratory animal. Antisera and antibodies against PEDV of the present invention can be used not only for diagnosing an animal suspected of being infected with PEDV, but can also be administered to the animal by mixing with feed or drinking water.

The present invention also provides a vaccine composition against PEDV. The vaccine composition comprises a PEDV epitope or an antibody against a PEDV epitope, further comprising a pharmacologically acceptable diluent or excipient. The vaccine composition of the present invention may include 100 to 1,000 mg / l of PEDV epitope, recombinant PEDV epitope protein or PEDV epitope antibody as an active ingredient, and may be administered 50 to 500 mg as a dose, but is not limited thereto. no.

The vaccine composition of the present invention can be used for oral or parenteral use, more preferably for oral, injection or spraying. The dosage form of the vaccine composition is preferably determined according to the method of use, and the dosage form includes PLASTERS, GRANULES, LOTIONS, LINIMENTS, LIMONADES, and powders. (POWDERS), SYRUPS, OPHTALMIC OINTMENTS, LIQUIDS AND SOLUTIONS, ELIXIRS, OINTMENTS, FLUIDEXTRACTS, Emulsion, EMULSIONS, SUSPENSION ), DECOCTIONS, INFUSIONS, OPTHALMIC SOLUTIONS, TABLETS, Suppositories (SUPPOSITIORIES), INJECTIONS, SPIRITS, CAPSULES, CREAMS, CREAMS, Troches, tinctures, and pastas. The present invention also provides a transformant comprising a PEDV epitope. The transformant may be a prokaryote or eukaryote, preferably a plant, plant cell or plant tissue.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

To determine the neutralizing epitope of PEDV, experiments were conducted based on the spike protein gene sequence of TEGV, a coronavirus that mediates acute enteritis that is lethal in piglets. The collagenase digested fragment, CO-26K, represents a 503-715 amino acid region in the TGEV spike protein, which has been shown to be an important epitope for virus neutralization by studies with monoclonal antibodies (Delmas, B., Gelfi, J). ., and Laude, H. (1986) J. Gen. Virol. 67, 1405-14180; Correa, I., Jimenez, G., Sune, C., Bullido, MJ, and Enjuanes, L. (1988) Virus Res. 10, 77-93; Delmas, B., Rasschaert, D., Godet, M., Gelfi, J., and Laude, H. (1990) J. Gen. Virol. 71, 1313-1323). It was also confirmed that TGEV and feline infectious peritonitis virus (FIPV) had high cross-neutralizing ability, and that the CO-26K region had 92% homology (Horzinek, MC, Lutz, H., and Pedersen, NC). (1982) Infect. Immun. 37, 1148-1155). In the spike protein of the PEDV Br1 / 87 strain, the amino acid sequence homologous to the CO-26K site, that is, the CO26K equivalent (hereinafter referred to as "COE"), showed very low homology (28%) with the CO-26K of TGEV. In particular, the homology between the amino acid sequence of spike protein 503-568 of PEDV and the corresponding TGEV spike protein 522-607 amino acid sequence was only 9%. This indicates that there is no cross-neutralizing force between TGEV and PEDV, and that COE in the PEDV spike protein is a very important factor for the neutralization of PEDV.

In order to confirm that COE in the PEDV spike protein is an epitope having a neutralizing ability, polyclonal antiserum was prepared as a recombinant protein, and specificity was confirmed.

Example 1: Recombinant Protein and Epitope Specific Antibody Production

Chemicals and experimental materials were purchased from Sigma Chemical Co. St. Louis, MO, USA and Falcon Labware Becton-Dickinson, Franklin Lakes, NJ, USA. CV777 strain of PEDV and Vero cell line for PEDV propagation were supplied by Dr. Shin-Young Kang (Chungbook National University, Chungjoo, Korea). Proliferation of PEDV in Vero cell lines was performed by Hoffmann, M. and Wyler, R. (1988) Propagation of the virus of porcine epidemic diarrhea in cell culture. J. Clin. Microbiol. 26, 2235-2239 . Was carried out accordingly.

(1) antigen production

The COE moiety expected to be the desired epitope in the PEDV spike protein was cloned and produced as a recombinant COE protein in an E. coli expression system.

The COE portion, a 504 bp Xho I / Bgl II fragment, was cloned from the PEDV Br1 / 87 strain and inserted into the pRSET-A (Invitrogen, The Netherlands) vector digested with the same restriction enzyme. Recombinant vectors were cloned and approximately 25 kDa recombinant COE protein was expressed and purified using a Ni-NTA agarose affinity column.

(2) polyclonal antiserum preparation

Polyclonal antiserum was prepared using the recombinant COE protein isolated above as an antigen.

50-100 ug of antigen was injected into mice mixed with complete Freund's adjuvant. Incomplete Freund's adjuvant was then inoculated twice in two week intervals by mixing with the same antigen and antisera were obtained from mice 4 days post immunization.

Example 2: Validation of PEDV Specific Response of Polyclonal Antisera

The polyclonal antiserum prepared in Example 1 was reacted with recombinant COE protein and whole virus protein to confirm PEDV recognition.

1 is a Western blot photograph using polyclonal antisera, lane 1 is electrophoresed with 0.5 ug of purified COE protein, and lane 2 is electrophoresed with whole virus (> 10 ¹² pfu).

The polyclonal antiserum, which can be seen in Figure 1, reacted at about 25 kDa and 110 kDa, which is consistent with the spike protein size of recombinant COE protein and PEDV, respectively. Since the control serum did not bind at all, and the polyclonal antiserum did not bind with other antigens of the PEDV, it could be seen that the polyclonal antiserum of the present invention specifically recognized PEDV.

Example 3 Verification of Neutralization Activity of Polyclonal Serum

Neutralizing activity of polyclonal antiserum was confirmed by Plaque reduction neutralization assay (Hofmann, M. and Wyler, R. (1989) Quantitation, biological and physicochemical properties of cell culture-adapted porcine epidemic diarrhea coronavirus ( PEDV) .Vet.Microbiol . 20, 131-142)

PEDV stock solution (5 × 10 ¹³ pfu / ml) was mixed with polyclonal antiserum or control syngeneic serum at a 10: 1 volume ratio and reacted at 37 ° C. for 1 hour. The mixture was serially diluted in VM1 medium (0.3% TPB, 0.02% YE, 30 mM HEPES, 1 mg / ml trypsine / MEM), added to Vero cell line, and reacted for 2 hours at room temperature to induce virus infection. The Vero cell line was prepared by rinsing with MEM lacking serum and washing with VM2 medium twice.

The medium was exchanged with the same volume of VM1 medium and incubated again at room temperature for 2 hours, and 4 ml of VM3 (0.3% TPB, 0.02% YE, 30 mM HEPES, 4% FBS / MEM) was added and cultured at 37 ° C for 2 days. Then, 0.9% noble agar dissolved in MEM was poured to form an upper layer on the medium, followed by incubation for 24 hours, and then 0.1% neutral red stain was added to visualize virus plaque formation.

Figure 2 is a graph showing the rate of inhibition of virus plaque formation by polyclonal anti-blood activity is derived by performing the same experiment five times. ** shown in FIG. 2 means P < 0.01.

In FIG. 2, the number of plaques formed in the control group not treated with polyclonal antisera, the group treated with neogenic serum and the group treated with polyclonal antisera was 452 ± 43, 385 ± 38 and 153 ± 41, respectively. It became. That is, polyclonal antiserum inhibited plaque formation by 66.2% ( P <0.01), but neogenic serum did not effectively inhibit plaque formation by PEDV.

Thus, the COE protein of PEDV not only acts as an epitope against PEDV, but polyclonal antiserum prepared with COE protein exhibits specific neutralizing activity for PEDV virus.

In addition, polyclonal antiserum in Figure 2 did not completely inhibit PEDV plaque formation can be estimated for the following reasons. Considering the experimental conditions with trypsin added, trypsin is an element that activates the receptor-ligand interaction between Vero cell line and PEDV, which may interfere with the action of the treated antibody. Next, since the antigen for polyclonal antiserum production was prepared using an E. coli expression system, polyclonal antiserum produced by an antigen lacking glycosylation may have a slightly lower antigen antibody affinity for COE of PEDV.

Example 4 Sequence Comparison of COE Sites of PEDV Br1 / 87 and PEDV CV777

Figure 3 shows the comparison of the amino acid sequence of the COE region of PEDV and TGEV, * indicates the amino acid is missing when compared with TGEV (Genebank M94101) and the dotted line is the same amino acid compared to PEDV Br1 / 87 (Genebank Z25483) It is shown.

The amino acid sequence of the COE region of PEDV CV777 is very similar to the amino acid sequence of the COE region of PEDV Br1 / 87, indicating that the portion inducing viral neutralizing antibodies during the PEDV virus week is well preserved.

As mentioned above, the PEDV epitope protein of the present invention can be used not only as a PEDV target antigen but also as a vaccine to effectively prevent and treat PEDV.

1 confirms the specificity of polyclonal antiserum against the swine epidemic diarrheal virus epitope of the present invention,

Figure 2 shows the plaque formation inhibition rate of the swine epidemic diarrhea virus by polyclonal antiserum of the present invention,

Figure 3 compares the amino acid of the swine epidemic diarrheal virus epitope of the present invention with another swine epidemic diarrheal virus strain and associated virus TGEV.

<110> JANG, Yong Suk YANG, Moon Sik KIM, Dae Hyuk <120> NEUTRALIZING EPITOPE FOR PORCINE EPIDEMIC DIARRHEA VIRUS AND USE THE SAME <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 140 <212> PRT P213 epitope sequence <400> 1 Val Thr Leu Pro Ser Phe Asn Asp His Ser Phe Val Asn Ile Thr Val 1 5 10 15 Ser Ala Ala Phe Gly Xaa Xaa Xaa Xaa Ala Asn Leu Val Ala Ser Asp 20 25 30 Thr Thr Ile Asn Gly Phe Ser Ser Phe Cys Val Asp Thr Arg Gln Phe 35 40 45 Thr Ile Xaa Leu Phe Tyr Asn Val Thr Xaa Ser Tyr Gly Tyr Val Ser 50 55 60 Lys Ser Gln Asp Ser Asn Cys Pro Phe Thr Leu Gln Ser Val Asn Asp 65 70 75 80 Tyr Leu Ser Phe Ser Lys Phe Cys Val Ser Thr Ser Leu Leu Ala Gly 85 90 95 Ala Cys Thr Ile Asp Leu Phe Gly Tyr Pro Ala Phe Gly Ser Gly Val 100 105 110 Lys Xaa Thr Ser Leu Tyr Phe Gln Phe Thr Lys Gly Glu Leu Ile Thr 115 120 125 Gly Thr Pro Lys Pro Leu Glu Gly Xaa Thr Asp Val 130 135 140 <210> 2 <211> 420 <212> DNA <213> sequence encoding PEDV epitope <400> 2 gttactttgc catcatttaa tgatcattct tttgttaata ttactgtctc tgcggctttt 60 ggtggtctta gtagtgccaa tctcgttgca tctgacacta ctatcaatgg gtttagttct 120 ttctgtgttg acactagaca atttaccatt acactgtttt ataatgttac aaacagttat 180 ggttatgtgt ctaaatcaca ggatagtaat tgtcctttca ccttgcaatc tgttaatgat 240 tacctgtctt ttagcaaatt ttgtgtttca accagccttt tggctggtgc ttgtaccata 300 gatctttttg gttaccctgc gttcggtagt ggtgttaagt tgacgtccct ttattttcaa 360 ttcacaaaag gtgagttgat tactggcacg cctaaaccac ttgaaggtat cacagacgtt 420 420 <210> 3 <211> 43 <212> DNA <213> primer <400> 3 gttactttgc catcatttaa tgandaacgt ctgtgatacc ttc 43

Claims (6)

A composition for inducing neutralizing antibodies against porcine epidemic diarrheal virus comprising a PEDV (porcine epidemic diarrhea virus) epitope of SEQ ID NO: 1, In SEQ ID NO: 1, Xaa at position 22 is Asp or Gly, Xaa at position 23 is Ser or Leu, Xaa in position 24 is Ser or Gly, Xaa at position 25 is Ser or Gly, Xaa at position 51 is Thr or Arg, Xaa at position 58 is Asn or Ser, Xaa at the 114 th position is Leu or Phe, Xaa at the 137th position is Ile or Val neutralizing antibody inducing against swine pandemic diarrheal virus. delete delete  As an antiserum against a porcine epidemic diarrhea virus (PEDV) obtained from an immunized animal by injecting a porcine epidemic diarrhea virus (PEDV) epitope of SEQ ID NO: 1, In SEQ ID NO: 1, Xaa at position 22 is Asp or Gly, Xaa at position 23 is Ser or Leu, Xaa in position 24 is Ser or Gly, Xaa at position 25 is Ser or Gly, Xaa at position 51 is Thr or Arg, Xaa at position 58 is Asn or Ser, Xaa at the 114 th position is Leu or Phe, Antiserum, where Xaa at position 137 is Ile or Val.  As an antibody against a porcine epidemic diarrhea virus (PEDV) obtained by injecting an epitope of a PEDV epitope of SEQ ID NO: 1 into an animal to obtain antiserum from an immunized animal, In SEQ ID NO: 1, Xaa at position 22 is Asp or Gly, Xaa at position 23 is Ser or Leu, Xaa in position 24 is Ser or Gly, Xaa at position 25 is Ser or Gly, Xaa at position 51 is Thr or Arg, Xaa at position 58 is Asn or Ser, Xaa at the 114 th position is Leu or Phe, The antibody at the 137th position is Ile or Val. A vaccine composition comprising a porcine epidemic diarrhea virus (PEDV) epitope of SEQ ID NO: 1, In SEQ ID NO: 1, Xaa at position 22 is Asp or Gly, Xaa at position 23 is Ser or Leu, Xaa in position 24 is Ser or Gly, Xaa at position 25 is Ser or Gly, Xaa at position 51 is Thr or Arg, Xaa at position 58 is Asn or Ser, Xaa at the 114 th position is Leu or Phe, The vaccine composition at the position 137 of the Xaa is Ile or Val.