KR102103013B1 - Vaccine composition for preventing or treating pathogenic Escherichia coli in pigs comprising Stx2eA-C-terminal fragment-(Stx2eB)5 recombinant protein, and F4+ enterotoxigenic E. coli and F18+ enterotoxigenic E. coli inactivated cells as a vaccine - Google Patents

Abstract

The present invention, in order to overexpress Stx2eA-C-terminal fragment-Stx2eB pentamer recombinant protein among Stx2e toxins, one of the causes of swine edema, performs cloning each of a Stx2eA-C-terminal fragment gene and a Stx2eB gene to pTWIN1 and pET30a, respectively, followed by transformation of vectors coliBL21(DE3)pLysS. Therefore, a Stx2eA-C-terminal fragment and a Stx2eB pentamer protein; and a heat killed antigen conducting a reaction chlorohexidine with Enterotoxigenic E. coli expressing F4 or Enterotoxigenic E. coli expressing F18 can be usefully used as a toxoid heat killed mixed vaccine which can prevent pathogenic E. coli infections such as swine edema disease, weaned pig diarrhea, etc.

Description

Vaccine composition for prevention or treatment of pathogenic Escherichia coli in pigs comprising Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein; and enterotoxin type E. coli expressing F4 and enterotoxin type E. coli expressing F18 mixed antigen {Vaccine composition for preventing or treating pathogenic Escherichia coli in pigs comprising Stx2eA-C-terminal fragment- (Stx2eB) 5 recombinant protein, and F4 + enterotoxigenic E. coli and F18 + enterotoxigenic E. coli inactivated cells as a vaccine}

The present invention is a Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein; And it relates to a vaccine composition for the prevention or treatment of pathogenic Escherichia coli in pigs comprising enterotoxin type E. coli expressing F4 and enterotoxin type E. coli expressing F18 mixed antigen.

In industrial animals, one of the biggest causes of mortality and increase in the rate of piglet growth is the result of mortality and reduced feed efficiency due to diarrhea due to digestive diseases. In order to prevent bacterial digestive diseases that cause serious economic losses to the pig industry, we are devoting much effort to developing effective vaccines worldwide. The mucous membrane of the digestive tract is the first place to contact the majority of disease-causing pathogens, and at the same time, it is a very important place as the forefront of defense against infection of these bacteria.

After weaning, diarrhea and edema disease after weaning continue to increase and preventive vaccines are being developed to prevent them, but products that are commercially available and sold are very rare. Escherichia coli diarrhea in piglets after weaning is mainly caused by F4 ⁺ and F18ac ⁺ Enterotoxigenic Escherichia coli (ETC), and edema is reported to be mainly caused by F18ab ⁺ and Stx2e + E. coli . .

In particular, swine edema is a major disease that has caused significant economic damage to pigs following foot and mouth disease (FMD) and porcine epidemic diarrhea (PED), and swine edema is an E. coli with Stx2e toxin ( Edema pathogen) is a bacterial disease caused by rapid multiplication in the upper intestinal tract and absorption of toxins into the blood. It is a bacterial disease that occurs frequently in piglets of 4 to 12 weeks of age and shows swelling of the eyelids, neurological symptoms, etc. We will die within. The mortality rate due to infection of edema germs is very high, 50 to 90%, and it is known that economic loss is very large because it causes a decrease in productivity due to recurrence or poor development. In Korea, vaccines for the prevention of swine edema are not commercially available, so they are treated with antibiotics, but after the onset, administration of antibiotics often misses the timing of treatment. In addition, the emergence of drug-resistant bacteria has been reported, and the development of effective preventive or therapeutic methods is urgently desired. For example, Korean Patent Registration No. 10-1743442 discloses a vaccine composition for the prevention or treatment of swine edema disease comprising a ghost Salmonella mutant expressing enterotoxin E. coli antigen as an active ingredient, and Korean Patent Publication No. 10 -2018-0114684 discloses a novel Stx2e epitope protein and porcine edema disease vaccine composition comprising the same.

Stx2e is known as a typical AB ₅ type toxin protein composed of A subunit (Stx2eA) with rRNA N-glycosidase activity and B subunit pentamer (Stx2eB) with receptor (globotetraosyl ceramide (Gb4)) binding ability. Stx2e absorbed from the intestine is in contact with the cell surface layer such as vascular endothelial cells by the B subunit pentamer, and the A subunit of Stx2e enters the target cytoplasm and inhibits protein synthesis of ribosomes, thereby causing symptoms of edema disease.

When the present inventors challenged after inoculating the piglets of Stx2eB, which is used as a vaccine for existing edema disease, to prevent swine edema disease and weaned pig diarrhea mainly caused by E. coli in piglets after weaning, It was confirmed that the antibody against edema disease was not formed properly and thus was killed. As a result of the study, it was confirmed that when the Stx2eB pentamer was inoculated as an antigen, the structure of the pentamer was not maintained and thus was not effective as a vaccine. Therefore, while studying the vaccine having an increased prophylactic or therapeutic effect by maintaining the pentameric structure of Stx2eB, the link site connecting Stx2eA and Stx2eB, except for the toxin portion that causes symptoms of edema disease in the base sequence of Stx2eA The Stx2eA c-terminal-fragment containing Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein linked to the Stx2eB pentamer was prepared from recombinant toxoid. Furthermore, the Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein and F4 ⁺ E. coli and F18 ⁺ E. coli were mixed with mycelium and inoculated into the piglets, and then challenged, resulting in an immune response than Stx2eB-pentameric antigen The present invention was completed by confirming that it effectively protects the pathogenic E. coli of pigs by inducing them well.

The present invention has been derived by the above-mentioned needs, the present inventors are the main cause of swine edema disease Stx2eB located in the C-terminal end of Stx2eB and mass expression of Stx2eB, Stx2eB forms a pentamer and the Stx2eA As the C-terminal Stx2eB binds the pentamer, the Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein is formed. The C-terminal of the Stx2eA has an effect of stably maintaining the Stx2eB pentameric form. In addition, the F4 ⁺ E. coli and F18 ⁺ E. coli strains were induced with chlorhexidine digluconate (Chlorhexidine digluconate or Chlorhexidine digluconate solution) and the Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein mixture vaccine. After inoculation in the challenge, the present invention was completed by confirming that it effectively protects pathogenic E. coli infection (including swine edema disease and weaned pig diarrheal disease) by inducing an immune response better than Stx2eB-pentameric antigen.

An object of the present invention is a Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein consisting of a C-terminal fragment monomer of Stx2eA consisting of the amino acid sequence of SEQ ID NO: 1 and a Stx2eB-pentamer consisting of the amino acid sequence of SEQ ID NO: 2. ; And intestinal toxin type E. coli expressing Fimbriae 4 (F4) and intestinal toxin type E. coli expressing Fimbriae 18 (F18), the intestine deposited with microorganism accession number KCTC13847BP (microbial name: Enterotoxigenic Escherichia coli (ETEC) F4, F18) Mixed strains of toxin-type E. coli, GI24, chlorhexidine digluconate, chlorhexidine digluconate solution, chlorhexidine diacetate, chlorhexidine acetate chlorhexidine diacetate hydrochloride (Chlorhexidine diacetate) A fungus reacted with any one selected from; It is to provide a vaccine composition for the prevention or treatment of pathogenic E. coli in pigs containing.

Another object of the present invention is a Stx2eA C-terminal fragment monomer consisting of the amino acid sequence of SEQ ID NO: 1 and a Stx2eA-C-terminal fragment consisting of the amino acid sequence of SEQ ID NO: 2 Stx2eA-C-terminal fragment-Stx2eB-pentamer Recombinant protein; And intestinal toxin type E. coli expressing Fimbriae 4 (F4) and intestinal toxin type E. coli expressing Fimbriae 18 (F18), the intestine deposited with microorganism accession number KCTC13847BP (microbial name: Enterotoxigenic Escherichia coli (ETEC) F4, F18) Mixed strains of toxin-type E. coli, GI24, chlorhexidine digluconate, chlorhexidine digluconate solution, chlorhexidine diacetate, chlorhexidine acetate chlorhexidine diacetate hydrochloride (Chlorhexidine diacetate) A fungus reacted with any one selected from; To provide a feed additive for the prevention of pathogenic E. coli in pigs.

Another object of the present invention is a Stx2eA C-terminal fragment monomer consisting of the amino acid sequence of SEQ ID NO: 1 and a Stx2eA-C-terminal fragment consisting of the amino acid sequence of SEQ ID NO: 2 Stx2eA-C-terminal fragment-Stx2eB-pentamer Recombinant protein; And intestinal toxin type E. coli expressing Fimbriae 4 (F4) and intestinal toxin type E. coli expressing Fimbriae 18 (F18), the intestine deposited with microorganism accession number KCTC13847BP (microbial name: Enterotoxigenic Escherichia coli (ETEC) F4, F18) Mixed strains of toxin-type E. coli, GI24, chlorhexidine digluconate, chlorhexidine digluconate solution, chlorhexidine diacetate, chlorhexidine acetate chlorhexidine diacetate hydrochloride (Chlorhexidine diacetate) A fungus reacted with any one selected from; It is to provide a method for enhancing immunity to pathogenic Escherichia coli in pigs comprising the step of inoculating the vaccine composition containing sows or piglets.

In order to solve the above problems, the C-terminal fragment monomer of Stx2eA consisting of the amino acid sequence of SEQ ID NO: 1 and the Stx2eA-C-terminal fragment consisting of the amino acid sequence of SEQ ID NO: 2 Stx2eA-C-terminal fragment-Stx2eB-pentamer Recombinant protein; And intestinal toxin type E. coli expressing Fimbriae 4 (F4) and intestinal toxin type E. coli expressing Fimbriae 18 (F18), the intestine deposited with microorganism accession number KCTC13847BP (microbial name: Enterotoxigenic Escherichia coli (ETEC) F4, F18) Mixed strains of toxin-type E. coli, GI24, chlorhexidine digluconate, chlorhexidine digluconate solution, chlorhexidine diacetate, chlorhexidine acetate chlorhexidine diacetate hydrochloride (Chlorhexidine diacetate) Any one of the selected reactants; It may provide a vaccine composition for the prevention or treatment of pathogenic E. coli in pigs containing.

The vaccine composition is aluminum hydroxide, GEL 01, GEL 02, IMS 1313 (VG), IMS 1313 (VG N), ISA 15A (VG), ISA 28R (VG), ISA 35 (VG), ISA 201 (VG), ISA 206 (VG), ISA 660 (VG), water-in-oil emulsion, incomplete Freund's adjuvant, alum, aluminum hydroxide, Toll-like receptor agonist (Toll-like receptor agonist), immunostimulatory oligonucleotides (Immunostimulatory oligonucleotide) and biological adjuvants (biological adjuvant) selected from the group consisting of one or more adjuvants (adjuvant) may be additionally included.

The concentration of the Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein may be 10 ug to 1,000 ug.

The cells may be mixed in an equal amount from 1 × 10 ⁸ cells / ml to 1 × 10 ¹¹ cells / ml, respectively.

The vaccine composition may be inoculated into piglets or sows.

The pathogenic E. coli of the pig may be edema disease of pigs, newborn pig diarrhea disease, premature diarrhea disease, 3-week-old diarrhea disease, baekri, mammalian pig diarrhea disease or weaned pig diarrhea disease.

A Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein consisting of a C-terminal fragment monomer of the amino acid sequence of SEQ ID NO: 1 and a Stx2eB-pentamer consisting of the amino acid sequence of SEQ ID NO: 2; And intestinal toxin type E. coli expressing Fimbriae 4 (F4) and intestinal toxin type E. coli expressing Fimbriae 18 (F18), the intestine deposited with microorganism accession number KCTC13847BP (microbial name: Enterotoxigenic Escherichia coli (ETEC) F4, F18) Mixed strains of toxin-type E. coli, GI24, chlorhexidine digluconate, chlorhexidine digluconate solution, chlorhexidine diacetate, chlorhexidine acetate chlorhexidine diacetate hydrochloride (Chlorhexidine diacetate) A fungus reacted with any one selected from; It may provide a feed additive for the prevention of pathogenic E. coli in pigs.

 The pathogenic E. coli of the pig may be edema disease of pigs, newborn pig diarrhea disease, premature diarrhea disease, 3-week-old diarrhea disease, baekri, mammalian pig diarrhea disease or weaned pig diarrhea disease.

The present invention also provides a Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein consisting of a C-terminal fragment monomer of Stx2eA consisting of the amino acid sequence of SEQ ID NO: 1 and a Stx2eB-pentamer consisting of the amino acid sequence of SEQ ID NO: 2; And intestinal toxin type E. coli expressing Fimbriae 4 (F4) and intestinal toxin type E. coli expressing Fimbriae 18 (F18), the intestine deposited with microorganism accession number KCTC13847BP (microbial name: Enterotoxigenic Escherichia coli (ETEC) F4, F18) Mixed strains of toxin-type E. coli, GI24, chlorhexidine digluconate, chlorhexidine digluconate solution, chlorhexidine diacetate, chlorhexidine acetate chlorhexidine diacetate hydrochloride (Chlorhexidine diacetate) A fungus reacted with any one selected from; It is possible to provide a method for enhancing immunity to pathogenic E. coli of pigs, comprising inoculating the vaccine composition comprising sows or piglets.

The pathogenic E. coli of the pig may be edema disease of pigs, newborn pig diarrhea disease, premature diarrhea disease, 3-week-old diarrhea disease, baekri, mammalian pig diarrhea disease or weaned pig diarrhea disease.

The recombinant Stx2eA-C-terminal fragment-pentameric Stx2eB protein of the present invention has the effect of increasing its structural stability than the commonly known pentameric Stx2eB protein, thereby increasing the production of antibodies as vaccines. In addition, as a kind of toxoid, it is effective in preventing edema disease of pigs by preventing Stx2e from attaching to effective cells by acting on the secreted Stx2e infected with E. coli that causes edema disease in pigs by inducing antisera to the attachment site without toxicity. It has a healing effect. In addition, enterotoxin type E. coli expressing F4 or enterotoxin type E. coli expressing F18 produces antibodies against these bacteria and continuously releases Stx2e, LT, and STa toxins in the intestinal tract F4 ⁺ E. coli and F18 ⁺ E. coli It has the effect of effectively preventing or treating pathogenic E. coli infectious diseases such as swine edema disease and weaned pig diarrhea by preventing the production of Stx2e, LT, and STa toxins that no longer produce edema disease and weaned pig diarrhea. The Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein and Stx2e + F4 ⁺ Escherichia coli and Stx2e + F18 ⁺ Escherichia coli cells were mixed and inoculated into piglets with a vaccine, and the antibody induction was confirmed, and only Stx2eB-pentameric antigen was inoculated. The immune response to each antigen was induced better than in one case, and it was effective to prevent pathogenic E. coli infection (including swine edema disease and weaned pig diarrhea) when challenged with pathogenic outdoor isolates.

1 is a schematic diagram of the Stx2eA-C-terminal fragment protein expression recombinant vector pTWIN1 used in the present invention.
2 is a schematic diagram of Stx2eB protein expression recombinant expression pET30 used in the present invention.
Figure 3 is the result of SDS-PAGE for confirming the expression of Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein of the present invention.
Fig. 4 also includes enterotoxin type E. coli expressing F4 (Stx2e alone or F4 expressing E. coli expressing Stx2e simultaneously) or enterotoxin type E. coli expressing F18 (including F18 expressing E. coli also expressing Stx2e alone or Stx2e) Electron microscopy before and after treatment with Chlorhexidine digluconate or Chlorhexidine digluconate solution or Chlorhexidine acetate or Chlorhexidine diacetate or Chlorhexidine diacetate salt hydrate.
5 is a result of analyzing the serum IgG antibody titer for Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein after inoculating the vaccine composition of the present invention into piglets.
6 is a result of analyzing the serum IgG antibody titer for K88ac and FedA (a component of F18 finbria) recombinant protein after inoculating the vaccine composition of the present invention into piglets.
Figure 7 after the vaccine composition of the present invention inoculated into piglets, outdoor toxicity F4 ⁺ Stx2e ⁺ LT ⁺ STa ⁺ STb ⁺ F4 ⁺ STEC and F18 ⁺ Stx2e ⁺ LT ⁺ STa ⁺ STb ⁺ F4 ⁺ STEC mixed to prevent after oral inoculation This is the result for the effect.

After weaning, diarrhea and edema disease after weaning continue to increase and preventive vaccines are being developed to prevent them, but few products are commercially available. The most widely studied vaccine for the prevention of edema disease is the recombinant Stx2eB pentameric protein. However, when the present inventors challenged the piglets after inoculating the recombinant Stx2eB pentameric protein, it was confirmed that edema disease was expressed without antibody induction. As a result of research on the reason, it was confirmed that the structure of the Stx2eB pentamer could not be maintained firmly and thus could not properly function as an antigen. Therefore, while studying the method to maintain the structure of the pentamer structure of Stx2eB, the researchers excluded the toxin that causes the symptoms of edema in the base sequence of Stx2eA, and included Stx2eA, including the site that connects Stx2eA and Stx2eB. When the c-terminal fragment of Stx2eB was combined with a pentamer, it was confirmed that the structure of the pentamer of Stx2eB is maintained firmly and serves as an antigen. Furthermore, enterotoxin type E. coli expressing F4 or enterotoxin type E. coli expressing F18 is chlorhexidine digluconate (Chlorhexidine digluconate or Chlorhexidine digluconate solution) or chlorhexidine diacetate (Chlorhexidine acetate or Chlorhexidine diacetate or Chlorhexidine diacetate salt hydrate using Chlorhexidine acetate or Chlorhexidine diacetate or Chlorhexidine diacetate) When inactivated and inoculated into piglets, it was confirmed that the bacteria were removed by the induced antibody and no longer produce toxins, and thus the inactivated cells of the bacteria were also essential for the prevention of pathogenic E. coli.

In addition, the Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein and Stx2e ⁺ F4 ⁺ Escherichia coli and Stx2e ⁺ F18 ⁺ Escherichia coli cells were mixed and inoculated into piglets with a vaccine to confirm whether the antibody was induced. By inducing the immune response to each antigen better than in the case of inoculating only the monomer, the present invention was confirmed by effectively defending against pathogenic E. coli infection (including swine edema disease and weaned pig diarrheal disease) when challenged with a pathogenic outdoor isolate. Completed.

The present invention is a Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein composed of a C-terminal fragment monomer of Stx2eA consisting of the amino acid sequence of SEQ ID NO: 1 and a Stx2eB-pentamer consisting of the amino acid sequence of SEQ ID NO: 2; And intestinal toxin type E. coli expressing Fimbriae 4 (F4) and intestinal toxin type E. coli expressing Fimbriae 18 (F18), the intestine deposited with microorganism accession number KCTC13847BP (microbial name: Enterotoxigenic Escherichia coli (ETEC) F4, F18) Mixed strains of toxin-type E. coli, GI24, chlorhexidine digluconate, chlorhexidine digluconate solution, chlorhexidine diacetate, chlorhexidine acetate chlorhexidine diacetate hydrochloride (Chlorhexidine diacetate) A fungus reacted with any one selected from; It may provide a vaccine composition for the prevention or treatment of pathogenic E. coli in pigs containing.

The Stx2eA-C-terminal fragment has the effect of increasing the role of the vaccine by binding to the pentameric Stx2eB after formation of the pentameric Stx2eB, in which the recombinant monomer Stx2eB subunit can serve as a vaccine, thereby maintaining the shape.

Stx2eB-pentamer containing the C-terminal fragment monomer of the Stx2eA; and enterotoxin type E. coli expressing F4 or enterotoxin type E. coli expressing F18, respectively, GI24, chlorhexidine digluconate (Chlorhexidine digluconate), The mixture of inactivated bacteria is reacted with any one selected from the group consisting of chlorhexidine digluconate solution, chlorhexidine diacetate, and chlorhexidine diacetate salt hydrate. It has the effect of enhancing the role, preferably chlorhexidine digluconate (Chlorhexidine digluconate) or chlorhexidine digluconate aqueous solution (Chlorhexidine digluconate solution) can be used.

The intestinal toxin type E. coli expressing Fimbriae 4 (F4) and the enterotoxin type E. coli expressing Fimbriae 18 (F18) are mixed strains, and the microorganism deposit number KCTC13847BP (microbial name: Enterotoxigenic Escherichia coli (ETEC) F4, F18) It may be a deposited E. coli strain.

Intestinal toxin type E. coli expressing Fimbriae 4 (F4) and enterotoxin type E. coli expressing Fimbriae 18 (F18) are selected from the group consisting of F4 ⁺ , F18 ⁺ , LT ⁺ , STa ⁺ , STb ⁺ and Stx2e ⁺ Any one or more toxins may be secreted, preferably any one or more toxins selected from the group consisting of Stx2e ⁺ , F4 ⁺ and F18 ⁺ , more preferably F4 ⁺ and Stx2e ⁺ , or F18 ⁺ And Stx2e ⁺ .

In addition, the Stx2eA-C-terminal fragment-Stx2eB-pentameric recombination protein and F4 ⁺ enterotoxin type E. coli and F18 ⁺ enterotoxin type E. coli inoculated with a vaccine were inoculated into piglets, and then confirmed whether the antibody was induced. -Induced immune responses to each antigen better than when inoculated with only the pentameric antigen, and effectively challenged the pathogenic E. coli infection when challenged with pathogenic outdoor isolates.

The vaccine composition may additionally include a carrier or an adjuvant. The adjuvant is aluminum hydroxide, GEL 01, GEL 02, IMS 1313 (VG), IMS 1313 (VG N), ISA 15A (VG), ISA 28R (VG), ISA 35 (VG), ISA 201 (VG), ISA 206 (VG), ISA 660 (VG), water-in-oil emulsion, incomplete Freund's adjuvant, alum, aluminum hydroxide, Toll-like receptor agonist ( agonist), immunostimulatory (Immunostimulatory) oligonucleotides, and any one or more adjuvants selected from the group consisting of biological adjuvants (adjuvant), most preferably IMS 1313 (VG).

The carrier includes any and all solvents, dispersion media, coating agents, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption retardants, and the like. Carriers, excipients, and diluents that may be included in the vaccine composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The composition of the present invention can be administered orally or parenterally (e.g., applied intramuscularly, intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, preferably orally or intramuscularly. However, it is not limited thereto. The vaccine composition may be inoculated into piglets or sows, but most preferably, it may be inoculated into piglets. In addition, the dosage of the composition may vary depending on the weight of the pig, age, sex, health status, diet, administration time, administration method, excretion rate and disease severity.

The dose of the Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein and Stx2e ⁺ F4 ⁺ E. coli and Stx2e ⁺ F18 ⁺ E. coli cell mixture is a mammal for the Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein For piglets or weaned piglets, it is preferable to administer about 1 ug to 1,000 ug per piglet, preferably about 30 ug to 200 ug, and most preferably 75 ug.

In addition, in the case of F4 ⁺ E. coli and F18 ⁺ E. coli cells, about 1 × 10 ⁷ cells to 1 × 10 ¹¹ cells per E. coli per piglet, preferably about 1 × 10 ⁸ cells to 1 × 10 ¹⁰ cells per E. coli As, most preferably, it is preferably administered at about 1 × 10 ⁹ cells per each E. coli.

In the present invention, the term "vaccine" refers to an immunogen or antigenic substance that is immune to a living body by injection or oral administration to a human or animal for the prevention of infectious diseases as a biological agent containing an antigen that immunizes the living body. In vivo immunity is largely divided into automatic immunity, in which immunity in vivo is automatically obtained after infection of the pathogen, and passive immunity, obtained by an externally injected vaccine. While autoimmunity is characterized by a long period of production of antibodies related to immunity and continuous immunity, passive immunization by vaccine acts immediately to treat infectious diseases, but has a disadvantage of poor persistence.

The vaccine composition includes stabilizers, emulsifiers, aluminum hydroxide, aluminum phosphate, pH adjusters, surfactants, liposomes, iscom adjuvants, synthetic glycopeptides, extenders, carboxypolymethylene, subviral particle adjuvants, cholera toxins , N, N-dioctadecyl-N ', N'-bis (2-hydroxyethyl) -propanediamine, monophosphoryl lipid A, dimethyldioctadecyl-ammonium bromide and any one selected from the group consisting of mixtures thereof The above second auxiliary agent may be further contained. In addition, the composition for the vaccine is an oral dosage form such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, and nasal dosage form such as drip or spray, and a sterile injection solution, respectively, according to a conventional method. It can be used in the form of a formulation. In the case of formulation, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient such as starch, calcium carbonate, sucrose in the lecithin-like emulsifier. Or it can be prepared by mixing lactose, gelatin, and the like. It is also possible to use lubricants, such as magnesium stearate, in addition to simple excipients. As a liquid preparation for oral administration, suspending agents, intravenous solutions, emulsions, syrups, etc. can be used. In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. Can be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous agents, suspensions, emulsions, and lyophilized preparations. Non-aqueous preparations and suspensions include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Penetrating agents suitable for formulation for intranasal administration are generally known to those skilled in the art. Such suitable formulations are formulated to be sterile, isotonic and buffered, preferably for stability and compliance. Formulations for intranasal administration are also prepared to stimulate mucus secretion in several aspects to maintain normal ciliary function, described in Remington's Pharmaceutical Science, 18th Ed., Mack Publishing Co., Easton, PA (1990). As indicated, suitable formulations are preferably isotonic, slightly buffered formulations that maintain a pH of 5.5 to 6.5, and most preferably include antimicrobial preservatives and suitable drug stabilizers.

The recombinant Stx2eA-C-terminal fragment-Stx2eB-pentameric protein and Stx2e ⁺ F4 ⁺ Escherichia coli and Stx2e ⁺ F18 ⁺ Escherichia coli mixtures alone or by mixing with an adjuvant such as aluminum sodium hydroxide, IMS1313 to muscle, subcutaneous, etc. Inoculation can be used as a vaccine.

The pathogenic E. coli of the pig may be edema disease of the pig, neonatal pig diarrhea disease, coarse diarrhea disease, 3-week-old diarrhea disease, baekri, maternal pig diarrhea disease or weaned pig diarrhea disease, preferably pig edema disease or weaned pig diarrhea disease.

In addition, the present invention is a Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein consisting of a C-terminal fragment monomer of Stx2eA consisting of the amino acid sequence of SEQ ID NO: 1 and a Stx2eB-pentamer consisting of the amino acid sequence of SEQ ID NO: 2; And intestinal toxin type E. coli expressing Fimbriae 4 (F4) and intestinal toxin type E. coli expressing Fimbriae 18 (F18), the intestinal deposit deposited with microorganism accession number KCTC13847BP (microbial name: Enterotoxigenic Escherichia coli (ETEC) F4, F18) It is possible to provide a feed additive for preventing pathogenic E. coli in pigs, including a fungus reacted with a mixed strain of toxin-type E. coli and chlorhexidine digluconate or an aqueous solution thereof (Chlorhexidine digluconate solution).

The pathogenic E. coli of the pig may be edema disease of the pig, neonatal pig diarrhea disease, coarse diarrhea disease, 3-week-old diarrhea disease, baekri, maternal pig diarrhea disease or weaned pig diarrhea disease, preferably pig edema disease or weaned pig diarrhea disease.

For the feed additive of the present invention, Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein and F4 ⁺ Escherichia coli and F18 ⁺ Escherichia coli or mixtures thereof are used in their original form or additionally, such as grains and by-products that are allowed for livestock. Added carriers, stabilizers, etc., and if necessary, organic acids such as citric acid, humic acid, adipic acid, lactic acid, and malic acid, or phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polymerized phosphate), or poly Natural antioxidants such as phenol, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid, antibiotics, and other additives may also be added. It may be in a suitable state such as granules, pellets, suspensions, etc. When the feed additive is supplied, it can be used alone or for livestock. To be supplied by mixed.

The present invention also provides a Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein consisting of a C-terminal fragment monomer of Stx2eA consisting of the amino acid sequence of SEQ ID NO: 1 and a Stx2eB-pentamer consisting of the amino acid sequence of SEQ ID NO: 2; And intestinal toxin type E. coli expressing Fimbriae 4 (F4) and intestinal toxin type E. coli expressing Fimbriae 18 (F18), the intestine deposited with microorganism accession number KCTC13847BP (microbial name: Enterotoxigenic Escherichia coli (ETEC) F4, F18) A method for inducing pathogenic Escherichia coli of pigs, comprising inoculating sows or piglets with a vaccine composition comprising a bacterium reacted with a mixed strain of toxin-type E. coli with chlorhexidine digluconate or an aqueous solution thereof (Chlorhexidine digluconate solution) It can provide a way to improve immunity.

The pathogenic E. coli of the pig may be edema disease of the pig, neonatal pig diarrhea disease, coarse diarrhea disease, 3-week-old diarrhea disease, baekri, maternal pig diarrhea disease or weaned pig diarrhea disease, preferably pig edema disease or weaned pig diarrhea disease.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, the content of the present invention is not limited to the following examples.

Example 1. Bacterial strain, plasmid and growth conditions

All strains and plasmids used in the present invention are listed in Table 1 below. STEC 150229 and HJL574 and HJL746, which are outdoor isolate strains isolated from suspected edema disease, were used as challenge strains. The recombinant Stx2eA-C-terminal fragment and Stx2eB protein were overexpressed from HJL573. STEC 150229, HJL573, HJL574 HJL746 and HJL954 were cultured at 37 ° C in LB liquid medium (Luria-Bertani broth; Becton, Dickinson and Company, USA) or LB agar medium. For overexpression, HJL954 was incubated at 18 ° C. for 24 hours after 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (Duchefa) was added.

Strains and vectors used in the present invention Strain / plasmid Explanation source Strain  Escherichia coli  HJL565 Wild type F18 ⁺ STa ⁺ ETEC isolate from piglet The present invention  HJL574 Wild type Stx2e ⁺ LT ⁺ STa ⁺ F18 ⁺ STEC isolate from piglet with edema disease The present invention  HJL593 Wild type F4ac ⁺ LT ⁺ STa ⁺ ETEC isolate from piglet The present invention  HJL746 Wild type Stx2e ⁺ LT ⁺ STa ⁺ STb ⁺ F4 ⁺ STEC isolate from piglet with edema disease The present invention  STEC150229 Wild type Stx2e ⁺ F18 ⁺ STEC isolate from piglet with edema disease The onset  HJL954 E. coli BL21 (DE3) pLysS with pStx2eA-C-terminal fragment-fusion and pStx2eB The present invention Plasmid  pStx2eA-C-terminal fragment-fusion pTWIN1 containing the gene for Stx2eA C-terminal (a.a. 215-319 of Stx2eA) fragment The present invention  pStx2eB pET30a containing the gene for Stx2eB The present invention

Example 2. Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein expression and purification

The recombinant HJL954 strain was inoculated into 100 ml of LB containing Ampicillin, Chloramphenicol and Kanamycin, and cultured overnight at a temperature of 37 ° C at 200 rpm. 1 ml LB containing ampicillin, chloramphenicol, and kanamycin was inoculated with 10 ml of the culture medium to inoculate O.D. Incubation was performed at a temperature of 37 ° C at a rate of 200 rpm until the value of 600 became 0.5. After adding IPTG to a final concentration of 0.8 mM, expression was induced for 16 hours at a speed of 150 rpm at a temperature of 18 ° C. The culture solution was centrifuged at 4000 rpm for 20 minutes to obtain cells overexpressing the protein. After crushing the cells by ultrasonic grinding, the supernatant was fractionated by centrifugation at 12,000 rpm for 20 minutes. The supernatant was filtered using a 0.45 μm syringe filter followed by pure purification using a FPLC system with HisTrap FF column connected. Protein was attached to the column using A buffer (10 mM Tris, pH 8.0, 300 mM NaCl), and the amount of B buffer (10 mM Tris, pH 8.0, 300 mM NaCl, 300 mM Imidazole) containing Imidazole was sequentially increased. Protein was eluted in the column. The purified protein was concentrated and stored at 4 ° C until use. As a result of westernizing the obtained Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein, it was confirmed that the Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein was formed (FIG. 3).

Example 3. F4 ⁺ ETEC, F18 ⁺ ETEC new concept fungus production

One colony of F4 ⁺ ETEC (HJL593) or F18 ⁺ ETEC (HJL565) strains was selected and inoculated into 200 ml of LB liquid medium, respectively, and then cultured at 37 ° C. until absorbance reached 0.8. Chlorhexidine digluconate or Chlorhexidine digluconate solution or Chlorhexidine diacetate prepared in cultured medium was added at 3 ul / ml to 20 ul / ml (3.18 mg / ml to 12.2 mg / ml) each. The ETEC strain was reacted at room temperature for 5 to 30 minutes to induce lysis. After the reaction, 100 μl of each reaction solution was inoculated into LB agar medium and broth, respectively, to confirm whether or not it was induced into living cells such as live bacteria. In addition, the confirmed lysis reaction solution was concentrated by centrifugation at 4,000 × g for 30 minutes, washed three times with sterile PBS, and finally diluted to 2 × 10 ⁹ cells / ml per each serotype and stored in refrigeration. .

Example 4. F4 ⁺ ETEC, F18 ⁺ ETEC Projection electron microscope (TEM) of new concept germ cells

The F4 ⁺ ETEC and F18 ⁺ ETEC new concept fungi prepared by the method of Example 3 were ajuvant IMS 1313 and Stx2eA-C-terminal fragment prepared in Example 2 -Stx2eB-pentamer 2.5% overnight before mixing with recombinant protein After linear fixation with glutaraldehyde, the bacterial pellet was washed 3 times with PBS and then fixed with 1% osmium tetraoxide in PBS for 2 hours. The fixed bacterial cells were washed 3 times with PBS and dehydrated in various series of ethanol (50%, 70%, 90%, 100%) for 15 minutes. After placing in acetone anhydrous for 20 minutes, these samples were transferred to 1: 1 and 1: 3 mixtures of acetone anhydrous and epoxy resin for 1 hour each, and overnight to pure epoxy resin. Ultrathin sections obtained using Ultramicrotome were post-stained with uranyl acetate and lead citrate. The specimen was observed with a transmission electron microscope (Hitachi H-7650, Japan). TEM was taken according to the method described in Lv et al (Lv et al. 2014). As shown in FIG. 2A, the F4 ⁺ ETEC and F18 ⁺ ETEC strains that were not treated with the antibacterial agent used in Example 3 were observed with intact cell membranes and cell contents filling the cells (FIG. 2A). On the other hand, the new concept of F4 ⁺ ETEC and F18 ⁺ ETEC induced by chlorhexidine digluconate (Chlorhexidine digluconate or Chlorhexidine digluconate solution) or chlorhexidine diacetate is empty with one perforated cell membrane as shown in Figure 2B. Cytoplasmic space was observed.

Example 5. Vaccination and sample collection

Pregnancy sows (n = 5) and piglets (n = 25) used in the present invention were purchased for farms that had no symptoms of edema disease and were negative for Stx2e in a domestic pig farm in Jeonbuk, Korea. Pregnant sows were usually vaccinated according to the vaccine program at the farm. Ten days before the expected delivery date, the sows were transferred to delivery cages with heat lamps and delivery frames for each pig. Pregnant sows were fed on a diet without antibiotics or other growth promoters. Piglets were raised with sows up to 21 days after birth. 25 piglets born from 5 pregnant sows were divided into 5 groups. When the piglets were 14 days old, Group A and B piglets were inoculated with 1 ml of sterile PBS, and in group C, the piglets were Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein and F4 ⁺ ETEC and F18 ⁺ ETEC new concept bacteria. As a vaccine mixed with IMS1313 adjuvant so as to be 1 × 10 ⁹ cells / ml, Stx2eA-C-terminal fragment-Stx2eB-omeric recombinant protein was intramuscularly inoculated with 50 ug, 75 ug, and 100 ug, respectively (0 week post prime immunization; WPPI) ). When the pigs were 3 weeks old, they were transferred to the specialized campus of Chonbuk National University, where 5 animals were transferred to each group to spend a week of adaptation. And at the age of 4 weeks, pigs from the same group were inoculated secondly with the same antigen (3 WPPI). Blood samples were collected prior to the second inoculation (2 WPPI) and before challenge (6 weeks old, 4 WPPI), respectively. All samples were stored at -70 ° C until use. All experimental animals were used in accordance with the guidelines of the Korea Animal Protection Association and approved by the Chonbuk National Animal Ethics Committee.

Example 6. ELISA immune response measurement

To test the immune-enhancing effect on each Stx2eA-C-terminal fragment-Stx2eB-pentamer and F4 (K88ac) and F18 (FedA) recombinant protein antigens, a pig IgG ELISA Quantitation Kit (Bethyl Lab Inc., USA) was used. The enzyme immunoassay (ELISA) was performed. Serum samples of piglets were diluted 1: 200 and used for IgG titers. The enzyme reaction was developed with o -phenylenediamine (Sigma-Aldrich, USA) to measure absorbance at 492 nm. Antibody responses to each Stx2eA-C-terminal fragment-Stx2eB-pentamer and F4 (K88ac) and F18 (FedA) recombinant protein antigens in piglet serum are disclosed in FIG. 4. Serum IgG titers for F4 (K88ac) and F18 (FedA) recombinant protein antigens in all vaccinated sow groups compared to control group A, group B (group inoculated with PBS) showed similar results to control. On the other hand, in the case of group CE, it was significantly increased compared to the control group (P≤0.05) (Fig. 4A). However, the serum IgG titer for the Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein antigen was similar to that of the control group, compared to the control group, group B (group inoculated with PBS), whereas 50 ug, Group CE group inoculated with 75 ug and 100 ug significantly increased (P≤0.05) compared to the control group (Fig. 4B). In particular, serum IgG titers in piglets of groups inoculated with 75 and 100 ug were significantly increased.

Example 7. Challenge infection

For challenge infection experiments, JHL574, JHL746, and STEC 150229 were prepared as challenge strains. When the piglets were 6 weeks old, each strain was prepared at 1 * 10 ⁹ CFU / ml, mixed in equal amounts, and orally inoculated with 3 ml of all piglets except group A piglets. Piglets were monitored for clinical signs and mortality daily for 14 days of inoculation. Pathogen Escherichia coli was isolated by collecting samples from the piglets with suspected edema disease or weaned piglet diarrheal disease symptoms, and F4 ⁺ E. coli or F18 ⁺ E. coli using the primers described in Table 2 below. Whether it was confirmed by PCR method.

 Size of primers and PCR products used in this experiment Primer Sequence Size (bp) F4 F TGA ATG ACC TGA CCA ATG GTG GAA CC 484 R GCG TTT ACT CTT TGA ATC TGT CCG AG F18 F TGG CAC TGT AGG AGA TAC CAT TCA GC 334 R GGT TTG ACC ACC TTT CAG TTG AGC AG LT F ACG GCG TTA CTA TCC TGT CTA TGT GC 275 R TTG GTC TCG GTC AGA TAT GTG ATT CT STa F GTC AGT CAA CTG AAT CAC TTG ACT CT 152 R CAT GGA GCA CAG GCA GGA TTA CAA CA Stx2e F CGG TAT CCT ATT CCC AGG AGT TTA CG 599 R GTC TTC CGG CGT CAT CGT ATA AAC AG

In group E of piglets (group inoculated with 100 ug), clinical symptoms and mortality were not observed until 14 days of challenge infection, while 4 out of 5 groups of group B piglets, who died, showed clinical symptoms and died of group C piglets (50 ug inoculation group died of 1 in 5 patients with clinical symptoms, but no clinical symptoms were observed in group D piglets (group inoculated with 75 ug) and group E piglets (group inoculated with 100 ug). Did (Table 3).

Clinical symptoms of piglets after challenge infection against outdoor pathogenic E. coli strains group Piglet The number of our piglets (%) A 5 0 B 5 4 (80) C 5 1 (20) D 5 0 E 5 0

<110> INDUSTRIAL COOPERATION FOUNDATION CHONBUK NATIONAL UNIVERSITY <120> Vaccine composition for preventing or treating porcine edema          disease comprising Stx2eA-C-terminal fragment- (Stx2eB) 5          recombinant protein as an antigen <130> 1064972 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of Stx2eA C-terminal fragment <400> 1 Pro Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Ile Ser Asn Val   1 5 10 15 Leu Pro Glu Tyr Arg Gly Glu Ala Gly Val Arg Val Gly Arg Ile Ser              20 25 30 Phe Asn Asn Ile Ser Ala Ile Leu Gly Thr Val Ala Val Ile Leu Asn          35 40 45 Cys His His Gln Gly Ala Arg Ser Val Arg Ala Val Asn Glu Glu Ser      50 55 60 Gln Pro Glu Cys Gln Ile Thr Gly Asp Arg Pro Val Ile Lys Ile Asn  65 70 75 80 Asn Thr Leu Trp Glu Ser Asn Thr Ala Ala Ala Phe Leu Asn Arg Lys                  85 90 95 Ser Gln Ser Leu Tyr Thr Thr Gly Glu             100 105 <210> 2 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of Stx2eB <400> 2 Lys Lys Met Phe Ile Ala Val Leu Phe Ala Leu Val Ser Val Asn Ala   1 5 10 15 Met Ala Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr Asn              20 25 30 Glu Asp Asn Thr Phe Thr Val Lys Val Ser Gly Arg Glu Tyr Trp Thr          35 40 45 Asn Arg Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr Gly      50 55 60 Met Thr Val Thr Ile Ile Ser Asn Thr Cys Ser Ser Gly Ser Gly Phe  65 70 75 80 Ala Gln Val Lys Phe Asn                  85 <210> 3 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for expressing Stx2eA C-terminal fragment in          vector <400> 3 Met Gly His His His His His His Pro Glu Asp Val Asp Leu Thr Leu   1 5 10 15 Asn Trp Gly Arg Ile Ser Asn Val Leu Pro Glu Tyr Arg Gly Glu Ala              20 25 30 Gly Val Arg Val Gly Arg Ile Ser Phe Asn Asn Ile Ser Ala Ile Leu          35 40 45 Gly Thr Val Ala Val Ile Leu Asn Cys His His Gln Gly Ala Arg Ser      50 55 60 Val Arg Ala Val Asn Glu Glu Ser Gln Pro Glu Cys Gln Ile Thr Gly  65 70 75 80 Asp Arg Pro Val Ile Lys Ile Asn Asn Thr Leu Trp Glu Ser Asn Thr                  85 90 95 Ala Ala Ala Phe Leu Asn Arg Lys Ser Gln Ser Leu Tyr Thr Thr Gly             100 105 110 Glu     <210> 4 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for expressing Stx2eB in vector <400> 4 Met Ser Glu Asp Thr Lys Ser Pro Lys Lys Lys Ser Ser Pro Ile Arg   1 5 10 15 Ala Asn Arg Leu Ala His Met Gly Ser Ala Thr Met Ala His His His              20 25 30 His His His Lys Lys Met Phe Ile Ala Val Leu Phe Ala Leu Val Ser          35 40 45 Val Asn Ala Met Ala Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser      50 55 60 Lys Tyr Asn Glu Asp Asn Thr Phe Thr Val Lys Val Ser Gly Arg Glu  65 70 75 80 Tyr Trp Thr Asn Arg Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln                  85 90 95 Leu Thr Gly Met Thr Val Thr Ile Ile Ser Asn Thr Cys Ser Ser Gly             100 105 110 Ser Gly Phe Ala Gln Val Lys Phe Asn         115 120 <210> 5 <211> 318 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of Stx2eA C-terminal fragment <400> 5 ccggaagacg tggacctcac tctgaactgg gggagaatca gcaatgtgct tccggagtat 60 cggggagagg ctggtgtcag agtggggaga atatccttta ataatatatc agcgatactt 120 ggtactgtgg ccgttatact gaattgccat catcagggcg cgcgttctgt tcgcgccgtg 180 aatgaagaga gtcaaccaga atgtcagata actggcgaca ggcccgttat aaaaataaac 240 aatacattat gggaaagtaa tacagcagca gcgtttctga acagaaagtc acagtcttta 300 tatacaactg gtgaatga 318 <210> 6 <211> 258 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of Stx2eB <400> 6 aagaagatgt ttatagcggt tttatttgca ttggtttctg ttaatgcaat ggcggcggat 60 tgtgctaaag gtaaaattga gttttccaag tataatgagg ataatacctt tactgtgaag 120 gtgtcaggaa gagaatactg gacgaacaga tggaatttgc agccattgtt acaaagtgct 180 cagctgacag ggatgactgt aacaatcata tctaatacct gcagttcagg ctcaggcttt 240 gcccaggtga agtttaac 258 <210> 7 <211> 350 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for expressing Stx2eA C-terminal fragment in          vector <400> 7 ccatggggca tcatcaccat caccatccgg aagacgtgga cctcactctg aactggggga 60 gaatcagcaa tgtgcttccg gagtatcggg gagaggctgg tgtcagagtg gggagaatat 120 cctttaataa tatatcagcg atacttggta ctgtggccgt tatactgaat tgccatcatc 180 agggcgcgcg ttctgttcgc gccgtgaatg aagagagtca accagaatgt cagataactg 240 gcgacaggcc cgttataaaa ataaacaata cattatggga aagtaataca gcagcagcgt 300 ttctgaacag aaagtcacag tctttatata caactggtga atgaggatcc 350 <210> 8 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for expressing Stx2eB in vector <400> 8 catatgagtg aggacacaaa atcaccgaaa aagaagtcga gcccaatccg tgccaataga 60 ctagcccaca tgggatccgc caccatggcc caccatcatc accaccataa gaagatgttt 120 atagcggttt tatttgcatt ggtttctgtt aatgcaatgg cggcggattg tgctaaaggt 180 aaaattgagt tttccaagta taatgaggat aataccttta ctgtgaaggt gtcaggaaga 240 gaatactgga cgaacagatg gaatttgcag ccattgttac aaagtgctca gctgacaggg 300 atgactgtaa caatcatatc taatacctgc agttcaggct caggctttgc ccaggtgaag 360 tttaacggat cc 372

Claims (10)

A Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein consisting of a C-terminal fragment monomer of the amino acid sequence of SEQ ID NO: 1 and a Stx2eB-pentamer consisting of the amino acid sequence of SEQ ID NO: 2; And
Intestinal toxin type E. coli expressing Fimbriae 4 (F4) and enterotoxin type E. coli expressing Fimbriae 18 (F18), enteric poison deposited with microorganism accession number KCTC13847BP (microbial name: Enterotoxigenic Escherichia coli (ETEC) F4, F18) Mixed strains of small E. coli and GI24, chlorhexidine digluconate, chlorhexidine digluconate solution, chlorhexidine diacetate, chlorhexidine acetate chlorhexidine diacetate salt hydrate consisting of a group of chlorhexidine diacetate salts of chlorhexidine diacetate A fungus that reacts any one selected;
Vaccine composition for the prevention or treatment of pathogenic E. coli in pigs comprising a. The method of claim 1, wherein the vaccine composition is aluminum hydroxide, GEL 01, GEL 02, IMS 1313 (VG), IMS 1313 (VG N), ISA 15A (VG), ISA 28R (VG), ISA 35 (VG), ISA 201 (VG), ISA 206 (VG), ISA 660 (VG), water-in-oil emulsion, incomplete Freund'adjuvant, alum, aluminum hydration Additional one or more adjuvants selected from the group consisting of aluminum hydroxide, toll-like receptor agonist, immunostimulatory oligonucleotide and biological adjuvant A vaccine composition for the prevention or treatment of pathogenic E. coli in pigs. The vaccine composition of claim 1, wherein the vaccine composition has a concentration of 10 ug to 1,000 ug of Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein. The vaccine composition for preventing or treating pathogenic Escherichia coli in pigs according to claim 1, wherein the germ cells are mixed in an amount of 1 × 10 ⁸ cells / ml to 1 × 10 ¹¹ cells / ml, respectively. The vaccine composition according to claim 1, wherein the vaccine composition is inoculated into piglets or sows, for prevention or treatment of pathogenic E. coli in pigs. The method of claim 1, wherein the pathogenic E. coli of the pig is edema disease of the pig, neonatal pig diarrhea disease, premature diarrhea disease, 3-week-old diarrhea disease, baekri, mammalian pig diarrhea disease, or weaned pig diarrhea disease, a vaccine composition for preventing or treating pathogenic E. coli in pigs . A Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein consisting of a C-terminal fragment monomer of the amino acid sequence of SEQ ID NO: 1 and a Stx2eB-pentamer consisting of the amino acid sequence of SEQ ID NO: 2; And
Intestinal toxin type E. coli expressing Fimbriae 4 (F4) and enterotoxin type E. coli expressing Fimbriae 18 (F18), enteric poison deposited with microorganism accession number KCTC13847BP (microbial name: Enterotoxigenic Escherichia coli (ETEC) F4, F18) Mixed E. coli strain and GI24, chlorhexidine digluconate, chlorhexidine digluconate solution, chlorhexidine diacetate, chlorhexidine acetate chlorhexidine diacetate salt group consisting of chlorhexidine diacetate salt hydrate (Chlorhexidine diacetate) A fungus that reacts any one selected;
Containing, feed additive for preventing pathogenic E. coli in pigs. The method of claim 7, wherein the pathogenic E. coli of the pig is edema disease of the pig, neonatal pig diarrhea disease, premature diarrhea disease, 3-week-old diarrheal disease, baekri, piglet diarrhea disease, or weaned pig diarrhea disease, feed additive for preventing pathogenic E. coli of pigs. A Stx2eA-C-terminal fragment-Stx2eB-pentameric recombinant protein consisting of a C-terminal fragment monomer of the amino acid sequence of SEQ ID NO: 1 and a Stx2eB-pentamer consisting of the amino acid sequence of SEQ ID NO: 2; And intestinal toxin type E. coli expressing Fimbriae 4 (F4) and intestinal toxin type E. coli expressing Fimbriae 18 (F18), the intestine deposited with microorganism accession number KCTC13847BP (microbial name: Enterotoxigenic Escherichia coli (ETEC) F4, F18) Mixed strains of toxin-type E. coli, GI24, chlorhexidine digluconate, chlorhexidine digluconate solution, chlorhexidine diacetate, chlorhexidine acetate chlorhexidine diacetate hydrochloride (Chlorhexidine diacetate) A method for enhancing immunity against pathogenic Escherichia coli in pigs, comprising inoculating sows or piglets with a vaccine composition comprising a bacterium reacted with any one selected from. 10. The method of claim 9, wherein the porcine pathogenic E. coli is a swine edema disease, neonatal pig diarrhea disease, premature diarrhea disease, 3-week-old diarrhea disease, baekri, mammalian pig diarrhea disease, or weaned pig diarrhea disease. .

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