KR102000120B1 - The composition of the enterotoxigenic Escherichia coli ghost vaccine candidate by recombinant P22 lysozyme-PMAP36 fusion protein against neonatal piglet colibacillosis - Google Patents

Abstract

Enterotoxigenic E. coli (ETEC) expressing F4 and F18 pimelia, The strain was induced to be ghosted by the P22 lysozyme-PMAP36 fusion protein, and the cells thus induced were evaluated as ETEC ghost vaccine. To assess the efficacy of this new prophylactic vaccine candidate, the candidate vaccine was inoculated intramuscularly with the piglets and confirmed for protection against E. coli. In other words, the piglets of group B were vaccinated with the respective gym vaccine containing Pimbria so as to be about 2 × 10 ⁹ cells at the second week of life, and the control group, the piglets of group A, were inoculated with PBS. Serum IgG levels in piglets of group B were significantly higher than those of group A pigs. Thereafter diarrhea and mortality were not observed in all piglets of Group B after challenge with wild-type ETEC, whereas diarrhea was observed in 70% of piglets in Group A and 30% mortality was observed. The results showed that the new vaccine could effectively protect E. coli from diarrhea after intramuscular injection of the piglets.

Description

(ETEC) ghost vaccine composition using a recombinant P22 lysozyme-PMAP36 fusion protein to prevent colony-forming diseases of pigs, rabbits, and pigs }

The present invention relates to a recombinant P22 lysozyme-PMAP36 fusion protein for preventing colicosis of ruminant piglets and an intestinal poison microbial (ETEC) ghost vaccine composition using the same.

Enterotoxigenic Escherichia E. coli , ETEC) causes diarrhea, especially diarrhea in newborn piglets and weaned pigs, and is considered one of the most common bacteria in the world and one of the most economically important diseases in the swine industry worldwide. ETEC usually attaches to the surface of intestinal epithelial cells using Pimbria. The four important pimbrids associated with ETEC are K88 (F4), K99 (F5), F6 (Fas) and F41, Among the F4 rm strains expressing Pimbria are known as K88ac + ETEC and F18 + ETEC. These ETEC strains secrete enterotoxins, heat-stable enterotoxins and heat-labile enterotoxins (LT) after adherence to the intestinal mucus, resulting in severe diarrhea in newborn piglets and piglets. Thus, these Pimbria are known to play an important role in ETEC diarrhea.

In general, most infections in the piglet piglets can be prevented by vaccination of the pregnant sows. Thus, a vaccine for the prevention of ETEC colicosis has been used in the veterinary medical field for pregnant sows. These vaccines are either a mixture of formalin-inactivated K88ab +, K88ac +, K88ad +, K99 +, F6 + and F41 + ETEC whole-cell bacterins (whole-cell bacterin) or purified ETEC fimbrial subunit vaccines Lt; / RTI > vaccine). However, traditional inactivated bacterial vaccines produced by chemical treatment of cultured bacteria can affect the physiochemical and structural properties of bacterial surface antigens. Thus, it is known to potentially inhibit defense immunity, and this inactivated vaccine only induces low-level and short-term immune responses. Thus, traditional inoculum vaccines require multiple inoculations with a strong adjuvant to induce effective defense immunity. However, the vaccines have major risks, such as allergic reactions and sarcomas at the vaccinated site. On the other hand, ghosted inactivated bacteria maintain the cell surface antigenic structure which retains the shape of living cells and bacterial adhesion characteristics intact. Ghosted bacteria also retain their unique adjuvant properties, which can lead to systemic and mucosal and cellular immune responses.

The use of bacterial ghost cells as vaccine candidates is a new and progressive approach to developing safe and effective vaccines against infectious diseases. It has been shown that oral or parenteral inoculation of these new ghost vaccine candidate substances in animals can effectively induce humoral and cellular immune responses. In addition, the portion of vaccine production that is an important part of immunogenicity is inactivated chemically and physically, without change, and thus retains the extracellular components that maintain immunogenicity such as bacteria.

Antimicrobial peptides (AMPs) found in most animals and plants play an important role in congenital immune responses and have broad-spectrum activity against these AMPs. These antimicrobial actions cause these AMPs to bind to the bacterial cell membrane and react quickly to create a hole in the cell membrane, causing the cellular component to escape, eventually leading to death. So far, eleven AMPs have been found in pigs, one of which has the most net positive charge to bind bacterial cell walls with porcine myeloid antimicrobial peptide-36 (PMAP-36) in pig marrow .

To date, eleven host defense peptides (HDP) or antimicrobial peptides (AMP) have been reported in pigs, including porcine myeloid antimicrobial peptide-36 (PMAP-36) Is known to have the highest positive charge. The α-helical domain (α-helical domain) of the PMAP36 N-terminal (PMAP36 N-terminal) is known as the active site. The peptide has the ability to penetrate the bacterial membrane through electron-bonding with positive charge-bearing peptides and negatively charged molecular components of the germinal cell membrane. Some bacteriophages endor lysine may have antimicrobial activity that can break down bacterial cell walls externally. Both of these substances attack the cell walls of both Gram-positive and negative bacteria, weakening the mechanical strength of the cell walls and eventually dissolving the cell walls. In particular, the activity of this enzyme on gram-negative bacteria is closely related to lipopolysaccharide (LPS), which has a negative charge in the cell membrane so that the enzyme can enter the membrane.

The object of the present invention is firstly the expression and purification of a recombinant P22-lysozyme-PMAP36 fusion protein using an over-expression system, followed by the use of this recombinant fusion protein to determine whether induction of ghosting of F4 + and F18 + The efficacy of the induced cells as a vaccine is confirmed by using the mammalian piglets.

The present invention relates to a recombinant gene of P22-lysozyme-PMAP36 consisting of the nucleotide sequence shown in SEQ ID NO: 1; And a P22-lysozyme-PMAP36 fusion protein comprising a recombinant gene of P22-lysozyme-PMAP36 consisting of the amino acid sequence shown in SEQ ID NO: 2.

Also provided is a microorganism transformant ghosted by a P22-lysozyme-PMAP36 fusion protein.

The microorganism transformant is an Escherichia coli strain deposited with the microorganism deposit number KCTC18534P (microorganism name: Escherichia coli HJL779).

Claims: What is claimed is: 1. A ghost vaccine composition for preventing colicosis in piglets, comprising P22-lysozyme-PMAP36 fusion protein as an effective ingredient.

 The present invention relates to a ghost vaccine composition for the prevention of colibacillosis of piglets, which is characterized in that the P22-lysozyme-PMAP36 fusion protein defends colicacosis caused by induction of ghosts of F4 + or F18 + ETEC strains causing diarrhea in the causal piglet. .

In the present invention, it was confirmed that intramuscular inoculation of the ETEC ghost vaccine with the mammal piglet effectively induces the systemic immune response. In addition, no clinical symptoms were observed after challenge with wild-type virulent ETEC strains after muscle inoculation in the mammalian piglets, whereas 70% of the control group piglets had diarrhea. Furthermore, mortality was observed in the control group. Based on these results, therefore, it is effective to prevent ETEC colibacillosis by inoculating muscle piglets with new vaccine candidates of the present invention.

Figure 1 shows the pET30a-P22 lysozyme-PMAP36 construct.
Figure 2 shows the expression of P22 lysozyme-PMAP36 fusion protein using 15% SDS-PAGE.
Figure 3 shows Western blot results of the purified P22 lysozyme-PMAP36 fusion protein.
Figure 4 shows circular dichroism (CD) spectroscopy for the identification of secondary structure of P22 lysozyme, PMAP36 peptide and P22 lysozyme-PMAP36 fusion protein.
FIG. 5 is an electron micrograph, FIG. 5A shows the P22 lysozyme-PMAP36 fusion protein untreated group, and FIG. 5B shows the P22 lysozyme-PMAP36 fusion protein treated group.
Fig. 6 shows the serum antibody titer for each antigen. Group A represents the control group and group B represents the vaccinated group.

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

Materials and methods

Bacteria and growth conditions used in the present invention

All bacterial strains used in the present invention are described in Table 1. F4 + (HJL593) and F18 + (HJL565) ETEC strains were used as strain for vaccine preparation and challenge infection . E. coli DH5α and E. coli BL21 (DE3) were used as overexpressant host strains for K88ac and FedA antigen production. All strains were cultured in LB broth and LB agar. The pQE31 and pET28a plasmids were also used to over-express the K88ac and FedA antigens, respectively, and the pET30a plasmid was used to overexpress the P22 lysozyme-PMAP36 fusion protein.

Table 1 below shows the strains and plasmids used in the study.

Strain / Plasmid Description source strains E. coli HJL593 Wild type F4ac (K88ac) ⁺ LT ^+, Sta ⁺ ETEC isolate from pig Lab stock
HJL565 Wild type F18 ⁺ Sta ⁺ ETEC isolate from pig Lab stock
DH5α 1, hsdR17 (SEQ ID NO: 1), dA (lacZ) 2, dA (rAF-lacZ), U169, phoA, glnV44, w80, D Promega
BL21 (DE3) F ^? , ompT, hsdSB (r ^? _B , m ^? _B ), dcm, gal, (DE3) Promega HJL667 DH5a K88ac in pQE31 This study HJL669 BL21 (DE3) fedA in pET28a This study HJL779 BL21 (DE3) P22 lysozyme-PMAP36 in pET30a This study Plasmids pQE31 IPTG-inducible expression vector; Am ^r QIAGEN pET28a IPTG-inducible expression vector; Km ^r Novagen pET30a IPTG-inducible expression vector; Km ^r Novagen

P22 lysozyme - PMAP 36 fusion  The gene coding for the protein Cloning

The modified fusion gene for PMAP 36 lysozyme protein was inserted chemically (Bioneer, Daejeon, South Korea) into a synthetic pET30a plasmid.

The recombinant protein was purified using the expression of E. coli BL21 (DE3) and the nickel-nitrilotriacetic acid-agar affinity purification process. The purified protein was identified by Western blot analysis.

All purified antigens were mixed in 50% glycerol and stored at -70 C until use.

The PMAP36 peptide gene was optimized for Escherichia coli codon so that it can be expressed well in E. coli, and was prepared in Bioneer with the lysozyme gene of Salmonella typhimurium bacteriophage P22 (hereinafter referred to as 'P22'). Each gene was amplified by PCR using each primer set (Table 2), and the thrombin cleavage site was added to the C-terminal of P22 lysozyme using PCR. The amplified P22 lysozyme and thrombin cleavage sites were digested with NdeI and BamHI and ligated into pET30a digested with the same restriction enzyme (hereinafter referred to as pET30a-P22 lysozyme). In addition, the amplified PMAP36 gene was digested with BamHI and NotI and ligated into pET30a-P22 lysozyme vector digested with the same restriction enzyme (Fig. 1). These ligation vectors (pET30a = P22 lysozyme-PMAP36) were transformed into E. coli DH5α.

Table 2 below shows the primers used for the cloning of the recombinant P22 lysozyme-PMAP36 fusion protein.

P22 lysozyme - PMAP36  Fusion protein expression and purification

The pET30a-P22 lysozyme-PMAP36 vector was transformed into E. coli BL21 (DE3) for expression. To over-express the recombinant P22 lysozyme-PMAP36 fusion protein from the transformed E. coli, one colony was inoculated into 100 ml of Luria-Bertani (LB) broth containing 50 μg / ml kanamycin and incubated at 37 ° C < / RTI > overnight. 10 ml of this culture was transferred to 1,000 ml of LB broth containing 50 μg / ml kanamycin and re-cultured until the OD600 reached 0.6. To this culture solution was added isopropyl-bD-thiogalactopyranoside (IPTG) at a concentration of 0.5 mM and cultured at 28 ° C for 24 hours to induce the expression of p22 lysozyme-PMAP36 fusion protein. The cultured cells were concentrated by centrifugation at 5,000 rpm for 15 minutes at 4 ° C, resuspended in 20 ml of lysis buffer (10 mM Tris, 1 M NaCl, pH 8.0), sonicated , 4 seconds rest for 4 minutes). After centrifugation at 4 ° C and 12,000 rpm for 25 minutes, the water-soluble protein was concentrated in the eluate, filtered through a syringe filter of 0.45 μm pore size, and then applied to an AKTA prime FPLC system (GE healthcare, USA) The column was transferred to the HHistrap FF colum (column), and purification was started. The reacted columm was washed with lysis buffer (Buffer A) and the soluble proteins were separated according to the gradient concentration of buffer B (10 mM Tris-HCl, 1 M NaCl, 300 mM Imidazole, pH 8.0). Each separated fraction was analyzed by 15% SDS-PAGE and the purified target protein was dialyzed using buffer C (PBS buffer, GE healthcare, USA) and centrifuged (cut-off 30 kDa, Amicon, Millipore, Germany). Finally, the concentration of the P22 lysozyme-PMAP36 fusion protein was measured by the Bradford protein assay.

Western Blotting (Western blotting)

Purified and concentrated P22 lysozyme-PMAP36 fusion protein was loaded by 15% SDS-PAGE and then transferred to PVDF membrane Millipore, Germany. The fusion proteins were then transferred to an anti-6-his polyclonal antibody -Hiz polyclonal antibody (BD, France) was diluted to 1: 6,000 and reacted with the primary antibody. After washing, HRP-conjugated goat anti-mouse IgG antibody (Enzo, USA) was diluted to 1: 12,000, reacted with secondary antibody, and the fusion protein band was confirmed with ECL solution (SurModics, USA).

Circular polarization Dichroism  Spectroscopy dichroism  spectroscopy)

In order to evaluate the secondary structure and folding properties of this fusion protein, the purified fusion protein was amplified by far-UV circular dichroism spectroscopy (JASCO J-1500 spectropolarimeter, wavelength range of 190 nm to 260 nm). 0.5 mg / ml of each sample (P22 lysozyme, PMAp36 peptide, and P22 lysozyme-PMAP36 fusion protein) was dissolved in buffer D (PBS buffer containing 50% Glycerol) It was measured at 298K using a cuvette.

Construction of F4 + and F18 + ETEC  백원자

One colony of F4 + or F18 + ETEC strains was selected and inoculated into 200 ml of LB liquid medium, respectively, and cultured at 37 ° C until the absorbance reached 0.3. The P22 lysozyme-PMAP36 fusion protein prepared in the cultured medium was added at a concentration of 100 μg / ml, and each ETEC strain was reacted at 37 ° C for 16 hours until lysis of the cells was induced. After the reaction, 100 μl of each reaction solution was inoculated into LB agar and cultured at 37 ° C for 24 hours to confirm whether or not the reaction solution was ghosted with or without colony formation. The ghost-progressed reaction solution was concentrated by centrifugation at 4000 × g for 30 minutes, washed three times with sterilized PBS, resuspended at a final concentration of 1 × 10 ⁹ cells / ml, and stored at -20 ° C.

Projection electron microscope TEM )

Samples for confirmation of progression of each ETEC ghost were prepared in the same manner as for the preparation of F4 + and F18 + ETEC ghost vaccines for bacterial samples (to observe membrane integrity and intracellular changes in TEM) before and after treatment with P22 lysozyme-PMAP36 fusion protein . TEM has been used to characterize Lv et al. (Lv, Y., Wang, J., Gao, H., Wang, Z., Dong, N., Ma, Q., et al., 2014. Antimicrobial properties and membrane- mechanism of a potential α? elical antimicrobial derived from cathelicidin PMAP-36. PLoS. One. 9, e86364.) was taken along in the described way.

Vaccination and Sick  Extraction

20 Yorkshire larvae were used in the present invention and all piglets were transferred to a barn with a heat lamp. Commercial diets containing no antibiotics or growth promoters were also fed. A total of 20 piglets were divided into two groups. All groups of piglets were vaccinated with intramuscular injection (once every 2 weeks of age). Group A pigs were inoculated with 2 ml of sterilized PBS as a control group, and Group B pigs were inoculated into 2 ml of PBS at 2 × 10 ⁹ cellsd each ghost vaccine mixture (F4 + alc F18 + ETEC was prepared to be 1 × 10 ⁹ cells each) . Blood samples were collected at 0 (pre-inoculation) and 2 (pre-challenge) WPI (week post immunization). All samples were stored at minus 70 ° C until used. The animal tests reported in this experiment were approved by the Chonbuk National Animal Ethics Board, which was accredited by the Korean Council on Animal Care.

An enzyme linked immunoassay (ELISA)

F4 and F18 ETEC fimbrial antigens (antigens of Pimbria) were purified from HJL667 and HJL669 according to the manufacturer's recommended method. To measure the optical density (OD) of ETEC fimbrial antigen-specific IgG in each serum, pig IgG (E100-104, Bethyl Lab Inc. Montgomery, TX, USA) ELISA quantitation kit was used according to the manufacturer's instructions . Serum samples were diluted to 400: 1 in the IgG concentration experiments. Enzyme reactions were studied with o- phenylenediamine (Sigma-Aldrich, St. Louis, Mo., USA) and measured by automated ELISA spectrophotometer at 492 nm (TECAN, Salzburg, Austria).

Challenge infection

The challenge infectious strains JHL593 and HJL565 were prepared according to the method described previously (Hur and Lee 2011; and 2013). All young pigs were challenged orally at 4 weeks of age with a 2 ml mixture (1 × 10 ⁹ CFU / ml). Diarrhea and mortality of all piglets were monitored for 14 days after the challenge. When diarrhea was observed after challenge infection, isolation and identification of challenged strains were carried out in the rectal swab of piglets. If one of the isolated strains contained one of at least one of the ETEC fimbriae genes challenged from the rectal swab using PCR using fimbria-specific primers (Table 3), it was considered to be diarrhea due to challenge infection.

The following Table 3 shows the primers used for identification of the electrically conductive infectious strains after the conductive infection.

Statistical analysis

All results were expressed as mean ± standard deviation. Mann-Whitney U-test was used to differentiate antibodies in the vaccinated, non-vaccinated group of serum. SPSS 16.0 (SPSS Inc., Chicago, IL, USA) was used for all analyzes and P ≤ 0.05 was considered statistically significant.

P22 lysozyme - PMAP36  Fusion protein expression

15% As a result of confirming the fusion protein expressed and purified by SDS-PAGE, it was confirmed that the fusion protein was expressed as shown in FIG. As a result of measuring the concentration, it was confirmed that 1.8 mg per 1 L was expressed.

Western Blotting (Western blotting)

Purified and concentrated P22 lysozyme-PMAP36 fusion protein was loaded on 15% SDS-PAGE and transferred to PVDF membrane (Millipore, Germany) using anti-6-his polyclonal antibody (BD, France) As a result, the fusion protein band was confirmed as shown in FIG.

Circular polarization Dichroism  Spectroscopy dichroism  spectroscopy)

In order to evaluate the secondary structure and folding properties of this fusion protein, the purified fusion protein was measured by far-UV circular dichroism (CD) spectroscopy (JASCO J-1500 spectropolarimeter, wavelength range of 190 nm to 260 nm) , And as shown in FIG. 4, two typical negative bands were observed at 208 and 222 nm in the PMAP36 and P22 lysozyme-PMAP36 fusion proteins, and the helical structure was confirmed. On the other hand, as shown in FIG. 4, no significant difference between PMAP36 and P22 lysozyme-PMAP36 was found, indicating that the helical structure of PMAP36 was not significantly influenced by fusion.

Projection electron microscope ( TEM )

As shown in FIG. 5, the ETEC strain not treated with the P22 lysozyme-PMAP36 fusion protein showed intact cell membranes and cell contents filled in the cells (FIG. 5A). On the other hand, each ETEC ghosted by the P22 lysozyme-PMAP36 fusion protein was observed to have a cytoplasmic space with one perforated cell membrane as shown in FIG. 5B.

Immune response induced after vaccination

The antibody titers of each of the zodiac serum IgG against the K88ac and FedA antigens in the vaccinated piglets are shown in FIG. Serum IgG titers against K88ac and FedA were significantly increased in the inoculated group compared to the control group (P ≤ 0.05).

In challenging infection  Defense Effect

All of the piglets were challenged orally at 4 weeks of age. None of the 10 newborn piglets in Group B had diarrhea, and no piglets were dead until 14 days after challenged infection. In contrast, diarrhea was observed in 7 out of 10 Group A children (70%) (3 days after challenged infection) and 3 (30%) died from severe diarrhea.

The digestive tract is the main space for digestion and nutrition absorption. In addition, intestinal blood plays an important role in preventing bacterial antigens and toxins from infecting the host. The intestinal status of the new money can be changed by several factors. Among such factors, suckling pigs and juveniles are particularly sensitive to many bacterial infections. The reason for this is the separation from the sows, the coexistence with other groups of pigeons that are similar in size to those of the same breed, and the stresses that are sensitive to many bacteria due to changes in feed and environment, not familiar mammals. Mucosal immunity is more important in defense against ETEC colibacillosis than systemic immunity. Some industrial sow vaccines for the prevention of ETEC colibacillosis for the mammal piglets have been marketed, but vaccines for the sow pigs have not been sold yet. However, recently, vaccines against edema diseases, which are mainly caused by pathogenic Escherichia coli, have begun to be marketed in weaned piglets. However, the formalin - inactivated vaccine to prevent colibacillosis in newborn piglets may alter the physicochemical and structural properties of bacterial surface antigens, so that formalin - inactivated ETEC vaccines may not be effective in preventing colibacillosis in newborn piglets. Therefore, it is necessary to develop new vaccines that do not alter the structural and physicochemical properties of bacteria. The ETEC ghost vaccine, which is derived from the P22 lysozyme-PMAP36 fusion protein, has been shown to retain native surface structures with high immunogenicity to the cell, making holes in the bacterial cell membrane to produce intracellular cytoplasmic substances It exits the cell and eventually becomes inactive. Therefore, ghost vaccine candidates derived from the P22 lysozyme-PMAP36 fusion protein can be used as a more advanced vaccine candidate in pigs. Therefore, in the present invention, the P22 lysozyme-PMAP36 fusion protein was induced to ghost by F4 + ETEC and F18 + ETEC inducing diarrhea in the weaned piglets. The ETEC ghost vaccine candidates thus induced were inoculated into the piglets, And defending against the challenge infectious strains.

As a result, a significant increase in the serum IgG of K88ac and FedA antigens was observed in the mixed inoculation group of F4 + ETEC and F18 + ETEC ghost vaccine. These results suggest that the IM vaccination of the new ETEC vaccine candidate has a protective immune response It can be effectively induced. To observe the efficacy of vaccine candidates, all piglets challenged with wild-type ETEC strains. Diarrhea and mortality were observed in 70% and 30% of the control infants in Group A after challenge infection in piglets. However, pigs in the vaccinated group showed no diarrhea and mortality in almost all piglets, suggesting that intramuscular injection of ghost vaccine candidates to the piglets could be effective in defending against ETEC colibacillosis in the piglet pigs.

In the present invention, it was confirmed that intramuscular inoculation of the ETEC ghost vaccine with the mammal piglet effectively induces the systemic immune response. In addition, no clinical symptoms were observed after challenging with wild-type virulent ETEC strains after muscle inoculation in the piglets, whereas 70% of the control group's piglets had diarrhea. Furthermore, mortality was observed in the control group. Therefore, based on these results, we show that intramuscular injection of mammal piglets with our new vaccine candidate can be effective in defending ETEC colibacillosis after the reason.

The present invention is a research funded by the Korean Research Foundation Basic Research Project funded by the government (Ministry of Education) in 2015 (No. 2015R1D1A1A02062126)

Korea Biotechnology Research Institute KCTC18534P 20161228

<110> komipharm          Chonbuk National University Industry-University Collaboration Foundation <120> The composition of the enterotoxigenic Escherichia coli ghost          vaccine candidate by recombinant P22 lysozyme-PMAP36 fusion          protein against neonatal piglet colibacillosis <130> c16-42 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 585 <212> DNA <213> Artificial Sequence <220> <223> P22-lysozyme-PMAP36 <400> 1 atgcaccatc atcaccatca catgcaaatc agcagtaacg gaatcaccag attaaaacgt 60 gaagaaggtg agagactaaa agcctattca gatagcaggg ggataccaac cattggggtt 120 gggcataccg gaaaagtgga tggtaattct gtcgcatcag ggatgacaat caccgccgaa 180 aaatcttctg aactgcttaa agaggatttg cagtgggttg aagatgcgat aagtagtctt 240 gttcgcgtcc cgctaaatca gaaccagtat gatgcgctat gtagcctgat attcaacata 300 ggtaaatcag catttgccgg ctctaccgtt cttcgccagt tgaatttaaa gaattaccag 360 gt; ccacggaggc ggcgagaaag agcgctgttc ttatcgctgg ttccgcgtgg atccggacga 480 tttagacgtt tacgtaaaaa aacccggaaa cgtctgaaaa agattgggaa agtgttgaaa 540 tggattcctc ctattgtcgg ttcaataccc ttaggttgtg gataa 585 <210> 2 <211> 194 <212> PRT <213> Artificial Sequence <220> <223> P22-lysozyme-PMAP36 amino acid sequence <400> 2 Met His His His His His Met Gln Ile Ser Ser Asn Gly Ile Thr   1 5 10 15 Arg Leu Lys Arg Glu Glu Gly Glu Arg Leu Lys Ala Tyr Ser Asp Ser              20 25 30 Arg Gly Ile Pro Thr Ile Gly Val Gly His Thr Gly Lys Val Asp Gly          35 40 45 Asn Ser Val Ala Ser Gly Met Thr Ile Thr Ala Glu Lys Ser Ser Glu      50 55 60 Leu Leu Lys Glu Asp Leu Gln Trp Val Glu Asp Ala Ile Ser Ser Leu  65 70 75 80 Val Arg Val Pro Leu Asn Gln Asn Gln Tyr Asp Ala Leu Cys Ser Leu                  85 90 95 Ile Phe Asn Ile Gly Lys Ser Ala Phe Ala Gly Ser Thr Val Leu Arg             100 105 110 Gln Leu Asn Leu Lys Asn Tyr Gln Ala Ala Ala Asp Ala Phe Leu Leu         115 120 125 Trp Lys Lys Ala Gly Lys Asp Pro Asp Ile Leu Leu Pro Arg Arg Arg     130 135 140 Arg Glu Arg Ala Leu Phe Leu Ser Leu Val Pro Arg Gly Ser Gly Arg 145 150 155 160 Phe Arg Arg Leu Arg Lys Lys Thr Arg Lys Arg Leu Lys Lys Ile Gly                 165 170 175 Lys Val Leu Lys Trp Ile Pro Pro Ile Val Gly Ser Ile Pro Leu Gly             180 185 190 Cys Gly        

Claims (5)

delete A microorganism transformant inactivating F4 + or F18 + ETEC strains causing diarrhea in the pigs, comprising F4 + or F18 + ETEC strains ghosted by P22 lysozyme-PMAP-36 fusion protein,
The P22 lysozyme-PMAP-36 fusion protein is a P22 lysozyme-PMAP-36 fusion protein expressed by SEQ ID NO: 2 expressed and purified by a recombinant gene of P22 lysozyme-PMAP-36 represented by SEQ ID NO: 1, The microorganism transformant according to any one of claims 1 to 3, wherein the microbial transformant is T. fumigatus bacteriophage P22 and the PMAP-36 is procine myeloid antimicrobial peptide-36. The microorganism transformant according to claim 2, wherein the microorganism transformant has a microorganism deposit number KCTC18534P (microorganism name: Escherichia E. coli HJL779). &lt; / RTI &gt; A ghost vaccine composition for prevention of colonic disease in pigs, which contains P22 lysozyme-PMAP-36 fusion protein according to claim 2 as an effective ingredient. The method according to claim 4, characterized in that the P22 lysozyme-PMAP-36 fusion protein defends colicacosis caused by induction of ghosts of F4 + or F18 + ETEC strains causing diarrhea in needy pigs Of a ghost vaccine composition for preventing colicosis.

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