KR101001023B1 - A vaccine for treating or preventing pasteurellosis using a yeast surface-expression system - Google Patents

Abstract

The present invention relates to a vaccine composition for the prevention or treatment of Pasteurella disease using a yeast surface expression system, specifically iii) nucleotides encoding the envelope peptide of Pasteurella multocida, ii) aureo Recombinant nucleotides for vaccine preparation for the prevention or treatment of Pasturelaosis comprising nucleotides encoding the Aureobasidin A peptide and iii) nucleotides encoding the heat-labile enterotoxin subunit (LTB) peptide, including the nucleotides The present invention relates to a recombinant expression vector and a vaccine composition for preventing or treating pasturela disease obtained by infecting the vector with a host cell. The vaccine composition prepared according to the present invention can induce an immune response by a simple immunization method such as an oral vaccine, an intradermal immune vaccine using a mucosal vaccine and a tattoo instrument.

Pasteurella, Poultry cholera, Yeast surface expression, Vaccine

Description

A vaccine for treating or preventing pasteurellosis using a yeast surface-expression system}

The present invention relates to a vaccine composition for treating or preventing pasteurellosis using a yeast surface expression system, and more specifically, iii) nucleotides encoding the outer membrane peptide of Pasteurella multocida, Ii) recombinant nucleotides for the manufacture of vaccines for the prevention or treatment of pasteurella, comprising nucleotides encoding the Aureobasidin A peptide and iii) nucleotides encoding the heat-labile enterotoxin subunit (LTB) peptide, The present invention relates to a recombinant expression vector comprising the nucleotide and a vaccine composition for preventing or treating pasturela obtained by infecting the vector with a host cell.

Pasteurellosis is a disease caused by infection with Pasteurella bacteria, and occurs in various livestock such as cattle, buffalo, sheep, pigs, rabbits, poultry, dogs, rodents and cats. Examples include fever, calf epidemic pneumonia, mastitis, pleural pneumonia, snuffles, poultry cholera, epidemic hemorrhagic sepsis, atrophic rhinitis and abscesses. The fungi that cause Pasteurellasis include Pasteurella multocida type A, B, D, E, F and Pasteurella hemorhaitika types A and T (P. haemolytica biotype A, T), Pasteurella pneumotropica (P. pneumotropica) is the type. Pasteurella multocida type A causes fever, calf influenza, mastitis in cattle, pleural pneumonia, mastitis, secondary infections in pigs, pleural pneumonia in rabbits, and poultry cholera in poultry. Causes Pasteurella multocida type B causes epidemic / hemorrhagic sepsis in cattle and buffaloes, and pasteurella multocida type C causes atrophic rhinitis in pigs. In addition, type E causes epidemic / bleeding sepsis in cattle and buffaloes in Africa, and type F is infected with turkeys. Pasteurella haemolytica type A causes transport fever and pneumonia in cattle, epidemic pneumonia and sepsis, and necrotizing breast cancer in sheep. Pasteurella haemolytica type T causes sepsis in sheep. In addition, Pasteurella pneumotropha causes pneumonia and abscess in rodents.

In particular, poultry cholera is an acute infectious disease of birds with a history of more than 200 years, which occurs in chickens, poultry, and turkeys. It is caused by Pasteurella multocida, first isolated by Pasteur about 100 years ago. It is a pathogen with a history of making vaccine for the first time in the world using this bacteria. Poultry cholera is a bacterial infectious disease in which clinical symptoms and lesions are caused by the toxin of this bacterium. It has been reported in Korea and is the first type of statutory infectious disease in Korea.

As mentioned above, the pathogen of poultry cholera is caused by Pasteurella multocida, which is classified as a Gram-negative double-staining monoclonal bacillus and a bacterium with a capillary. Bacteria with a capillary are known to be highly pathogenic, and pathogenicity among strains has been investigated. The culture of the bacterium grows well in blood and dextrose starch media, but not in MacConkey media. Serotype analysis of poultry cholera was carried out using the gel diffusion precipitin test, and there were 16 serotypes, which were found to be serotypes 1, 3, and 3x4. It is reported that serotypes are most isolated.

The acute form suddenly dies of sepsis and is common in the summer, with mucus draining from the mouth and cyanosis appearing in the crust or broth. Chronic forms are common in autumn and winter, and are converted to chronic forms after acute elapsed, or occur when infected with low-pathogenic strains. If the lesion is acute, there are often no specific lesions. Acute primary lesions are hemorrhages of the heart, line, intestinal membrane, and abdominal adipose tissue. It is deformed. In chronic form, inflammatory exudates can be seen in eyes and nose, and inflamed sites can be seen in cheese-like inflammatory exudates. Recovered chickens are immunized against the same strain and cannot be reinfected. However, once infected chickens are known to carry germs permanently, elimination of the carrier system is the most important in controlling poultry cholera.

Treatment of poultry cholera requires a continuous program of medications, which has the disadvantage of being higher than the cost of vaccination. In addition, many sulfa-based drugs and other antibiotics are effective in reducing mortality, but mortality resumes when medication is discontinued. In addition, while drugs such as sulfaquinoxaline can achieve a better effect, they result in lower egg laying rates in laying hens, and even make them completely egg-free, which poses a risk of caution.

Poultry cholera vaccines are not completely effective, but repeated injections at 2 weeks of age are known to be sufficient to impart an immune response to chickens, but they are not effective against cross-protection between various serotypes. Oil vaccines are used to immunize breeders, and because they are so stressful, they result in a lower egg production rate. Probiotic vaccines are given in chickens by Wing web vaccination. In laying hens and breeders 10-10 weeks of age are given. In the United States, the CU strain is used as a live vaccine strain. Live vaccines are more protective than vaccinated vaccines, and are known to exert broad defenses against most serotypes.

Thus, there has been a need for effective vaccines that can be used to provide protective immunity for the prevention and treatment of poultry cholera.

Lee, J. S., K. S. Shin et al., (2000) Nat. Biotechnol. 18 (6): 645-8 suggested the availability of a vaccine by expressing the antigen on the surface of Salmonella, but Salmonella was a prokaryote, which was not similar to the protein expressed in humans, which are eukaryotes. In addition, Schreuder, MP, C. Deen, et al., (1996) Vaccine 14 (5): 383-8 uses yeast that can be glycosylated to make proteins more similar to proteins expressed in eukaryotes. The expression of proteins on the surface of the yeast showed the possibility of oral vaccines through the immune response resulting from immunization by the oral route of administration. However, the selectivity of the transgenic yeast surface-expressed antigenic protein of the antigen protein loses expression as it is passaged successively, which means that the gene of the antigenic protein is episomally maintained in the yeast, and a selection marker is selected for nutrient needs. This is because the selection power is not strong on the basis of that. In addition, since the codon optimization of the gene encoding the yeast surface expression protein was not performed, the protein expression rate was remarkably low, and when used as a vaccine, the protein expressed on the surface was likely to be degraded by various factors. The immunity was relatively low. Therefore, there is an increasing demand for a masking technique that can help to improve the immune method and stabilize the surface expression protein to overcome this.

Accordingly, the present inventors apply a conventional yeast surface expression system to select only the antigenic epitope peptide portion of the envelope protein of Pasteurella multocida type A which is a poultry cholera bacterium as an antigen, and fuse the LTB protein to increase immunity. In order to efficiently express the yeast surface expression system and increase the selectivity of the transformed yeast, an existing expression vector was modified to integrate the improved expression vector into yeast genomic DNA. Yeast was prepared by stably expressing the antigenic protein on the surface of the yeast, it was confirmed that it can be applied to pasturella including poultry cholera by confirming the excellent immune effect by administering it in various routes such as oral and dermal routes The present invention was completed.

The object of the present invention is iii) nucleotides encoding the envelope peptide of Pasteurella multocida, ii) nucleotides comprising the aureobasidin A resistance gene and iii) heat-labile To provide a recombinant nucleotide for vaccine production for the prevention or treatment of pasteurella, comprising a nucleotide encoding the enterotoxin subunit) peptide, an expression vector comprising the same and a host cell transformed with the expression vector.

Still another object of the present invention is to prepare a method for preventing or treating pasteurosis, including a process of transforming and amplifying the expression vector into a host cell, and preventing or treating pasturelaosis produced by the method. It relates to a vaccine composition for.

For this purpose, the present invention, in one embodiment, includes: i) a nucleotide encoding the envelope peptide of Pasteurella mult ocida, ii) an aureobasidin A resistance gene. To provide a recombinant nucleotide for vaccine production for the prevention or treatment of pasteurella, including nucleotides, and iii) nucleotides encoding the heat-labile enterotoxin subunit (LTB) peptide.

As used herein, the term "pasteurella" is a disease caused by infection with Pasteurella bacteria, and occurs in various livestock such as cattle, buffalo, sheep, pigs, rabbits, poultry, dogs, rodents, and cats. Pasteurella multocida types A, B, D, E, F and Pasteurella hemorhaitika types A and T (P. haemolytica biotype A, T), It includes all diseases caused by Pasteurella pneumotropica type. This specifically includes, for example, transport fever, calf pandemic pneumonia, mastitis, pleural pneumonia, snuffles, poultry cholera, epidemic hemorrhagic sepsis, atrophic rhinitis and abscesses and the like. In a preferred embodiment, the vaccine according to the invention is for the prevention or treatment of poultry cholera.

The nucleotide encoding the envelope peptide of Pasteurella multocida used in the present invention is used as an antigen. Preferably, the envelope protein of Pasteurella multocida, which is a poultry cholera, has high hydrophobicity as a macromolecule. Since it is difficult to express on the yeast surface, only the epitope portion of the envelope protein can be selected and obtained in order to increase the surface expression rate. The length of the nucleotides is preferably 140 to 180 bp, more preferably 169 bp. In one preferred embodiment, the present invention uses Pasteurella multocida A type. In an embodiment of the present invention, L2 and L5, which are estimated to have high hydrophilicity and antigenicity, are obtained from the epidermal H gene of Pasteurella multocida serotype A: 3 through antigenic epitope prediction. The area was used.

In addition, the nucleotide containing the Aureobasidin A resistance gene used in the present invention is introduced to use Aureobasidin A as a selection marker to improve the selection ability of yeast cells capable of surface expression. Preferably, it is AUR1 gene. In one embodiment of the present invention, the restriction enzyme site inside the AURC-1 selection marker gene, which is an aureobasidin A resistance gene, was cut and then transformed into yeast in the form of a linear plasmid.

In addition, the nucleotides encoding the LTB (heat-labile enterotoxin subunit) peptide used in the present invention, by using the strong GM1 ganglioside binding activity of the LTB protein selective, target-oriented antigen to cells expressing GM1 receptor Used to deliver protein and boost immunity. The preferred size of the nucleotide is 300 to 350 bp, more preferably 321 bp. In one embodiment of the present invention, a gene of heat-labile enterotoxin subunit B (LTB) isolated from Enterotoxigenic Escherichia coli (ETCT) was used. In a preferred embodiment, the nucleotides of the invention are nucleotides having the nucleic acid sequence set forth in SEQ ID NO: 1.

In another aspect, the present invention provides an expression vector comprising the nucleotide.

The term “vector” includes plasmid vectors, cosmid vectors, bacteriophage vectors and adenovirus vectors, retroviral vectors and the like as a means for expressing a gene of interest in a host cell, and is preferably a plasmid vector. Vectors capable of effectively expressing foreign proteins in host cells, particularly in yeast systems, include promoters, extracellular secretory sequences, cytokine genes and transcription termination signal sequences. In a preferred embodiment, the expression vector of the present invention transforms the host cell by integrating into genomic DNA of the host cell. In a specific embodiment of the present invention, pAURYD-peptide and pAURYD-LTB vectors were prepared by cloning the ompH (A: 3) gene or LTB gene into pAURYD having the configuration shown in FIG. 7, respectively, and the pAURYD-LTB vector. After restriction using the enzyme was cloned into the pAURYD-peptide plasmid vector was prepared pAURYD-fusion expression vector.

When the expression vector of the present invention is introduced into the host cell, the expression vector is not present in the form of plasmid in the host cell but is integrated into the genomic DNA of the host cell, thereby stably pasteurella multoci, an antigenic peptide on the surface of the host cell. To allow expression of multiple envelope peptide-LTB fusion recombinant proteins.

In another aspect, the present invention provides a host cell transformed with the expression vector. Preferably it is yeast, More preferably, Saccharomyces cerevisiae is EBY 100.

The host cell of the present invention expresses Pasteurella multosid envelope membrane peptide-LTB fusion recombinant protein on the cell surface by the expression vector. Specifically, the recombinant protein can be expressed as shown in FIG. 23.

In another aspect, the present invention is to transform the expression vector into a host cell to express the outer membrane peptide-LTB (heat-labile enterotoxin subunit) fusion peptide of Pasteurella multocida A It provides a method for producing a vaccine for the prevention or treatment of Pasteurellasis comprising.

In still another aspect, the present invention provides a method for preventing Pasteurella disease, which comprises the outer membrane peptide-LTB (heat-labile enterotoxin subunit) fusion peptide of Pasteurella multocida , A therapeutic vaccine composition is provided.

Vaccine compositions according to the invention can be used in formulated in suspension, solution or emulsion, etc., well known to those skilled in the art, pharmaceutically or veterinary acceptable. Preferred pharmaceutically acceptable carriers may include acid sterilized aqueous or non-aqueous solutions, suspensions and emulsions suitable for ingestion, inhalation or administration to the rectum or vagina by suppositories. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils (eg olive oil), and specific organic esters (eg ethyl oleate). Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions (including saline and buffered media). Preservatives and other additives may also be included, such as antibacterial agents, antioxidants, chelating agents and inert gases.

In addition, the vaccine composition according to the present invention can appropriately select a route of administration that is medically or veterinary. As an example, possible routes of administration include oral, inhalation, rectal, nasal, intradermal or intravenous administration, which may vary depending on the dosage, the tissue to which the vaccine is administered, the titer of the vaccine and the specific therapeutic requirements of the subject to be immunized, and the economics. can do. In particular, the vaccine composition according to the present invention exhibits an effective immunization effect by oral administration, intranasal or dermal administration, as shown in Figs. 21A and 21B. .

The vaccine composition according to the invention can also be provided alone or as a component of a multivalent vaccine, ie in combination with other vaccines. The other vaccine may be a known live or dead vaccine against Pasteurella, preferably poultry cholera.

In addition, as another aspect, the present invention is (a) nucleotide) nucleotide encoding the envelope peptide of Pasteurella multocida , ii) nucleotide comprising aureobasidin A resistance gene And iii) a recombinant nucleotide comprising a nucleotide encoding a heat-labile enterotoxin subunit (LTB) peptide;

(b) transforming the recombinant vector into yeast;

(c) culturing the transformed yeast to express the recombinant protein on the yeast surface

It provides a yeast surface expression system comprising.

By using the yeast surface expression system of the present invention, it is possible to develop a vaccine that is economical, efficient and user-friendly against infectious pathogens of animals and poultry as described above.

The vaccine composition prepared according to the present invention can induce an immune response by a simple immunization method such as an oral vaccine, an intradermal immune vaccine using a mucosal vaccine and a tattoo instrument.

Hereinafter, the present invention will be described in detail with reference to the following examples, but the following examples are provided to help the understanding of the present invention, and the scope of the present invention is not limited thereto.

Example 1. Plasmid DNA Construction

The OmpH (A: 3) gene is obtained by separating the envelope H gene sequence of Pasteurella multocida serotype A: 3. In order to clone the gene into pYD1, PCR primers were prepared as shown in FIG. 1. PCR products obtained by PCR were cloned into pYD1. And BamHI and NotI enzyme restriction (enzyme restriction) was confirmed whether cloning. At this time, the size of ompH (A: 3) was 1210bp (Fig. 2). This was commissioned to core bio, the sequencing results were confirmed (Fig. 3). The resulting clone was named “pYD1-ompH”, which is a clone that does not integrate into the yeast chromosome.

pYD1 vector is expressed in plasmid form in yeast, resulting in low stability during yeast cleavage and integration of antibiotic selection and genes into yeast genomic DNA to complement selectivity of tryptophan nutritional requirements. Was selected. The strategy is as follows. PCR primers were constructed by dividing the region from the GAL1 promoter of the pYD1 vector to the MATα transcription termination region into two fragments. At this time, new enzyme sites Sma I, Sal I, SacII, and Nde I were added to the primers for multiple cloning sites (MCS) to function as new vectors. After performing PCR and sequencing the sequences of the two fragments obtained through the TA cloning kit (invitrogen ™), the Sac of pAUR101 (Takara Bion, Otsu, Japan), a vector for introducing the yeast chromosome from the TA clone, was confirmed. A restriction enzyme and a ligase were inserted between I and Sph I sites (see FIGS. 4 to 5). The pAUR101 vector does not autonomously replicate in yeast and is stably preserved and maintained only in the state of recombination in chromosomes. Yeast chromosome with high efficiency by cutting the restriction enzyme site inside the AUR1-C selection marker gene, which is an Aureobasidin A resistance gene, and transforming the yeast in the form of a linear plasmid It can be introduced into the ( aur1 ) site.

The resulting pAURYD plasmid was integrated into yeast genomic DNA, resulting in resistance to aureobasidin A in YPD medium and expressing the target protein on the cell surface in 2% Galactose medium. Let's go. The plasmid thus prepared was commissioned to core bio, and the sequencing result was confirmed (see FIGS. 6 to 7). The resulting clone was named “pAURYD” and then used as a vector for cloning the antigen gene.

In order to increase the expression rate on the surface of the yeast in the ompH (A: 3) gene, only the epitope portion was selected from the OmpH (A: 3) gene. PCR primers were prepared for cloning two L2 and L5 regions of high antigenicity, referring to the hydrophilic region and the literature, from the portions obtained through antigenic epitope prediction (see FIGS. 8 to 9). . PCR was performed for each of primer sets # 1 and # 2 using ompH (A: 3) as a template (denaturing-94 ° C., 1 min, annealing-54 ° C., 30 sec, extension (extension-72 ° C., 1 min) Each PCR product obtained by performing PCR was purified using a PCR purification kit, followed by overlapping PCR using two products as templates (denaturing- 94 ° C., 1 min, annealing-54 ° C., 30 sec, extension-72 ° C., 1 min.) The product thus obtained was cloned into pAURYD and then cloned by enzyme restriction, where the peptide size was 169 bp. (Fig. 10) This was submitted to core bio sequencing, and the sequencing result was aligned with the original sequence for sequencing (Fig. 11). The clone obtained was “pAURYD-peptide. ".

A primer was constructed to clone the gene of heat-labile enterotoxin subunit B (LTB) isolated from Enterotoxigenic Escherichia coli ( ETEC) into pAURYD vector. In this case, the GPGP (ggc-) corresponding to the sense primer may be used to generate glycine-proline-glycine-proline linker when fusion of LTB to the antigen gene cloned in pAURYD. ccg-ggc-ccg) was added to the sequence (Fig. 12), PCR was performed using this primer (denaturing-94 ℃, 1 min, annealing-58 ℃, 30 sec, extension-72 ℃, 1 min). PCR products were cloned into XhoI and BstI sites of pAURYD. And enzyme restriction (enzyme restriction) was confirmed whether cloning. LTB size was 321 bp (FIG. 13). This was commissioned to core bio, the sequencing results were confirmed (Fig. 14). The resulting clone was named “pAURYD-LTB”.

Proteins fused to peptides and LTBs were restricted to enzymes in the pAURYD-LTB plasmid vector for expression on the surface of the yeast and cloned into the pAURYD-peptide plasmid vector. And enzymatic restriction was confirmed whether cloning (see Fig. 15 and 16a). The clone obtained by fusion of OmpH peptide and LTB was named “pAURYD-fusion.” Thus, cloning constructs a construct in which the peptide and LTB previously made are connected by GPGP linker.

A primer was constructed to clone the gene of heat-labile enterotoxin subunit B (LTB) isolated from Enterotoxigenic Escherichia coli (ETEC) into pAURYD vector. (FIG. 16B), PCR was performed using this primer (denaturing-94 ° C., 1 min, annealing-54 ° C., 30 sec, extension-72 ° C., 1 min). PCR products were cloned into XhoI and BstBI sites of pAURYD. And enzyme restriction confirmed cloning. LTB size was 309 bp. This was commissioned to core bio, and the sequencing result was confirmed (16c). The resulting clone was named pAURYD-LTB (NoGP).

Example 2. Transformation of yeast

pAURYD vector clones were transformed into saccharomyces cerevisiae EBY100 strain. A single colony of EBY100 was inoculated in 2 ml of YPD broth and incubated at 250 rpm for 30 hours at 30 ° C., and then inoculated with 500 μl of cells in 50 ml of YPD broth until OD600 = 1.2 at 250 rpm at 30 ° C. After down the cells at 1000xg and resuspended with 10 ml of Solution A (LiAc, Tris-HCl, EDTA), the cells were resuspended and resuspended with 1 ml of Solution A. After incubation, 5 μg of StuI cut DNA and 150 μg of salmon testes DNA treated with 100 ° C. and 10 min and 850 μl of solution B (solution A, 40% PEG) were incubated at the same conditions for 30 minutes. Immediately after the heat shock (heat shock) for 15 minutes at 42 ℃ waited for 10 minutes at room temperature and then cell down to 5000rpm and 5ml of YPD was put in and waited overnight at 30 ℃. After cell down, the cells were resuspended in 1 ml of YPD, and 200 µl of which was plated on a 0.5 mg / ml YPD plate. After 3 days, colonies having resistance to Aureobasidin A were identified.

Example 2-1. Transformed Yeast Assay by PCR

After inoculating the transformed yeast in 2ml of YPD, grown for 24 hours at 30 ℃, 250rpm, down the cells in the E-tube, washing the pellet once with 1ml of distilled water and then lysis buffer ( 2% (v / v) Triton X-100, 1% (v / v) SDS, 100mM NaCl, 10mM Tris base pH 8.0, 1mM EDTA pH 8.0) and resuspended in glass beads, 200ul of acid-washed (sigma, Cat No. G8772) and 200ul of 25: 24: 1 phenol: chloroform: isoamyl alcohol (Bioneer Cat. No. C-9017) were added and vortexing was performed for 1 minute. Then, 200 μl of TE buffer (10 mM Tris base pH 8.0, 1 mM EDTA pH 8.0) was added, mixed well through inversion, and centrifuged at 13200 rpm for 5 minutes to separate only the supernatant. The separated supernatant was mixed with 1 ml of 100% ethanol, centrifuged at 13200 rpm for 5 minutes, the resulting pellet was washed with 70% ethanol, dried well and the pellet was dissolved in 50ul of distilled water. (yeast) DNA was extracted. Thus obtained DNA was quantified and PCR (denaturing-94 ° C, 1 min, annealing-54 ° C, 1min, extension-72 ° C, 1min) was performed using 50ng of DNA as a template. The primers used to identify the clones were prepared primers that can be annealed on both sides of the multiple cloning site (MCS) of the pYD1 vector (FIG. 17A). The band size of each matching DNA can be obtained from (Fig. 17b). Also, DNA such as genomic DNA PCR was performed by performing colony PCR with a single colony transformed using this primer. Transformed yeast was assayed by confirming the band size (FIG. 17C).

Example 3. Induction of transformed yeast

A single colony was inoculated into 2 ml of YPD containing 0.5 μg / ml of Aureobasidin A, and then 1 ml of cells were lowered into the E-tube at 30 ° C. and 250 rpm at OD600 = 2 to 5, followed by induction medium (0.67%). Resuspend in 10ml YNB (with ammonium sulfate, without amino acids), 10ml, 0.5% Casamino acids, 2% glucose or galactose, 0.01% tryptophan, It was induced for 48 hours at 250 rpm.

Example 4. Immunofluorescence assay of transformed yeast

The transformed yeast was induced with galactose medium followed by IFA. At this time, since the V5 tag is fused to the 3 'end of the antigen gene, monoclonal anti V5 antibody (invitrogen ™) is used as the first antibody (room temperature, 2 hours), and Alexa Fluor 488 is used as the second antibody (room temperature, 2). Time). Unlike the negative control group EBY100, the transformed yeast showed a circular signal on the cell surface. (FIGS. 17D and 18)

Example 5. GM1 binding assay of transformed yeast

In order to measure the activity of the protein expressed on the surface of the yeast, a property of binding to GM1 ganglioside, one of the characteristics of the LTB protein, was used. LTB proteins are not known to bind GM1 gangliosides when they are monomers. The method is similar to ELISA and GM1 ganglioside (Calbiochem cat. No. 345724) was coated with 0.15 ug / well, 4 ° C, 16 hours. Afterwards, the yeast expressing fusion with LTB and the number of yeast expressing only pAURYD as a control are equally adjusted, and put into each well through serial dilution and react with lysate. In order to see the E-tube, glass beads and cell pellets were added 1: 1, 400ul of Tris-Cl (pH 8.0) was added and vortex was performed three times for 30 seconds, followed by a centrifuge. Supernatant was separated through to give lysate. The lysate is also diluted in a well coated with GM1 and reacted at 37 ° C for 1 hour, and then used as a primary antibody such as ELISA through poly anti LTB antibody at 37 ° C. After the reaction for 1 hour, the anti-rabbit IgG antibody conjugated with HRP as a secondary antibody was reacted at 37 ° C for 1 hour, and the amount of color reaction was determined by TMB solution. The amount of bound yeast was measured by using a spectrophotometer. It was confirmed that a stronger binding affinity was observed in yeast expressed in fusion (fusion) than in yeast expressed only in LTB alone (FIG. 19).

Example 6. Immunization and ELISA

Transformed yeast was induced to confirm expression via IFA and then mice were immunized. Mice used BALB / C (4 weeks old) and each route of administration was performed by oral, rectal, intranasal, sublingual, intradermal route. Immunization using the oral route of administration (FIG. 20A) was administered in the same amount for each individual using a stainless-steel animal needle (Zonde), intrarectal (FIG. 20C), sublingual (FIG. 20B) and intranasal ( In the case of intranasal, Figure 20a, c) route of administration, mice were anesthetized and immunization was performed. Intradermal route (FIG. 20B) was performed using a tattoo machine. Immunization was carried out according to the scheme shown in FIG. 19, blood was collected from orbital cavity veins of experimental animals according to the scheme, and serum was isolated to verify whether antigen-specific antibody reactions appeared by IgG ELISA. In addition, after collecting fecal, IgA was obtained from fecal through extraction buffer (PBS, 0.01% sodium azide, 5% FBS, protease inhibitor cocktail), and then antigen-specific antibody reaction in the intestinal mucosa through IgA ELISA. Check if this appears. At this time, ompH (A: 3) protein purified from bacteria was coated (FIG. 21). In the case of oral immunization at low doses, the amount of specific antibodies was slightly increased when compared with the negative group and the serum before immunization at 6 weeks after immunization (FIG. 21A). Intranasal administration also showed a slight increase in the amount of specific antibodies when compared with the negative group and the serum before immunization at 6 weeks after immunization (FIG. 21A). In the immunization using the intradermal route, the highest amount of specific antibody was observed in ompH (A: 3) peptide-LTB fusion construct at 5 weeks. A: 3) was also shown to increase the amount of specific antibodies in the immunized group (Fig. 21b) ompH (A: 3) peptide-LTB fusion construct results in the largest increase in the amount of specific antibodies in the immunization group Appears to be associated with strong binding affinity in yeasts in which fusion constructs have been expressed in previously performed GM1 binding assays (FIG. 19). Samples with the highest levels in GM1 binding assays are presumed to have specific targeting to cells in which GM1 gangliosides are distributed in vivo in mice. Immunization of pYD-ompH with or without purified LTB protein in the rectum and nasal cavity showed no immune adjuvant effect by LTB, but the overall response of serum IgG antibody specific for OmpH antigen increased over time. Observation was possible (FIG. 21C). The amount of fecal IgA that can be used to determine the efficacy of mucosal vaccines was not found in the LTB-immunoadjuvant effect in both the rectal and intranasal immunization groups. Was observed (FIG. 21C).

The present invention relates to a vaccine composition for the prevention or treatment of Pasteurella disease using a yeast surface expression system, and more specifically iii) nucleotides encoding the envelope peptide of Pasteurella multocida, ii) Recombinant nucleotides for vaccine production for the prevention or treatment of pasteurella, comprising nucleotides encoding the Aureobasidin A peptide and nucleotides encoding the heat-labile enterotoxin subunit (LTB) peptide, the nucleotides It relates to a recombinant expression vector comprising and a vaccine composition for preventing or treating Pasteurellasis obtained by infecting the vector with a host cell.

The vaccine composition prepared by the present invention can induce an immune response by a simple immunization method such as an oral vaccine, an intradermal immune vaccine using a mucosal vaccine and a tattoo instrument, and can be used in various ways such as poultry, cattle, pigs, rabbits, and the like. It is useful for various pasteurellasis against livestock and can be widely activated industrially.

1 shows the sequence of a pYD1-ompH (A: 3) PCR primer.

Figure 2 shows the cloned results of pYD1-ompH (A: 3) by yeast cleavage.

Figure 3 shows the pYD1-ompH (A: 3) sequencing results.

4 illustrates a cloning strategy for pAURYD.

5 shows the sequence of a pAURYD PCR primer.

FIG. 6A shows the pAURYD sequencing results and FIG. 6B shows the sequence of the pAURYD.

7 shows a pAURYD vector construct map.

Figure 8 shows the antigenic prediction of ompH (A: 3) for pAURYD-peptide construction.

9 shows the sequence of a pAURYD-peptide PCR primer.

10 shows the results of pAURYD-peptide cloning by yeast cleavage.

Figure 11 shows the results of pAURYD-peptide sequencing and its sequence.

12 shows the sequence of a pAURYD-LTB PCR primer.

Figure 13 shows the results of pAURYD-LTB cloning by yeast cleavage.

Figure 14 shows the results of pAURYD-LTB sequencing and its sequence.

15 shows the results of pAURYD-fusion cloning by yeast cleavage.

16A shows the results of pAURYD-fusion sequencing.

Figure 16b shows a primer prepared for cloning the gene of heat-labile enterotoxin subunit B (LTB) to the pAURYD vector.

Figure 16c shows the results of sequencing the product of the result of the PCR using a primer prepared for cloning to the pAURYD vector using a restriction enzyme.

Figure 17a shows the result of preparing a primer for identifying the clone.

FIG. 17B shows the respective DNA band sizes that can emerge from the transformed yeast by performing PCR.

Figure 17c shows the result of confirming the DNA band size by performing colony PCR with a single colony transformed using a primer prepared for assaying the transformed yeast by PCR.

17D shows IFA results of pAURYD clones of transformed yeast.

18 shows confocal microscopy images of pAURYD clones of transformed yeast.

Figure 19 shows GM1 binding assay of surface expressed yeast.

20A and B are tables showing the results of the generation of antigen-specific antibodies when administered by the respective routes of administration (oral, intranasal, intradermal).

21A shows serum IgG ELISA results after oral administration and intranasal administration.

Figure 21B shows the serum IgG ELISA results in the intradermal administration.

21C shows serum IgG and IgA ELISA results.

22 shows a schematic of the pAURYD constructs.

Figure 23 shows a schematic of the yeast surface expression system of the OmpH peptide-LTB fusion construct.

<110> SUNGKYUNKWAN UNIVERSITY Foundation for Corporate Collaboration <120> A fowl cholera vaccine using a yeast surface-expression system <130> PA9706-0372KR <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 508 <212> DNA <213> Artificial Sequence <220> <223> AURYD-fusion sequencing result <400> 1 ggtaccaagt aaagatgtga aagataaaga gggtaaagtt aaccaaccaa tcggtaacga 60 agttcatgct aaaggcccgg gcccgagcca aaaatatgta aaacagaaag ttgaacaacc 120 tcagccttta cctcctggtc aagtagagcg tttcaaagat gaaaaagaaa aagctctcga 180 gggcccgggc ccggctcccc agtctattac agaactatgt tcggaatatc gcaacacaca 240 aatatatacg ataaatgaca agatactatc atatacggaa tcgatggcag gtaaaagaga 300 aatggttatc attacattta agagcggcgc aacatttcag gtcgaagtcc cgggcagtca 360 acatatagac tcccaaaaaa aagccattga aaggatgaag gacacattaa gaatcacata 420 tctgaccgag accaaaattg ataaattatg tgtatggaat aataaaaccc ccaattcaat 480 tgcggcaatc agtatggaaa acttcgaa 508 <210> 2 <211> 1047 <212> DNA <213> Artificial Sequence <220> <223> Pastereurella multocida type A OMPH sequence <400> 2 atgaaaaaga caatcgtagc attagcagtc gcagcagtag cagcaacttc agcaaacgca 60 gcaacagttt acaatcaaga cggtacaaaa gttgatgtaa acggttctgt acgtttactt 120 cttaaaaaag aaaaagatat gcgtggtgat ttaattgata acggttcacg cgtttctttc 180 aaagcatctc atgacttagg tgaaggctta agcgtattag gttatgcaga acttcgtttt 240 agtaaagatg tgaaagataa agagggtaaa gttaaccaac caatcggtaa cgaagttcat 300 gctaaacgtc tttatgcggg ttttgcgtat gaaggtttag gtacattaac ttttggtaac 360 caattaacaa ttggtgatga tgttggtgtg tctgattaca cttattttaa cagtggtatc 420 aatggcgtac ttattactag tggtcaaaaa gcaattaact tcaaatcagc agagttcaac 480 ggtttcacat ttggtggtgc gtatgtgttc tcaggcgatg caaacaaaga tgcattacgt 540 gatggtcgcg gtttcgtagt agcaggttta tacaacagac aaatcggtga tgttggtttt 600 gcattcgaag caggctatag ccaaaaatat gtaaaacaga aagttgaaca acctcagcct 660 ttacctcctg gtcaagtaga gcgtttcaaa gatgaaaaag aaaaagcttt cttggtcggt 720 gcagaattat cgtacgctgg tttagcgctt ggtgttgact acgcacaatc taaagtgact 780 aacgtagatg gtaaaaaacg tgcacttgaa gtgggtttaa actatgacct taatgataaa 840 gcgaaagttt atacagactt catctgggaa aaagaaggtc ctaaaggtga tgttgaaaga 900 actcgcactg tagctgtagg ttttggttac aaacttcaca aacaagttga aacctttgtt 960 gaaggtgctt ggggaagaac aaaagatgca gatggtaaaa caacaaaaga taatgtagtt 1020 ggtacaggtt tacgcgtaca cttctaa 1047 <210> 3 <211> 1118 <212> DNA <213> Artificial Sequence <220> <223> pAURYD sequence fragment # 1-result <400> 3 cgccagctat ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag 60 ctcggatcca ctagtaacgg ccgccagtgt gctggaattc ggcttgagct cacggattag 120 aagccgccga gcgggtgaca gccctccgaa ggaagactct cctccgtgcg tcctcgtctt 180 caccggtcgc gttcctgaaa cgcagatgtg cctcgcgccg cactgctccg aacaataaag 240 attctacaat actagctttt atggttatga agaggaaaaa ttggcagtaa cctggcccca 300 caaaccttca aatgaacgaa tcaaattaac aaccatagga tgataatgcg attagttttt 360 tagccttatt tctggggtaa ttaatcagcg aagcgatgat ttttgatcta ttaacagata 420 tataaatgca aaaactgcat aaccacttta actaatactt tcaacatttt cggtttgtat 480 tacttcttat tcaaatgtaa taaaagtatc aacaaaaaat tgttaatata cctctatact 540 ttaacgtcaa ggagaaaaaa ccccggatcg gactactagc agctgtaata cgactcacta 600 tagggaatat taagctaatt ccctacttca tacattttca attaagatgc agttacttcg 660 ctgtttttca atattttctg ttattgcttc agttttagca caggaactga caactatatg 720 cgagcaaatc ccctcaccaa ctttagaatc gacgccgtac tctttgtcaa cgactactat 780 tttggccaac gggaaggcaa tgcaaggagt ttttgaaaat gggtcgggat ctgtacgacg 840 atgacgataa ggtacccccg ggtcgactag taatattaca aatcagtaac gtttgtcagt 900 aattgcggtt ctcacccctc aacgactagc aaaggcagcc ccataaacac acagtatgtt 960 tttaagcttc tgcaggctag tggtggtggt ggttctggtg gtggtggttc tggtggtggt 1020 ggttctgcta gcatgactgg tggacagcag ccgaattctg cagatatcca tcacactggc 1080 ggccgctcga gcatgcatct agagggccca attcgccc 1118 <210> 4 <211> 842 <212> DNA <213> Artificial Sequence <220> <223> pAURYD sequence fragment # 2-result <400> 4 attacgccag ctatttaggt gacactatag aatactcaag ctatgcatca agcttggtac 60 cgagctcgga tccactagta acggccgcca gtgtgctgga attcggctta ctagtccgcg 120 gcatatggcg gccgctcgag tctagagggc ccttcgaagg taagcctatc cctaaccctc 180 tcctcggtct cgattctacg cgtaccggtc atcatcacca tcaccattga gtttaaaccc 240 gctgatctga taacaacagt gtagatgtaa caaaatcgac tttgttccca ctgtactttt 300 agctcgtaca aaatacaata tacttttcat ttctccgtaa acaacatgtt ttcccatgta 360 atatcctttt ctatttttcg ttccgttacc aactttacac atactttata tagctattca 420 cttctataca ctaaaaaact aagacaattt taattttgct gcctgccata tttcaatttg 480 ttataaattc ctataattta tcctattagt agctaaaaaa agatgaatgt gaatcgaatc 540 ctaagagaat tgcatgcaag ccgaattctg cagatatcca tcacactggc ggccgctcga 600 gcatgcatct agagggccca attcgcccta tagtgagtcg tattacaatt cactggccgt 660 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc 720 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca 780 acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc 840 gg 842 <210> 5 <211> 181 <212> DNA <213> Artificial Sequence <220> <223> pAURYD-peptide sequence <400> 5 ggtaccaagt aaagatgtga aagataaaga gggtaaagtt aaccaaccaa tcggtaacga 60 agttcatgct aaaggcccgg gcccgagcca aaaatatgta aaacagaaag ttgaacaacc 120 tcagccttta cctcctggtc aagtagagcg tttcaaagat gaaaaagaaa aagctctcga 180 g 181 <210> 6 <211> 333 <212> DNA <213> Artificial Sequence <220> <223> pAURYD-LTB sequece <400> 6 ctcgagggcc cgggcccggc tccccagtct attacagaac tatgttcgga atatcgcaac 60 acacaaatat atacgataaa tgacaagata ctatcatata cggaatcgat ggcaggcaaa 120 agagaaatgg ttatcattac atttaagagc ggcgcaacat ttcaggtcga agtcccgggc 180 agtcaacata tagactccca aaaaaaagcc attgaaagga tgaaggacac attaagaatc 240 acatatctga ccgagactaa aattgataaa ttatgtgtat ggaataataa aacccccaat 300 tcaattgcgg caatcagtat ggaaaacttc gaa 333  

Claims (29)

A fusion of nucleotides encoding the outer membrane peptide of Pasteurella multocida to pAUR101 containing an aureobasidin A resistance gene and a nucleotide encoding a heat-labile enterotoxin subunit (LTB) peptide. Recombinant nucleotide construct inserted. The recombinant nucleotide construct of claim 1, wherein the fusion encoding the envelope peptide of Pasteurella multocida and the nucleotide encoding the LTB peptide have a nucleic acid sequence of SEQ ID NO: 1. An expression vector comprising the recombinant nucleotide construct of claim 1. Yeast transformed with the expression vector of claim 3. delete The yeast of claim 4, wherein the yeast expresses on the surface a peptide encoded by a fusion of nucleotides encoding the envelope membrane peptide of Pasteurella multocida and a nucleotide encoding the LTB peptide. The yeast of claim 6, wherein the pasteurella multocida is selected from the group consisting of pasteurella multocida types A, B, D, E and F. The yeast of claim 6, wherein the nucleotide encoding the LTB peptide is derived from Enterotoxigenic Escherichia coli (ETEC). The yeast of claim 6, wherein the fusion is a nucleotide encoding the envelope membrane peptide of Pasteurella multocida and a nucleotide encoding the LTB peptide is linked by a linker. 10. The yeast of claim 9, wherein the linker is a linker consisting of nucleotides encoding glycine-proline-glycine-proline. Pasteurase comprising transforming the expression vector of claim 3 to yeast to express a peptide encoded by a fusion of a nucleotide encoding the envelope membrane peptide of Pasteurella multocida and a nucleotide encoding the LTB peptide. Method of producing a vaccine for the prevention or treatment of Vaccine composition for the prevention or treatment of pasteurella comprising the yeast of claim 4. The vaccine composition of claim 12, wherein the composition is for oral or intradermal administration. (a) transforming the yeast with a vector comprising the recombinant nucleotide construct of claim 1; (b) expressing a recombinant peptide for the prevention or treatment of pasteurella on the yeast surface, comprising culturing the transformed yeast. A method for delivering Pasteurella multocida envelope membrane peptides to GM1 receptor expressing cells using the yeast of claim 4. The pAURYD-fusion construct of claim 1, shown in FIG. 22. A vaccine composition for the prevention or treatment of pasteurella, comprising a recombinant peptide expressed on the surface of yeast according to the method of claim 14. The vaccine composition of claim 13 wherein said intradermal administration is administered using a tattoo instrument. delete delete delete The method according to claim 11, wherein the pasteurella is a disease caused by a fungus selected from the group consisting of Pasteurella multocida type A, B, D, E and F. 23. The method of claim 22, wherein the pasteurella is poultry choline. The vaccine composition according to claim 12, wherein the pasteurella is a disease caused by a fungus selected from the group consisting of Pasteurella multocida type A, B, D, E and F. 25. The composition of claim 24, wherein the pasteurella is a poultry choleline vaccine composition. 15. The method of claim 14, wherein the pasteurella is a disease caused by a fungus selected from the group consisting of Pasteurella multocida type A, type B, type D, type E and type F. The method of claim 26, wherein the pasteurella is poultry choleline. 18. The vaccine composition according to claim 17, wherein the pasteurella is a disease caused by a fungus selected from the group consisting of Pasteurella multocida type A, B, D, E and F. 29. The composition of claim 28, wherein said pasteurella is a poultry choleline vaccine composition.

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