KR101303282B1 - Bacterial mutants devoid of AB type exotoxin by causing deficiency of toxin B-subunit and use thereof - Google Patents

Abstract

The present invention relates to a strain lacking the AB type bacterial toxin, and its use, characterized in that the toxin B-subunit of the AB type bacterial toxin (AB ₅ toxin) is missing, and more specifically, Bacterial exotoxin, an AB-type that combines one or more B-subunit proteins that bind to a cytotoxic receptor with one A-subunit that is toxic to form a holotoxin. In pathogenic bacteria producing the A-subunit protein, which has an enzymatically active site of toxicity, in the mutant that stops the functional expression of the toxin B-subunit, the cytoplasm is expressed in the cytoplasm, but the AB subunit of the toxin is It does not exist in the periplasm, which is connected to the unit-to-unit and secretion outside the cell, and the toxicity of the outer membrane vesicles (OMV) secreted by the mutant is significantly higher than that of the normal strain. It was found that the strains lacking the AB type bacterial toxin by the toxin B-subunit deficiency of the present invention, and the secreted outer membrane vesicles (OMV) produced therefrom are useful for vaccine compositions for infectious diseases. Can be used.

Description

Bacterial mutants devoid of AB type exotoxin by causing deficiency of toxin B-subunit and use according to toxin Ｂ-subunit deletion of AB type bacterial toxin

The present invention relates to a strain lacking the AB type bacterial toxin by a toxin B-subunit deletion of the AB type bacterial toxin, a method for producing the strain, a method for preparing the outer membrane vesicles using the strain, and a use thereof.

Escherichia coli , E. coli ) O157 is a deadly bacterium that causes death of hemolytic colitis or hemolytic uremic syndrome in humans, especially children and elderly people with low immune system. However, in most cases, development of diagnostic methods such as detection of antibody (IgM) and Stx antibody (IgG) against bacterial O antigen (LPS) in patient sera has been undertaken. Moreover, the treatment of E. coli O157 infection with antibiotics may increase the frequency of hemolytic uremic syndrome, due to the bacterial lysis by antibiotics and the release of intracellular Stx.

Vaccine using LPS has been attempted to induce anti LPS IgG antibodies to kill bacteria, which also increases the frequency of hemolytic uremic syndrome due to the release of toxins contained in bacteria. Therefore, new methods for the development of E. coli O157 vaccines are being explored. In addition, research on new methods for the development of vaccines for the prevention of other infectious diseases, as well as for the development of vaccines against E. coli , is steadily underway.

The classic vaccines for infectious diseases to date are largely divided into killed vaccines that inactivate the infecting organisms and live-attenuated cells that have been attenuated without killing the cells. The distinction is based on differential induction of immune responses to defend against pathogens' infection and proliferation patterns in the host. Therefore, the prophylactic vaccine of infectious diseases caused by intracellular pathogens is developed as a live vaccine or DNA vaccine that promotes the induction of effective cell-mediated immunity, and protects against pathogens proliferating outside the host cell. In order to induce humoral immunity through antibodies secreted into body fluids, it is essential to use a dead vaccine or a subunit vaccine prepared by extracting only the major cell surface antigen components from the cells. Developing is commonplace.

However, in the development of such a vaccine, the problems of efficacy and safety have always been an obstacle. A representative problem is the stability problem that always comes up in the development of live vaccines. Probiotic vaccine candidates, although attenuated, are still a viable pathogen, and therefore probiotic vaccines that stimulate cellular immunity because of the risk of proliferation and activation of live bacteria used as vaccine strains in vivo, resulting in symptoms similar to natural infection. Although its efficacy is large, it is difficult to obtain development and marketing authorization due to stability issues. Therefore, there is a great need to develop a non-proliferative vaccine material in vivo that exhibits a cellular immune induction effect similar to that of a live vaccine, while excluding side effects caused by a live vaccine. In addition, although the bacteriophage vaccine and subunit vaccines are non-proliferative vaccine substances in vivo, they are effective in humoral immunity and are not considered in diseases requiring cellular immune induction. The problem with the dead bacterium vaccine is that since all of the bacterium are used as vaccines, many kinds of intracellular proteins are included in addition to the cell surface antigen components effective for defense against infection, which may cause side effects such as inducing an autoimmune response. Subunit vaccines usually contain only one type of antigenic component, have low immunogenicity, and are difficult to induce cellular immune responses, and therefore, cost-effective ones have to be mixed with expensive antigenic adjuvants to overcome them.

As one of the next-generation non-cellular vaccine candidates to compensate for the shortcomings of these non-proliferative vaccines in recent years, there is an increasing interest and research on nano-scale outer mebrane vesicles (OMV) naturally produced in the outer membrane of Gram-negative bacteria. There is a trend. This is because the outer membrane endoplasmic reticulum contains natural antigenic components composed of complex glycolipids such as outer membrane protein and lipopolysaccharide (LPS), so that it can be used as a natural nano-bio material to obtain multifunctional outer proteins or carbohydrates. This is because it can be used as an excellent material for bio-nano vaccines when combined with technology that can display on the surface of nanoparticles.

However, these multifunctional engineered OMVs are still in the field of unexplored research, although they may increase medical / industrial applicability when applied to vaccine development and targeted cell-specific drug delivery. This is because the OMV inherently contains various components derived from the outer membrane and periplasm of Gram-negative bacteria, and thus, safety issues that may arise in vivo applications are emerging. In order to develop OMV's biotechnology, it is urgent to solve OMV safety issues.

On the other hand, the toxicity of OMV can be divided into two aspects, one of which is endotoxicity by LPS, which is a major component of OMV, which is a toll-like receptor in which the lipid A portion of LPS is an animal cell receptor. This is because it is recognized as a strong inflammatory stimulus by the toll-like receptor 4-MD2 complex and causes sepsis. In order to reduce the toxicity of the OMV, a mutant that changed the lipid A portion of the LPS was prepared, and studies to reduce the toxicity of OMV by greatly reducing the toxicity by LPS have been conducted (Kim et al ., 2009, BBA). Biomembranes, 1788: 2150-2159.

A second aspect of the toxicity of OMV is the toxicity of the exotoxins of bacteria contained and secreted in the OMV, including Shiga toxin (STx), heat-labile toxin (LT), and LT toxin. Cholera toxin (CT), which is very similar in structure and function, is a representative example of the AB type toxin. The AB type toxin (AB ₅ toxin) is a protein aggregate of one molecule of A-subunit with an enzymatically active site of toxicity and a molecule of B-subunit of five molecules that binds to the cell receptor without toxicity. The pentamer is characterized in that it is coalesced into one complete toxin (holotoxin) in a non-covalent bond. In order to eliminate the toxicity caused by these exotoxins, by constructing a mutant lacking the toxic toxin A-subunit, an engineered OMV containing only the toxin B-subunit having no toxicity but having antigenicity was generated. Animal testing confirmed that the overall toxicity of the prepared OMV was very low (Kim et al ., 2010, FEMS Immunol. Med. Microbiol., 58: 412-420), and efforts to develop a new method for attenuating OMV. This is happening steadily.

In an effort to find a new method aimed at attenuating this OMV, the present inventors have found toxin B-subunit in cigar toxin (Stx) and dimeric toxin (LT) among AB type bacterial toxins. Toxin A-subunit protein with enzymatic activity that is toxic in the missing mutant is unstable in periplasm and is not secreted by OMV. The present invention was completed by making a mutant lacking the toxin B-subunit gene and confirming that it was removed, and revealing that the produced outer membrane vesicles (OMV) can be usefully used as a vaccine composition for infectious diseases. It was.

An object of the present invention is a strain lacking the AB type bacterial toxin, characterized in that the toxin B-subunit of the AB type bacterial toxin (AB ₅ toxin), method for producing the strain, the It is to provide a method for producing outer membrane vesicles (OMV) in a strain, and a vaccine composition for infectious diseases containing the produced outer membrane vesicles as an active ingredient.

In order to achieve the above object, the present invention provides a strain lacking the AB type bacterial toxin, characterized in that the toxin B-subunit of the AB type bacterial toxin (AB ₅ toxin) is missing.

The present invention also provides a method for preparing a strain lacking the AB type bacterial toxin, comprising the following steps:

1) preparing a vector comprising a toxin B-subunit gene of AB type bacterial toxin;

2) Deleting a DNA fragment of 100 to 200 bp into the toxin B-subunit gene ORF (open reading frame) in the vector prepared in step 1) and inserting an antibiotic resistance gene cassette into the deleted DNA portion Preparing homologous DNA for mutagenesis; And

3) amplifying and purifying the homologous DNA prepared in step 2), and then introducing the AB-type bacterial toxin into the strain.

The present invention also provides a method for producing an outer membrane endoplasmic reticulum (OMV) lacking a toxin from a strain lacking an AB type bacterial toxin comprising the following steps:

1) culturing the strain of claim 1;

2) separating the cell precipitate and the culture supernatant by centrifuging the cultured strain of step 1); And

3) separating the outer membrane vesicles (OMV) from the culture supernatant of step 2).

In addition, the present invention provides a vaccine composition for infectious diseases containing the outer membrane vesicles (OMV) produced using the strain as an active ingredient.

Strain lacking AB type bacterial exotoxin due to toxin B-subunit deficiency of the present invention is due to unstable toxin A-subunit in periplasm. Since no toxicity is naturally eliminated, secreted vesicles produced from the strain (OMV) can be usefully applied to vaccine compositions for infectious diseases.

FIG. 1 shows the analysis of periplasmic protein and outer membrane vesicle (OMV) fractions isolated from various mutants lacking the toxin B-subunit gene (StxB):
(A) electron micrograph of outer membrane vesicle (OMV);
(B, C, D) Western blot analysis of periplasmic-derived protein and enveloped vesicle samples; And
(E) Western blot analysis of periplasmic protein samples:
PPE: Periplasm Protein Extract Fraction;
OMV: bacterial epithelial vesicles;
BAP: Bacterial alkaline phosphatase (periplasmic protein);
WT: wild type Sakai O157 strain;
DM: msbB double mutant ( msbB gene deletion variant);
STx1B: B subunit of cigatoxin type 1;
STx2B: B subunit of cigatoxin type 2;
STx2A: A subunit of cigatoxin type 2;
LTA: A subunit of dimeric enterotoxin;
Δ lt-B mutant: B -subunit deletion mutant of dimeric enterotoxin;
Δ degP :: Cm mutant: a mutant strain lacking the DegP protein gene and inserted with the chloramphenicol (Cm) resistance gene; And
α-STx2A: antibody against STx2A.
Figure 2 shows the presence of toxin A-subunit of the recombinant mutant deficient in the toxin B-subunit in E. coli producing Shigatoxin and E. coli producing dimeric toxin through Western blot analysis:
Sakai-WT: wild type Sakai O157 strain;
WT / ΔStx2B: Stx2B gene deletion mutant strain of wild type (wild type) Sakai O157 strain;
WT / ΔStx2B / degP :: Cm: wild type (wild type) mutants lacking the Stx2B gene and DegP protein gene of the Sakai O157 strain and inserted the chloramphenicol (Cm) resistance gene;
α-STx2A: antibody against STx2A;
ETEC H10407: Enterotoxin E. coli;
Δlt-B mutant: B-subunit deletion mutant of dimeric intestinal toxin;
α-LTA: an antibody against the A subunit of a dimeric enterotoxin; And
α-BAP: Antibody against Bacterial alkaline phosphatase.
Figure 3 is a result of confirming the cytotoxicity of the outer membrane endoplasmic reticulum produced by the method of the present invention using Shigatoxin-sensitive Vero cell line.

The present invention provides a strain lacking the AB type bacterial toxin, characterized in that the toxin B-subunit of the AB type bacterial toxin (AB ₅ toxin) is deleted.

The AB type bacterial toxins include Shiga toxin, heat-labile toxin, cholera toxin, Campylobacter jejuni enterotoxin, and pertussis toxin. And Shiga-like toxin (shiga-like toxin or verotoxin) is preferably any one selected from the group consisting of, but is not limited thereto.

The AB type bacterial toxin is Shiga toxin-producing E. coli (STEC), which produces Shigatoxin, enterotoxin type E. coli (ETEC), which produces dimeric toxin, enteritis Vibrio bacteria that produce cholera toxin. (Vibrio cholerae ), Campylobacter jejuni, Bordetella pertussis and Shigella dysenteriae is preferably derived from any one of the strains selected from the group consisting of It is not limited to this.

The E. coli Shiga toxin is the acid production in the deposit accession No. KCTC 11344BP E.coli O157: H7 Sakai or accession No. KCTC 11346BP deposited in an E.coli O157: the msb B1 / B2 gene from msb H7 Sakai strain deficient Sakai- It is preferably DM, but is not limited thereto.

The enterotoxin E. coli is preferably Escherichia coli ETEC strain (Strain) H10407, but is not limited thereto.

The toxin B-subunit is Stx-1B (Shiga toxin 1B subunit) having the nucleotide sequence set forth in SEQ ID NO: 7, Stx-2B (Shiga toxin 2B subunit) having the nucleotide sequence set forth in SEQ ID NO: 8, SEQ ID NO: 9 Coding from any of the genes selected from the group consisting of LT-B (heat-labile toxin B subunit) having a nucleotide sequence as set forth below and CT-B (cholera toxin B subunit) having a nucleotide sequence as set forth in SEQ ID NO: 10 It is preferably a polypeptide, but is not limited thereto.

The strain lacking the AB type bacterial toxin is E. coli O157: H7 Sakai-DM / Δ stx2B Mutant (accession number: KCTC18220P), E. coli O157: H7 Sakai-WT / Δ stx2B mutant (Accession Number: KCTC18221P) or E. coli O157: H7 Sakai-DM / Δ stx1B / Δ stx2B Mutant (Accession Number: KCTC18222P) is preferably, but not limited to.

In a specific embodiment of the present invention, Shiga toxin-producing E. coli (STEC) producing E. coli O157: H7 Sakai, a Sakai-DM strain or a thermophilic (heat instability) toxin ( LT; E. coli enterotoxins small (enterotoxigenic E. coli having a producing ability of a heat-labile toxin); ETEC) in E.coli the B- subunit (subunit) part of the operon structural genes coding for the homologous gene from exotoxin strain H10407 By artificially deleting by an allelic exchange method, recombinant mutants lacking the toxin B-subunit were prepared and deposited on September 02, 2011, at the Korea Institute of Bioscience and Biotechnology. [ E. coli O157: H7 Sakai-DM / Δ stx2B Mutant (accession number: KCTC18220P), E. coli O157: H7 Sakai-WT / Δ stx2B mutant (Accession Number: KCTC18221P) or E. coli O157: H7 Sakai-DM / Δ stx1B / Δ stx2B mutant (Accession No .: KCTC18222P)] (see Table 1).

Further, in another specific embodiment of the present invention, the PPE (Periplastism-derived protein) and outer membrane vesicles (OMV) samples of the prepared toxin B-subunit-deficient mutants were A-subunit As a result of Western blot using an antibody that specifically reacts to, it was confirmed that A-subunits were not detected in PPE and outer membrane vesicles (see FIGS. 1 and 2).

In another specific embodiment of the present invention, the outer membrane endoplasmic reticulum (OMV) in a strain lacking AB type bacterial exotoxin due to the toxin B-subunit deletion of the present invention Was isolated and its cytotoxicity was confirmed using Shigatoxin sensitive Vero cell line. As a result, when treated with OMV isolated and purified from the Shigatoxin-expressing strain, it showed a concentration-dependent cytotoxicity, compared to the control group not treated with OMV, and treated with OMV separated and purified from the strain lacking the B-subunit In one case, it did not show cytotoxicity (see FIG. 3), and it was confirmed that OMV secreted from the toxin B-subunit-deficient strain did not show cytotoxicity.

In view of these facts, the deletion of the toxin B-subunit commonly found in AB-type exotoxin-secreting bacteria results in the absence of the toxin A-subunit in periplasm, resulting in strains and outer membranes lacking exotoxin. The endoplasmic reticulum (OMV) can be produced, which, unlike the outer membrane vesicles secreted from normal strains, can be seen that the toxicity is significantly removed.

The present invention also provides a method for preparing a strain lacking the AB type bacterial toxin, comprising the following steps:

1) preparing a vector comprising a toxin B-subunit gene of AB type bacterial toxin;

2) Deleting a DNA fragment of 100 to 200 bp into the toxin B-subunit gene ORF (open reading frame) in the vector prepared in step 1) and inserting an antibiotic resistance gene cassette into the deleted DNA portion Preparing homologous DNA for mutagenesis; And

3) amplifying and purifying the homologous DNA prepared in step 2), and then introducing the AB-type bacterial toxin into the strain.

AB type bacterial toxin of step 1) is Shiga toxin, heat-labile toxin, cholera toxin, Campylobacter jejuni enterotoxin, perchusis toxin (pertussis toxin) and Shiga-like toxin (shiga-like toxin or verotoxin) is preferably any one selected from the group consisting of, but is not limited thereto.

AB type bacterial toxin of step 1) is produced by Shiga toxin-producing E. coli (STEC), bipyrogen-producing E. coli (enterotoxigenic E. coli ; ETEC), cholera toxin Derived from any one strain selected from the group consisting of Vibrio cholerae , Campylobacter jejuni , Bordetella pertussis , and Shigella dysenteriae Preferably, but not limited to.

Toxin B-subunit gene of step 1) is Stx-1B (Shiga toxin 1B subunit) having the nucleotide sequence of SEQ ID NO: 7, Stx-2B (Shiga toxin 2B subunit) having the nucleotide sequence of SEQ ID NO: 8 ), Any one selected from the group consisting of LT-B (heat-labile toxin B subunit) having a nucleotide sequence as set out in SEQ ID NO: 9 and CT-B (cholera toxin B subunit) having a nucleotide sequence as set out in SEQ ID NO: 10 One is preferred, but is not limited thereto.

 In general, the vector of step 1) may be any cloning vector used in the art, and it is preferable to use pUC18, but is not limited thereto.

The antibiotic of step 3) may select E. coli used in the art. All of the general antibiotics may be used, and any one selected from the group consisting of ampicilline, tetracycline, and chloramphenicol may be used. Preferably, but not limited to, chloramphenicol (chloramphenicol) is more preferably, but is not limited thereto.

The strains of step 3) include Shiga toxin-producing E. coli (STEC), E. coli (enterotoxigenic E. coli ; ETEC), and cholera toxin. It is preferably one selected from the group consisting of Vibrio cholerae , Campylobacter jejuni , Bordetella pertussis and Shigella dysenteriae , but is not limited thereto. I never do that.

In a specific embodiment of the present invention, when the toxin B-subunit is commonly deleted in AB-type exotoxin-secreting bacteria, the toxin A-subunit is not present in the periplasm, and as a result, the strain that lacks the toxin And the outer membrane endoplasmic reticulum (OMV) can be produced, which was confirmed that the toxicity is significantly removed unlike the outer membrane endoplasmic reticulum secreted from the normal strain, through which, the present invention is usefully used in the production method of strains lacking AB type bacterial toxin Can be.

The present invention also provides a method for producing an outer membrane endoplasmic reticulum (OMV) lacking a toxin from a strain lacking an AB type bacterial toxin comprising the following steps:

1) culturing the strain of claim 1;

2) separating the cell precipitate and the culture supernatant by centrifuging the cultured strain of step 1); And

3) separating the outer membrane vesicles (OMV) from the culture supernatant of step 2).

In a specific embodiment of the present invention, when the toxin B-subunit is commonly deleted in AB-type exotoxin-secreting bacteria, the toxin A-subunit is not present in the periplasm, and as a result, the strain that lacks the toxin And it can produce the outer membrane endoplasmic reticulum (OMV), which was confirmed that the toxicity is significantly removed unlike the outer membrane endoplasmic reticulum secreted from the normal strain, through which, the present invention is the outer membrane vesicle lacking the toxin from the strain lacking AB type bacterial toxin It can be usefully used in the production of (OMV).

In addition, the present invention provides a vaccine composition for infectious diseases containing the outer membrane vesicles (OMV) produced using the strain of the present invention as an active ingredient.

The infectious disease is preferably a disease caused by pathogenic bacteria that secrete AB type toxin, but is not limited thereto.

The infectious disease is preferably any one selected from the group consisting of hemolytic colitis, hemolytic uremic syndrome, diarrhea and enteritis, but is not limited thereto.

In a specific embodiment of the present invention, in a specific embodiment of the present invention, if the toxin B-subunit is commonly deleted in AB-type exotoxin-secreting bacteria, the toxin A-subunit is not present in the periplasm. As a result, it was possible to produce strains and outer membrane endoplasmic reticulum (OMV) lacking the toxin, which was confirmed that the toxicity is significantly removed unlike the outer membrane endoplasmic reticulum secreted from the normal strain, through which the outer membrane vesicles produced by the method of the present invention ( OMV) can be usefully used in vaccine compositions for infectious diseases.

The vaccine containing the outer membrane vesicles produced by the method of the present invention as an active ingredient may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

The pharmaceutical dosage forms of the vaccines may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds, as well as in a suitable collection.

Vaccine compositions comprising the outer membrane vesicles produced by the method of the present invention, respectively, oral formulations, external preparations, suppositories, and sterile injections of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. according to conventional methods. It can be formulated and used in the form of a solution. Carriers, excipients and diluents that may be included in the compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, 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. When formulated, they are formulated using commonly used fillers, weights, binders, wetting agents, disintegrating agents, diluents or excipients of the surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, or the like. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the vaccine composition comprising the enveloped vesicles produced by the method of the present invention may vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration of the drug. The amount can be administered once to several times a week. In addition, the dosage may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

< Example  1> Intestinal hemorrhage  Escherichia coli STEC ( Shiga toxin - producing E. coli ) Δ stx1B / Δ s tx 2B Construction of Mutants

For the production of recombinant bacterial mutants lacking the toxin B-subuint, E. coli O157: H7, which secretes two forms of Shigatoxin, Shigatoxin 1 (STx1) and 2 (STx2), is among the enterococci. Shiga toxin-producing E. coli Δ stx1B / Δ stx2B mutants were prepared using Sakai (KCTC 11344BP) or Sakai-DM strain (KCTC 11346BP) as the parental strain.

Specifically, in order to prepare mutants through allelic exchange method, which is a homologous gene recombination technique by targeting the stx1B and stx2B genes present on the chromosome, first, a transgenic DNA having homology with the normal gene (mutated alleles) were constructed in the pUC18 cloning vector. Construction of the mutagenic DNA was basically performed by PCR to amplify DNA fragments containing the stx1B and stx2B genes, and cloned by inserting the respective DNA fragments into the pUC18 plasmid. These stx1B and to the stx2B clone as a template Fw1B-Kpn (CTGTCATAAT GGTACC GGGATTCAGCGAAG; SEQ ID NO: 1) and Re1B-Sal (TCAGGC GTCGAC AGCGCACTTGCTG; SEQ ID NO: 2) primers and Fw2B-SacI (CCTGTGAATCA GAGCTC CGGATTTGCT; SEQ ID NO: 3) and Inverse PCR using Re2B-Sal (GCACAATCC GTCGAC ATTGCATTAACAG; SEQ ID NO: 4) primer pairs results in mutations that result in deletion of 180 bp fragments within the open reading frame (ORF) of each stxB gene, followed by the missing DNA portion A chloramphenicol (Cm, chloramphenicol) resistance gene cassette was inserted into the target DNA for mutagenesis.

Then, the amplified and purified homologous DNA (mutated allele) prepared above, and then reported previously (Kim et al ., 2009, BBA-Biomembranes, 1788: 2150-2159; Kim et al ., 2010 , FEMS Immunol.Med.Microbiol., 58: 412-420), by inserting into the E. coli O157: H7 sakai strain or Sakai-DM strain containing the pKD46 expression vector, STEC (Shiga toxin-producing E. coli ) stx1B / stx2B mutants were constructed and E. coli O157: H7 Sakai-DM / Δ stx2B Mutant (accession number: KCTC18220P), E. coli O157: H7 Sakai-WT / Δ stx2B mutant (accession number: KCTC18221P) and E. coli O157: H7 Sakai-DM / Δ stx1B / Δ stx2B A mutant (accession number: KCTC18222P) was deposited on September 02, 2011, at the Korea Institute of Bioscience and Biotechnology.

In addition, the strain produced in the present invention and the plasmid used are as shown in Table 1 below.

< Example  2> Small toxin  Escherichia coli ETEC ( enterotoxigenic E. coli ) Δ lt -B :: Cm  Construction of Mutants

For the production of recombinant bacterial mutants lacking the toxin B-subuint, enterotoxigenic E. coli (ETEC) having the ability to produce heat-labile toxin (LT) The H10407 strain (ACTC 35401) was used as a parental strain to prepare an enterotoxigenic E. coli Δ lt-B :: Cm mutant.

Specifically, DNA fragments containing the lt-B gene were cloned into the pUC18 plasmid from the E. coli (ETEC) H10407 strain. For the introduction of mutations in the gene lt -B LTB-BamF (CTATCATATAC GATGGCAGG GGATCC; SEQ ID NO: 5) primers and LTB-SalR (GAGAGGATA GTCGAC GCCGTAAATAA; SEQ ID NO: 6), modified by using a primer (denaturing): 94- 30 sec, combined (annealing): Inverse PCR was performed by repeating 32 cycles with 55-30 seconds and extension: 70- 1 minute, which resulted in the deletion of approximately 120 bp DNA fragments inside the lt-B ORF. Next, the PCR reaction product was confirmed by agarose gel electrophoresis. Then, the restriction enzyme was inserted into a Sal I and a Cm-cassette DNA with the same enzymes treated with Bam HI cut, thus completing lt-B :: In order to facilitate the search for using antibiotic resistance in the defect mutant By inserting the Cm allele DNA into the ETEC H10407 strain containing the pKD46 expression vector by electroporation, an ETEC (enterotoxigenic E. coli ) Δ lt-B :: Cm mutant was prepared, which was then ETEC It was named / dLT-B.

Example 3 Confirmation of A-Subunit Existence in PPE of Mutant Produced in the Present Invention

Western blot confirmed the presence or absence of the A-subunit in the periplasmic protein extract (PPE) of the mutants of the present invention prepared in <Example 1> and <Example 2> It was.

Specifically, the Western blot was subjected to SDS-PAGE of the PPE sample extracted according to the manual of the PeriPreps ^™ Periplasting Kit (Epicentre) on a 15% gel, and then blotted to the nitrocellulose membrane. Reacted with an anti-STx2A monoclonal antibody (clone 11E10, SantaCruz Biotech) or an anti-LT-A antibody (ab62446, abcam) that specifically binds to the A-subunit with the primary antibody, and then the feoxidases with the secondary antibody A-subunit was confirmed by reaction with (peroxidase) conjugated rabbit anti-mouse IgG (Sigma, USA).

Here, bacterial alkaline phosphatase (BAP) was used as a control to check whether the protein fraction of periplasm was properly formed as an example of a typical periplasmic protein.

As a result, wild-type (WT) and Sakai-DM (DM) strains detected the A-subunit in the extracted PPE sample (Fig. 1B), and confirmed the presence of STx2A in the strain lacking STx1B and STx2B, respectively, Since STx2A is not present only in strains lacking STx2B (even if STx1B is present), it has been confirmed that hybrid holotoxins of type 1 and 2 of cigar toxins do not exist in vivo (FIG. 1C). ), It was confirmed that STx2A does not exist in the OMV of the strain lacking both STx1B and STx2B (FIG. 1D).

In addition, it was shown that STx1B was present in the strain lacking STx2B (lane 1), and STx1B was not present as expected in the strains lacking STx1B and STx2B at the same time, so that the exact variants with identical phenotypes of each mutant were produced. Confirmation (FIG. 1E).

In addition, as a result of confirming the presence of STx2A in the PPE sample of each strain, it was found that STx2A does not exist in the strain lacking STx2B (lane 2) and also in the strain lacking the deg P gene (lane 3). It was confirmed that DegP, a periplasmic protease, did not affect the proteolysis of STx2A (FIG. 2A). In a mutant lacking LT-B in the E. coli H10407 strain producing S. a. (lane 2) It was confirmed that LT-A is not present in the protein fraction of periplasm (FIG. 2B).

Through this, it was found that in the strain deficient in the B subunits of Shigatoxin and dimeric toxin, the secretion of the A-subunit in the periplasm derived protein was suppressed.

Example 4 Isolation of Outer Membrane vesicles (OMV) and its Cytotoxicity

OMV (outer membrane vesicle) was isolated from the mutants of the present invention prepared in <Example 1> and <Example 2>, and its cytotoxicity was confirmed.

Specifically, 1 liter of LB medium was inoculated with E. coli, shake cultured at 37 ° C. for 19 hours, and then centrifuged to separate cell precipitate and culture supernatant. Then, in order to separate the OMV contained in the culture supernatant, the culture supernatant fractions were filtered through a membrane filter (0.22 uM pore-size) to remove the strains and precipitates contained in the culture supernatant, and the filtered supernatant was In order to isolate and concentrate the protein or OMV contained in the supernatant having a molecular weight of about 100 kDa or more, a QuixStand filter concentrator was used. Thereafter, the concentrated sample was precipitated using an ultrafast centrifuge, and then the OMV contained in the precipitate was suspended in a PBS buffer, followed by sucrose-gradient ultrafast centrifugation. Only fractions were separated.

Then, in order to measure the cytotoxicity of the purified purified OMV, Shigatoxin-sensitive Vero cell line was treated with DMEM-complete (10% FBS, 2 mM _L -glutamine, penicillin-streptomycin) medium at 37 ° C., 5%. Under CO ₂ conditions, the cell line (4 × 10 ⁶ cells / well) and the OMV sample (5 ug up to 4.8 ng diluted in 2 × 10 steps) were treated together in a 96-well plate to propagate and then subjected to cytotoxicity for 2 days. Cell changes were observed.

As a result, when Vero cell lines treated with OMV isolated and purified from Shigatoxin-expressing strains exhibited concentration-dependent cytotoxicity, compared to controls without OMV treatment, B-subunit-deficient isolates were purified. When treated with OMV, even when treated with the highest concentration of 5 ug did not show cytotoxicity as in the control (Fig. 3), through which, the OMV secreted from the toxin B-subunit-deficient strain showed no cytotoxicity Was confirmed.

Example 5 Observation of Outer Membrane vesicles (OMV) Using Electron Microscopy (TEM)

To observe OMV under an electron microscope, OMV samples were adsorbed onto a grid, stained with 2% uranyl acetate for several seconds, blotted with filter paper, and dried to dry CM20 TEM (Philips, The Netherlands) typical. The size and shape of the OMV were observed using an electron microscope.

As a result, it was confirmed that OMV produced and secreted in E. coli has various sizes having a diameter of about 20-200 nm and has a vesicle form surrounded by a spherical lipid bilayer membrane.

As seen above, the strain lacking exotoxin by the toxin B-subunit deficiency of the AB type bacterial toxin prepared in the present invention is naturally toxic and thus secreted from the strain. The produced outer membrane vesicles (OMV) can be used as a safe vaccine candidate, as well as vaccine antigen carriers that deliver foreign antigens through the production of recombinant variants fused / expressed with outer membrane proteins to introduce foreign antigens. Or it may be usefully used as a drug delivery vehicle (drug delivery vehicle) that can deliver the drug to a specific target cell.

Korea Biotechnology Research Institute KCTC11344BP 20080619 Korea Biotechnology Research Institute KCTC11345BP 20080619 Korea Biotechnology Research Institute KCTC11346BP 20080619 Korea Biotechnology Research Institute KCTC18220P 20110902 Korea Biotechnology Research Institute KCTC18221P 20110902 Korea Biotechnology Research Institute KCTC18222P 20110902

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Claims (18)

Shigatoxin, pyrogenic toxin or cholera characterized by a deficiency of the toxin B-subunit of Shiga toxin, heat-labile toxin or Cholera toxin. Strain lacking toxin.
delete The method according to claim 1, wherein the Shiga toxin is Shiga toxin-producing E. coli (STEC), the dimeric toxin is enterotoxin type E. coli (enterotoxigenic E. coli; ETEC) , The cholera toxin is a group consisting of enteritis Vibrio bacteria (Vibrio cholerae ), Campylobacter jejuni , Bordetella pertussis and Shigella dysenteriae producing cholera toxin Strains, characterized in that derived from any one strain selected from.
4. The strain according to claim 3, wherein the E. coli producing Shigatoxin is E. coli 0157: H7 Sakai deposited with accession number KCTC 11344BP or Sakai-DM deposited with accession number KCTC 11346BP.
The method of claim 3, wherein the enterotoxin E. coli is Escherichia coli ) A strain characterized in that the ETEC strain (strain) H10407.
According to claim 1, wherein the toxin B - subunit is Stx-1B (Shiga toxin 1B subunit) having a nucleotide sequence of SEQ ID NO: 7, Stx-2B (Shiga toxin 2B having a nucleotide sequence of SEQ ID NO: 8) subunit), LT-B (heat-labile toxin B subunit) having the nucleotide sequence set forth in SEQ ID NO: 9, and CT-B (cholera toxin B subunit) having the nucleotide sequence set forth in SEQ ID NO: 10 Strain, characterized in that the polypeptide is encoded from any one gene.
The method of claim 1, wherein the strain lacking cigar toxin, dipytoxin or cholera toxin is E. coli O157: H7 Sakai-DM / Δ stx2B mutant deposited with accession number KCTC 18220P, E deposited with accession number KCTC 18221P. .coli O157: H7 Sakai-WT / Δ stx2B mutant or E. coli O157: H7 Sakai-DM / Δ stx1B / Δ stx2B mutant deposited with accession number KCTC 18222P.
1) preparing a vector comprising a toxin B-subunit gene of cigatoxin, dimeric toxin or cholera toxin;
2) Deleting a DNA fragment of 100 to 200 bp into the toxin B-subunit gene ORF (open reading frame) in the vector prepared in step 1) and inserting an antibiotic resistance gene cassette into the deleted DNA portion Preparing homologous DNA for mutagenesis; And
3) Amplification and purification of the homologous DNA prepared in step 2), followed by introducing into the strain secreting cigar toxin, dimeric toxin or cholera toxin, lacking cigatoxin, dimeric toxin or cholera toxin Method of producing isolated strains.
delete The method according to claim 8, wherein the Shiga toxin of step 1) is Shiga toxin-producing E. coli (STEC), and the dimeric toxin is enterotoxin type E. coli (enterotoxigenic E. coli; ETEC), the cholera toxin is enteritis Vibrio cholerae , Campylobacter jejuni , Bordetella pertussis and Shigella dysenteriae producing cholera toxin. The production method characterized in that it is derived from any one strain selected from the group consisting of.
The method of claim 8, wherein the toxin of the step 1) B- subunit Stx gene having the nucleotide sequence represented by SEQ ID NO: 7 -1B (Shiga toxin subunit 1B), Stx- having the nucleotide sequence represented by SEQ ID NO: 8 2B (Shiga toxin 2B subunit), CT-B (cholera toxin B subunit) having the nucleotide sequence described by the LT-B (heat-labile toxin B subunit) and SEQ ID NO: 10 comprising the nucleotide sequence represented by SEQ ID NO: 9 in The production method, characterized in that any one selected from the group consisting of.
The method of claim 8, wherein the vector of step 1) is pUC18.
The method of claim 8, wherein the antibiotic of step 2) is chloramphenicol.
According to claim 8, wherein the strain of step 3) is Shiga toxin-producing Escherichia coli (Shiga toxin-producing E. coli ; STEC), E. coli (enterotoxigenic E. coli ; ETEC) producing febrile toxin, cholera One selected from the group consisting of toxin-producing Vibrio cholerae , Campylobacter jejuni , Bordetella pertussis , and Shigella dysenteriae The manufacturing method characterized by the above-mentioned.
1) culturing the strain of claim 1;
2) separating the cell precipitate and the culture supernatant by centrifuging the cultured strain of step 1); And
3) Toxin-deficient outer membrane vesicles (OMV) from the strain lacking cigatoxin, dipyrotoxin or cholera toxin, comprising the step of separating the outer membrane vesicle (OMV) from the culture supernatant of step 2) Production method.
The vaccine composition for infectious diseases containing the outer membrane vesicles (OMV) produced using the strain of claim 1 as an active ingredient.
17. The vaccine composition of claim 16, wherein the infectious disease is caused by a pathogenic bacterium that secretes cigatoxin, dimeric toxin or cholera toxin.
The vaccine composition according to claim 16, wherein the infectious disease is any one selected from the group consisting of hemolytic colitis, hemolytic uremic syndrome, diarrhea and enteritis.

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