CN106999562A - Immune composition for yochubio and preparation method thereof - Google Patents
Immune composition for yochubio and preparation method thereof Download PDFInfo
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- CN106999562A CN106999562A CN201580064645.4A CN201580064645A CN106999562A CN 106999562 A CN106999562 A CN 106999562A CN 201580064645 A CN201580064645 A CN 201580064645A CN 106999562 A CN106999562 A CN 106999562A
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- Prior art keywords
- orientia tsutsugamushi
- outer membrane
- yochubio
- membrane vesicles
- tsutsugamushi
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/0233—Rickettsiales, e.g. Anaplasma
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/29—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Richettsiales (O)
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- C12N1/20—Bacteria; Culture media therefor
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The present invention relates to immune composition for yochubio and preparation method thereof.The immune composition for yochubio of the present invention can effectively cause immune response due to the structure with bacterial derivation, and thus it can effectively cause immunogenicity for immune composition at present using Orientia Tsutsugamushi, the immune composition of yochubio can be prevented by thus providing.
Description
The cross reference of related application
This application claims the korean patent application 10-2014- submitted in Korean Intellectual Property Office on October 29th, 2014
0148624 and the korean patent application 10-2015-0150499 submitted on October 28th, 2015 priority, the disclosure of which
It is incorporated herein by reference.
Invention field
This disclosure relates to be directed to the immunogenic composition of yochubio (scrub typhus) and preparation method thereof, and more
It is related to for immunogenic composition of yochubio and preparation method thereof, the immunogenic composition for yochubio body
Contain the material from Orientia Tsutsugamushi (Orientia tsutsugamushi) and can be than conventional Orientia Tsutsugamushi
It is safer and effectively induce immune response, so that the composition is suitable for Clinical practice.
Background of invention
Yochubio causes death in the annual appearance in about 6,000 patients of South Korea, unless entered with antibiotic
The appropriate treatment of row.Therefore, social economical burden is huge.Further, since climate change for its potential diffusion, it is necessary to carry out accurate
It is standby.Therefore, in the urgent need to providing the vaccine for yochubio.
Yochubio occurs in the whole world, particularly in Malaysia, Thailand, Philippine, China, Japan and Australia.
In South Korea, it is found that many patients for being referred to as strange sick (strange malady) in rural area have yochubio.
Yochubio mainly occurs in the fall, and it mainly with the mite class trombiculid (mite chigger) as medium
Density is related.The October frequently occurred from the larva of leptotrombidium pallidium (Leptotrombidium pallidum) is whole to November
Individual South Korea often causes yochubio.
In addition, Orientia Tsutsugamushi is gramnegative bacterium, and known parasitism survive in it is (obligate in its host cell
Cytozoon) in.In addition, Orientia Tsutsugamushi is the genic organisms of yochubio, and cause acute and chronic infection,
Its symptom includes such as pneumonia, encephalitis, fash, fever and headache.In addition, the symptom and signal of yochubio and other fever diseases
Such as leptospirosis, mouse typhus are similar with malaria, therefore are very difficult to them and yochubio differentiation.Yochubio it is flat
The equal death rate is 6%, and it is a kind of serious disease, and the death rate is 60% in the case of without antimicrobial therapy.Have
The antimicrobial therapy reduction death rate of effect, but people may due to treatment delay starts and dead or the elderly may be by
It is dead in severe complications such as shock, respiratory failure or encephalitis.After they treat, general malaise or myalgia are sustainable
Several months.Therefore, in the urgent need to exploitation can effectively prevent the immunogenic composition of yochubio.
In the routine techniques on foregoing description, Korean Patent Publication 2002- disclosed in 15 days March in 2002
0020281 discloses a kind of method of use recombinant protein antigen diagnosis of tsutsugamushi disease, and the recombinant protein antigen is used by cultivating
The bacterial strain of carrier conversion comprising DNA is obtained, and the DNA is determined by two or more antigens for being derived from Orientia Tsutsugamushi
The gene connection of the protein of cluster.
The content of the invention
The disclosure contemplates the problem of solving described above, and first purpose to be realized by the disclosure is to provide pin
There is immunogenic composition to yochubio and preparation method thereof, the immunogenic composition for yochubio bacterium to spread out
Raw structure and it can effectively induce immune response.
Second purpose that the disclosure is realized is to provide the pharmaceutical composition for preventing and treating yochubio.
The 3rd purpose that the disclosure is realized is to provide the use for preparing the pharmaceutical composition for being used to prevent and treat yochubio
On the way.
The 4th purpose that the disclosure is realized is to provide the method for preventing and treating yochubio, and it passes through to there is this to need
The individual wanted gives the pharmaceutical composition for being used to prevent and treat yochubio of effective dose.
Present disclose provides the immunogenic composition for yochubio, it includes the outer membrane vesicle from Orientia Tsutsugamushi
Bubble.
According to the disclosure embodiment, the average diameter of outer membrane vesicles can be 20nm to 200nm.
According to another embodiment of the disclosure, immunogenic composition can include the 20kDa of Orientia Tsutsugamushi
To 110kDa albumen.
According to another embodiment of the disclosure, immunogenic composition can further include Orientia Tsutsugamushi
56kDa albumen (type specific antigens 56).
According to another embodiment of the disclosure, outer membrane vesicles albumen and 56kDa from Orientia Tsutsugamushi are included
The immunogenic composition of albumen can have cooperative effect to immunogenicity.
In addition, present disclose provides the method for preparing the immunogenic composition for yochubio, it includes：Use illness
Parasitosis Orientia infection cell, cell of the culture through infection and the step of obtain the cell through infection of culture；From the training
The step of foster cell through infection obtains the culture supernatants without the cell through infection；And filter and separate
The step of outer membrane vesicles of the culture supernatants to obtain the purifying of Orientia Tsutsugamushi.
Can be with according to another embodiment of the disclosure, the step of the outer membrane vesicles for the purifying for obtaining Orientia Tsutsugamushi
Including：With described in the membrane filtration in the hole of the average diameter with 0.1 μm to 0.3 μm the step of culture supernatants；Addition is folded
Sodium nitride and carry out the step of ultrafiltration is to obtain concentrate；And carry out the filtering, concentration and resuspension of the concentrate to obtain
The step of obtaining the outer membrane vesicles of Orientia Tsutsugamushi.
According to another embodiment of the disclosure, can by the filtering of the concentrate, concentration and be resuspended the step of enter
Row 2 to 6 times.
According to another embodiment of the disclosure, the outer membrane vesicles of the purifying of Orientia Tsutsugamushi can include illness
The 20kDa of parasitosis Orientia is to 110kDa albumen.
In addition, present disclose provides the pharmaceutical composition for preventing yochubio, it is included from Orientia Tsutsugamushi
Outer membrane vesicles.
In addition, present disclose provides the pharmaceutical composition for preventing yochubio, it is included from Orientia Tsutsugamushi
Outer membrane vesicles and 56kDa albumen (type specific antigens 56).
In addition, present disclose provides the purposes for preparing the prevention and treatment agent for yochubio, it is described to be directed to tsutsugamushi mite
The prevention and treatment agent of disease includes the outer membrane vesicles from Orientia Tsutsugamushi.
Present disclose provides the method for preventing and treating yochubio, it includes giving effectively individuals in need
The outer membrane vesicles from Orientia Tsutsugamushi of amount.
There is the immunogenic composition for yochubio of the disclosure structure of bacterial derivation to be answered so that effective induction is immune
Answer, and therefore than the more effective inducing immunogenic of immunogenic composition using conventional Orientia Tsutsugamushi.Therefore, this public affairs
Open to provide and can prevent the immunogenic composition of yochubio.
Brief description
From carried out with reference to accompanying drawing it is described in detail below can be more clearly understood the disclosure it is above and other in terms of, feature and
Further advantage, wherein：
Fig. 1 shows the cell mass infected with Orientia Tsutsugamushi with electron microscope observation embodiment 1 in embodiment 4
The result of grain；
Fig. 2 is the TEM image of the outer membrane vesicles for the Orientia Tsutsugamushi for obtaining and purifying in embodiment 3；
Fig. 3 is shown using FS15 monoclonal antibodies to being obtained and pure in Orientia Tsutsugamushi lysis thing and embodiment 3
The result of the western engram analysis of the outer membrane vesicles of the Orientia Tsutsugamushi of change；
Fig. 4 show using the polyclonal antibody separated from the serum of tsutsugamushi mite patient to Orientia Tsutsugamushi lysis thing with
And the result of the western engram analysis of the outer membrane vesicles for the Orientia Tsutsugamushi for obtaining and purifying in embodiment 3；
Fig. 5 is shown using FS15 monoclonal antibodies to the outer membrane from Orientia Tsutsugamushi that is purified by immunoprecipitation
The result of the western engram analysis (using the polyclonal antibody separated from the serum of tsutsugamushi mite patient) of vesica；
Fig. 6 is the image of the outer membrane vesicles of the Orientia Tsutsugamushi for the serial dilution observed by immunofluorescence；And
Fig. 7 shows the experimental group (G1 handled with 56kDa as antigen：OMV administration groups, G2：56kDa administration groups, G3：
OMV+56kDa administration groups, and G4：PBS+Alum administration groups) every kind of serum elisa assay result.
Embodiment
Hereinafter, it will be described in the disclosure.
As discussed above, yochubio causes very huge social economical burden, and due to climate change, it is necessary to be directed to
Its potential propagation is prepared.Therefore, in the urgent need to providing the vaccine for yochubio.
Thus, present disclose provides the immunogenic composition for yochubio, it is included from Orientia Tsutsugamushi
Outer membrane vesicles.
The Orientia Tsutsugamushi of the disclosure can be the bacterium as described in scientific name Orientia Tsutsugamushi, and can be with
Boryong serotypes Orientia Tsutsugamushi (NCCP No.14794) preferably as described in example 1 above.
Outer membrane vesicles can have 20nm to 200nm average diameter, and immunogenic composition can include tsutsugamushi mite
The 20kDa of sick Orientia to 110kDa protein.
In the exemplary of the disclosure, the outer membrane vesicles lysis thing of Orientia Tsutsugamushi is subjected to Western
Blotting.It was found that protein has 42kDa, 47kDa, 56kDa, 70kDa, 80kDa and 110kDa size.In the another of the disclosure
In one exemplary, it has already been proven that when the protein with 56kDa sizes is mixed with outer membrane vesicles albumen, its
Play significantly higher immunogenicity effect.Thus, present disclose provides the immunogenic composition for yochubio, except from
Outside the outer membrane vesicles of Orientia Tsutsugamushi, the immunogenic composition also includes 56kDa outer membrane proteins.
In addition, present disclose provides the method for preparing the immunogenic composition for yochubio, it includes：Use yochubio
Orientia infection cell, cell of the culture through infection and the step of obtain the cell through infection of culture；From the culture
The step of cell through infection obtains the culture supernatants without the cell through infection；And filter and separate described
The step of outer membrane vesicles of the culture supernatants to obtain the purifying of Orientia Tsutsugamushi, thus solve asking as described above
Topic.Therefore, the disclosure have provide can be safely used for Clinical practice for yochubio immunogenic composition and its
The effect of preparation method, the immunogenic composition, which contains, has been free of endotoxin effect from the material of Orientia Tsutsugamushi
Lipopolysaccharides (LPS) and/or peptide glycan.
First, describe with Orientia Tsutsugamushi infection cell, culture the cell through infection and obtain culture through infection
Cell.
Cell is not particularly limited, as long as being commonly used for culture and purification of bacterial.It is, for example, possible to use such as intravascular
Chrotoplast (ECV304), fibroblast (L929), VERO cells, L-929 cells, hel cell, MRC5 cells, HeLa cells,
The cell such as bhk cell and McCoy cells.
Infection is not particularly limited, as long as it is the common method using pathogenic bacteria infection cell.Infection can be with excellent
Cell mixing and Orientia Tsutsugamushi were gated, and cultivates 100 hours to 200 hours to carry out at 35 DEG C to 40 DEG C.Infection can
With more preferably by with 1:The ratio of 3 to 10 cell compartments mixes Orientia Tsutsugamushi and bacterium, and in 36 DEG C to 38 DEG C trainings
150 hours to 180 hours are supported to carry out.
When being cultivated in the temperature less than 35 DEG C, the growth rate of Orientia Tsutsugamushi can be reduced, and cause to have
The problem of effect ground is cultivated.When being cultivated in the temperature more than 40 DEG C, there may be host cell and Orientia tsutsugamushi
The problem of body grows.
In addition, when culture be less than 100 hours, due to the limitation of mycelial growth, the generations of outer membrane vesicles can be with
Reduction, and when culture is performed for more than 200 hours, it can cause the destabilization of the outer membrane vesicles of gained.
Next, obtaining the culture supernatants without the cell through infection from the cell through infection of culture.
The method for obtaining the culture supernatants without the cell through infection from the cell through infection of culture is not special
Limitation, as long as it is the common method that supernatant and culture cell are separated from culture medium, but it can be preferably with 10,000rpm
Centrifuged 10 minutes to 60 minutes to 20,000rpm, and it is further preferred that it can centrifuge 15 points with 11,000rpm to 18,000rpm
Clock was to 40 minutes.
When it less than 10,000rpm to centrifuge, the partial destruction of the cell through infection can be mixed into culture supernatant
In liquid.When it is centrifuged with the rotating speed more than 20,000rpm, it may be difficult to obtain of the outer membrane vesicles of Orientia Tsutsugamushi
Grain.
In addition, when centrifugation be less than 10 minutes, the cell through infection or the particle from the cell through infection can be with
Mix to cause the difficulty that the outer membrane vesicles of Orientia Tsutsugamushi are purified with the outer membrane vesicles of Orientia Tsutsugamushi.When centrifugation exceedes
At 60 minutes, it is understood that there may be the problem of outer membrane vesicles of Orientia Tsutsugamushi lose.
Next, the culture supernatants without the cell through infection obtained as described above are filtered and separated, to obtain
Obtain the outer membrane vesicles of the purifying of Orientia Tsutsugamushi.
Filtering is not particularly limited, as long as the method that it is typically used for broth filtrate, but it is preferred that can be with apparatus
There is a filter membrane in the hole of 0.1 μm to 0.3 μm of average diameter, more preferably the filter membrane in the hole with 0.15 μm -0.28 μm of average diameter
Filtered.
When the average diameter in the hole of filter membrane is less than 0.1 μm, of the outer membrane vesicles of loss Orientia Tsutsugamushi may occur in which
Grain the problem of, and when filter membrane hole average diameter more than 0.3 μm when, the material of the cell derived through infection may get dirty
Dye.
Separation is not particularly limited, as long as it is the common method for obtaining sediment in the mixture, but preferably, it with
100,000g to 250,000g, more preferably 120,000g to 220,000g are centrifuged.
It can include according to the embodiment of the disclosure, the step of the outer membrane vesicles for the purifying for obtaining Orientia Tsutsugamushi：
The step of with the membrane filtration culture supernatants in the hole with 0.1 μm to 0.3 μm of average diameter；Addition sodium azide is simultaneously carried out
The step of ultrafiltration is to obtain concentrate；And carry out the filtering, centrifugation and resuspension of concentrate to obtain the outer of Orientia Tsutsugamushi
The step of membrane vesicle.
The addition of sodium azide is not particularly limited, as long as it is can be added to the usual amounts of culture medium, it is preferred that
Ground it can with the cumulative volume relative to solution by volume 0.005% to 0.05% amount add.It can be with relative to molten
The cumulative volume of liquid by volume 0.01% to 0.03% amount addition.
When the cumulative volume relative to solution is by volume less than 0.005% addition sodium azide, it can be by except tsutsugamushi mite
The external microorganism pollution in sick east.When adding sodium azide with amount by volume more than 0.05%, due to mistake in purifying
Many crosses sodium nitride, might have various problems, the purifying is next process.
Ultrafiltration is not particularly limited, as long as it can be generally used for separation of bacterial or cell.Filtering, which is preferably used, to be had
The filter membrane in average diameter 50kDa to 500kDa hole is carried out.More preferably using the hole with average diameter 70kDa to 200kDa
Hollow-fibre membrane is filtered.
When the filter membrane using the hole for being less than 50kDa with average diameter, extend concentration time to cause Orientia tsutsugamushi
The deformation of the outer membrane vesicles of body.When the filter membrane using the hole for being more than 500kDa with average diameter, it can cause yochubio
The loss of the outer membrane vesicles particle of Orientia.
Do not limited especially by the filtering of concentrate, centrifugation and the method that the outer membrane vesicles for obtaining Orientia Tsutsugamushi are resuspended
System, as long as the method that it is typically used for separation and/or purification of bacterial from culture medium and/or nutrient solution.
According to another embodiment of the disclosure, by using the filter membrane in the hole with 0.05 μm to 0.4 μm of average diameter
Filtering and concentrating thing, is centrifuged with 100,000g to 250,000g, then concentrate is resuspended to carry out with phosphate buffered saline (PBS) (PBS)
Filtering, centrifugation and resuspension.
According to another embodiment of the invention, filtering, centrifugation and can carry out the step of concentrate is resuspended once or
Repeatedly, preferably twice is to six times.
In addition, the step of obtaining the outer membrane vesicles of the Orientia Tsutsugamushi of the disclosure can use immunoprecipitation method to obtain
Obtain the highly purified outer membrane vesicles of Orientia Tsutsugamushi.
According to another embodiment of the disclosure, come from using FS15 mouse monoclonal antibodies and Protein G magnetic bead
The high-purity Orientia Tsutsugamushi outer membrane vesicles of other compositions.
The outer membrane vesicles of the Orientia Tsutsugamushi obtained by method as discussed above can have 20nm's to 200nm flat
Equal diameter, and can the 20kDa containing Orientia Tsutsugamushi to 110kDa protein, and preferably comprise 56kDa albumen.
In addition, the outer membrane vesicles of Orientia Tsutsugamushi contain 56kDa protein, it is the main anti-of Orientia Tsutsugamushi
Original, and known be primarily present on surface and do not contain lipopolysaccharides (LPS) and/or peptide glycan, it is therefore contemplated that clinical real
It is used as very safe immunogenic composition in trampling.
The disclosure provides the pharmaceutical composition for preventing yochubio, and it includes the outer membrane vesicle from Orientia Tsutsugamushi
Bubble.
In addition, the disclosure provides the pharmaceutical composition for preventing yochubio, it includes outer from Orientia Tsutsugamushi
Membrane vesicle and 56kDa albumen (type specific antigens 56).
The Orientia Tsutsugamushi of the disclosure can be the bacterium as described in scientific name Orientia Tsutsugamushi, and can be with excellent
Elect Boryong serotypes Orientia Tsutsugamushi (NCCP No.14794) as described in example 1 above as.
The 56kDa albumen of the disclosure refers to be present in the 56kDa albumen in the outer membrane of Orientia Tsutsugamushi, preferably by SEQ
ID NO：The protein of 1 base sequence coding.
The pharmaceutical composition of the disclosure can also include pharmaceutically acceptable carrier and auxiliary material.Term " auxiliary material " is effect
For the material of the immunogenicity of the immunogenic composition of the enhancing disclosure.Immunogenicity auxiliary material can strengthen when individually giving
To the immune response of weak immunogene antigen, for example, in the absence of exempting from that antibody titer or weak antibody titer or inducing cell are mediated
Epidemic disease response, increases the antibody titer to antigen, and reduce the antigen dose for the immune response effectively realized in individual.Therefore,
Auxiliary material generally works in increase immune response, and this is known in those skilled in the art.Increase composition validity
Suitable excipients include the following, but are not limited to：
(1) aluminium salt (Alum), such as aluminium hydroxide, aluminum phosphate, aluminum sulfate；(2) oil in water emulsion preparation is (for example, contain
Have or without other specific immunostimulant, such as muramyl peptide (defined below) or bacterial cell wall components), such as (a)
MF59, it includes 5% squalene, 0.5%Tween 80 and 0.5%Span 85 (MTP-PE optionally containing various amounts), and
With microfluidization device such as 110Y types microfluidization device (Microfluidics:Newton, Massachusetts, USA) it is configured to Asia
Micron particles (referring to International Patent Application Publication WO90/14837), (b) SAF, it includes 10% squalene, 0.4%Tween
80th, 5%pluronic block polymers L121 and thr-MDP, and miniflow turn to less than micron emulsion or by be vortexed shake
Swing and be formulated as the emulsion with larger granularity, (c) 2% squalene, 0.2%Tween 80 and (such as United States Patent (USP) 4, in 912,094
Disclosed, the bacteria cell wall that one or more are selected from the group：3-O- deacylation monophosphoryl lipid As (MPLTM), trehalose two
Mycolate (TDM) and cell wall skeleton (CWS), preferably containing MPL+CWS (DetoxTM) RibiTMAdjuvant systems (RAS)
(Corixa, Hamilton, Montana, USA)；Montanide ISA (d)；(3) saponin(e auxiliary material, such as Quil A or
STIMULON TMQS-21(Antigenics:Framingham, Massachusetts, USA) (see, for example, United States Patent (USP) 5,
057,540) (above-mentioned substance can be used), or from the particle of its generation, such as ISCOM is ((by cholesterol, saponin(e, phosphatide and amphiphilic
The immunostimulating compound that the combination of albumen is formed) and Iscomatrix (essentially identical with the ISCOM without protein)；
(4) bacteria lipopolysaccharide, synthesis lipid A analogue are (for example, aminoalkyl aminoglucose phosphate compound (AGP) or derivatives thereof
Or the like (Corixa is purchased from, and in United States Patent (USP) 6, disclosed in 113,918) (it is used as a kind of AGP, 2- as described above
[(R) -3- tetradecanoyloxytetradecanoyl bases amino] ethyl 2- deoxidation -4-O- phosphonos -3-O- [(R) -3- myristoyl base epoxides
Myristoyl base] [(R) -3- tetradecanoyloxytetradecanoyl base amino-b-D- glucopyranosides, also referred to as 529 (claimed -2- in the past
For RC529), it is configured to aqueous form or stable emulsion)；(5) synthetic polyribonucleotides, such as few nucleosides containing the motif containing CpG
Sour (United States Patent (USP) 6,207,646)；(6) cell factor, such as interleukins (such as IL-1, IL-2, IL-4, IL-5, IL-6,
IL-7, IL-12, IL-15, IL-18 etc.), interferon (such as interferon), granulocyte macrophage colony stimulating factor (GM-
CSF), macrophage colony stimulatory factor (M-CSF), TNF (TNF), costimulation molecules B7-1 and B7-2；(7)
Complement, for example, complement component C3 d tripolymer.
Vaccine combination can be mixed with pharmaceutically acceptable excipient.The excipient can include water, salt solution, Portugal
Grape sugar, glycerine, ethanol, its mixture.In addition, vaccine combination can include other auxiliary substance, such as wetting agent, emulsification
Agent, pH buffer etc..The vaccine combination can be with the shape of solution, powder, aerosol, capsule, enteric coatel tablets, capsule or suppository
Formula is given.
The approach of giving includes but is not limited to intraperitoneal, intravenous, intramuscular, subcutaneous, oral, part, transmucosal, intranasal, lung
Interior, rectum etc..When oral give, because peptide is digested, oral cavity composition should be coated with active medicine or prepare to protect it
From the degraded in stomach.Furthermore, it is possible to by the way that medicine group can be given by any device of active delivery to target cell
Compound.
Dosage can with formulation, give approach, age, health, weight, seriousness, Current treatment protocols, treatment number of times etc.
And change, and can change with the immunogenicity expressed when giving immunogenic composition, and can be by this area
Technical staff is readily determined.For example, when directly giving in musculature, immunologically or prophylactically effective dose is about 1 μ g to about
5mg, preferably from about 10 μ g to about 2mg.
The vaccine combination of the disclosure can individually be given or be combined with other therapeutic agents and be given.When combination is given, its
Can simultaneously or successively it be given with conventional therapy agent.
The composition of the disclosure can pass through one or more methods known to persons of ordinary skill in the art, such as stomach
Outside, transmucosal, intramuscular, intravenous, intracutaneous, intranasal, subcutaneous and intraperitoneal are given in individual, can correspondingly prepare.
The composition of the disclosure can be configured to single-dose vials, multiple dose vials or prefilled injection liquid.
Hereinafter, reference implementation example and comparative example are more fully described to the structure and effect of the disclosure.However, these
Embodiment is only for being explained in greater detail the example of the disclosure, and the scope of the present disclosure is not limited to these embodiments.
[embodiment 1]
The cell culture and infection of Orientia Tsutsugamushi
By Orientia Tsutsugamushi Boryong serotypes (CDC of South Korea, NCCP No.14794) inoculation
Cultivate 7 days to lure into ECV-304 cells (Blood vessel endothelial cell line, Cell Line Service, Germany), and at 37 DEG C
Lead bacterial multiplication.With containing 10% (v/v) hyclone (by Corning Cellgro, USA manufacture) M199 culture mediums (by
Welgene Inc., Korea manufacture) the ECV-304 cells that infect of culture Orientia Tsutsugamushi.
The culture of the ECV-304 cells through infection is measured by indirect immunofluoresent assay (IFA).When confirmation infection
When rate is more than 90%, then the ECV304 cells in five 150mm tissue culture dishes of subinfection.Again in 30 150mm tissue trainings
Support infection in ware (being manufactured by SPL, Korea) and confirm as the cell infected through the above way, for largely producing yochubio east
The outer membrane vesicles of cube.
The cell through infection of culture is suspended in culture medium, and 30 minutes are centrifuged with 13,000rpm with separation at 4 DEG C
Culture supernatants without the cell through infection and the cell granule infected with Orientia Tsutsugamushi.
[embodiment 2]
The purifying of Orientia Tsutsugamushi
The EVC-304 cells infected with Orientia Tsutsugamushi of embodiment 1 are resuspended in containing in 250mM TS buffer solutions
In the 33mM tri hydrochlorides of middle expression and the solution of pH 7.4 of sucrose, and cell is ground 1 minute to lure using 1mm beades
Orientia Tsutsugamushi in guided cell comes out from cell.The Orientia Tsutsugamushi of acquisition is centrifuged 3 points at 4 DEG C with 1200rpm
Clock obtains the suspension of Orientia Tsutsugamushi to remove cell fragment.The Orientia tsutsugamushi liquid suspension of acquisition is added
To 90%percoll, until obtaining concentration 40%percoll (being manufactured by GE Life Science, USA), and with 14,
000rpm centrifuges 60 minutes layers to separate cell-derived debris layer and be only made up of bacterium.Collect the Orientia tsutsugamushi obtained
Body layer, washed twice, be resuspended in sterile phosphate buffered saline with phosphate buffered saline (PBS) (PBS), then with liquid nitrogen frozen with
Obtain the Orientia Tsutsugamushi of purifying.
[embodiment 3]
The purifying of the outer membrane vesicles of Orientia Tsutsugamushi
Filtering (is manufactured) by Corning corporation, USA using the filtration system with 0.22 μm of hole average diameter
The culture supernatants without the cell through infection that 900ml is obtained in embodiment 1.Add to it by volume 0.02%
Sodium azide, and using QuixStandBenchtop systems (by GE Healthcare care life sciences, USA
Manufacture) and 100kDa hollow-fibre membranes mixture is concentrated into 50ml.Then, concentrate is filtered through 0.22 μm of syringe filter
Device (is manufactured) by Pall Life Science, USA, and filtrate is centrifuged into 3 hours to obtain tsutsugamushi mite with 150,000g at 4 DEG C
Outer membrane vesicles granule derived from sick Orientia.Then, the Orientia Tsutsugamushi outer membrane vesicles granule of acquisition is resuspended in 1ml phosphoric acid
In salt buffer salt solution (PBS), so that it is more purified, and 0.22 μm of syringe filter is filtered through (by Pall Life
Science, USA are produced).
Sucrose solution is gradually accumulated in the super filter tube with cumulative volume 13.2ml with 2.5M sucrose comprising 3ml, 3ml
The 0.6M sucrose of 1.6M sucrose and 3ml.Then, filtrate 1ml being resuspended in phosphate buffered saline (PBS) is carefully placed in sucrose
In gradient.Then, pipe is centrifuged 20 hours with 200,000g at 4 DEG C using Beckman SW 41Ti rotors, and layer from it
Isolate each 1ml.Dodecyl is carried out to the solution of separation with 120V with 10% SDS-polyacrylamide acrylamide gel
Sodium sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), continues 20 minutes 1 hour.The a piece of gel that will be obtained by electrophoresis
It is immersed in Coomassie blue stain solution (INVITROGEN) 30 minutes, is then immersed in distilled water 30 minutes to decolourize, so
Protein band pattern is confirmed afterwards.For another, by protein band with 110V voltage transfer to polyvinylidene fluoride
(PVDF) film is up to 10 minutes 1 hour, then in room temperature by itself and the complementary FS15 antibody responses 1 hour for tsutsugamushi antigen, institute
It is primary antibody (anti-TSA56, the type specificity membranous antigen of Orientia Tsutsugamushi, Inha University (Inha to state FS15 antibody
University) medical college's department of microbiology).Then, buffered with the phosphate buffered saline (PBS) (PBST) with 0.1%Tween 20
Liquid is washed three times, and small with FS15 reactions 1 at 25 DEG C with secondary antibody (anti-mouse IgG, JACKSON Lab. conjugated FITC)
When.Then, washed 3 times with PBST buffer solutions, by enhanced chemiluminescence (ECL) solution (by GE Life Science, USA
Manufacture) and (its be can with secondary antibody HRP (horseradish peroxidase) react substrate solution) be sprayed to film, then by x-ray
(KODAK) film is exposed to light 1 minute to observe strip pattern.
Then western engram analysis are carried out.Thus, the separation solution for showing the distribution of similar protein matter is mixed and 4
DEG C 3 hours are centrifuged to obtain tsutsugamushi mite outer membrane vesicles (OMV) particle with 150,000g.The tsutsugamushi mite OMV granules of acquisition are resuspended in and contained
In the phosphate buffered saline (PBS) for having 1X protease inhibitor cocktails (being manufactured by Sigma-Aldrich, USA).Use DC albumen
Matter determines the tsutsugamushi mite OMV granules that reagent (being manufactured by Bio-Rad, USA) quantifies to be resuspended, and in -70 DEG C of storages.
[embodiment 4]
Observe Orientia Tsutsugamushi bubble and Orientia Tsutsugamushi OMV
4-1：Observe Orientia Tsutsugamushi bubble
In order to observe Orientia Tsutsugamushi bubble, by the cell granule infected with Orientia Tsutsugamushi of embodiment 1 with containing
2.5% glutaraldehyde for having 0.1M phosphate (pH 7.4) fixes 2 hours.The EVC-304 that washing Orientia Tsutsugamushi infects is thin
Born of the same parents' granule, is subsequently placed in 1% osmium tetroxide 30 minutes, then with ethanol dehydration, is subsequently placed in epoxy resin (commodity
Name：Epon 812, by Electron Microscope Sciences manufacture) Beam capsules in.Use ultramicrotome
(UltraE is manufactured by Reichert-Jung, USA) cutting ultra thin is cut into slices, and is contaminated with uranyl acetate and citrate
Color.Then observe cell to confirm with electron microscope (CM200 transmission electron microscopes (Phillips, Netherlands))
With the infection of Orientia Tsutsugamushi.Observation result is shown in Fig. 1.
As shown in fig. 1, the cell granule infected with electronics observation by microscope Orientia Tsutsugamushi.It is thus identified that
Outer membrane vesicles are secreted from the cell surface infected with Orientia Tsutsugamushi.In addition, in the cell week infected with Orientia Tsutsugamushi
Surround and watch to observe and think the monofilm vesica from Orientia Tsutsugamushi.
In addition, the size of outer membrane vesicles is about 130nm, shown in such as Fig. 1.
4-2：Orientia Tsutsugamushi OMV observation
For the OMV of the Orientia Tsutsugamushi of observing the purifying obtained in embodiment 3, the illness of the purifying of embodiment 3 is sampled
The part of parasitosis Orientia is placed in 200 mesh charcoal coated copper grid (CF200-Cu carbon films, Electron as sample
Microscopy Sciences) in 5 minutes.Copper grid is coloured with 1% uranyl acetate.Hereafter, it is dried in atmosphere,
And with CM200 transmission electron microscopes (TEM is manufactured by Phillips, Netherlands) observation.Fig. 2 shows observation knot
The TEM photos of fruit.
As shown in Fig. 2, it is able to confirm that OMV derived from the Orientia Tsutsugamushi of purifying has spherical form, it is relatively various
Size is 50nm to 150nm, and OMV constituted with monofilm.
[embodiment 5]
The confirmation of the outer membrane vesicles albumen of Orientia Tsutsugamushi
The Orientia Tsutsugamushi of the purifying obtained in embodiment 2 in the phosphate buffer aqueous solution of ice bath, and
10 30kHz ultrasonic waves are subjected to up to 10 by Vltrasonic device (types of Sonic Dismemrator 300 are manufactured by Fisher, USA)
Second.Hereafter, centrifuged 5 minutes with 12,000rpm, kit (DC protein determinations are determined using dihomocinchonine acid protein
Reagent, Bio-Rad, USA) measurement Orientia Tsutsugamushi lysis thing protein concentration, then by its -70 DEG C storage.
Carry out western blot method to confirm in embodiment 3 using the FS15 antibody with the complementation of Orientia Tsutsugamushi antigen
The protein size of the outer membrane vesicles (OMV) of the Orientia Tsutsugamushi of acquisition, and whether it be derived from Orientia Tsutsugamushi
Real outer membrane vesicles albumen.The result is shown in Fig. 3.
In addition, as outer membrane vesicles (OMV) using the Orientia Tsutsugamushi of the disclosure, using in yochubio patients serum
The polyclonal antibody (the complementary antibody with the antigen of Orientia Tsutsugamushi) contained carries out western blot method, and shows in Fig. 4
The result is shown.
From the serum purified polyclonal antibodies of tsutsugamushi mite patient to carry out Western blot analysis.The illness cultivated from patient
The genotype of parasitosis Orientia is accredited as Boryong genotype by Genotech (Daejeon, Korea company).
As shown in Fig. 3, it is thus identified that contain outer membrane vesicles egg in the Orientia Tsutsugamushi lysis thing separated from above-mentioned substance
In vain, and in embodiment 3 protein purified is derived from the outer membrane vesicles (OMV) of Orientia Tsutsugamushi.In addition, further acknowledging
42kDa, 47kDa, 56kDa, 70kDa, 80kDa and 110kDa protein band.
As shown in Fig. 4, western blot method is carried out using the polyclonal antibody contained in yochubio patients serum.Cause
This, it is thus identified that Orientia Tsutsugamushi lysis thing contains the Orientia Tsutsugamushi antigen protein of all size, and divides in embodiment 3
From the outer membrane vesicles from Orientia Tsutsugamushi be made up of protein such as predominantly 56kDa and other antigens.
In other words, shown as described above in result, the outer membrane vesicles of Orientia Tsutsugamushi seem to combine to be contained in patients serum
The polyclonal antibody for Orientia Tsutsugamushi.Therefore, may induction when the yochubio vaccine combination using the disclosure
The antibody of 56kDa and other oroteins for Orientia Tsutsugamushi, so as to improve for the immune of Orientia Tsutsugamushi.
In other words, mainly the Orientia Tsutsugamushi of the outer membrane protein of 56kDa sizes is contained in the cell exocrine through infection
Outer membrane vesicles, this represent they be suitable for prepare yochubio immune composition.
[embodiment 6]
Immunoprecipitation analysis and the analysis of western blot method
In order to which the protein contained in the outer membrane vesicles (OMV) to the Orientia Tsutsugamushi separated in embodiment 3 is exempted from
Epidemic disease is precipitated, and FS15 mouse monoclonal antibodies and 10 μ l Protein Gs magnetic beads (being manufactured by BioLabs, UK) are mixed, and 25 DEG C
With the lower culture of 60rpm stirrings 1 hour.Hereafter, with IP buffer solutions (include 25mM tri- (hydroxymethyl) aminomethane, pH 7.5,
150mM NaCl, 2.5mM ethylenediamine tetra-acetic acids (EDTA), 0.05%Triton X-100, and 0.01%NaN3) wash mixture
Wash three times, therefore obtain the G magnetic beads for being combined with FS15 antibody.
Each obtained above is combined with to the G magnetic beads of FS15 antibody and the outer membrane vesicles and illness of Orientia Tsutsugamushi respectively
Parasitosis Orientia lysis thing is mixed, and mixes 24 hours (or staying overnight) under agitation to obtain the solution of mixing at 4 DEG C.Use IP
Buffer solution washs the mixed solution of gained four times, and is washed with the phosphate-buffered aqueous solution.
(50mM Tris-Cl pH 6.8,100mM dithiothreitol (DTT)s (DTT), 2%0 are included in reproducibility sample buffer
Sodium dialkyl sulfate (SDS), 0.1% bromophenol blue, 10% glycerine) at 100 DEG C granule is heated 10 minutes to dissolve in solution, so
10 minutes are centrifuged to obtain soluble protein with 14,000rpm at 4 DEG C afterwards.
Some soluble proteins obtained in -20 DEG C of storages, and some carry out 10% lauryl sodium sulfate-poly- third
Acrylamide gel electrophoresis (SDS-PAGE).In addition, the polyclonal antibody contained in serum using tsutsugamushi mite patient is carried out
Western blot method.It is used as comparative sample implementation experiment by loading the sample of the immunoprecipitation without experience embodiment 6.
Western blot method is carried out according to scheme same as Example 3.Horseradish peroxidase is used at 25 DEG C
(HRP) conjugated secondary antibody (being manufactured by Jackson Immunoresearch Laboratories, USA) is small by secondary antibody culture 1
When.Hereafter, the film of culture is washed three times with PBST solution, and with enhanced chemiluminescence (ECL) solution (by GE Life
Science, USA are manufactured) expansion, and result is shown in Fig. 5.
As shown in Figure 5, immunoprecipitation is carried out with FS15 antibody, and is carried out using antibody derived from patients serum
Western blot method, and thereby confirmed that the size with 42kDa, 47kDa, 56kDa, 70kDa, 80kDa and 110kDa
Protein is main to express in Orientia Tsutsugamushi lysis thing, and these are very similar to 22,47,56,70 and 110kDa, its
It is known as the major outer membrane protein of Orientia Tsutsugamushi.For the outer membrane vesicles from Orientia Tsutsugamushi, it is thus identified that 56kDa eggs
Informal voucher band (known it be major antigen and be primarily present on its surface) is also in the outer membrane vesicles from Orientia Tsutsugamushi
Upper dense list reaches.
56kDa albumen plays an important role in the pathogenesis of Orientia Tsutsugamushi, and is primarily present in yochubio east
In the cell membrane of cube.In other words, as one of most important antigen of Orientia Tsutsugamushi, it plays attachment and penetration cell
Effect.Therefore, the antibody for 56kDa albumen act as neutrality antibody, and is played an important role in defence is immune.
[embodiment 7]
The separation and purifying of 56kDa recombinant proteins
7-1：The amplification of 56 kDa (type specific antigens 56) gene
In order to expand gene region (SEQ NO ID corresponding with the 88th to the 479th amino acid of 56kDa albumen:1),
Tsutsugamushi mite full genome, which is used, as the gene amplification (PCR) of template by using following primer pair expands the gene regions to be obtained
Domain.
[table 1]
| Primer | Nucleotide sequence | SEQ ID NO. |
| The forward primers of TSA 56 | 5`-GTGAATTCGTCGACAGAGCAGAGCATAGGT-3` | 2 |
| The reverse primers of TSA 56 | 5`-GTAAGCTTCTCGAGTCAATACCCTTTAACATCC-3` | 3 |
7-2：56kDa clone and overexpression
With EcoR I and Xho I restriction enzymes (TAKARA, Japan) processing pRSET-A carriers (Invitrogen, USA) with
By in the gene insertion vector of amplification, for being overexpressed the 56kDa albumen combined with 6 histidine residues, and connection is used
Enzyme (NEB biolab, UK) linker because and carrier.In order to obtain recombinant protein, BL21 (DE3) Escherichia coli are transfected, and
The even spread on the LB agar plates containing ampicillin antibiotic, then allows it to be stood overnight in 37 DEG C.Select several bacterium colonies
One of, and be placed in the LB fluid nutrient mediums that 6ml contains ampicillin, and in 37 DEG C in the case where being stirred with 200rpm
Overnight incubation.The 5ml saturation Escherichia coli being prepared as above are placed in 500ml LB fluid nutrient mediums, and add 0.2mM
IPTG (SIGMA, USA), for being overexpressed in 0.5 absorbance in 600nm.Stirred the mixture for 3 hours with 200rpm at 37 DEG C.
7-3：The purifying of 56kDa albumen
The Escherichia coli being overexpressed are collected by being centrifuged 15 minutes with 5000rpm, and are suspended in 30ml lysis buffers
(include 50mM Tris pH=8.0,100mM NaCl, 0.1%NaN3, 0.1%TX-100,0.1mM PMSF, and 1mM DTT)
In.Suspension is destroyed three times up to 10 seconds using sonic disruption device (70%), then centrifuges 20 minutes to obtain with 12,000rpm
The protein mixture of Escherichia coli containing recombinant protein in granule part.It is slow in 10ml dissolvings for purification of recombinant proteins
Suspension granule in fliud flushing (PBS, 0.5mM DTT, 0.05%TX-100,8M UREA), then, is used sonic disruption device (70%)
Suspension is destroyed three times up to 10 seconds, then centrifuged 20 minutes with 12,000rpm.Therefore, supernatant is obtained.Supernatant is poured into
On post filled with the sepharose 4B (Invitrogen, USA) (volume of sepharose 4B is 4ml) combined with Ni-NTA, and
40ml wash solutions (identical with the composition of lysis buffer, pH=6.3) are wherein flowed through three times to eliminate unnecessary protein.
In order to obtain the protein that desired 56kDa histidines are combined, by 4ml every kind of elution buffer (compositions with lysis buffer
Identical, pH=4.5) be added to it is each to collect protein in 6 pipes.Each cut is carried out to use 10%SDS-PAGE gels
Electrophoresis, and pass through pipe of the selection with appropriate amount and purity and purify 56kDa albumen.In order to successively remove 8M from solution
UREA, puts it into the osmotic pressure vinyl bag (PIERCE, USA) with 3kDa holes, then carries out dialysis 24 in each concentration
Hour, during which reduced with the same composition of the solution with being used in purifying with 4M, 2M, 1M, 0.5M, 0.25M and 0.1M order
UREA concentration.After dialysis, it is 0.05EU/ml (Genescript, USA) that endotoxin, which is removed to its concentration,.
[embodiment 8]
Zoopery on the immunogenicity of 56kDa albumen
8-1：The preparation of experimental animal
In embodiment 8, the Orientia Tsutsugamushi of purifying for carrying out zoopery to confirm to obtain in embodiment 2 it is immune
Originality.According to the research therein bibliography similar to the research of the disclosure, using every group of 3 to 5 mouse, and therefore originally
Experiment uses every group of 5 mouse.
Mouse is 6 to 8 week old female Balb/c mouse, and it is purchased from Orientbio.It is being suitable for clean animal facility
Environmental standard SPF (no-special pathogen) facility in raise the mouse of purchase.In accordance with the ethics purposes on experimental animal
3R principles.Zoopery is carried out under the regulation of the Animal Experimental Ethical committee of Inha University.
8-2：For the zoopery for the immunogenicity for assessing the outer membrane vesicles from Orientia Tsutsugamushi
In order to assess the immunogenicity for the outer membrane vesicles for being derived from Orientia Tsutsugamushi, animal is carried out according to following experimental program
Experiment.
[table 2]
Experimental program for OMV and 56kDa immunogenicity
| NO. | Experimental group | Medicine is constituted | Inoculation interval |
| 1 | OMV | OMV (80 μ g)+2%Alum (50ul) | With 2 Wednesdays time |
| 2 | 56kDa | 56kDa (20 μ g)+2%Alum (50ul) | With 2 Wednesdays time |
| 3 | OMV+56kDa | OMV (80 μ g)+56kDa (20 μ g)+2%Alum (50ul) | With 2 Wednesdays time |
| 4 | Alum+PBS | 2%Alum (50ul)+PBS (50ul) | With 2 Wednesdays time |
| 5 | PBS | PBS(100ul) | With 2 Wednesdays time |
OMV described in table 2 is derived from the outer membrane vesicles of Orientia Tsutsugamushi, and IA is immune auxiliary material, and uses
Aluminium salt (Alum) is used as the immune auxiliary material in this experiment.In addition, OMV represents the outer membrane vesicles albumen purified in embodiment 3,And
56kDa is the outer membrane protein of the Orientia Tsutsugamushi purified in embodiment 7.In addition, PBS represents phosphate buffered saline (PBS).
It is each with 1 with 2% aluminium glue auxiliary material (Invitrogen) and PBS respectively:OMV and 56kDa that 1 dilution is used above, extremely
Final volume is 100ul.Scheme according to described in above-mentioned table 2 carries out being subcutaneously injected three times with 2 weekly intervals.Blood is gathered after 2 weeks
Sample, and pass through ELISA and IFA analysis and evaluation immunogenicities.Immunogenicity confirmation side is used as using immunofluorescence and ELISA
Case confirms immunogenicity.
8-3：Confirm immunogenicity using immunofluorescence scheme
First, immunogenicity is confirmed using immunofluorescence.In the slide of bacterium of the coating with proteantigen thereon
In make serial dilution to 1:1280 mice serum reaction.After the anti-mouse IgG reactions combined with FITC, pass through fluorescence microscopy
The presence of art observation antibody formation or shortage.
Therefore, the photo with micro- sem observation is shown in Fig. 6.Shown in following article table 3, it is small that the useful OMV of institute is injected
Mouse shows the positive findings of Orientia Tsutsugamushi, and their most of displays 1:640 or more high-titer.The opposing party
Face, the response in the control group with Alum and PBS processing not to Orientia Tsutsugamushi.In other words, it is thus identified that OMV can be lured
Guide pin is shown in such as table 3 to the immunogenicity of Orientia Tsutsugamushi.
[table 3]
Use immunogenicity result of the immunofluorescence after OMV inoculations
8-4：Confirm immunogenicity using ELISA
Next, as ELISA experimental method, the protein confirmed for immunogenicity being coated with 96 orifice plates and is resisted
Original, by mice serum serial dilution to 1:1280, and mixture is assigned in 96 orifice plates and reacted.Then combine HRP
Anti-mouse IgG react.HRP substrate is added, and is reacted.Finally, in 450nm wavemeter (BioTek
Instruments, USA) measure and observe absorbance.
Thus, in using the ELISA of 56kDa albumen tests, the mean OD value for the mouse group 1 injected with OMV is 0.25,
And the mean OD value with the group 2 of 56kDa protein injections is 0.39, shown in such as Fig. 7.Give the group 3 of OMV and 56kDa albumen
Mean OD value be 0.93, its mean OD value obtained from than giving each of OMV and 56kDa albumen is much higher.When logical
When crossing Colby formula Evaluated effects, the numerical value of measurement is 0.93, and as depicted in Equation 1 below, it is increased by expected value
45.3% numerical value, so as to confirm cooperative effect.
[equation 1]
Therefore, as the result of mouse experiment group 1, it is thus identified that immunogenicity of the OMV inductions for 56kDa albumen.In addition,
It is used as the result of mouse experiment group 3, it is thus identified that formation of the OMV to the immunogenicity for the 56kDa albumen given together plays collaboration and made
With.
Such as find out from embodiment described above, it is thus identified that the outer membrane vesicles from Orientia Tsutsugamushi contain 56kDa
Antigen and various other antigens, the 56kDa antigens are the most important antigen for Orientia Tsutsugamushi.Furthermore, it is possible to make
With from Orientia Tsutsugamushi outer membrane vesicles (it is known its be free of outer membrane in liposome (LPS) and/or peptide glycan, despite
Gram negative strain) it is used as the safer vaccine or vaccine auxiliary material in clinical practice.
Sequence table
<110>Inha University Industry Cooperation Agency
<120>Immune composition for yochubio and preparation method thereof
<130> PF-B1873-CN
<140> PCT/KR2015/011486
<141> 2015-10-29
<150> 10-2014-0148624
<151> 2014-10-29
<150> 10-2015-0150499
<151> 2015-10-28
<160> 3
<170>PatentIn version 3s .5
<210> 1
<211> 1176
<212> DNA
<213>Artificial sequence
<220>
<223>The Br56 sequences of truncation
<400> 1
agagcagagc taggtgttat gtaccttaga aatataagcg ctgaggttga agtaggtaaa 60
ggcgaggtag attctaaagg tgagataaag gcagattctg gaggtgggac agatgctcct 120
atacgtaagc cgtttaaact tacaccacct cagcctacta tgatgcctat aagtatagct 180
gatcgtgact ttgggattga tattcctaac atacctcagg cgcaagcgca agctgcacag 240
cctccgctta atgatcagaa gcgtgctgca gctaggatcg cttggttaaa gaattgtgct 300
ggtattgact atatggtgaa ggatcctaat aatcctgggc atatgatggt aaatccggtg 360
ttgttaaata ttccacaggg caaccctaat cctgttggac agccaccgca gcgagcaaat 420
cagcctgcaa attttgcgat acataaccat gagcaatgga ggagtttggt agttggtctt 480
gctgcattat caaatgctaa taaacctagc gcttctcctg tcaaagtttt gagtgacaaa 540
attattcaga tatatagtga tataaagcca tttgctgata tagctggtat taatgttcct 600
gatactggtt tgcctaatag tgcatctatc gaacagatac agagtaaaat ccaagaatta 660
ggtgatacat tggaagaact cagagattct tttgatgggt atattaataa tgcttttgtt 720
aatcagatac acttgaattt tgtcatgccg ccgcaagcac agcaacagca ggggcaaggg 780
cagcaacagc aagctcaagc tacagcgcaa gaagcagtag cagcagcagc tgttaggctt 840
ttaaatggca gtgatcagat tgcgcagtta tataaagatc ttgttaaatt gcagcgtcat 900
gcaggaatta ggaaagccat ggaaaaatta gctgcccaac aagaagaaga tgcaaagaat 960
caaggtaaag gtgactgcaa gcagcaacaa ggagcatctg aaaaatctaa agaaggaaaa 1020
gtcaaagaaa cagagtttga tctgagtatg gttgttggcc aagttaaact ctatgctgac 1080
ctagttacaa ctgaatcatt ctcaatatac ggtggtgttg gtgcagggtt agcttatact 1140
tatggaaaaa tagataataa ggatgttaaa gggtat 1176
<210> 2
<211> 30
<212> DNA
<213>Artificial sequence
<220>
<223>TSA56 forward primers
<400> 2
gtgaattcgt cgacagagca gagcataggt 30
<210> 3
<211> 33
<212> DNA
<213>Artificial sequence
<220>
<223>TSA56 reverse primers
<400> 3
gtaagcttct cgagtcaata ccctttaaca tcc 33
Claims (15)
1. a kind of immunogenic composition for yochubio, it, which is included, is derived from Orientia Tsutsugamushi (Orientia
Tsutsugamushi outer membrane vesicles).
2. the immunogenic composition of claim 1, wherein the outer membrane vesicles have 20~200nm average diameter.
3. the immunogenic composition of claim 1, wherein 20kDa of the composition comprising the Orientia Tsutsugamushi is extremely
110kDa albumen.
4. the immunogenic composition of claim 3, it further includes the 56kDa albumen (types of the Orientia Tsutsugamushi
Specific antigen 56).
5. the immunogenic composition of claim 4, wherein the composition includes the outer membrane for being derived from Orientia Tsutsugamushi
Vesicle protein, and the 56kDa albumen has synergy to immunogenicity.
6. a kind of method for being used to prepare the immunogenic composition for yochubio, it includes：
Orientia Tsutsugamushi infection cell is used, the cell through infection is cultivated, and obtain the cell through infection of culture；
The culture supernatants without the cell through infection are obtained from the cell through infection of the culture；And
Filter and separate the culture supernatants with the outer membrane vesicles for the purifying for obtaining Orientia Tsutsugamushi.
7. the method for claim 6, wherein the step of obtaining the outer membrane vesicles of the purifying of Orientia Tsutsugamushi includes：
With culture supernatants described in the membrane filtration in the hole of the average diameter with 0.1 μm to 0.3 μm；
Sodium azide is added, and carries out ultrafiltration to obtain concentrate；And
The filtering, concentration and resuspension of the concentrate is carried out to obtain the outer membrane vesicles of Orientia Tsutsugamushi.
8. the method for claim 7, wherein the step of filtering of the concentrate, concentration and resuspension is carried out 2~6 times.
9. the outer membrane vesicles of the purifying of the method for claim 6, wherein Orientia Tsutsugamushi include Orientia Tsutsugamushi
20kDa is to 110kDa albumen.
10. a kind of pharmaceutical composition for being used to prevent yochubio, it includes the outer membrane vesicles from Orientia Tsutsugamushi.
11. a kind of pharmaceutical composition for being used to prevent yochubio, it includes outer membrane vesicles and 56kDa from Orientia Tsutsugamushi
Albumen (type specific antigens 56).
12. a kind of purposes for being used to prepare the prevention and treatment agent for yochubio, the prevention and treatment for yochubio
Agent includes the outer membrane vesicles from Orientia Tsutsugamushi.
13. a kind of method for preventing and treating yochubio, it, which includes giving effective dose to individuals in need, is derived from
The outer membrane vesicles of Orientia Tsutsugamushi.
14. a kind of purposes for being used to prepare the prevention and treatment agent for yochubio, the prevention and treatment for yochubio
Agent includes outer membrane vesicles and 56kDa albumen (type specific antigens 56) from Orientia Tsutsugamushi.
15. a kind of method for preventing and treating yochubio, it, which includes giving effective dose to individuals in need, is derived from
The outer membrane vesicles and 56kDa albumen (type specific antigens 56) of Orientia Tsutsugamushi.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR20140148624 | 2014-10-29 | ||
| KR10-2014-0148624 | 2014-10-29 | ||
| KR1020150150499A KR101742236B1 (en) | 2014-10-29 | 2015-10-28 | immunogenic compositions against Scrub typhus and method of producing thereof |
| KR10-2015-0150499 | 2015-10-28 | ||
| PCT/KR2015/011486 WO2016068613A1 (en) | 2014-10-29 | 2015-10-29 | Immune composition for scrub typhus and method for preparing same |
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| CN106999562A true CN106999562A (en) | 2017-08-01 |
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| CN201580064645.4A Pending CN106999562A (en) | 2014-10-29 | 2015-10-29 | Immune composition for yochubio and preparation method thereof |
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| Country | Link |
|---|---|
| JP (1) | JP6440857B2 (en) |
| KR (1) | KR101742236B1 (en) |
| CN (1) | CN106999562A (en) |
Families Citing this family (2)
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|---|---|---|---|---|
| KR102029252B1 (en) * | 2018-07-13 | 2019-10-08 | 한국기초과학지원연구원 | Diagnostic composition of scrub typhus and diagnosis method of scrub typhus using the same |
| KR102132964B1 (en) * | 2018-08-03 | 2020-07-13 | 이진수 | Method for Diagnosing Scrub Typhus Using Exosome derived from Orientia tsutsugamushi |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101115501A (en) * | 2004-09-03 | 2008-01-30 | 诺华疫苗和诊断有限公司 | Improvements relating to meningococcal outer membrane vesicles |
| CN104602702A (en) * | 2012-09-18 | 2015-05-06 | 诺华股份有限公司 | Outer membrane vesicles |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2003234538A1 (en) * | 2002-05-15 | 2003-12-02 | The United States Of America As Represented By The Secretary Of The Navy | Orientia tsutsugamuchi truncated recombinant outer membrane protein (r47) and (r56) vaccines diagnostics and therapeutics for scrub typhus and HIV infection. |
-
2015
- 2015-10-28 KR KR1020150150499A patent/KR101742236B1/en active IP Right Grant
- 2015-10-29 JP JP2017543685A patent/JP6440857B2/en active Active
- 2015-10-29 CN CN201580064645.4A patent/CN106999562A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101115501A (en) * | 2004-09-03 | 2008-01-30 | 诺华疫苗和诊断有限公司 | Improvements relating to meningococcal outer membrane vesicles |
| CN104602702A (en) * | 2012-09-18 | 2015-05-06 | 诺华股份有限公司 | Outer membrane vesicles |
Non-Patent Citations (2)
| Title |
|---|
| OH YOUN KIM等: "Preparation of Outer Membrane Vesicle from Escherichia coli", 《BIO-PROTOCOL》 * |
| SUCHISMITA CHATTOPADHYAY等: "Scrub Typhus Vaccines: Past History and Recent Developments", 《HUMAN VACCINES》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| JP6440857B2 (en) | 2018-12-19 |
| KR101742236B1 (en) | 2017-05-31 |
| JP2017535603A (en) | 2017-11-30 |
| KR20160052363A (en) | 2016-05-12 |
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