CN106220737A - Fusion protein and the application in treatment C difficile-associated disease thereof - Google Patents
Fusion protein and the application in treatment C difficile-associated disease thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1282—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Clostridium (G)
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Abstract
The invention discloses fusion protein and the application in treatment C difficile-associated disease thereof.The invention provides a kind of compositions, be made up of clostridium difficile A toxin C end receptor binding domain TcdA, clostridium difficile B toxin C end receptor binding domain TcdB, clostridium difficile spore antigen BclA3 and clostridium difficile adhesion molecule CD0873.The experiment proves that said composition immune mouse, had good immunogenicity, its antiserum has the good neutralization activity for toxin, can substantially reduce the clostridium difficile field planting at intestinal, and mice can be protected to survive in C. difficile infection.
Description
Technical field
The present invention relates to biological technical field, particularly relate to fusion protein and in treatment C difficile-associated disease
Application.
Background technology
Clostridium difficile (Clostridium difficile, C.difficile), Firmacutes, clostridium mesh, belong to gram
Positive anaerobic spore-bearing bacilli, is widely present in natural environment, only infects mammal, propagated by fecal oral route.Its spore
Vitality is strong, can resist majority disinfectant and antibacterial, cause its can in health care environments wide-scale distribution.Become doctor
The topmost pathogen of institute, causes substantial amounts of economic loss every year, is more than 25% antibiotic-associated diarrhea
The cause of disease of (Antibiotic-associated diarrhea, AAD).Slight C. difficile infection can cause clostridium difficile abdomen
Rushing down (Clostridium difficile-associated diarrhea, CDAD), self limiting is suffered from diarrhoea, and severe patient can develop into
Septicemia, toxic megacolon, peritonitis and pseudomembranous enteritis (pseudomembranous colitis, PMC), break out sexuality
The fatality rate of dye reaches more than 50%.Simultaneously because the existence of spore, the spore in rehabilitation patient's body may again be sprouted and make patient
Relapse rate.Propagate in main institute before C. difficile infection, extended to Community Communication.This bacterium is found first from 1978
With pseudomembranous enteritis (pseudomembranous colitis, PMC) about drawing attention later.Within 2003, play ribosome 027
Type (being called for short RT027) new strains, in multiple countries outbreak of epidemic, causes mortality case, and China also reports Sporadic cases.
RT027 is because morphing, and infectiousness and drug resistance enhancing, the mortality rate caused, sickness rate were also above in the past.A lot of countries are by difficult
The monitoring cause of disease that difficult clostridium must be reported as Notifiable disease.Vaccine is considered as to control CDI (C.difficile infection)
The most basic effective measures.
The most important virulence factor of clostridium difficile is enterotoxin toxin A (308kDa) and cytotoxin toxin B (270kDa).
In recent years both toxin and the construction features of gene, pathogenesis, regulatory mechanism etc. are all achieved breakthrough.Poison
The C-end of element A and toxin B is all made up of multiple repetitive sequences, is toxin identification and the functional areas combining cell receptor, is referred to as being subject to
Body land (receptor-binding domain, RBD), the number of repetitive sequence directly affects toxin and is subject to cell surface
The adhesion of body is strong and weak.Research shows, the toxicity controlling both toxin is effective to resisting CDI, prepares around both toxin
Protective antigen or antibody also demonstrate that there is good effect.
With elongated and in wavy bending filament on some thalline, 1-2 root at least, at most up to hundreds of.This
A little filaments are referred to as flagellum, are the organ of locomotion of antibacterial.Flagellum antibacterial cause a disease in play a significant role: (1) promote antibacterial
Host cell is sticked；(2) antibacterial motor capacity to the more rich place of nutrition is strengthened；(3) biomembranous formation is promoted；(4)
Promote the transhipment of virulence factor cross-cell membrane；(5) by activating TLR-5 signal path regulation immunoreation etc..Research in the past shows
Clostridium difficile flagellum plays a significant role in bacteria planting.The FliC of clostridium difficile genome F1 section coding and cap albumen
FliD is the structural protein with complete function flagellum.C. difficile infection patients serum can detect anti-FliC and
The antibody of FliD exists.
Some antibacterial (bacillus cereus, clostridium, minority coccus etc.) is in its growth promoter later stage, in intracellular shape
The circle become or ellipse, heavy wall, water content are low, the hypopus structure of strong stress resistance, referred to as spore.Clostridium difficile spore
Diameter more than thalline diameter, making whole thalline is fusiformis, is positioned in the middle of thalline.The shape of the pathogenic dependence spore of clostridium difficile
Become.The clostridium difficile that can not form spore cannot be at intestinal colonisation, can not horizontal transmission.Spore can also resist antibiotic and
The attack of host immune system.Clostridium difficile spore divides multilamellar, the most respectively theca interna, bacterium parietal layer, cortical layer, adventitia
Layer, coating layer and outer wall layer.BclA3 is the constituent that outer wall layer is important, plays important in clostridium difficile sporulation
Effect.
Some special constructions of bacterium surface existence and related protein, have the work making bacterial adhesion to host target cells
With, referred to as adhesin (adhesin).Adhesion is pathogen contact and the first step of infection cell, closely related with pathogenic.With
Finding toward research, compared to low pathogenic strain clostridium difficile, high pathogenic strain can preferably be attached on host target cells.CD0873 is
Nearest newfound clostridium difficile adhesin, plays a significant role in clostridium difficile causes a disease.
Summary of the invention
It is an object of the present invention to provide a kind of product.
The present invention provide product, including following 1)-6) in any one:
1) fusion protein compositions, described fusion protein compositions is made up of fusion protein A and fusion protein B；Described melt
Hop protein A includes clostridium difficile A toxin C end receptor binding domain TcdA and clostridium difficile B toxin C end receptor binding domain TcdB；Institute
State fusion protein B and include following at least one albumen: clostridium difficile spore antigen BclA3, clostridium difficile adhesion molecule CD0873 and
Clostridium difficile flagellin FliD；
2) fusion protein, described fusion protein is by clostridium difficile A toxin C end receptor binding domain TcdA, clostridium difficile B toxin
C end receptor binding domain TcdB and following at least one protein fusion composition: clostridium difficile spore antigen BclA3, clostridium difficile are glutinous
Attached molecule CD0873 and clostridium difficile flagellin FliD；
3) protein composition, described protein composition is by clostridium difficile A toxin C end receptor binding domain TcdA, clostridium difficile B
Toxin C end receptor binding domain TcdB and following at least one albumen composition: clostridium difficile spore antigen BclA3, clostridium difficile are glutinous
Attached molecule CD0873 and clostridium difficile flagellin FliD；
4) DNA molecular, is 2) non-coding DNA molecules of described fusion protein
5) DNA molecular compositions, by 1) shown in the non-coding DNA molecules and 3 of fusion protein A in fusion protein compositions)
The non-coding DNA molecules composition of the fusion protein B in shown fusion protein compositions；
6) containing 4) or 5) expression cassette, recombinant vector, recombinant bacterium or transgenic cell line.
In the said goods, in described fusion protein, each albumen is all connected by connection peptides.
In the said goods, 1) in fusion protein compositions,
Described fusion protein A is by clostridium difficile A toxin C end receptor binding domain TcdA, connection peptides and clostridium difficile B toxin C
End receptor binding domain TcdB merges the albumen obtained；
Described fusion protein B is melted by clostridium difficile spore antigen BclA3, connection peptides and clostridium difficile adhesion molecule CD0873
Close the albumen obtained；
3) protein composition, for following 3)-A to 3) in-E any one:
3)-A by clostridium difficile A toxin C end receptor binding domain TcdA, clostridium difficile B toxin C end receptor binding domain TcdB and
Clostridium difficile spore antigen BclA3 forms；
3)-B by clostridium difficile A toxin C end receptor binding domain TcdA, clostridium difficile B toxin C end receptor binding domain TcdB and
Clostridium difficile adhesion molecule CD0873 forms；
3)-C by clostridium difficile A toxin C end receptor binding domain TcdA, clostridium difficile B toxin C end receptor binding domain TcdB and
Clostridium difficile flagellin FliD forms；
3)-D by clostridium difficile A toxin C end receptor binding domain TcdA, clostridium difficile B toxin C end receptor binding domain TcdB,
Clostridium difficile spore antigen BclA3 and clostridium difficile adhesion molecule CD0873 composition；
3)-E by clostridium difficile A toxin C end receptor binding domain TcdA, clostridium difficile B toxin C end receptor binding domain TcdB,
Clostridium difficile spore antigen BclA3 and clostridium difficile flagellin FliD composition；
3)-F by clostridium difficile A toxin C end receptor binding domain TcdA, clostridium difficile B toxin C end receptor binding domain TcdB,
Clostridium difficile spore antigen BclA3, clostridium difficile adhesion molecule CD0873 and clostridium difficile flagellin FliD composition.
In the said goods, described fusion protein A) aminoacid sequence be following a or b:
A is successively by described clostridium difficile B toxin C end receptor binding domain TcdB aminoacid sequence, described connection from N-terminal
Peptide amino acid sequence, described clostridium difficile A toxin C end receptor binding domain TcdA aminoacid sequence form；
B is successively by described clostridium difficile A toxin C end receptor binding domain TcdA aminoacid sequence, described connection from N-terminal
Peptide amino acid sequence, described clostridium difficile B toxin C end receptor binding domain TcdB aminoacid sequence form；
Described fusion protein B) aminoacid sequence be following c or d:
C is successively by described clostridium difficile spore antigen BclA3 aminoacid sequence, described connection peptides aminoacid from N-terminal
The albumen composition of sequence and described clostridium difficile adhesion molecule CD0873 aminoacid sequence composition；
D is successively by described clostridium difficile adhesion molecule CD0873 aminoacid sequence, described connection peptides aminoacid from N-terminal
The albumen composition of sequence and described clostridium difficile spore antigen BclA3 aminoacid sequence composition.
In the said goods, the aminoacid sequence of described clostridium difficile B toxin C end receptor binding domain TcdB is sequence 4, and it is compiled
The nucleotides sequence of code DNA molecular is classified as sequence 3,
The aminoacid sequence of described clostridium difficile A toxin C end receptor binding domain TcdA is sequence 2, its non-coding DNA molecules
Nucleotides sequence is classified as sequence 1,
The aminoacid sequence of described clostridium difficile spore antigen BclA3 is sequence 6, the nucleotides sequence of its non-coding DNA molecules
It is classified as sequence 5,
The aminoacid sequence of described clostridium difficile adhesion molecule CD0873 is sequence 8, the nucleotides sequence of its non-coding DNA molecules
It is classified as sequence 7；
The aminoacid sequence of described clostridium difficile flagellin FliD is sequence 10, the nucleotides sequence of its non-coding DNA molecules
It is classified as sequence 9；
The aminoacid sequence of described connection peptides is sequence 12, and the nucleotides sequence of its non-coding DNA molecules is classified as sequence 11.
The said goods has 1)-3) at least one function: 1) prevent and/or treat C difficile-associated disease；2) press down
The field planting of intestinal clostridium difficile spore processed；3) diagnosis C difficile-associated disease.
The said goods is medicine or test kit.
By 1 in above-mentioned product)-6) in any one is also the scope of protection of the invention as antibody prepared by antigen.
Above-mentioned antibody is polyclonal antibody.
Above-mentioned product or above-mentioned antibody have 1 in preparation)-3) at least one function test kit in application also
It is the scope of protection of the invention: 1) prevent and/or treat C difficile-associated disease；2) determining of intestinal clostridium difficile spore is suppressed
Plant；3) diagnosis C difficile-associated disease.
In above-mentioned application, described C difficile-associated disease is the disease that C. difficile infection causes.
Clostridium difficile A toxin C end receptor binding domain TcdA, by 859 aminoacid of 2577bp nucleotide coding；
Clostridium difficile B toxin C end receptor binding domain TcdB, by 514 aminoacid of 1542bp nucleotide coding；
Clostridium difficile spore antigen BclA3, by 659 aminoacid of 1977bp nucleotide coding；
Clostridium difficile adhesion molecule CD0873, by 317 aminoacid of 951bp nucleotide coding；
Clostridium difficile flagellin FliD, by 505 aminoacid of 1518bp nucleotide coding；
Described clostridium difficile is clostridium difficile ATCC 43255.
The experiment proves that, by clostridium difficile A toxin C end receptor binding domain, the C end receptor binding domain of B toxin,
The fusion protein that spore antigen BclA3 and clostridium difficile adhesion molecule CD0873 is formed has good immunogenicity, and it lures
The antibody led can neutralize clostridium difficile toxin, and mice can be protected to survive in C. difficile infection.
Accompanying drawing explanation
Fig. 1 is mouse infection clostridium difficile 24h rear intestinal spore number.
Fig. 2 is survival condition after mouse infection clostridium difficile.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Embodiment 1, different protein composition and functional verification thereof
One, the preparation of different protein compositions
1, the encoding gene of synthetic proteins
TcdA aminoacid sequence is sequence 2 in sequence table, and the nucleotides sequence of its encoding gene is classified as sequence 1；
TcdB aminoacid sequence is sequence 4 in sequence table, and the nucleotides sequence of its encoding gene is classified as sequence 3；
BclA3 aminoacid sequence is sequence 6 in sequence table, and the nucleotides sequence of its encoding gene is classified as sequence 5；
CD0873 aminoacid sequence is sequence 8 in sequence table, and the nucleotides sequence of its encoding gene is classified as sequence 7；
FliD aminoacid sequence is sequence 10 in sequence table, and the nucleotides sequence of its encoding gene is classified as sequence 9；
The nucleotide sequence of the encoding gene of synthetic TcdA, TcdB, BclA3, CD0873 and FliD.
2, the clone of albumen, expression and purification.
The nucleotide sequence of the encoding gene of synthetic TcdA, TcdB, BclA3, CD0873 and FliD, encodes each
Gene insert respectively expression plasmid pET22b (+) Nde I and the Xho I restriction enzyme site of (Merk Millipore, 69744-3CN)
Between, obtain recombinant vector；
Recombinant vector pET22b (+)-TcdA be by albumen TcdA encoding gene insert expression plasmid pET22a (+) Nde
The recombinant vector obtained between I and Xho I site, express with thrombin restriction enzyme site and histidine-tagged fusion protein
TcdA；
Recombinant vector pET22a (+)-TcdB be by albumen TcdB+BclA3 encoding gene insert expression plasmid pET22b (+)
BamH I and Xho I site between the recombinant vector that obtains, express with thrombin restriction enzyme site and histidine-tagged melting
Hop protein TcdB；
Recombinant vector pET22b (+)-BclA3 be by fusion protein BclA3 encoding gene insert expression plasmid pET22b (+)
Nde I and Xho I site between the recombinant vector that obtains, express with thrombin restriction enzyme site and histidine-tagged fusion
Protein B clA3；
Recombinant vector pET22b (+)-CD0873 be by fusion protein CD0873 encoding gene insert expression plasmid pET22b
The recombinant vector obtained between the Nde I of (+) and Xho I site, express with thrombin restriction enzyme site and histidine-tagged
Fusion protein CD0873；
Recombinant vector pET22b (+)-FliD be by fusion protein F liD encoding gene insert expression plasmid pET22b (+)
The recombinant vector obtained between Nde I and Xho I site, express with thrombin restriction enzyme site and histidine-tagged fusion egg
White FliD；
Respectively above-mentioned recombinant vector is converted after expressing bacterium BL31 (DE3), obtain recombinant bacterium BL31/pET22b (+)-
TcdA, recombinant bacterium BL31/pET22b (+)-TcdB, recombinant bacterium BL31/pET22b (+)-BclA3, recombinant bacterium BL31/pET22b
(+)-CD0873, recombinant bacterium BL31/pET22b (+)-FliD；
Above-mentioned each recombinant bacterium IPTG (final concentration 0.1mM) is induced (20 DEG C, 4h), and 8000r/min is centrifuged 15min and receives
Collection thalline, sonicated cells (300w, ultrasonic 30s, intermittently 30s, 30 circulations).Afterwards, 8000r/min is centrifuged 15min receipts
HisTrap FF crude 5ml prepacked column on collection supernatant, is purified (seeing GE Products service manual).
Chromatographic column HisTrap FF crude 5ml (GE Healthcare) of above-mentioned purification, the combination buffer of employing
Formula is 7.6g sodium phosphate, 58.5g sodium chloride, 2.72g imidazoles, and hydrochloric acid is adjusted to pH 7.4, is settled to 1L.The elution buffer used
Formula of liquid is 7.6g sodium phosphate, 58.5g sodium chloride, 27.2g imidazoles, and hydrochloric acid is adjusted to pH 7.4, is settled to 1L, and elution flow rate is
1ml/min, elution time is 5min；
Loading recombinant bacterium BL31/pET22b (+)-TcdA ultrasonication supernatant, collect the eluent of 1 column volume,
To with thrombin restriction enzyme site and histidine-tagged albumen TcdA；
Loading recombinant bacterium BL31/pET22b (+)-TcdB ultrasonication supernatant, collect the eluent of 1 column volume,
To with thrombin restriction enzyme site and histidine-tagged albumen TcdB；
Loading recombinant bacterium BL31/pET22b (+)-BclA3 ultrasonication supernatant, collect the eluent of 1 column volume,
To with thrombin restriction enzyme site and histidine-tagged protein B clA3；
Loading recombinant bacterium BL31/pET22b (+)-CD0873 ultrasonication supernatant, collect the eluent of 1 column volume,
Obtain with thrombin restriction enzyme site and histidine-tagged PROTEIN C D0873；
Loading recombinant bacterium BL31/pET22b (+)-FliD ultrasonication supernatant, collect the eluent of 1 column volume,
To with thrombin restriction enzyme site and histidine-tagged albumen FliD；
Above-mentioned albumen through thrombin enzyme action effect can remove for affinity purification histidine-tagged, specific as follows:
The eluent obtained by above-mentioned each eluting respectively adds the amount of the thrombin of 125 units, 4 DEG C of enzymes according to 1mg albumen
Cut through night, retain super filter tube through different molecular weight after enzyme action and be centrifuged, it is thus achieved that the not destination protein of tape label, SDS-PAGE detects mesh
The molecular weight of albumen, result is as follows:
The molecular weight of destination protein TcdA is 94.5kD, and concentration is 1.2mg/ml；
The molecular weight of destination protein TcdB is 56.5kD, and concentration is 1.4mg/ml；
The molecular weight of destination protein BclA3 is 72.4kD, and concentration is 0.9mg/ml；
The molecular weight of destination protein CD0873 is 34.8kD, and concentration is 1.1mg/ml；
The molecular weight of destination protein FliD is 55.5kD, and concentration is 1.3mg/ml.
3, the preparation of protein composition
By the albumen of above-mentioned 2 preparations according to combining mixing as follows, obtain destination protein compositions:
(1) protein composition TcdA+TcdB: for being that 1:1 is uniformly mixed so as to obtain compositions by TcdA and TcdB according to mass ratio；
(2) protein composition TcdA+TcdB+BclA3: for being that 1:1:1 mixes by TcdA, TcdB and BclA3 according to mass ratio
Even obtain compositions；
(3) protein composition TcdA+TcdB+CD0873: for being 1:1:1 by TcdA, TcdB and CD0873 according to mass ratio
It is uniformly mixed so as to obtain compositions；
(4) protein composition TcdA+TcdB+FliD: for by TcdA, TcdB and FliD according to mass ratio be 1:1:1 mixing
Obtain compositions；
(5) protein composition TcdA+TcdB+BclA3+CD0873: for by TcdA, TcdB, BclA3, CD0873 according to matter
Amount ratio is uniformly mixed so as to obtain compositions for 1:1:1:1；
(6) protein composition TcdA+TcdB+BclA3+FliD: for by TcdA, TcdB, BclA3, FliD according to mass ratio
It is uniformly mixed so as to obtain compositions for 1:1:1:1；
(7) protein composition TcdA+TcdB+CD0873+FliD: for by TcdA, TcdB, CD0873, FliD according to quality
Ratio is uniformly mixed so as to obtain compositions for 1:1:1:1；
(8) protein composition TcdA+TcdB+BclA3+CD0873+FliD: for by TcdA, TcdB, BclA3, CD0873,
FliD is that 1:1:1:1:1 is uniformly mixed so as to obtain compositions according to mass ratio.
Two, the immunocompetence of protein composition
Prepared by toxin: in CCFA flat board, picked clones, clostridium difficile ATCC 43255 streak inoculation is inoculated in 800ml
In BHI culture medium, it is statically placed in anaerobic culture box and cultivates about 72h；8000g, takes supernatant after 4 DEG C of centrifugal 20min；It is slowly added to grind
The ammonium sulfate of milled, makes final concentration of 60%, 4 DEG C, stands 24h；8000g, 4 DEG C of centrifugal 20min；Take precipitation；By resolution of precipitate
In 10ml 10mM Tris-Cl (pH7.5)；Lysate is added in bag filter, in 500ml 10mM PBS (pH7.2) or
10mM Tris-Cl (pH 7.5) 4 DEG C dialysis, three times, each 6h；By toxin filtration sterilization good for PBS, subpackage ,-20 DEG C
Freezen protective.The toxin HiTrap DEAE FF column chromatography that Tris-Cl dialyses, obtains clostridium difficile ATCC43255
Toxin (comprises A, B toxin).
The albumen using an above-mentioned preparation is tested as follows:
6 week old BALB/C mice random packet, the respectively following combination of immunity: (1) protein composition TcdA+TcdB 10 μ g
Group；(2) protein composition TcdA+TcdB+BclA3 10 μ g group；(3) protein composition TcdA+TcdB+CD0873 10 μ g group；
(4) protein composition TcdA+TcdB+FliD10 μ g group；(5) protein composition TcdA+TcdB+BclA3+CD087310 μ g group；
(6) protein composition TcdA+TcdB+BclA3+FliD 10 μ g group；(7) protein composition TcdA+TcdB+CD0873+FliD
10 μ g groups；(8) protein composition TcdA+TcdB+BclA3+CD0873+FliD 10 μ g group；(9) placebo group, normal saline.
Intramuscular injection, every 100 μ l.0d injects initial immunity, and 14d injects booster immunization.The preparation immunity of 21d tail vein blood
Serum.
Take the Vero cell that growth conditions is good, be in logarithmic (log) phase, PBS twice；With 0.25% trypsin in 37
DEG C digestion 3min, adds and is centrifuged 5min containing 1000rpm after blood serum medium, go culture medium；Resuspended carefully with DMEM (containing 10%FBS)
Born of the same parents' precipitation makes single cell suspension, suitably instills blood counting chamber after dilution and counts under inverted microscope；Cell is diluted to
105/ ml, in 96 porocyte culture plates, every hole adds 100 μ l, arranges 3 multiple holes, 37 DEG C, 5%CO2Overnight incubation in incubator；
Every hole adds toxin (the whole content 4CD that DMEM (containing 10%FBS) dilutes50) 50 μ l and the immune serum 50 μ l of serial dilution, 37
DEG C, 5%CO2Hatch 24h；Outwelling culture medium, PBS twice, every hole adds 100 μ l DMEM (without FBS) and 15 μ lMTT
(5mg/ml is dissolved in PBS), 37 DEG C, 5%CO2, place 4h；Outwelling culture medium and MTT, every hole adds 150 μ l DMSO, and level is shaken
Bed shake 10min makes crystallization fully dissolve, and enzyme connection detector surveys OD490, right for blank with the hole only adding DMEM, MTT, DMSO
According to, with only add cell, DMEM, MTT, DMSO hole as negative control；Calculate each experimental group cell proliferation inhibition rate, with survival rate
For vertical coordinate, serum diluting multiple is abscissa mapping, with the serum titer in 50% and during cytotoxicity (i.e. 50% survival)
For IC50Value.
Result is as shown in table 1,
Table 1 is the IC of the serum that different group fusion protein immunization obtains50Value
From the above, it can be seen that obtain after the 5th histone compositions TcdA+TcdB+BclA3+CD0873 combination immunity
In serum and activity best.
Therefore protein composition TcdA+TcdB+BclA3+CD0873 is the combination that immune effect is best, then different albumen
Put in order and whether immune effect had an impact, following experiment is groped.
Embodiment 2, different fusion protein compositions and application thereof
One, the preparation of fusion protein compositions
1, the preparation of fusion protein
1) fusion protein TcdA-TcdB is successively by TcdA, connection peptides (G3S1)3, TcdB composition, its aminoacid sequence is from N
Successively by TcdA aminoacid sequence, (G3S1)3Aminoacid sequence, TcdB aminoacid sequence form；Fusion protein TcdA-TcdB's
The nucleotide sequence of encoding gene from 5 ' ends successively by the nucleotide sequence, (G of TcdA encoding gene3S1)3The core of encoding gene
Nucleotide sequence, the nucleotide sequence composition of TcdB encoding gene；
2) fusion protein TcdB-TcdA is successively by TcdB, connection peptides (G3S1)3, TcdA composition, its aminoacid sequence is from N
Successively by TcdB aminoacid sequence, (G3S1)3Aminoacid sequence, TcdA aminoacid sequence form；Fusion protein TcdB-TcdA's
The nucleotide sequence of encoding gene from 5 ' ends successively by the nucleotide sequence, (G of TcdB encoding gene3S1)3The core of encoding gene
Nucleotide sequence, the nucleotide sequence composition of TcdA encoding gene；
3) fusion protein CD0873-BclA3 is successively by CD0873, connection peptides (G3S1)3Form with BclA3, its aminoacid sequence
Arrange from N successively by CD0873 aminoacid sequence, (G3S1)3Aminoacid sequence, BclA3 aminoacid sequence form；Fusion protein
The nucleotide sequence of CD0873-BclA3 encoding gene from 5 ' ends successively by the nucleotide sequence of CD0873 encoding gene,
(G3S1)3The nucleotide sequence of encoding gene, the nucleotide sequence composition of BclA3 encoding gene；
4) fusion protein BclA3-CD0873 is successively by BclA3, connection peptides (G3S1)3Form with CD0873, its aminoacid sequence
Arrange from N successively by BclA3 aminoacid sequence, (G3S1)3Aminoacid sequence, CD0873 aminoacid sequence form；Fusion protein
The nucleotide sequence of BclA3-CD0873 encoding gene from 5 ' ends successively by the nucleotide sequence of BclA3 encoding gene,
(G3S1)3The nucleotide sequence of encoding gene, the nucleotide sequence composition of CD0873 encoding gene.
2, the clone of fusion protein, expression and purification.
Synthetic above-mentioned 1)-4) shown in the nucleotide sequence of encoding gene of fusion protein, by each fusion protein
Encoding gene insert respectively expression plasmid pET22b (+) Nde I and the Xho I enzyme action of (Merk Millipore, 69744-3CN)
Between site, obtain recombinant vector；
Recombinant vector pET22b (+)-TcdA-TcdB be by fusion protein TcdA-TcdB encoding gene insert expression plasmid
PET22a (+) Nde I and Xho I site between the recombinant vector that obtains, express with thrombin restriction enzyme site and histidine
The fusion protein TcdA-TcdB of label；
Recombinant vector pET22a (+)-TcdB-TcdA be by fusion protein TcdB-TcdA encoding gene insert expression plasmid
PET22b (+) BamH I and Xho I site between the recombinant vector that obtains, express with thrombin restriction enzyme site and histidine
The fusion protein TcdB-TcdA of label；
Recombinant vector pET22b (+)-CD0873-BclA3 be by fusion protein CD0873-BclA3 encoding gene insert table
Reach plasmid pET22b (+) Nde I and Xho I site between the recombinant vector that obtains, express with thrombin restriction enzyme site and
Histidine-tagged fusion protein CD0873-BclA3；
Recombinant vector pET22b (+)-BclA3-CD0873 be by fusion protein BclA3-CD0873 encoding gene insert table
Reach plasmid pET22b (+) Nde I and Xho I site between the recombinant vector that obtains, express with thrombin restriction enzyme site and
Histidine-tagged fusion protein BclA3-CD0873.
Respectively above-mentioned recombinant vector is converted after expressing bacterium BL31 (DE3), obtain recombinant bacterium BL31/pET22b (+)-
TcdA-TcdB, recombinant bacterium BL31/pET22a (+)-TcdB-TcdA, recombinant bacterium BL31/pET22b (+)-CD0873-BclA3, weight
Group bacterium BL31/pET22b (+)-BclA3-CD0873；
Above-mentioned each recombinant bacterium IPTG (final concentration 0.1mM) is induced (20 DEG C, 4h), and 8000r/min is centrifuged 15min and receives
Collection thalline, sonicated cells (300w, ultrasonic 30s, intermittently 30s, 30 circulations).Afterwards, 8000r/min is centrifuged 15min receipts
HisTrap FF crude 5ml prepacked column on collection supernatant, is purified (seeing GE Products service manual).
Chromatographic column HisTrap FF crude 5ml (GE Healthcare) of above-mentioned purification, the combination buffer of employing
Formula is 7.6g sodium phosphate, 58.5g sodium chloride, 2.72g imidazoles, and hydrochloric acid is adjusted to pH 7.4, is settled to 1L.The elution buffer used
Formula of liquid is 7.6g sodium phosphate, 58.5g sodium chloride, 27.2g imidazoles, and hydrochloric acid is adjusted to pH 7.4, is settled to 1L, and elution flow rate is
1ml/min, elution time is 5min；
Loading recombinant bacterium BL31/pET22b (+)-TcdA-TcdB ultrasonication supernatant, collect the eluting of 1 column volume
Liquid, obtain with thrombin restriction enzyme site and histidine-tagged fusion protein TcdA-TcdB；
Loading recombinant bacterium BL31/pET22a (+)-TcdB-TcdA ultrasonication supernatant, collect the eluting of 1 column volume
Liquid, obtain with thrombin restriction enzyme site and histidine-tagged fusion protein TcdB-TcdA；
Loading recombinant bacterium BL31/pET22b (+)-CD0873-BclA3 ultrasonication supernatant, collect washing of 1 column volume
De-liquid, obtain with thrombin restriction enzyme site and histidine-tagged fusion protein CD0873-BclA3；
Loading recombinant bacterium BL31/pET22b (+)-BclA3-CD0873 ultrasonication supernatant, collect washing of 1 column volume
De-liquid, obtain with thrombin restriction enzyme site and histidine-tagged fusion protein BclA3-CD0873.
Above-mentioned fusion protein through thrombin enzyme action effect can remove for affinity purification histidine-tagged, the most such as
Under:
Respectively the eluent that above-mentioned each eluting obtains is added according to 1mg fusion protein the amount of the thrombin of 125 units, 4
DEG C enzyme action overnight, retains super filter tube through different molecular weight after enzyme action and is centrifuged, it is thus achieved that the not purpose fusion protein of tape label, SDS-
The molecular weight of PAGE testing goal fusion protein, result is as follows:
The molecular weight of purpose fusion protein TcdA-TcdB is 151kD, and concentration is 1.2mg/ml；
The molecular weight of purpose fusion protein TcdB-TcdA is 151kD, and concentration is 1.5mg/ml；
The molecular weight of purpose fusion protein CD0873-BclA3 is 107kD, and concentration is 0.9mg/ml；
The molecular weight of purpose fusion protein BclA3-CD0873 is 107kD, and concentration is 1.1mg/ml.
3, the preparation of fusion protein compositions
By the fusion protein of above-mentioned 2 preparations according to combining mixing as follows, obtain fusion protein compositions:
(1) TcdA-TcdB+BclA3-CD0873: fusion protein TcdA-TcdB and fusion protein BclA3-CD0873 is pressed
It is 1:1 mixing according to mass ratio, obtains fusion protein compositions；
(2) TcdA-TcdB+CD0873-BclA3: fusion protein TcdA-TcdB and fusion protein CD0873-BclA3 is pressed
It is 1:1 mixing according to mass ratio, obtains fusion protein compositions；
(3) TcdB-TcdA+CD0873-BclA3: fusion protein TcdB-TcdA and fusion protein CD0873-BclA3 is pressed
It is 1:1 mixing according to mass ratio, obtains fusion protein compositions；
(4) TcdB-TcdA+BclA3-CD0873: fusion protein TcdB-TcdA and fusion protein BclA3-CD0873 is pressed
It is 1:1 mixing according to mass ratio, obtains fusion protein compositions.
Two, the immunocompetence of fusion protein
Identical with the method for the two of embodiment 1, except for the difference that immunogen is the above-mentioned one purpose fusion protein combination obtained
Thing, specific as follows:
6 week old BALB/C mice random packet, respectively with following antigen intramuscular immunity: (1) TcdA-TcdB+BclA3-
CD0873 10μg；(2)TcdA-TcdB+CD0873-BclA3 10μg；(3)TcdB-TcdA+CD0873-BclA3 10μg；(4)
TcdB-TcdA+BclA3-CD0873 10μg；(5) placebo group, normal saline；Every 100 μ l.0d injects initial immunity,
14d injects booster immunization.21d tail vein blood prepares immune serum.
Take the Vero cell that growth conditions is good, be in logarithmic (log) phase, PBS twice；With 0.25% trypsin in 37
DEG C digestion 3min, adds and is centrifuged 5min containing 1000rpm after blood serum medium, go culture medium；Resuspended carefully with DMEM (containing 10%FBS)
Born of the same parents' precipitation makes single cell suspension, suitably instills blood counting chamber after dilution and counts under inverted microscope；Cell is diluted to
105/ ml, in 96 porocyte culture plates, every hole adds 100 μ l, arranges 3 multiple holes, 37 DEG C, 5%CO2Overnight incubation in incubator；
Every hole adds toxin (the whole content 4CD that DMEM (containing 10%FBS) dilutes50) 50 μ l and the immune serum 50 μ l of serial dilution, 37
DEG C, 5%CO2Hatch 24h；Outwelling culture medium, PBS twice, every hole adds 100 μ l DMEM (without FBS) and 15 μ lMTT
(5mg/ml is dissolved in PBS), 37 DEG C, 5%CO2, place 4h；Outwelling culture medium and MTT, every hole adds 150 μ l DMSO, and level is shaken
Bed shake 10min makes crystallization fully dissolve, and enzyme connection detector surveys OD490, right for blank with the hole only adding DMEM, MTT, DMSO
According to, with only add cell, DMEM, MTT, DMSO hole as negative control；Calculate each experimental group cell proliferation inhibition rate, with survival rate
For vertical coordinate, serum diluting multiple is abscissa mapping, with the serum titer in 50% and during cytotoxicity (i.e. 50% survival)
For IC50Value.
Result is as shown in table 2,
Table 2 is the IC of the serum that the fusion protein compositions immunity of different group obtains50Value
From the above, it can be seen that obtain after the 4th group of fusion protein compositions TcdB-TcdA+BclA3-CD0873 combination immunity
In the serum obtained with activity preferably, protection is shown as high.
The immune protective of embodiment 3, protein composition and fusion protein compositions
One, protein composition and the immune protective of fusion protein compositions
6 week old BALB/C mice are randomly divided into 4 groups, carry out following intramuscular injection respectively immune: (1) normal saline group；(2)
The protein composition TcdA+TcdB 10 μ g of embodiment 1；(3) the protein composition BclA3+CD0873 10 μ g of embodiment 1；(4)
The fusion protein compositions TcdB-TcdA+BclA3-CD0873 10 μ g of embodiment 2；Every 100 μ l.0d injects left lower limb flesh
Meat, 14d injects right leg muscle；The spore of the 28th day oral cavity gavage 100CFU clostridium difficile ATCC 43255 infects.Sense
Contaminate first 7 days, mice drinking-water adds 0.5mg/ml cefoperazone (every two days Fresh, mice freely drinks water)；Process 5 days
After, use aquesterilisa instead；Infect after 2 days.
Infect latter 24 hours, dissect mice, collect mouse Colon content, process 20 minutes in 65 DEG C after PBS dilutes,
Then be applied to TCCFA (taurocholate cefoxitin serine cefoxitin-cycloserine-fructose agar) flat board, 37 DEG C cultivate 24 hours after count difficult
Difficult clostridium spore number.
Result is as shown in Figure 1, it can be seen that the protein composition containing BclA3 and CD0873 or fusion protein compositions
The field planting of intestinal clostridium difficile spore can be significantly reduced.
Two, protein composition and the immune protective of fusion protein compositions
6 week old BALB/C mice are randomly divided into 4 groups, carry out following intramuscular injection respectively immune: (1) normal saline group；(2)
The protein composition TcdA+TcdB 10 μ g of embodiment 1；(3) the protein composition BclA3+CD0873 10 μ g of embodiment 1；(4)
The fusion protein compositions TcdB-TcdA+BclA3-CD0873 10 μ g of embodiment 2；Every 100 μ l.0d injects left lower limb flesh
Meat, 14d injects right leg muscle.Within 28th day, oral cavity gavage 200CFU clostridium difficile ATCC 43255 spore infects.Infect
First 7 days, mice drinking-water adds 0.5mg/ml cefoperazone (every two days Fresh, mice freely drinks water)；After processing 5 days,
Use aquesterilisa instead；Infect after 2 days.Often 10 mices of group.
Record mouse survival situation every day.Result is as shown in fig. 2, it can be seen that group (1) mice is the most dead；Group (4) is little
Mus can survive in this infection experiment, shows the fusion protein compositions TcdB-TcdA+BclA3-CD0873 of embodiment 2
Good immunoprotection can be provided after immunity.
Claims (10)
1. a product, including following 1)-6) in any one:
1) fusion protein compositions, described fusion protein compositions is made up of fusion protein A and fusion protein B；Described fusion egg
White A includes clostridium difficile A toxin C end receptor binding domain TcdA and clostridium difficile B toxin C end receptor binding domain TcdB；Described melt
Hop protein B includes following at least one albumen: clostridium difficile spore antigen BclA3, clostridium difficile adhesion molecule CD0873 and difficult
Clostridium flagellin FliD；
2) fusion protein, described fusion protein is by clostridium difficile A toxin C end receptor binding domain TcdA, clostridium difficile B toxin C end
Receptor binding domain TcdB and at least one protein fusion following composition: clostridium difficile spore antigen BclA3, clostridium difficile stick point
Sub-CD0873 and clostridium difficile flagellin FliD；
3) protein composition, described protein composition is by clostridium difficile A toxin C end receptor binding domain TcdA, clostridium difficile B toxin
C end receptor binding domain TcdB and at least one albumen following composition: clostridium difficile spore antigen BclA3, clostridium difficile stick point
Sub-CD0873 and clostridium difficile flagellin FliD；
4) DNA molecular, is 2) non-coding DNA molecules of described fusion protein
5) DNA molecular compositions, by 1) shown in the non-coding DNA molecules and 3 of fusion protein A in fusion protein compositions) shown in
The non-coding DNA molecules composition of the fusion protein B in fusion protein compositions；
6) containing 4) or 5) expression cassette, recombinant vector, recombinant bacterium or transgenic cell line.
Product the most according to claim 1, it is characterised in that: in described fusion protein, each albumen is all by connection peptides even
Connect.
Product the most according to claim 1 and 2, it is characterised in that:
1) in fusion protein compositions,
Described fusion protein A is subject to by clostridium difficile A toxin C end receptor binding domain TcdA, connection peptides and clostridium difficile B toxin C end
Body land TcdB merges the albumen obtained；
Described fusion protein B is merged by clostridium difficile spore antigen BclA3, connection peptides and clostridium difficile adhesion molecule CD0873
The albumen arrived；
3) protein composition, for following 3)-A to 3) in-E any one:
3)-A is by clostridium difficile A toxin C end receptor binding domain TcdA, clostridium difficile B toxin C end receptor binding domain TcdB and difficult
Clostridium spore antigen BclA3 forms；
3)-B by clostridium difficile A toxin C end receptor binding domain TcdA, clostridium difficile B toxin C end receptor binding domain TcdB and sticks
Molecule CD0873 forms；
3)-C is by clostridium difficile A toxin C end receptor binding domain TcdA, clostridium difficile B toxin C end receptor binding domain TcdB and flagellum
Albumen FliD forms；
3)-D is by clostridium difficile A toxin C end receptor binding domain TcdA, clostridium difficile B toxin C end receptor binding domain TcdB, difficult
Clostridium spore antigen BclA3 and clostridium difficile adhesion molecule CD0873 composition；
3)-E is by clostridium difficile A toxin C end receptor binding domain TcdA, clostridium difficile B toxin C end receptor binding domain TcdB, difficult
Clostridium spore antigen BclA3 and clostridium difficile flagellin FliD composition；
3)-F is by clostridium difficile A toxin C end receptor binding domain TcdA, clostridium difficile B toxin C end receptor binding domain TcdB, difficult
Clostridium spore antigen BclA3, clostridium difficile adhesion molecule CD0873 and clostridium difficile flagellin FliD composition.
Product the most according to claim 3, it is characterised in that:
Described fusion protein A) aminoacid sequence be following a or b:
A is successively by described clostridium difficile B toxin C end receptor binding domain TcdB aminoacid sequence, described connection peptides ammonia from N-terminal
Base acid sequence, described clostridium difficile A toxin C end receptor binding domain TcdA aminoacid sequence form；
B is successively by described clostridium difficile A toxin C end receptor binding domain TcdA aminoacid sequence, described connection peptides ammonia from N-terminal
Base acid sequence, described clostridium difficile B toxin C end receptor binding domain TcdB aminoacid sequence form；
Described fusion protein B) aminoacid sequence be following c or d:
C is successively by described clostridium difficile spore antigen BclA3 aminoacid sequence, described connection peptides aminoacid sequence from N-terminal
Albumen composition with described clostridium difficile adhesion molecule CD0873 aminoacid sequence composition；
D is successively by described clostridium difficile adhesion molecule CD0873 aminoacid sequence, described connection peptides aminoacid sequence from N-terminal
Albumen composition with described clostridium difficile spore antigen BclA3 aminoacid sequence composition.
5. according to described product arbitrary in claim 1-4, it is characterised in that:
The aminoacid sequence of described clostridium difficile B toxin C end receptor binding domain TcdB is sequence 4, the nucleoside of its non-coding DNA molecules
Acid sequence is sequence 3,
The aminoacid sequence of described clostridium difficile A toxin C end receptor binding domain TcdA is sequence 2, the nucleoside of its non-coding DNA molecules
Acid sequence is sequence 1,
The aminoacid sequence of described clostridium difficile spore antigen BclA3 is sequence 6, and the nucleotides sequence of its non-coding DNA molecules is classified as
Sequence 5,
The aminoacid sequence of described clostridium difficile adhesion molecule CD0873 is sequence 8, and the nucleotides sequence of its non-coding DNA molecules is classified as
Sequence 7；
The aminoacid sequence of described clostridium difficile flagellin FliD is sequence 10, and the nucleotides sequence of its non-coding DNA molecules is classified as
Sequence 9；
The aminoacid sequence of described connection peptides is sequence 12, and the nucleotides sequence of its non-coding DNA molecules is classified as sequence 11.
6. according to described product arbitrary in claim 1-5, it is characterised in that: described product has 1)-3) and at least one
Function: 1) prevent and/or treat C difficile-associated disease；2) field planting of intestinal clostridium difficile spore is suppressed；3) diagnose difficult
Clostridium relevant disease.
7. according to described product arbitrary in claim 1-6, it is characterised in that: described product is medicine or test kit.
8. by 1 in described product arbitrary in claim 1-7)-6) in any one antibody prepared as antigen.
9. in claim 1-7, arbitrary described product or the antibody described in claim 8 have 1 in preparation)-3) at least one
Application in the test kit of kind function: 1) prevent and/or treat C difficile-associated disease；2) suppression intestinal clostridium difficile spore
Field planting；3) diagnosis C difficile-associated disease.
Application the most according to claim 9, it is characterised in that: described C difficile-associated disease is C. difficile infection
The disease caused.
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| CN110607314A (en) * | 2019-10-31 | 2019-12-24 | 四川农业大学 | TcdB RBD gene, recombinant RBD protein and application |
| CN110691609A (en) * | 2017-03-15 | 2020-01-14 | 诺瓦瓦克斯股份有限公司 | Methods and compositions for inducing an immune response against clostridium difficile |
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| CN103608464A (en) * | 2010-12-29 | 2014-02-26 | 卡恩基因公司 | Clostridium difficile antigens |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110691609A (en) * | 2017-03-15 | 2020-01-14 | 诺瓦瓦克斯股份有限公司 | Methods and compositions for inducing an immune response against clostridium difficile |
| US11938179B2 (en) | 2017-03-15 | 2024-03-26 | Novavax, Inc. | Methods and compositions for inducing immune responses against Clostridium difficile |
| CN110691609B (en) * | 2017-03-15 | 2024-04-19 | 诺瓦瓦克斯股份有限公司 | Methods and compositions for inducing an immune response against clostridium difficile |
| CN110607314A (en) * | 2019-10-31 | 2019-12-24 | 四川农业大学 | TcdB RBD gene, recombinant RBD protein and application |
| CN110607314B (en) * | 2019-10-31 | 2022-09-23 | 四川农业大学 | TcdB RBD gene, recombinant RBD protein and application |
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