CN106692963A - Combined vaccine for preventing staphylococcus aureus infection and tetanus - Google Patents
Combined vaccine for preventing staphylococcus aureus infection and tetanus Download PDFInfo
- Publication number
- CN106692963A CN106692963A CN201710037501.0A CN201710037501A CN106692963A CN 106692963 A CN106692963 A CN 106692963A CN 201710037501 A CN201710037501 A CN 201710037501A CN 106692963 A CN106692963 A CN 106692963A
- Authority
- CN
- China
- Prior art keywords
- staphylococcus aureus
- vaccine
- sasa
- ser
- tent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- 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
- A61K39/08—Clostridium, e.g. Clostridium tetani
-
- A—HUMAN NECESSITIES
- 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
- A61K39/085—Staphylococcus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/70—Multivalent vaccine
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a bivalent immunogen combined vaccine for preventing staphylococcus aureus infection and tetanus. The combined vaccine consists of recombinant staphylococcus aureus surface protein A (SasA) and tetanus neurotoxin C fragment (TeNT-Hc). The antibody response induced by the combined immunization of SasA and TeNT-Hc in a mouse is stronger than that of single component, and a synergistic effect is realized; and moreover, in a mouse staphylococcus aureus and tetanus toxin counteracting model, the combined vaccine shows relatively high protection property.
Description
Technical field
The invention belongs to genetic engineering and immunological technique field, more particularly it relates to a kind of contain golden yellow
Staphylococcal surface protein A (Staphylococcus aureussurface protein A, SasA) and lockjaw Nervous toxicity
The bivalent immunogenic composition of plain C fragments (C fragment of tetanus neurotoxin, TeNT-Hc) and its resisting
Application in staphylococcus aureus and tetanus infection.
Background technology
Staphylococcus aureus and clostridium tetani are distributed widely in nature, any trauma patient for having an open wound
There is the possibility for infecting both pathogenic bacteria.Staphylococcus aureus can result in Skin and soft tissue infection, and fatal
Septicemia and aggressive complication.Lockjaw is caused by obligate anaerobe clostridium tetani, and its neurotoxin for producing can
Invasion and attack nervous system, causes generalized muscle spasm even death by suffocation.Although the lockjaw incidence of disease of developed country is relatively low,
Worldwide, the tetanic death rate is 6-72%.In addition, in the limited developing country of medical and health conditions, it is newborn
Youngster and puerpera are vulnerable to infection of staphylococcus aureus and lockjaw during childbirth.
Combined vaccine is the conventional strategy of two or more pathogenic infections of prevention and control simultaneously, can simplify vaccination regimen, is increased
Plus user's compliance, reduce vaccine inoculation cost.However, not having also at present for infection of staphylococcus aureus and tetanic
The research report of combined vaccine.Anti-Staphylococcus aureus infect and tetanic combined vaccine is applied to the group that hurts sb.'s feelings that is easily wound,
Including sportsman, soldier and police, and the developing country women of child-bearing age.
Staphylococcus aureus surface albumin A (SasA), is the Cell wall anchored proteins of staphylococcus aureus, with 2,
271 amino acid.SasA recombinant proteins immune mouse can protect the lethal of staphylococcus aureus to attack poison.Additionally, SasA bases
Because of the generally existing in clinical strains, SasA albumen can also be expressed in vivo during S. aureus L-forms infect.Tetanus toxin
Molecular weight is 150kDa, with tri- domains of A, B, C, respectively N- ends endopeptidase domain, indexable heavy chain structure
Domain and C- ends acceptor combination heavy chain structure (methods of preparing tetanus C fragments, TeNT-Hc).There are some researches show with nerve
The restructuring TeNT-Hc for saving glycosides fat binding activity can substitute existing tetanus toxoid vaccine (tetanus toxoid, TT).
In security, production convenience and homogeneity, TeNT-Hc is better than tetanus toxoid vaccine.
These early-stage Studies show that recombinant protein SasA and TeNT-Hc can be used as staphylococcus aureus and lockjaw bars
The candidate vaccine component of bacterium.For vaccination regimen is simplified, increase user's compliance, reduce vaccine inoculation cost, lifting vaccine is protected
The consideration of shield rate, this area exist to can and meanwhile effectively prevent infection of staphylococcus aureus and tetanic combined vaccine
Demand, but there is presently no find with obvious cooperative effect combined vaccine.Combined vaccine is not equal to any vaccine
Arbitrarily mixing, preparing combined vaccine need to consider following factor：(1) whether the immunogenic response effect of various antigens can be influenceed；(2)
Not whether there is incompatibility between synantigen or interfere；(3) whether antagonistic effect can occur between different antigenic components；
(4) whether vaccine other compositions (such as adjuvant) are appropriate with the mixed proportion of various antigens.In addition, the formulation of combined vaccine, such as
Liquid or freeze dried powder, and their mixing later duration, can all have influence on the stability of vaccine.
When being used in combination for SasA and TeNT-Hc whether there is antagonism, i.e., the antibody response for producing in vivo and
Whether protectiveness can interfere still does not understand also.There is substantially collaboration effect it is an object of the invention to provide one kind and higher exempt from
Epidemic disease protective rate for preventing infection of staphylococcus aureus and tetanic bivalent combined vaccine and preparation method thereof.
The content of the invention
Based on foregoing invention purpose, the invention provides a kind of bivalent immunogene combined vaccine, it is used to protect host to support
The anti-disease caused by staphylococcus aureus and/or clostridium tetani, the vaccine includes staphylococcus aureus surface egg
White A and methods of preparing tetanus C fragments.
In a preferred technical scheme, the staphylococcus aureus surface albumin A and methods of preparing tetanus C pieces
Duan Junwei gene recombinant proteins.
In a technical scheme being more highly preferred to, the staphylococcus aureus surface albumin A (SasA) is what is truncated
Albumen, the albumen is the sequence (GenBank of MASA252 plants of the Staphylococcus Aureus according to report：
BX571856.1), design primer amplification 142-999bp is the sequence of purpose fragment, is the 28-333 bit aminos of full-length proteins
Sour section.As shown in SEQ ID NO.1, amino acid sequence is as shown in SEQ ID NO.2 for its nucleotide sequence.
It is further preferable that the amino acid sequence such as SEQ ID NO.4 of the methods of preparing tetanus C fragments (TeNT-Hc)
It is shown, sequencing result (the GeneBank sequence numbers according to domestic 64008 plants of clostridium tetani C.Tetani velogen strains：
AF154828), optimization is analyzed to its nucleotide sequence, optimization is as shown in SEQ ID NO.3.
Again for preferably, in the combined vaccine, the staphylococcus aureus surface albumin A and lockjaw Nervous toxicity
Plain C fragments exist respectively as independent component.
In a preferred technical scheme, the staphylococcus aureus surface albumin A and methods of preparing tetanus C pieces
The weight proportion of section is 1:1.
In a technical scheme being more highly preferred to, the staphylococcus aureus surface albumin A and lockjaw Nervous toxicity
Plain C fragments are aluminium hydroxide and are adsorbed.
At one again in preferred technical scheme, the aluminium hydroxide and staphylococcus aureus surface albumin A and break
The weight proportion of neurotoxin C fragments of catching cold is 75:1:1.
In being again preferred technical scheme at one, the vaccine is prepared as capsule, freeze-dried or injection.
In a preferred technical scheme, the staphylococcus aureus surface albumin A and methods of preparing tetanus C pieces
Section recombinant protein is prepared as capsule respectively, and the composition of every kind of capsule is recombinant protein, N- acryloxies succinyl Asia
Amine (NAS), acrylamide (AAm), methylene diacrylamide (Bis), its portfolio ratio are 1：20：3000：400, it is every kind of afterwards
Capsule is adsorbed by aluminium hydroxide again.
Present invention also offers a kind of method for preparing above-mentioned combined vaccine, methods described step is：Take the golden yellow
Staphylococcal surface protein A and methods of preparing tetanus C fragments mix.
The SasA and the combined vaccine of TeNT-Hc that the present invention is provided are immunized big in the antibody response of mouse Immune inducing in vivo generation
In one-component.Receive immune the 2nd, 4 and 6 weeks, the SasA that combined immunization (SasA+TeNT-Hc) induction is produced in mouse
IgG potency is respectively immune 3.083 times (p=0.0210), 3.013 times (p=0.0053) and the 3.365 times of (p of SasA one pack systems
=0.0077).The TeNT-Hc IgG potency that combined immunization (SasA+TeNT-Hc) induction is produced is respectively TeNT-Hc one pack systems
Immune 2.183 times (p=0.0342), 2.594 times (p=0.0124) and 2.377 times (p=0.0089), show SasA and
The antibody response that the combined immunization induction of TeNT-Hc is produced has cooperative effect.In mouse staphylococcus aureus and lockjaw
Toxin is attacked in malicious model, and combined vaccine also shows that protectiveness higher.SasA one pack systems (p=0.0467) and TeNT-Hc+
The combined immunization (p=0.0001) of SasA all has protectiveness to mouse, and the protecting effect of combined immunization is higher than SasA single groups
Divide (p=0.0417), show that there is collaboration to make for protection of the combined immunization of TeNT-Hc+SasA to infection of staphylococcus aureus
With.In addition, TeNT-Hc one pack systems (p<0.0001) with the combined immunization (p of TeNT-Hc+SasA<0.0001) all have to mouse
Protectiveness, and the protecting effect of combined immunization is higher than TeNT-Hc one pack systems, shows the combined immunization of TeNT-Hc+SasA to broken wound
The protection of wind also has synergy.And, SasA and TeNT-Hc combined vaccines the repairing by Nano capsule that the present invention is provided
Decorations, heat endurance is further improved, and protection potency also obtain significant raising.
Brief description of the drawings
Fig. 1 .SasA and TeNT-Hc+SasA combined vaccine are in the horizontal column analysis of IgG antibody that mouse Immune inducing in vivo is produced
Figure；
The horizontal column of IgG antibody that Fig. 2 .TeNT-Hc and TeNT-Hc+SasA combined vaccine are produced in mouse Immune inducing in vivo
Analysis chart；
Fig. 3 .SasA and TeNT-Hc+SasA combined vaccine are deposited to mouse infection of staphylococcus aureus model protection
Curve map living；
The survival curve of protectiveness of Fig. 4 .TeNT-Hc and the TeNT-Hc+SasA combined vaccine in mouse lockjaw model
Figure；
The horizontal column analysis of SasA IgG antibodies that Fig. 5 .TeNT-Hc+SasA and the induction of Nano capsule combined vaccine are produced
Figure；
The TeNT-Hc antibody level column analysis charts that Fig. 6 .TeNT-Hc+SasA and the induction of Nano capsule combined vaccine are produced
Specific embodiment
Further describe the present invention with reference to specific embodiment, advantages of the present invention and feature will be with description and
It is apparent.But these embodiments are only exemplary, do not constitute any limitation to protection scope of the present invention.
The staphylococcus aureus of the restructuring of embodiment 1. truncates the preparation of SasA albumen
Staphylococcus aureus about recombinating truncates the acquisition of SasA albumen referring to Chinese patent application
CN102268444A, it is of the invention so that the disclosure of CN102268444A is incorporated into description of the invention in the form of quoting,
Preparation method is illustrated briefly below.
1. according to the sequence (GenBank of MASA252 plants of the Staphylococcus Aureus for reporting：
BX571856.1), design primer amplification 142-999bp is the sequence of purpose fragment, its amino acid sequence such as SEQ ID NO.2 institutes
Show, be the 28-333 amino acids sections of full-length proteins.NdeI and XhoI digestions position is separately added at the two ends of SasA sequences
Point, and terminator codon is removed, introduce 6 sequences of histidine of coding at 3 ' ends.
2. after SasA gene NdeI and the XhoI double digestions for obtaining will be expanded, connect into same with the double enzymes of NdeI and XhoI
On expression vector pET21a (+) after cutting, competent escherichia coli cell DH5 α, 37 DEG C of overnight incubations are converted.Next day picking list
It is cloned in 5mL LB (Amp+) culture medium, 37 DEG C, 220rpm culture 12h extract plasmid, correct plasmid is sequenced and is named as
pET21a-SasA。
3. staphylococcus aureus truncates Bacillus coli expression and the Western-blot identification of SasA
Correct pET21a (+) carrier connected into SasA genes, conversion competent escherichia coli cell BL21(DE3), picking
In 5mL LB (Amp+) fluid nutrient medium, 37 DEG C, 220rpm is cultivated during to OD600nm ≌ 0.6 monoclonal, is added final concentration of
The IPTG of 1mM, 28 DEG C, 220rpm continues to cultivate 6h.5,000g, 4 DEG C are collected by centrifugation thalline, with the resuspended rear ultrasonic broken bacterium of PBS.
12,000g, 4 DEG C of centrifuging and taking supernatants, row SDS-PAGE electrophoresis identifies the expression of the SasA albumen that size is 40kDa, Western-
The bolt result verifications albumen can His tag monoclonal antibodies anti-with mouse there is specific combination.
4. the seed liquor 1L of SasA albumen is expressed, in 30L fermentation tanks of transferring, is cultivated during to OD600nm ≌ 0.6, added
The IPTG of final concentration of 1mM, 37 DEG C, 300rpm continues to cultivate 6h.10,000g, 4 DEG C are collected by centrifugation thalline, use 20mMTris-
The broken bacterium of homogenate after HCl buffer solutions (pH8.5) are resuspended.20,000g, 4 DEG C are collected by centrifugation supernatant.Supernatant cross QFF posts carry out it is cloudy from
Son is exchanged, efflux 20mM NaH2PO4, ni-sepharose purification is carried out after 3 times of dilutions of 0.5M NaCl (pH7.4), use 20mM
NaH2PO4, 0.5M NaCl, 0.5M imidazoles (pH7.4) carries out continuous gradient wash-out.By after two-step purifying, destination protein SasA
Good purifying is obtained, up to more than 85%, destination protein is ready for use on following exempting to purity in being finally stored in PBS
Epidemic disease is tested.
The preparation of methods of preparing tetanus C fragments (TeNT-Hc) of the restructuring of embodiment 2.
About the acquisition of TeNT-Hc albumen that recombinates referring to Chinese patent CN101880675B, shape of the present invention to quote
Be incorporated into the disclosure of CN101880675B in description of the invention by formula, and preparation method is illustrated briefly below.
1. according to sequencing result (the GeneBank sequence numbers of domestic 64008 plants of clostridium tetani C.Tetani velogen strains：
AF154828), the Tet-Hc sequences to 451Aa are analyzed optimization, and optimization carries out full base as shown in SEQ ID NO.3
Because of synthesis.
2. tetanus toxin subunit vaccine Hc expression vector establishments
By the Tet-Hc genes after optimum synthesis with EcoRI and XhoI double digestions after, connect and use EcoRI and XhoI into same
On expression vector pET32a (+) after double digestion, competent escherichia coli cell DH5 α, 37 DEG C of overnight incubations are converted.Next day chooses
Monoclonal is taken in 5mLLB (Amp+) culture medium, 37 DEG C, 220rpm culture 12h extract plasmid, EcoRI and XhoI double digestions mirror
Determine the insertion of genes of interest, and send sequencing.Correct plasmid is sequenced and is named as pET32a-Tet-Hc.
3. the Bacillus coli expression of tetanus toxin recombinant subunit vaccine Hc and Western-blot are identified
Correct pET32a (+) carrier connected into Tet-Hc genes, conversion competent escherichia coli cell BL21(DE3), choose
Monoclonal is taken in 5mLLB (Amp+) fluid nutrient medium, 37 DEG C, 220rpm is cultivated during to OD600nm ≌ 0.6, add final concentration
It is the IPTG of 0.2mM, 28 DEG C, 220rpm continues to cultivate 6h.5,000g, 4 DEG C are collected by centrifugation thalline, broken with the resuspended rear ultrasounds of PBS
Bacterium.12,000g, 4 DEG C of centrifuging and taking supernatants, row SDS-PAGE electrophoresis identifies the expression of the Hc albumen that size is 50KD.Western-
Blot results confirm that the albumen can occur specific combination with mouse anti-tetanus monoclonal antibody.
4. the Escherichia coli fermentation of tetanus toxin subunit vaccine Hc and purifying
The seed liquor 1L of Hc albumen is expressed, in 30L fermentation tanks of transferring, is cultivated during to OD600nm ≌ 0.6, added dense eventually
The IPTG for 0.2mM is spent, 28 DEG C, 300rpm continues to cultivate 6h.10,000g, 4 DEG C are collected by centrifugation thalline, use 20mMTris-HCl
The broken bacterium of homogenate after buffer solution (pH8.5) is resuspended.20,000g, 4 DEG C are collected by centrifugation supernatant.Supernatant crosses QFF posts and carries out anion friendship
Change, after being balanced with 20mMTris-HCl buffer solutions (pH8.5), carry out gradient with the Tris-HCl buffer solutions containing 0-0.5MNaCl and wash
It is de-.Eluent containing destination protein adds the (NH of final concentration of 0.5M4)2SO4Afterwards, cross phenyl hydrophobic post to carry out next step pure
Change.(NH of the destination protein containing concentration 0.5-0M4)2SO4Tris-HCl buffer solutions carry out gradient elution.Contain destination protein
Eluent desalting column displacement buffer solution be 20mMNaAc (pH4.0) after, crossing SP posts carries out cation exchange, and destination protein is used
20mMNaAc (pH4.0) buffer solution containing 0-0.5MNaCl carries out gradient elution.By three steps after purification, the purpose egg without label
White Hc has obtained good purifying, and up to more than 95%, yield is finally stored in PBS to purity in more than 300mg/L, destination protein
Following immunization experiment is ready for use in buffer solution.
The immunogenicity research of embodiment 3.SasA and TeNT-Hc combined vaccine
Following antigen is adsorbed respectively with 0.75mg aluminum hydroxide adjuvants：10 μ grSasA, 10 μ gTeNT-Hc, 10 μ grSasA+
10μgTeNT-Hc.The 0.75mg aluminum hydroxide adjuvants of unadsorbed antigen are used as negative control.At the 0th, 2 and 4 weeks, above-mentioned four groups
Antigen is respectively adopted intraperitoneal injection mode immunization. Female BALB/c mouse (6-8 weeks age), every group 10.In the 2nd, 4 and 6 weeks tails
Venous puncture blood sample simultaneously extracts serum.
The serum antibody titer of the IgG of antigentic specificity is analyzed by enzyme linked immunosorbent assay (ELISA) (ELISA).2 μ g/ml's
4 DEG C of rSasA or TeNT-Hc are overnight coated with 96 hole elisa plates, and coating buffer solution is 50mM carbonate buffer solutions (pH9.6).With containing
The PBS for having 2% (w/v) bovine serum albumin(BSA) incubates closing 1 hour at 37 DEG C.The serum of gradient dilution is added into elisa plate, 37
DEG C incubate 1 hour, then cleaned 3 times with PBST (PBS containing 0.05%Tween 20).Add horseradish peroxidase-labeled
Goat anti-mouse IgG antibody, 37 DEG C incubate 1 hour, PBST clean 3 times.Add TMB (3,3', 5,5'- tetramethyl benzidines
Dihydrochloride) substrate dark in incubate 10 minutes.2M sulfuric acid color development stopping is reacted, and the light at 450nm is read in ELIASA
Absorption value (A450).Antibody positive reaction is set as A450More than the twice of non-immune serum average value.ELISA antibody titers are represented
To show the highest serum dilution factor of positive reaction.Statistical analysis uses bilateral non-paired t test.
At the 2nd, 4 and 6 weeks, the SasA IgG potency that combined immunization (SasA+TeNT-Hc) induction is produced was respectively that SasA is mono-
Component immune 3.083 times (p=0.0210), 3.013 times (p=0.0053) and 3.365 times (p=0.0077) (Fig. 1).Joint
The TeNT-Hc IgG potency that immune (SasA+TeNT-Hc) induction is produced is respectively 2.183 times of immune (p of TeNT-Hc one pack systems
=0.0342), 2.594 times (p=0.0124) and 2.377 times (p=0.0089) (Fig. 2).Result shows SasA's and TeNT-Hc
The antibody response that combined immunization induction is produced has cooperative effect.
The protectiveness of TeNT-Hc+SasA combined vaccines in mouse infection of staphylococcus aureus model of embodiment 4. is ground
Study carefully
Incubated overnight staphylococcus aureus strains USA300,1:100 are diluted in TSB nutrient solutions, 37 DEG C of cultures to logarithm
Mid-term.6 weeks after initial immunity, malicious 3 immune mouse are attacked in abdominal cavity, and it is 3 × 10 to attack toxic agent amount9The USA300 of CFU.At 5 days
Time in observation mouse survival situation, using log rank Mantel-Cox check analysis survival curves.Result is control group
Mouse is all dead, the combined immunization (p=0.0001) of SasA one pack systems (p=0.0467) and TeNT-Hc+SasA to mouse all
Protecting effect with protectiveness, and combined immunization is higher than SasA one pack systems (p=0.0417) (Fig. 3).Result shows TeNT-Hc
Protection of the combined immunization of+SasA to infection of staphylococcus aureus has synergy.
The protection Journal of Sex Research of the TeNT-Hc+SasA combined vaccines in mouse lockjaw model of embodiment 5.
6 weeks after initial immunity, hypodermic injection 2 × 103LD50Methods of preparing tetanus attack malicious 3 immune mouse.
Observation mouse survival situation in the time of 5 days, using log rank Mantel-Cox check analysis survival curves.Result is right
All dead, the TeNT-Hc one pack systems (p according to group mouse<0.0001) with the combined immunization (p of TeNT-Hc+SasA<0.0001) it is right
Mouse all has protectiveness (Fig. 4), and the protecting effect of combined immunization is higher than TeNT-Hc one pack systems, but because of mouse survival rate
90% is above, statistically indifference.Result shows that the combined immunization of TeNT-Hc+SasA has collaboration to tetanic protection
Effect.
The THERMAL STABILITY of embodiment 6.SasA and TeNT-Hc Nano capsule combined vaccine
Embodiment 5 has confirmed the recombinant subunit combined vaccine of SasA and TeNT-Hc to infection of staphylococcus aureus
There is good protective effect with lockjaw, the combined vaccine can be prepared as solution injection, freeze-dried etc. and be used.
But the heat endurance for giving the traditional vaccine of these medicine types is not good, if lacking cold chain, the antigenicity and validity of vaccine
Extreme influence can be subject to.The present invention attempts being used for Nano capsule the preparation of vaccine, it is possible to increase vaccine is under normal temperature condition
Stability, so that the potential transport for reducing vaccine and preservation cost.
SasA and TeNT-Hc Nano capsules are referred to as n (SasA) and n (TeNT-Hc), and preparation method is as follows：
SasA or TeNT-Hc recombinant proteins are diluted in 50mM borate buffer solutions (pH 8.5), with N- acryloxy ambers
Amber acid imide (NAS) incubates 2 hours altogether in room temperature, and (every kind of recombinant protein is 1 with NAS mol ratios：20).It is subsequently added acryloyl
(mol ratio of every kind of recombinant protein and AAm, Bis is 1 for amine (AAm), methylene diacrylamide (Bis)：3000：400), add
Appropriate ammonium persulfate and tetramethylethylenediamine trigger Raolical polymerizable, room temperature reaction to be dialysed after 1 hour and change liquid to PBS bufferings
Liquid.
The SasA+TeNT-Hc and n (SasA)+n (TeNT-Hc) that equal proportion is mixed are preserved 7 days under the conditions of 37 DEG C, it
Adsorbed with aluminum hydroxide adjuvant afterwards.At the 0th and 2 week, above-mentioned 2 groups of antigen was respectively adopted intraperitoneal injection mode immunization. Female BALB/c
Mouse (6-8 weeks age), every group 10.Extract blood sample and extract serum in the 4th week tail vein.Exempted from by enzyme-linked as described above
The serum antibody titer of epidemic disease determining adsorption (ELISA) analysis antigentic specificity IgG.Nano capsule combined vaccine n (SasA)+n
(TeNT-Hc) the SasAIgG potency and TeNT-Hc potency that induction is produced are respectively 27.86 times of immune (p of SasA+TeNT-Hc<
0.001) (1 is SasA+TeNT-Hc in Fig. 5, Fig. 5, and 2 is n (SasA)+n (TeNT-Hc)) and 32.00 times of (p<0.001) (Fig. 6,
1 is SasA+TeNT-Hc in Fig. 6, and 2 is n (SasA)+n (TeNT-Hc)).Result shows that Nano capsule modification is significantly improved
The heat endurance of SasA and TeNT-Hc combined vaccines.
Nano capsule be it is a kind of nanometer-material-modified technology is carried out to large biological molecule, can be poly- on recombinant protein surface
Close out polymer protection layer.The experimental result of embodiment 6 confirms SasA the and TeNT-Hc Nano capsules joint epidemic disease that the present invention is provided
Seedling has stronger stability, can further improve the protection potency of TeNT-Hc+SasA combined vaccines.
Sequence table
<110>Biologic Engineering Inst., Academy of Millitary Medical Sciences of P.L.A
<120>One kind is for preventing infection of staphylococcus aureus and tetanic combined vaccine
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 882
<212> DNA
<213> Staphylococcus aureus
<400> 1
atgagtcata gtttagtgag tcaagataat caaagcatta gtaaaaaaat aacgggatac 60
ggactgaaaa ctacagcggt tattggtggt gcattcacag taaacatgtt gcatgaccag 120
caagcttttg cggcttctga tgcaccttta acatctgaat taaacacgca aagtgaaacc 180
gtaggcaatc aaaactcaac gacaatcgaa gcatcaacat caacaaccga ttctactagc 240
gtaacgaaaa atagcagttc ggaacaaaca tcaaatagtg acacagtctc aagtgaaaag 300
tctgaaaatg tcacttcgac aactaatagt acaagcaatc aacaagaaaa attgacatct 360
acatctgaat caacatcccc aaagaatact acatcaagtt ctgatactaa atctgtaaca 420
tcaatttcaa gtacagacca acaaactaat acatcaacaa atcaaagtac tgcatcaaat 480
actacttcac aaagcacaac gccagcttca gccaatttaa acaaaactag tacaacgtca 540
accagcacag cacctatcaa acttcgaact ttcagtcgat tagctatgtc aacttttgca 600
tcagctgcaa cgacaagtgc agtaaccgct aatacgatta cggttaataa agataacctc 660
aaacaatata tgacaacgtc aggtaatgct acttatgatc aaagtaccgg tattgtgacg 720
ttaacgcagg atgcatacag ccaaaaaggt gctattacat taggaacacg tattgactct 780
aataaaagtt ttcatttttc tggaaaagta aatttaggta ataaatacga aggtaatgga 840
aatggtggag atggtatcct cgagcaccac caccaccacc ac 882
<210> 2
<211> 294
<212> PRT
<213> Staphylococcus aureus
<400> 2
Met Ser His Ser Leu Val Ser Gln Asp Asn Gln Ser Ile Ser Lys Lys
1 5 10 15
Ile Thr Gly Tyr Gly Leu Lys Thr Thr Ala Val Ile Gly Gly Ala Phe
20 25 30
Thr Val Asn Met Leu His Asp Gln Gln Ala Phe Ala Ala Ser Asp Ala
35 40 45
Pro Leu Thr Ser Glu Leu Asn Thr Gln Ser Glu Thr Val Gly Asn Gln
50 55 60
Asn Ser Thr Thr Ile Glu Ala Ser Thr Ser Thr Thr Asp Ser Thr Ser
65 70 75 80
Val Thr Lys Asn Ser Ser Ser Glu Gln Thr Ser Asn Ser Asp Thr Val
85 90 95
Ser Ser Glu Lys Ser Glu Asn Val Thr Ser Thr Thr Asn Ser Thr Ser
100 105 110
Asn Gln Gln Glu Lys Leu Thr Ser Thr Ser Glu Ser Thr Ser Pro Lys
115 120 125
Asn Thr Thr Ser Ser Ser Asp Thr Lys Ser Val Thr Ser Ile Ser Ser
130 135 140
Thr Asp Gln Gln Thr Asn Thr Ser Thr Asn Gln Ser Thr Ala Ser Asn
145 150 155 160
Thr Thr Ser Gln Ser Thr Thr Pro Ala Ser Ala Asn Leu Asn Lys Thr
165 170 175
Ser Thr Thr Ser Thr Ser Thr Ala Prp Ile Lys Leu Arg Thr Phe Ser
180 185 190
Arg Leu Ala Met Ser Thr Phe Ala Ser Ala Ala Thr Thr Ser Ala Val
195 200 205
Thr Ala Asn Thr Ile Thr Val Asn Lys Asp Asn Leu Lys Gln Tyr Met
210 215 220
Thr Thr Ser Gly Asn Ala Thr Tyr Asp Gln Ser Thr Gly Ile Val Thr
225 230 235 240
Leu Thr Gln Asp Ala Tyr Ser Gln Lys Gly Ala Ile Thr Leu Gly Thr
245 250 255
Arg Ile Asp Ser Asn Lys Ser Phe His Phe Ser Gly Lys Val Asn Leu
260 265 270
Gly Asn Lys Tyr Glu Gly Asn Gly Asn Gly Gly Asp Gly Ile Leu Glu
275 280 285
His His His His His His
290
<210> 3
<211> 1353
<212> DNA
<213> Clostridium tetani
<400> 3
aaaaaccttg attgttgggt cgacaacgaa gaagacatcg atgttatcct gaaaaagtct 60
accattctga acttggacat caacaacgat attatctccg acatctctgg tttcaactcc 120
tctgttatca catatccaga tgctcaattg gtgccgggca tcaacggcaa agctatccac 180
ctggttaaca acgaatcttc tgaagttatc gtgcacaagg ccatggacat cgaatacaac 240
gacatgttca acaacttcac cgttagcttc tggctgcgcg ttccgaaagt ttctgcttcc 300
cacctggaac agtacgacac taacgagtac tccatcatca gctctatgaa gaaatactcc 360
ctgtccatcg gctctggttg gtctgtttcc ctgaagggta acaacctgat ctggactctg 420
aaagactccg cgggcgaagt tcgtcagatc actttccgcg acctgtctga caagttcaac 480
gcgtacctgg ctaacaaatg ggttttcatc actatcacta acgatcgtct gtcttctgct 540
aacctgtaca tcaacggcgt tctgatgggc tccgctgaaa tcactggtct gggcgctatc 600
cgtgaggaca acaacatcac tcttaagctg gaccgttgca acaacaacaa ccagtacgta 660
tccatcgaca agttccgtat cttctgcaaa gcactgaacc cgaaagagat cgaaaaactg 720
tataccagct acctgtctat caccttcctg cgtgacttct ggggtaaccc gctgcgttac 780
gacaccgaat attacctgat cccggtagct tacagctcta aagacgttca gctgaaaaac 840
atcactgact acatgtacct gaccaacgcg ccgtcctaca ctaacggtaa actgaacatc 900
tactaccgac gtctgtacag cggcctgaaa ttcatcatca aacgctacac tccgaacaac 960
gaaatcgatt ctttcgttcg ctctggtgac ttcatcaaac tgtacgtttc ttacaacaac 1020
aacgaacaca tcgttggtta cccgaaagac ggtaacgctt tcaacaacct ggacagaatc 1080
ctaagagtag gttacaacgc tccgggtatc ccgctgtaca aaaaaatgga agctgttaaa 1140
ctgcgtgacc tgaaaaccta ctctgttcag ctgaaactgt acgacgacaa agatgcttct 1200
ctgggtctgg ttggcaccca caacggtcag atcggtaacg acccgaaccg tgacatcctg 1260
atcgcttcta actggtactt caaccacctg aaagacaaaa ccctgacctg cgactggtac 1320
ttcgttccga ccgatgaagg ttggaccaac gac 1353
<210> 4
<211> 451
<212> PRT
<213> Clostridium tetani
<400> 4
Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile
1 5 10 15
Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile
20 25 30
Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala
35 40 45
Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn
50 55 60
Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn
65 70 75 80
Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys
85 90 95
Val Ser Ala Ser His Leu Glu Gln Tyr Asp Thr Asn Glu Tyr Ser Ile
100 105 110
Ile Ser Ser Met Lys Lys Tyr Ser Leu Ser Ile Gly Ser Gly Trp Ser
115 120 125
Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala
130 135 140
Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Ser Asp Lys Phe Asn
145 150 155 160
Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp Arg
165 170 175
Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly Ser Ala
180 185 190
Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn Ile Thr Leu
195 200 205
Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val Ser Ile Asp Lys
210 215 220
Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys Leu
225 230 235 240
Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe Trp Gly Asn
245 250 255
Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro Val Ala Tyr Ser
260 265 270
Ser Lys Asp Val Gln Leu Lys Asn Ile Thr Asp Tyr Met Tyr Leu Thr
275 280 285
Asn Ala Pro Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg
290 295 300
Leu Tyr Ser Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn
305 310 315 320
Glu Ile Asp Ser Phe Val Arg Ser Gly Asp Phe Ile Lys Leu Tyr Val
325 330 335
Ser Tyr Asn Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn
340 345 350
Ala Phe Asn Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro
355 360 365
Gly Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu
370 375 380
Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asp Ala Ser
385 390 395 400
Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro Asn
405 410 415
Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu Lys Asp
420 425 430
Lys Thr Leu Thr Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly Trp
435 440 445
Thr Asn Asp
450
Claims (11)
1. a kind of bivalent immunogene combined vaccine, it is used to protect host to resist by staphylococcus aureus and/or lockjaw bar
Microbial disease, it is characterised in that the vaccine includes staphylococcus aureus surface albumin A and methods of preparing tetanus C
Fragment.
2. vaccine according to claim 1, it is characterised in that the staphylococcus aureus surface albumin A and lockjaw
Neurotoxin C fragments are gene recombinant protein.
3. vaccine according to claim 2, it is characterised in that the staphylococcus aureus surface albumin A is what is truncated
Albumen, its amino acid sequence is as shown in SEQ ID NO.2.
4. vaccine according to claim 3, it is characterised in that the amino acid sequence of the methods of preparing tetanus C fragments
As shown in SEQ ID NO.4.
5. vaccine according to claim 4, it is characterised in that in the combined vaccine, the staphylococcus aureus
Surface protein A and methods of preparing tetanus C fragments exist respectively as independent component.
6. vaccine according to claim 5, it is characterised in that the staphylococcus aureus surface albumin A and lockjaw
The weight proportion of neurotoxin C fragments is 1:1.
7. vaccine according to claim 6, it is characterised in that the staphylococcus aureus surface albumin A and lockjaw
Neurotoxin C fragments are aluminium hydroxide and are adsorbed.
8. vaccine according to claim 7, it is characterised in that the aluminium hydroxide and staphylococcus aureus surface albumen
The weight proportion of A and methods of preparing tetanus C fragments is 75:1:1.
9. according to any described combined vaccines of claim 1-8, it is characterised in that the vaccine is prepared as capsule, freezes
Dry agent or injection.
10. according to any described vaccine of claim 9, it is characterised in that the staphylococcus aureus surface albumin A and broken
Cold neurotoxin C fragment recombinant proteins are prepared as capsule respectively, and the composition of every kind of capsule is recombinant protein, N- propylene
Acyloxysuccinimide, acrylamide, methylene diacrylamide, its portfolio ratio are 1：20：3000：400, every kind of glue afterwards
Capsule is adsorbed by aluminium hydroxide again.
A kind of 11. methods for preparing combined vaccine described in claim 5, it is characterised in that take the staphylococcus aureus table
Face albumin A and methods of preparing tetanus C fragments mix.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2016112360435 | 2016-12-28 | ||
| CN201611236043 | 2016-12-28 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106692963A true CN106692963A (en) | 2017-05-24 |
| CN106692963B CN106692963B (en) | 2020-12-22 |
Family
ID=58906965
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710037501.0A Active CN106692963B (en) | 2016-12-28 | 2017-01-19 | Combined vaccine for preventing staphylococcus aureus infection and tetanus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106692963B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109651491A (en) * | 2019-01-16 | 2019-04-19 | 上海赛伦生物技术股份有限公司 | A kind of immunization method that can improve horse antitetanus immunoglobulins titre |
| CN112638937A (en) * | 2018-07-31 | 2021-04-09 | 斯诺雷托克斯私人有限公司 | Pegylated tetanus neurotoxin and hypotonia treatment |
| WO2023134783A3 (en) * | 2022-01-12 | 2023-09-14 | 广东粤港澳大湾区国家纳米科技创新研究院 | Aluminum nanocrystal delivery system, and self-assembled particle adjuvant vaccine based on binding of aluminum nanocrystal delivery system and vaccine antigen molecule |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4511478A (en) * | 1983-11-10 | 1985-04-16 | Genetic Systems Corporation | Polymerizable compounds and methods for preparing synthetic polymers that integrally contain polypeptides |
| US6676958B2 (en) * | 2001-06-19 | 2004-01-13 | Advanced Bioadjuvants, Llc | Adjuvant composition for mucosal and injection delivered vaccines |
| US20050220812A1 (en) * | 2002-02-26 | 2005-10-06 | Titball Richard W | Screening process |
| CN101128215A (en) * | 2004-09-22 | 2008-02-20 | 葛兰素史密丝克莱恩生物有限公司 | Immunogenic composition for use in vaccination against staphylococcei |
| CN102268444A (en) * | 2010-06-03 | 2011-12-07 | 军事医学科学院微生物流行病研究所 | Expression of truncated SasA protein of staphylococcus aureus in escherichia coli and application thereof |
-
2017
- 2017-01-19 CN CN201710037501.0A patent/CN106692963B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4511478A (en) * | 1983-11-10 | 1985-04-16 | Genetic Systems Corporation | Polymerizable compounds and methods for preparing synthetic polymers that integrally contain polypeptides |
| US6676958B2 (en) * | 2001-06-19 | 2004-01-13 | Advanced Bioadjuvants, Llc | Adjuvant composition for mucosal and injection delivered vaccines |
| US20050220812A1 (en) * | 2002-02-26 | 2005-10-06 | Titball Richard W | Screening process |
| CN101128215A (en) * | 2004-09-22 | 2008-02-20 | 葛兰素史密丝克莱恩生物有限公司 | Immunogenic composition for use in vaccination against staphylococcei |
| CN102268444A (en) * | 2010-06-03 | 2011-12-07 | 军事医学科学院微生物流行病研究所 | Expression of truncated SasA protein of staphylococcus aureus in escherichia coli and application thereof |
Non-Patent Citations (8)
| Title |
|---|
| ROBERT S. DAUM等: "Progress Toward a Staphylococcus aureus Vaccine", 《CLINICAL INFECTIOUS DISEASES》 * |
| YI SQ等: "Immunity induced by Staphylococcus aureus surface protein A was protective against lethal challenge of Staphylococcus aureus in BALB/c mice", 《MICROBIOL IMMUNOL.》 * |
| YU R等: "A conformational change of C fragment of tetanus neurotoxin reduces its ganglioside-binding activity but does not destroy its immunogenicity", 《CLIN VACCINE IMMUNOL.》 * |
| YU R等: "Enhanced expression of soluble recombinant tetanus neurotoxin Hc in Escherichia coli for use as a tetanus vaccine candidate", 《IMMUNOBIOLOGY》 * |
| 冯美卿: "《生物技术制药》", 31 January 2016, 中国医药科技出版社 * |
| 杨正时等: "《人及动物病原细菌学》", 31 March 2003, 河北科学技术出版社 * |
| 王进: "《病原生物学与免疫学》", 28 February 2007, 郑州大学出版社 * |
| 谢茂超等: "金黄色葡萄球菌组分疫苗研究进展", 《微生物学免疫学进展》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112638937A (en) * | 2018-07-31 | 2021-04-09 | 斯诺雷托克斯私人有限公司 | Pegylated tetanus neurotoxin and hypotonia treatment |
| CN109651491A (en) * | 2019-01-16 | 2019-04-19 | 上海赛伦生物技术股份有限公司 | A kind of immunization method that can improve horse antitetanus immunoglobulins titre |
| CN109651491B (en) * | 2019-01-16 | 2021-02-02 | 上海赛伦生物技术股份有限公司 | Immunization method capable of improving titer of immunoglobulin for resisting tetanus of horses |
| WO2023134783A3 (en) * | 2022-01-12 | 2023-09-14 | 广东粤港澳大湾区国家纳米科技创新研究院 | Aluminum nanocrystal delivery system, and self-assembled particle adjuvant vaccine based on binding of aluminum nanocrystal delivery system and vaccine antigen molecule |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106692963B (en) | 2020-12-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103495161B (en) | A kind of mixture and preparation method thereof of polynary pneumococcal capsular polysaccharide-protein conjugate | |
| Williamson et al. | A genetically engineered vaccine against the alpha-toxin of Clostridium perfringens protects mice against experimental gas gangrene | |
| CN100460013C (en) | Oral administration recombinant helicobacterpylori vaccine and preparing method thereof | |
| JPH03502687A (en) | Respiratory syncytial viruses: vaccines and diagnostics | |
| CN104069504A (en) | Method for enhancing immunogenicity of proteoglycan protein conjugate | |
| CN106421772A (en) | Novel human rotavirus strains and vaccine | |
| Han et al. | A recombinant mutant abrin A chain expressed in Escherichia coli can be used as an effective vaccine candidate | |
| CN108135992A (en) | Immunogenic fusion proteins | |
| CN106692963A (en) | Combined vaccine for preventing staphylococcus aureus infection and tetanus | |
| CN108066755B (en) | Genetic engineering subunit vaccine for resisting sheep echinococcosis infection and preparation method and application thereof | |
| CN101880675B (en) | High-level expression of tetanus toxin receptor binding domain Hc in Escherichia coli and application | |
| CN106350527B (en) | One kind can be in the highly expressed diphtheria toxin mutation of bacterium coli solubility | |
| JP3169608B2 (en) | Preparation and use of formalin-killed colony-forming factor antigen (CFA) -expressing E. coli for inoculation against enteric infections / diarrhea caused by enterotoxin-producing E. coli in humans | |
| JP2015509713A (en) | Pili protein and composition | |
| CN104861049A (en) | Acinetobacter baumannii 1 A1S-1969 recombinant protein and preparation method and application thereof | |
| CN102816246A (en) | Human cytomegalo virus immunogen fusion protein as well as preparation method and usage thereof | |
| CN102993308B (en) | Methicillin-resistant staphylococcus aureus (MRSA) vaccine recombinant protein antigen HI2 and preparation method and application thereof | |
| CN102151332A (en) | Helicobacter pylori epitope vaccine, design method thereof, preparation method thereof and application thereof | |
| CN108774628A (en) | Synthesis causes the colibacillus engineering and purposes of neonatal meningitis Escherichia coli Glycoprotein binding vaccine | |
| CN104861050A (en) | Acinetobacter baumannii zinc dependent oligopeptide A1S-1610 recombinant protein and preparation method and application thereof | |
| CN103127498A (en) | Recombination antigen composition, vaccine and carrier and method for preparing antigen composition | |
| CN103333254A (en) | Purifying method of methicillin-resistant staphylococcus aureus MRSA recombinant protein vaccine I1C | |
| CN110382519A (en) | The completely new recombinant protein antigen of Orientia Tsutsugamushi and the vaccine composition for using above-mentioned recombinant protein antigen | |
| CN102977214B (en) | Recombinant protein HF2 used for methicillin-resistant staphylococcus aureus (MRSA) vaccine, and preparation method and application thereof | |
| CN106397602A (en) | A molecular adjuvant enhanced type protein engineered vaccine for chicken Marek's disease |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |