CN105582531A - Acinetobacter baumannii and subunit protein combined vaccine and preparation method thereof - Google Patents
Acinetobacter baumannii and subunit protein combined vaccine and preparation method thereof Download PDFInfo
- Publication number
- CN105582531A CN105582531A CN201610053758.0A CN201610053758A CN105582531A CN 105582531 A CN105582531 A CN 105582531A CN 201610053758 A CN201610053758 A CN 201610053758A CN 105582531 A CN105582531 A CN 105582531A
- Authority
- CN
- China
- Prior art keywords
- pld
- smpa
- vaccine
- restructuring
- preparation
- 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
- A61K39/104—Pseudomonadales, e.g. Pseudomonas
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
-
- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55505—Inorganic adjuvants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Mycology (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides an acinetobacter baumannii and subunit protein combined vaccine and a preparation method thereof. According to the preparation method, acinetobacter baumannii SmpA (small protein A) represented as SEQ ID No.1 and an acinetobacter baumannii PLD (phospholipase D) nucleotide sequence represented as SEQ ID No.2 are connected to expression vectors respectively, SmpA and PLD recombinant plasmids are established respectively, recombinant SmpA and PLD proteins are subjected to induced expression respectively, and then the two proteins are mixed with adjuvants for preparation of the vaccine. Based on immunoinformatics and reverse genetics, antigenicity and immunogenicity of outer membrane protein components of the multi-drug-resistant acinetobacter baumannii are analyzed, gene cloning, protein expression, purification and immunological research are performed, the effective outer membrane protein components are screened out and combined, the safety and the effectiveness of the vaccine are improved, and the vaccine has more realistic and more attractive application prospect compared with conventional vaccines.
Description
Technical field
The invention belongs to molecular biology and immunology crossing domain, is a kind of Acinetobacter bauamnnii combined subunit egg specificallyWhite vaccine and preparation method thereof.
Background technology
Acinetobacter bauamnnii is present in hospital environment and in the field planting of patient's multiple location, cause causing Nosocomial Pneumonia, bloodstream infection,Abdominal cavity infection, meningitis, skin soft-tissue infection and urinary infection etc. Due to its powerful acquisition drug resistance and clone's propagationAbility, Acinetobacter bauamnnii has become Global prevalence and has been the common pathogen of height resistance. In China, Acinetobacter bauamnniiThrough becoming one of modal pathogen of hospital infection, within 2013, Chinese CHINET Surveillance of antibiotic resistance in bacterial isolates shows not lever of Bao ManBacterium recall rate is 11.97%, is only second to EHEC and Klebsiella Pneumoniae, and wherein great majority are that multi-drug resistant bacteria is (to iminesThe resistant rate of training south and Meropenem is respectively 62.8% and 59.4%; To cefoperazone sulbactam, minocycline and left oxygen fluorineThe resistant rate of Sha Xing is respectively 36.4%, 41.8% and 43.4%). The appearance of full resistance Acinetobacter bauamnnii and being on the increaseBecome clinical another significant challenge facing, XDR Acinetobacter bauamnnii is mainly distributed in CICU or Burn Ward, aobviousWork increases patient's hospitalization cost, extends length of patient stay, and causes infected patient to be absorbed in the predicament that can use without medicine. Current newEffectively antibacterials research and development are serious lags behind, and new treatment means are targetedly very urgent. In recent years, domestic research mainly focuses onIn the research of monitoring Acinetobacter bauamnnii drug resistance dynamic change and resistance mechanism, foreign study has been contained resistance mechanism, has been caused a diseaseThe aspects such as mechanism, resistance variation, about the research of vaccine comprise inactivated vaccine, 1 outer-membrane protein vaccine, genetic vaccine andCapsular polysaccharide vaccine. The people's such as McConnell, Bomerger result of study shows IWC, and (inactivatedwholecell goes outLive full bacterium), OMC (outermembranecomplex outer membrane protein composite), OMV (outermembranevesicleOuter membrane vesicles) the mouse invasion and attack that all can resist Acinetobacter bauamnnii of immunity. But because ingredient is various and complicated, giving birth toIn product process, be difficult to standardization vaccine dose; And lipopolysaccharides (endotoxin) too high levels wherein, can produce that obvious poison is secondary to be doneWith. Corresponding recombinant protein vaccine such as Bap (biofilmassociatedprotein biomembrane GAP-associated protein GAP), OmpA(outermembraneproteinA outer membrane protein A) and Ata (trimericautotransporterprotein tri-Aggressiveness is from transport protein) be conservative gene expression product, there is high homology, and it is motionless to have covered the Bao Man of major partBacillus strain, has showed good immune effect, and composition is single, reproducible, product differences between batches are little, it is thin to be easy to controlThe advantages such as bacterium endotoxin content, but single component vaccine possibility immunoprotection deficiency, and easily Acinetobacter bauamnnii is placed in powerfulUnder selection pressure, inducing cell film target protein is lowered, and occurs that immune evasion causes vaccine ultimate failure.
The multiple medium and small albumin A of gram-negative bacteria genome (smallproteinA, SmpA) nearly all has homology to express, SmpAIt is one of Acinetobacter bauamnnii cell membrane important component; SmpA is considered to maintain the important structure that cell membrane is complete, is adventitia shapeImportant functional accessory in one-tenth process. Phospholipase D (phospholipaseD, PLD) is proved the mistake at pathogen Blood spreadIn journey, bring into play important effect, meanwhile, studies have shown that PLD is the important virulence factor of Acinetobacter bauamnnii. Reverse immunologyPrompting SmpA and PLD can be used as a good protein vaccine target spot. In addition, SmpA and PLD all can induce body to produceImmune response, SmpA antibody can destroy the integrality of Acinetobacter bauamnnii cell membrane in body, and PLD antibody can deactivation PLD activity,Lower Acinetobacter bauamnnii virulence in body, both synergies can be brought into play more perfect protective effect.
Summary of the invention
The object of this invention is to provide a kind of Acinetobacter bauamnnii combined subunit protein vaccine and preparation method thereof. Utilize immunity letterBreath is learned and reverse genetics, analyzes antigenicity and the immunogenicity of the outer membrane protein composition of multidrug resistant Acinetobacter bauamnnii, carries outGene cloning, protein expression, purifying and immunology research, filter out effective outer membrane protein composition Acinetobacter bauamnnii adventitiaAlbumen smallproteinA (SmpA), Acinetobacter bauamnnii phospholipase D (PLD), and combined, the safety of vaccine improvedProperty and validity, vaccine had more real and tempting application prospect more in the past.
A kind of Acinetobacter bauamnnii combined subunit protein vaccine is by the Acinetobacter bauamnnii SmpA as shown in SEQIDNo.1Acinetobacter bauamnnii PLD nucleotide sequence with as shown in SEQIDNo.2, is connected into respectively expression vector, builds respectively SmpAWith PLD recombinant plasmid, (protein sequence is as SEQIDNo.3 and SEQID for abduction delivering restructuring SmpA and PLD albumen respectively, then vaccine prepared by two kinds of albumen mixing adjuvants No.4).
The preparation method of described Acinetobacter bauamnnii combined subunit protein vaccine:
(1) according to SmpA, PLD gene order, the synthetic SmpA of design, PLD aligning primer, at two ends, primer dividesDo not introduce the restriction enzyme site of Nde I and Xho I, the synthetic SmpA of PCR and PLD gene;
(2) reclaim purified pcr product, TA is cloned into carrier pMD18-T, builds cloning vector pMD18-SmpAAnd pMD18-PLD, and be transformed in E.coliBL21, PCR screens positive transformant, extracts plasmid in a small amount, through enzymeCut qualification order-checking;
(3) with NdeI and XhoI double digestion check order correct pMD18-SmpA and pMD18-PLD, with the pET28b cutting through same enzymePlasmid connects with T4DNA ligase, construction expression plasmid pET28b-SmpA and pET28b-PLD, and be transformed into expressionIn host E.coliBL21 (DE3), PCR screens positive transformant;
(4) by positive list colony inoculation in containing in the LB culture medium of kanamycins, IPTG abduction delivering restructuring SmpA and restructuring PLDAlbumen;
(5) SDS-PAGE and WesternBlotting Analysis and Identification recombinant protein;
(6) adopt ion exchange chromatography purification of recombinant proteins and remove endotoxin;
(7) after the SmpA albumen of restructuring and restructuring PLD albumen mix, then be mixed with vaccine with adjuvant.
After step (7) is specifically mixed the SmpA protein 25 μ g of restructuring and restructuring PLD albumen 5 μ g, then with 1% hydrogen-oxygenChange aluminum solutions in mass ratio 1:1 is mixed with vaccine.
ATCC19606 source SmpA gene order and PLD gene order that the present invention retrieves in GenBank, SmpA eggIn vain (open gene title ABAYE2921, GeneBankID:CAM87743.1) and PLD albumen (open gene title A1S_2989,GenebankID：ABO13392.1)。
Construction of recombinant plasmid of the present invention:
(1) the ATCC19606 source SmpA gene order and the PLD gene order that in GenBank, retrieve, the synthetic SmpA of designWith the primer of PLD sequence, and introduce respectively Nde I and Xho I restriction enzyme site at two ends primer;
(2) taking the correct plasmid that checks order as template, carry out pcr amplification with pfu enzyme, introduce restriction enzyme site;
(3) reaction condition: 94 DEG C of denaturation 5min, 94 DEG C of 30s, 51 DEG C of 30s, 72 DEG C of 30s, 30 circulations, 72 DEG CExtend 10min, after 1.5% gel electrophoresis, reclaim purified pcr product;
(4) with Nde I and Xho I double digestion PCR product, products therefrom and the pET28b plasmid T4DNA cutting through same enzymeLigase connects and is converted into expressive host E.coli.
Prokaryotic expression and the purge process of recombinant protein of the present invention are specific as follows:
(1) the pET28b plasmid of getting 1ul restructuring transforms BL21 (DE3), leaves standstill on ice after 2min and be coated with and contain after 42 DEG C of thermal shock 90sThe culture medium flat plate of 30ug/mL kanamycins, 37 DEG C of overnight incubation;
(2) single bacterium colony that picking has transformed the expression bacterial strain BL21 (DE3) of recombinant plasmid contains 30ug/mL card that is mould in 4mL is housedIn the test tube of the LB culture medium of element, 37 DEG C, 220rpm incubated overnight;
(3) by the bacterium liquid of cultivation by volume the ratio of 1:100 be inoculated in LB fluid nutrient medium, add 30ug/mL kanamycins,37 DEG C, 220rpm cultivates, in the time that OD value reaches 0.6 left and right, and the IPTG that interpolation final concentration is 0.2mM, 37 DEG C, 220rpm,5h, centrifugal collecting cell thalline;
(4) collect thalline and use PBS buffer solution to suspend;
(5) SDS-PAGE electrophoresis detection;
(6) the bacterium thalline of collecting is dissolved with broken Buffer, broken Buffer formula is 50mMTris, 300mMNaCl,0.1%TritonX-114, pH=8.0, ultrasonication thalline in ice bath, power 500W, 25min, ultrasonic 2s, time-out 6s isA circulation; Ultrasonic complete, 12000rpm/min, 4 DEG C of centrifugal 20min, abandon supernatant, and precipitation is again with the broken Buffer that adds ureaDissolve (8M urea, 50mMTris, 300mMNaCl, 0.1%TritonX-114, pH=8.0) ultrasonication bacterium in ice bathBody, power 500W, 25min, ultrasonic 2s, suspending 6s is a circulation; Ultrasonic complete, 12000rpm/min, 4 DEG C are centrifugal20min, supernatant is for affinity chromatography;
(7) get 10mLNi-NTA, with the BindingBuffer cleaning balance pillar of 10 times of bed volumes, flow velocity 5mL/min, BindingBuffer formula is: 8M urea, 50mMTris, 300mMNaCl, pH=8.0;
(8) sample upper prop, flow velocity is 2mL/min;
(9) after end of the sample, rinse pillar with the endotoxin buffer that goes of 10 times of column volumes, remove endotoxin buffer formula: 8M urineElement, 50mMTris, 300mMNaCl, 1%TritonX-114, pH=8.0;
(10) clean pillar, flow velocity 2mL/min with the WashBuffer of 10 times of volumes; WashBuffer formula is: 8M urea,50mMTris, 300mMNaCl, 20/50 imidazoles, pH=8.0;
(11) ElutionBuffer wash-out, flow velocity 2mL/min, collects eluent; ElutionBuffer formula is: 8M urea,50mMTris, 300mMNaCl, 500mM imidazoles, pH=8.0.
Immune mouse after attempting two kinds of albumen to mix with aluminum hydroxide adjuvant respectively in the embodiment of the present invention, collects the rear mouse of immunitySerum, carries out ELISA and detects Acinetobacter bauamnnii SmpA and PLD antibody titer, finds after SmpA and PLD immunity in bodyAll produce higher antibody horizontal. Then immune mouse after two kinds of albumen being mixed with aluminum hydroxide adjuvant together, mouse atomizationSuck the Acinetobacter bauamnnii of clinical separation, observe 72h, in mouse lung tissue bacterial load in combined immunization group compared with control groupReduce by 102Doubly, be better than alone PLD immune group (10 times) and alone SmpA immune group (almost unchanged), and scorching in serumInflammation factor level significantly reduces, and lung tissue inflammatory cell infiltration obviously reduces. Show that this combined vaccine has higher immunogeneProperty. And mouse resisting anteserum after SmpA and PLD immunity is entered in Mice Body to resistance to clinical separation by tail vein injectionMedicine Acinetobacter bauamnnii infects, and finds 15 days survival rates 100% of mouse of SmpA and PLD associating serum passive immunity, PLD groupBe that 83.3%, SmpA group is 50%, control group is 0%. Show that SmpA and PLD antiserum have passive protection effect.
Advantage of the present invention and good effect
(1) zoopery shows that SmpA and PLD combined vaccine are no matter in active immunity or passive immunity has all showed preferablyImmune effect;
(2) have that preparation is simple, output is high, impurity is few, antigenicity is good, the advantage that bad reaction is few;
(3) Acinetobacter bauamnnii is low to the more single vaccine incidence of the escape from immune of SmpA and PLD combined vaccine in theory;
(4) clinical practice may significantly be reduced in the incidence of each intensive care unit Acinetobacter bauamnnii, saves patient's cost of hospitalizationWith and shorten length of patient stay.
Brief description of the drawings
Fig. 1 is protein SDS-PAGE analysis chart,
A is that SmpA protein SDS-PAGE analysis chart, B are that SmpA albumen affinity chromatography SDS-PAGE analysis chart, C are SmpAAlbumen WesternBlot analysis chart;
D is that PLD protein SDS-PAGE analysis chart, E are that PLD albumen affinity chromatography SDS-PAGE analysis chart, F are PLD albumenWesternBlot analysis chart;
Antibody titer when Fig. 2 is variable concentrations antigen immune mouse;
Fig. 3 is that atomization sucks bacterium colony number in the homogenate of latter 72 hours each group lung tissues of infection;
Fig. 4 is each group and infects cytokine concentrations in rear 72h BAL fluid (left side) and serum (right side);
Fig. 5 respectively organizes survival rate after injecting serum infection.
Detailed description of the invention
Be intended to further illustrate the present invention below in conjunction with embodiment, but not limit the scope of the invention.
Embodiment 1: construction of recombinant plasmid
(1) according to SmpA, PLD gene order, the synthetic SmpA of design, PLD aligning primer, at two ends, primer dividesDo not introduce the restriction enzyme site of Nde I and Xho I, the synthetic SmpA of PCR and PLD gene;
(2) reclaim purified pcr product, TA is cloned into carrier pMD18-T (purchased from the great Bioisystech Co., Ltd in Shanghai), buildsCloning vector pMD18-SmpA and pMD18-PLD, and be transformed in E.coliBL21, PCR screens positive transformant,Extract in a small amount plasmid, cut qualification order-checking through enzyme;
(3) with NdeI and XhoI double digestion check order correct pMD18-SmpA and pMD18-PLD, with the pET28b cutting through same enzymePlasmid (purchased from Bei Nuo bio tech ltd, Shanghai) connects with T4DNA ligase, construction expression plasmid pET28b-SmpAAnd pET28b-PLD, and be transformed in expressive host E.coliBL21 (DE3), PCR screens positive transformant.
Embodiment 2: the prokaryotic expression of fusion and purifying (Fig. 1)
(1) get the pET28b plasmid conversion BL21 (DE3) that 1ul recombinates, after 42 DEG C of thermal shock 90s, after standing 2min, be coated with flat board on ice(30ug/mL kanamycins), 37 DEG C of overnight incubation;
(2) picking is expressed single bacterium colony (4mLLB culture medium, 30ug/mL kanamycins) 37 in test tube of bacterial strain BL21 (DE3)DEG C, 220rpm incubated overnight;
(3) the bacterium liquid of cultivation is inoculated in respectively in 4mlLB culture medium by the volume ratio of 1:100, that is mould to add 30ug/mL cardElement, 37 DEG C, 220rpm cultivates;
(4) in the time that OD value reaches 0.6 left and right, add respectively the IPTG that final concentration is 0.2mM, 20 DEG C are spent the night, and 37 DEG C, 220rpmInduction 5h, do not add IPTG derivant as negative control;
(5) collect thalline and use PBS buffer solution to suspend;
(6) SDS-PAGE electrophoresis detection (Figure 1A, D);
(7) induction in a large number: the bacterium liquid that step (2) is cultivated is inoculated in by the volume ratio of 1:100 in the LB fluid nutrient medium of 4L,Add 30ug/mL kanamycins, 37 DEG C, 220rpm cultivates, and in the time that OD value reaches 0.6 left and right, interpolation final concentration is 0.2mMIPTG, 37 DEG C, 220rpm, 5h, centrifugal collecting cell thalline;
(8) by the broken Buffer for bacterium thalline (50mMTris, 300mMNaCl, 0.1%TritonX-114, pH=8.0) collectingDissolve ultrasonication thalline in ice bath, power 500W, 25min (ultrasonic 2s, suspending 6s is a circulation). It is ultrasonic complete,12000rpm/min, 4 DEG C of centrifugal 20min, abandon supernatant, precipitation with add urea broken Buffer (8M urea, 50mMTris,300mMNaCl, 0.1%TritonX-114, pH=8.0) dissolve, ultrasonication thalline in ice bath, power 500W, 25min is (superSound 2s, suspending 6s is a circulation). Ultrasonic complete, 12000rpm/min, 4 DEG C of centrifugal 20min, supernatant is for affinity chromatography(Figure 1B, E);
(9) get 10mLNi-NTA, with the BindingBuffer cleaning balance pillar of 10 times of bed volumes, flow velocity 5mL/min;
(10) sample (lysate supernatant) upper prop, flow velocity is 2mL/min;
(11), after end of the sample, remove endotoxin buffer (8M urea, 50mMTris, 300mMNaCl, 1%Triton with 10 times of column volumesX-114, pH=8.0) flushing pillar;
(12) clean pillar, flow velocity 2mL/min with the WashBuffer of 10 times of volumes;
(13) ElutionBuffer wash-out, flow velocity 2mL/min, collects eluent, and WB analyzes (Fig. 1 C, F).
Note: BindingBuffer (8M urea, 50mMTris, 300mMNaCl, pH=8.0)
WashBuffer (8M urea, 50mMTris, 300mMNaCl, 20/50 imidazoles, pH=8.0)
ElutionBuffer (8M urea, 50mMTris, 300mMNaCl, 500mM imidazoles, pH=8.0)
Embodiment 3:SmpA and PLD respectively animal immune select suitable dosage
SmpA and the PLD of restructuring respectively mix according to mass ratio 1:1 with 1% aluminium hydroxide solution with 5/25/125 μ g, to mixLiquid carried out respectively 3 hypodermic injection immunity at the 0th day, the 7th day, the 21st day, diabetic mice is raised in SPF level environment.After last immunity, the ELISA of antibody horizontal is carried out in blood sampling in 2 weeks, and antigen coated when ELISA detects is the restructuring SmpA of 10 μ gAnd PLD, testing result confirms that antibody titer reaches 103-106, confirm to have produced high titre specific antibody, and select SmpA25 μG and PLD5 μ g are as vaccine dose (Fig. 2).
Embodiment 4: the active immunity experiment of combined vaccine
SmpA and the PLD of restructuring respectively mix according to mass ratio 1:1 with 1% aluminium hydroxide solution with 25,5 μ g, with mixed liquorCarried out respectively 3 hypodermic injection immunity at the 0th day, the 7th day, the 21st day, diabetic mice is raised in SPF level environment.After last immunity, after 2 weeks, carry out the Acinetobacter bauamnnii of the resistance to multiple medicines atomization of clinical separation and infect, mouse lung tissue bacterial loadCombined immunization group reduces by 10 compared with control group2Doubly, be better than alone PLD immune group (10 times) and alone SmpA immune group (almost withoutChange), and in serum, inflammatory factor level significantly reduces, and lung tissue inflammatory cell infiltration obviously reduces. Show this associating epidemic diseaseSeedling has higher immunogenicity (Fig. 3,4).
Embodiment 5: the passive immunity experiment of combined vaccine
The mouse resisting anteserum 100 μ L that learn from else's experience after three immunity, tail vein injection enters in Mice Body, after 1h, injects through lumbar injection again106CFU Acinetobacter bauamnnii bacterium liquid, infects and mouse was observed in latter 15 days, SmpA and PLD associating serum passive immunity15 days survival rates 100% of mouse, PLD group is that 83.3%, SmpA group is 50%, control group is 0% (Fig. 5). Show SmpAThere is passive protection effect with PLD antiserum.
Claims (3)
1. Acinetobacter bauamnnii combined subunit protein vaccine, is characterized in that, is by not lever of the Bao Man as shown in SEQIDNo.1Bacterium SmpA and the Acinetobacter bauamnnii PLD nucleotide sequence as shown in SEQIDNo.2, be connected into respectively expression vector, builds respectivelySmpA and PLD recombinant plasmid, abduction delivering restructuring SmpA and PLD albumen respectively, then prepared by two kinds of albumen mixing adjuvantsVaccine.
2. the preparation method of Acinetobacter bauamnnii combined subunit protein vaccine claimed in claim 1, is characterized in that,
(1) according to SmpA, PLD gene order, the synthetic SmpA of design, PLD aligning primer, at two ends, primer dividesDo not introduce the restriction enzyme site of Nde I and Xho I, the synthetic SmpA of PCR and PLD gene;
(2) reclaim purified pcr product, TA is cloned into carrier pMD18-T, builds cloning vector pMD18-SmpAAnd pMD18-PLD, and be transformed in E.coliBL21, PCR screens positive transformant, extracts plasmid in a small amount, through enzymeCut qualification order-checking;
(3) with NdeI and XhoI double digestion check order correct pMD18-SmpA and pMD18-PLD, with the pET28b cutting through same enzymePlasmid connects with T4DNA ligase, construction expression plasmid pET28b-SmpA and pET28b-PLD, and be transformed into expressionIn host E.coliBL21 (DE3), PCR screens positive transformant;
(4) by positive list colony inoculation in containing in the LB culture medium of kanamycins, IPTG abduction delivering restructuring SmpA and restructuring PLDAlbumen;
(5) SDS-PAGE and WesternBlotting Analysis and Identification recombinant protein;
(6) adopt ion exchange chromatography purification of recombinant proteins and remove endotoxin;
(7) after the SmpA albumen of restructuring and restructuring PLD albumen mix, then be mixed with vaccine with adjuvant.
3. the preparation method of Acinetobacter bauamnnii combined subunit protein vaccine according to claim 2, is characterized in that step(7) be by after the SmpA protein 25 μ g of restructuring and the 5 μ g mixing of restructuring PLD albumen, then press matter with 1% aluminium hydroxide solutionAmount is mixed with vaccine than 1:1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610053758.0A CN105582531B (en) | 2016-01-27 | 2016-01-27 | Acinetobacter baumannii combined subunit protein vaccine and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610053758.0A CN105582531B (en) | 2016-01-27 | 2016-01-27 | Acinetobacter baumannii combined subunit protein vaccine and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105582531A true CN105582531A (en) | 2016-05-18 |
| CN105582531B CN105582531B (en) | 2020-06-12 |
Family
ID=55922677
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610053758.0A Active CN105582531B (en) | 2016-01-27 | 2016-01-27 | Acinetobacter baumannii combined subunit protein vaccine and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105582531B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110713524A (en) * | 2019-10-31 | 2020-01-21 | 湖北工业大学 | High-sensitivity acinetobacter baumannii antigen Elisa determination kit |
| CN110713523A (en) * | 2019-10-31 | 2020-01-21 | 湖北工业大学 | Detection strip for qualitatively detecting specific antibody of acinetobacter baumannii in human serum |
| CN110950939A (en) * | 2019-12-04 | 2020-04-03 | 南京医科大学第二附属医院 | Acinetobacter baumannii Omp22 recombinant multi-antigen epitope polypeptide and application thereof |
| CN111471616A (en) * | 2020-04-07 | 2020-07-31 | 中国人民解放军陆军军医大学 | Acinetobacter baumannii outer membrane vesicle and preparation method and application thereof |
| CN111876509A (en) * | 2020-09-10 | 2020-11-03 | 广州医科大学附属第一医院（广州呼吸中心） | PCR method for detecting high-toxicity acinetobacter baumannii |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104725492A (en) * | 2015-04-18 | 2015-06-24 | 吉林大学 | Immunity-protective Acinetobacter baumannii surface antigen SurAl |
| CN104861049A (en) * | 2015-04-24 | 2015-08-26 | 中国人民解放军第三军医大学 | Acinetobacter baumannii 1 A1S-1969 recombinant protein and preparation method and application thereof |
| CN104877019A (en) * | 2015-04-24 | 2015-09-02 | 中国人民解放军第三军医大学 | Protein of acinetobacter baumannii hypothetical protein A1S_1523 as well as preparation method and application of protein |
| CN105111288A (en) * | 2015-07-24 | 2015-12-02 | 中国医学科学院医学生物学研究所 | Acinetobacter baumannii subunit vaccine antigen protein, and applications thereof |
-
2016
- 2016-01-27 CN CN201610053758.0A patent/CN105582531B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104725492A (en) * | 2015-04-18 | 2015-06-24 | 吉林大学 | Immunity-protective Acinetobacter baumannii surface antigen SurAl |
| CN104861049A (en) * | 2015-04-24 | 2015-08-26 | 中国人民解放军第三军医大学 | Acinetobacter baumannii 1 A1S-1969 recombinant protein and preparation method and application thereof |
| CN104877019A (en) * | 2015-04-24 | 2015-09-02 | 中国人民解放军第三军医大学 | Protein of acinetobacter baumannii hypothetical protein A1S_1523 as well as preparation method and application of protein |
| CN105111288A (en) * | 2015-07-24 | 2015-12-02 | 中国医学科学院医学生物学研究所 | Acinetobacter baumannii subunit vaccine antigen protein, and applications thereof |
Non-Patent Citations (3)
| Title |
|---|
| DANILO G. MORIEL ET AL.: "Identification of Novel Vaccine Candidates against Multidrug-Resistant Acinetobacter baumannii", 《PLOS ONE》 * |
| HOSSEINGHOLI EZ ET AL.: "In silico analysis of Acinetobacter baumannii phospholipase D as a subunit vaccine candidate", 《ACTA BIOTHEOR》 * |
| 李海涛等: "鲍曼不动杆菌疫苗研究进展", 《中国感染与化疗杂志》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110713524A (en) * | 2019-10-31 | 2020-01-21 | 湖北工业大学 | High-sensitivity acinetobacter baumannii antigen Elisa determination kit |
| CN110713523A (en) * | 2019-10-31 | 2020-01-21 | 湖北工业大学 | Detection strip for qualitatively detecting specific antibody of acinetobacter baumannii in human serum |
| CN110713523B (en) * | 2019-10-31 | 2023-02-03 | 湖北工业大学 | Detection strip for qualitatively detecting acinetobacter baumannii specific antibody in human serum |
| CN110713524B (en) * | 2019-10-31 | 2023-02-03 | 湖北工业大学 | High-sensitivity acinetobacter baumannii antigen Elisa determination kit |
| CN110950939A (en) * | 2019-12-04 | 2020-04-03 | 南京医科大学第二附属医院 | Acinetobacter baumannii Omp22 recombinant multi-antigen epitope polypeptide and application thereof |
| CN111471616A (en) * | 2020-04-07 | 2020-07-31 | 中国人民解放军陆军军医大学 | Acinetobacter baumannii outer membrane vesicle and preparation method and application thereof |
| CN111876509A (en) * | 2020-09-10 | 2020-11-03 | 广州医科大学附属第一医院（广州呼吸中心） | PCR method for detecting high-toxicity acinetobacter baumannii |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105582531B (en) | 2020-06-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105582531A (en) | Acinetobacter baumannii and subunit protein combined vaccine and preparation method thereof | |
| CN100999550B (en) | Tubercle branch bacillus fusion protein and application thereof | |
| CN111018995A (en) | B, T cell epitope tandem fusion vaccine for African swine fever | |
| CN102068690A (en) | Polyvalent pneumococcal capsular polysaccharide conjugate vaccine and preparation method thereof | |
| CN103386128B (en) | Tuberculosis subunit vaccine containing unite adjuvant | |
| CN104292339A (en) | Recombinant protein containing SARS virus RBD antigen and baculovirus displaying RBD protein | |
| CN109055412A (en) | A kind of pig circular ring virus-mycoplasma pneumoniae bigeminy subunit vaccine and preparation method thereof | |
| CN105111288A (en) | Acinetobacter baumannii subunit vaccine antigen protein, and applications thereof | |
| CN104098700B (en) | Mycobacterium tuberculosis fusion protein EAMMH, its structure, expression and purification process and its application | |
| CN102058881B (en) | Gene recombinant vaccine for preventing enterovirus 71 infection and preparation method thereof | |
| CN102286100B (en) | SEB (staphylococcal enterotoxin B) resisting immune globulin F(ab')2 and preparation method thereof | |
| CN102580074B (en) | Riemerella anatipestifer-escherichia coli outer membrane protein bivalent vaccine and preparation method thereof | |
| CN105497884B (en) | Application of the outer membrane protein Omp22 as Acinetobacter bauamnnii vaccine target spot | |
| CN108026538A (en) | The preparation method of the epitheca protein of porcine circovirus 2 type and the medical composition containing the epitheca protein | |
| CN102240399B (en) | Application of siniperca chuatsi ISKNV (Infectious Spleen and Kidney Necrosis Virus) ORF093 protein | |
| CN104004068A (en) | Paralichthys olivaceus beta nodavirus capsid protein with immune protection function and preparing method thereof | |
| CN101914144B (en) | Staphylococcus aureus capsular polysaccharide and protein conjugate and preparation method and application thereof | |
| CN105646681A (en) | Preparation method and application of staphylococcus aureus alpha-hemolysin subunit vaccine for dairy cows | |
| CN105481953B (en) | Target cell specificity fusion protein and vaccine combination as porcine reproductive and respiratory syndrome virus vaccine antigen | |
| CN101757618B (en) | Recombined subunit vaccine of haemaphysalis concinna and preparation method thereof | |
| KR20150123356A (en) | Vaccine Composition for Orientia tsutsugamushi | |
| CN103936842B (en) | Pneumolysin mutants and the application as mucosal adjuvant thereof | |
| CN107129527A (en) | A kind of Malian drainage protective antigens HP0623 and preparation method thereof | |
| CN107828810B (en) | A kind of fusion and its preparing the application in pneumovax | |
| CN102977214B (en) | Recombinant protein HF2 used for methicillin-resistant staphylococcus aureus (MRSA) vaccine, and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CB03 | Change of inventor or designer information |
Inventor after: Pan Pinhua Inventor after: Li Haitao Inventor after: Niu Ruichao Inventor before: Pan Pinhua Inventor before: Li Haitao |
|
| CB03 | Change of inventor or designer information |