CN104877019A - Protein of acinetobacter baumannii hypothetical protein A1S_1523 as well as preparation method and application of protein - Google Patents
Protein of acinetobacter baumannii hypothetical protein A1S_1523 as well as preparation method and application of protein Download PDFInfo
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- CN104877019A CN104877019A CN201510197921.6A CN201510197921A CN104877019A CN 104877019 A CN104877019 A CN 104877019A CN 201510197921 A CN201510197921 A CN 201510197921A CN 104877019 A CN104877019 A CN 104877019A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
-
- 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
- A61K39/0208—Specific bacteria not otherwise provided for
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1217—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Neisseriaceae (F)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag
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- General Health & Medical Sciences (AREA)
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- Engineering & Computer Science (AREA)
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- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biomedical Technology (AREA)
- Cell Biology (AREA)
- Mycology (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Analytical Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to recombinant protein of A1S_1523 as well as a preparation method and application of the recombinant protein. The recombinant protein comprises A1S_1523 mature peptide, and an amino acid sequence of the recombinant protein is shown in SEQ ID NO. 3. The recombinant protein disclosed by the invention is high in expression quantity and convenient to separate and purify, is efficient and safe, can be directly matched with adjuvants for use, and can be used for preparing acinetobacter baumannii infection resistant subunit vaccines and related detection kits; proven by animal experiments, gene engineering recombinant monovalent subunit vaccines have good immune protective effects on acinetobacter baumannii infection resistance; and the recombinant protein can be used for laying a foundation for further researching combined vaccines and multi-subunit fusion vaccines, and can also play important roles in the development and application of prevention and treatment vaccines and diagnostic kits.
Description
Technical field
The present invention relates to biological technical field, particularly Acinetobacter bauamnnii putative protein A1S_1523 albumen and preparation method and application.
Background technology
Acinetobacter bauamnnii (Acinetobacter baumanni i) is a kind of common non-fermentative gram-negative bacilli, be widespread in nature, can normally be colonizated in human skin and cavity, for conditioned pathogen, mainly pneumonia, bloodstream infection, skin and soft tissue infection, endocarditis and meningitis etc. can be caused.Along with increasing year by year of its Resistant strain recall rate, Acinetobacter bauamnnii has become a kind of important pathogenic bacteria in the world, according to the report of U.S. NHSN (Nat ional Healthcare Safety Network), between 2009 to 2010 years, in the Acinetobacter bauamnnii bacterial strain that its all kinds of ward infections monitored are separated to, multidrug resistant (MDR) strain has accounted for 65%.China CHINET Bacterial resistance surveillance network data shows, and in 14 teaching hospitals of 10 provinces and cities of China in 2010, the Acinetobacter bauamnnii of clinical separation accounts for 16.11% of all gram negative bacillis, ranks the 3rd.In addition, Acinetobacter bauamnnii is also the important pathogenic bacteria being subject to War injury infections relating during soldier fights, and has very high recall rate in the wounded that U.S. army gets into action in Iraq, Kuwait and Afghanistan.
Because Acinetobacter bauamnnii resistance strengthens year by year, normal antibiotics is not obvious to its therapeutic action, new treatment plan or novel active drug is found to become instant task to the infection resisting the infection of Acinetobacter bauamnnii, particularly Multi-drug resistant Acinetobacter baumannii.Between new microbiotic slower development, and Acinetobacter bauamnnii resistance adapts to rapidly, many microbe research scholars start the immunogenicity attempting utilizing Acinetobacter bauamnnii full bacterium adventitia outer membrane vesicles or outer membrane protein, induction body produces the immunity of anti-Acinetobacter bauamnnii, from angle diverse with microbiotic, seek the effective new tool to overriding resistance Acinetobacter bauamnnii.
At present, multiple research is verified: deactivation full bacterial outer membrane protein outer membrane protein vesica and adventitia have good immunogenicity and antigenicity, the infection resisting Acinetobacter bauamnnii of their immune mouse and invasion and attack, but consider from aspects such as securities, outer membrane protein is more safer than full bacterium, therefore, outer membrane protein may be one of optimal candidate antigen of anti-Acinetobacter bauamnnii.
Summary of the invention
The invention provides a kind of Acinetobacter bauamnnii putative protein A1S_1523 recombinant protein, this expression of recombinant proteins amount is high, is convenient to separation and purification, highly effective and safe, can directly and adjuvant with the use of, the subunit vaccine infected for the preparation of anti-Acinetobacter bauamnnii and relevant detection kit.
The present invention utilizes reverse vaccinology to filter out a kind of putative protein A1S_1523, and this albumen is made up of 196 amino-acid residues, and its aminoacid sequence is as shown in SEQ ID NO.5, and its DNA sequence dna is as shown in SEQ ID NO.6.Utilize information biology to carry out structural-functional analysis to A1S_1523 albumen, find that A1S_1523 is a kind of secretory protein, the signal peptide be made up of 1-21 amino acids guides secretion to born of the same parents outer (shown in accompanying drawing 6).The present invention utilizes the mature peptide of A1S_1523 albumen, designs a kind of subunit vaccine.
Technical scheme of the present invention is specific as follows:
Acinetobacter bauamnnii A1S_1523 recombinant protein, comprises A1S_1523 mature peptide, and the aminoacid sequence of described mature peptide is as shown in SEQ ID NO.1.
The aminoacid sequence of described recombinant protein is as shown in SEQ ID NO.3.
Described recombinant protein is by label protein GST amalgamation and expression, and described label protein is blended in the N end of described A1S_1523 albumen.
The aminoacid sequence of described recombinant protein is also included in the aminoterminal of SEQ ID NO.1 and/or carboxy terminal deletion, substitute and/or with the addition of 1-20 amino-acid residue there is immunogenic variant, this variant and described aminoacid sequence have the identity of at least 80%.
This recombinant protein is by amalgamation and expression and enzyme adds GPLGS five amino acid at aminoterminal according to claim 1 after cutting is formed.
The polynucleotide of coding A1S_1523 recombinant protein.
The preparation method of A1S_1523 recombinant protein, comprises the following steps:
1) primer designing PCR is as follows:
Forward primer
5'-CGC
GGATCCGACTATAAAATGATCCAACACA-3'
BamHⅠ
Reverse primer
5'-TTAT
GCGGCCGCTTATTTTTTAGCTGCACTAGCC-3'
NotⅠ
2) step 1 is used) primer that designs to be gone out to encode A1S_1523 albumen goal gene fragment by pcr amplification;
3) step 2) gene fragment clone of gained to expression vector, be then converted into Host Strains;
4) Host Strains after Induction Transformation expresses A1S_1523 recombinant protein;
5) purification of recombinant proteins.
Expression vector or host cell, comprise polynucleotide or the host cell of coding A1S_1523 recombinant protein.
Described carrier is pGEX-6P-2 expression vector; Described host cell is intestinal bacteria, and described intestinal bacteria are XL1-Blue.
The antibody of anti-A1S_1523 albumen.
The application of A1S_1523 recombinant protein of the present invention in the medicine that preparation prevents or treatment Acinetobacter bauamnnii infects.And
A1S_15230 recombinant protein is preparing the application in Acinetobacter bauamnnii detection kit.
The present invention adopts this protective antigen of genetic engineering technique clonal expression A1S_1523 recombinant protein, and expression amount is high, is convenient to separation and purification, and highly effective and safe.A1S_1523 recombinant protein can directly and adjuvant (as Al (OH)
3adjuvant, AlPO
4adjuvant, MF59, AS03, AS04, incomplete Freund's adjuvant, complete Freund's adjuvant etc.) with the use of, preferred AlPO
4adjuvant is used for intramuscular injection immunity.
The expression method of genetically engineered recombinant protein of the present invention has following 6 advantages:
1.A1S_1523 albumen and from A1S_1523 albumen all not for recombinant subunit vaccine field, effectively can cause protective immune response by excitating organism, thus resist Acinetobacter bauamnnii lethal infection;
Expression plasmid abduction delivering in prokaryotic expression system (intestinal bacteria) of 2.A1S_1523 albumen, expression amount is high, and quality safety is controlled;
3. select pGEX-6p-2 expression vector, A1S_1523 recombinant protein, with fusion protein form expression, maintains its original space conformation to greatest extent;
4. just containing a GST label in the fusion rotein expressed by, this label just becomes the mark of protein purification, make that purification condition is gentle, step simply, does not need adding of denaturing agent, thus the albumen after purifying can keep its space conformation and immunogenicity to greatest extent;
5.A1S_1523 expressing fusion protein rate is about 30%, and the A1S_1523 fusion rotein purity after purifying is greater than 95%;
6.A1S_1523 fusion rotein can produce specific antibody by induced animal.
The subunit vaccine utilizing A1S_1523 fusion rotein of the present invention to prepare carries out immunization by subcutaneous (muscle) injecting pathway, and excitating organism produces IgG antibody and cellullar immunologic response.And confirm through experimentation on animals, described genetically engineered restructuring monovalent subunit vaccine has the immune protective effect that good anti-Acinetobacter bauamnnii infects.For further combined vaccine and the fusion bacterin research of many subunits lay the first stone, development and application simultaneously for preventing and treating vaccine and diagnostic kit have important effect.
In order to make the object of the invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Accompanying drawing explanation
Fig. 1 is the pcr amplification result of A1S_1523 gene fragment, wherein, and swimming lane M: nucleic acid (DNA) molecular weight standard (Marker); The pcr amplification product of swimming lane 1-2:A1S_1523 base gene fragment (540bp);
Fig. 2 is that the enzyme of expression vector pGEX-6p-2-A1S_1523 cuts qualification result: wherein, swimming lane M: nucleic acid (DNA) molecular weight standard (Marker); Swimming lane 4-6: the recombinant expression plasmid pGEX-6p-2-A1S_1523 qualification result after enzyme is cut, wherein swimming lane 4-6 all represents that enzyme cuts fragment 4000bp and the 540bp of rear separation;
Fig. 3 represents inducible protein expression of results under differing temps: wherein, swimming lane M: Protein Marker (Marker); Swimming lane 1:1 recombinant bacterial strain at 16 DEG C after abduction delivering, the fusion rotein obtained in supernatant; Swimming lane 2:1 recombinant bacterial strain at 30 DEG C after abduction delivering, the fusion rotein obtained in supernatant.
Fig. 4 represents the fusion rotein that No. 1 recombinant bacterial strain obtains after abduction delivering at 30 DEG C, wherein, and swimming lane M: Protein Marker (Marker); Swimming lane 1: after enzyme is cut, the fusion rotein (a) of non-specific binding on glutathione-Sepharose 4B, the enzyme (b) of specific binding on glutathione-Sepharose 4B and GST label (b), target protein (c); Swimming lane 2: after enzyme is cut, the target protein (shown in arrow) of acquisition; Swimming lane 3: before enzyme is cut, acquisition containing A1S_1523-GST fusion rotein (arrow shown in);
Fig. 5 represents the target protein after purifying, wherein, and swimming lane M: Protein Marker (Marker); Swimming lane 1: after purifying, obtains the A1S_1523 recombinant protein that No. 1 recombinant bacterial strain is expressed; Swimming lane 2: after purifying, obtains the A1S_1523 recombinant protein that No. 2 recombinant bacterial strains are expressed;
Fig. 6 utilizes online signal peptide analysis software http://www.cbs.dtu.dk/services/SignalP-4.0/ to predict the outcome figure to A1S_1523 protein signal peptide, and result shows that 1-21 amino acid is signal peptide sequence;
After the order-checking of Fig. 7 recombinant expression vector and the DNA sequence dna comparing result of putative protein, result show DNA sequence dna and theory completely the same.
Embodiment
Bacterial strain used in the present invention and all ingredients as follows:
1. bacterial strain
The strain of Acinetobacter bauamnnii 17978 international standard is provided by American ACT T;
2. reagent
Plasmid pGEX-6p-2 (being purchased from GE company), pET-22b (being purchased from Novagen company) and coli strain XL-1blue (being purchased from general as spit of fland company) are preserved by applicant microorganism teaching and research room;
PrimeSTAR HS DNA Polymerase, DNA Marker, DNA Ligat ion Mix, restriction enzyme BamH I and Not I, albumen Marker are Dalian TakaRa Products;
It is U.S. Omega Products that plasmid extraction kit and gel reclaim test kit;
It is sky root Products that bacterial genomes extracts test kit, ultra-thin recovery test kit and nitrite ion;
Glutathione-Sepharose Glutathione Sepharose 4B is U.S. GE Healthcare Products.
Embodiment 1: the clone of Acinetobacter bauamnnii A1S_1523 albumen
1. first according to Acinetobacter bauamnnii 17978 type strain A1S_1523 aminoacid sequence, applying biological information software carries out structural analysis, and analytical results see accompanying drawing 6, thus determines the gene fragment of the A1S_1523 albumen needing amplification.
2., according to analytical results, adopt the gene fragment that PCR method is template amplification A1S_1523 albumen with Acinetobacter bauamnnii 17978 full-length genome, amplification step is as follows:
1) design PCR primer as follows, be respectively SEQ ID NO.7-8 (underscore shows restriction enzyme site base sequence)
Forward primer PA1S1523B1:SEQ ID NO.7
5'-CGC
GGATCCGACTATAAAATGATCCAACACA-3'
BamHⅠ
Reverse primer PA1S1523N2:SEQ ID NO.8
5'-TTAT
GCGGCCGCTTATTTTTTAGCTGCACTAGCC-3'
NotⅠ
The nucleotide sequence SEQ ID NO.2 of A1S_1523 protein amino acid sequence shown in coding SEQ ID NO.1 is carried out pcr amplification as goal gene fragment by the present embodiment.
2) Acinetobacter bauamnnii 17978 bacterial strain taking out preservation in-80 DEG C of freezers is coated on special LB solid medium, in 37 DEG C of overnight incubation, picking list colony inoculation is cultivated 8 hours in LB liquid nutrient medium again, with reference to bacterial genomes extraction agent box extracting full-length genome.
3) with Acinetobacter bauamnnii 17978 complete genome DNA for template PCR amplifications A1S_1523 protein gene fragment
PCR system:
Pcr amplification reaction condition 98 DEG C of denaturation 40s, 94 DEG C of sex change 10s, 60 DEG C of annealing 30s, 72 DEG C extend 1min, and 30 circulations, 72 DEG C extend 10min completely.Use the sepharose of 1% to detect pcr amplification result after completion of the reaction, pcr amplification result as shown in Figure 1.
4) use gel to reclaim test kit and reclaim A1S_1523PCR product.
The qualification of 3.PCR product and clone, step is as follows:
1) BamH I and Not I enzyme cut pGEX-6P-2 plasmid and A1S_1523PCR product
Endonuclease reaction system:
37 DEG C of enzymes cut 3h.
2) PCR primer that ultra-thin recovery test kit reclaims pGEX-6P-2 plasmid and cuts through BamH I and Not I enzyme is used.
3) connect and transform
Measure goal gene enzyme by ultraviolet spectrophotometer and cut back to close product nucleic acid concentration: 58ng/ μ l, pGEX-6P-2 enzyme cuts back to close product nucleic acid concentration: 48ng/ μ l, according to carrier with exogenous sequences mole number generally than being 1:2-10, design following ligation system.
Ligation system:
Mixing, 16 DEG C connect 1h.
4) get 3 pipe intestinal bacteria XL-1blue competent cells from-80 DEG C of refrigerators, the first pipe adds pGEX-6P-2 plasmid, makes positive control; Second pipe adds DNA and connects product; 3rd pipe does not add foreign DNA, makes negative control.Ice bath 30min, thermal shock 90s in 42 DEG C of metal baths, rapid ice bath 2min.Add 600ulLB blank cultures, mixing, is placed in 37 DEG C of shaking table 200rp jolting 1h.
Each pipe, with the centrifugal 5min. of 5000rpm room temperature, discards 400ul supernatant, more resuspended thalline, gets 100 μ l and coats Amp resistance LB flat board.Flat-plate inverted is placed in 37 DEG C of incubators and cultivates 24h.
5) screening of the positive recombinant plasmid of pGEX-6p-2/A1S_1523, qualification
1. negative control plates does not have bacterium colony to occur; Positive control flat board covers with bacterium colony, illustrates that competent cell makes correct, credible result.Picking transformation plate separates good bacterium colony, and be inoculated in Amp resistance LB substratum, 37 DEG C of shaking culture are spent the night;
2. plasmid extraction: carry out with reference to plasmid extraction kit specification sheets;
3. plasmid DNA carries out BamH I and Not I double digestion;
Double digestion reaction system:
37 DEG C of enzymes cut 2h;
4. the agarose gel electrophoresis of 1% detects double digestion result, and as shown in Figure 2, visible swimming lane 1-4 sample is the pGEX-6p-2/A1S_1523 recombinant plasmid successfully constructed to result;
5. pGEX-6p-2/A1S_1523 recombinant plasmid is sent to the order-checking of Beijing Hua Da genome company, and as shown in Figure 7, the sequence of the goal gene fragment of visible recombinant plasmid is correct to sequencing result comparison result.
Embodiment 2: the qualification of Acinetobacter bauamnnii-17978A1S_1523 albumen abduction delivering, purifying and expression-form in prokaryotic expression system-intestinal bacteria
1. target protein abduction delivering
1) get double digestion and identify that two correct recombined engineering bacterial classification pGEX-6P-2-A1S_1523/XL-1blue bacterium liquid 100 μ L add in the TB substratum of 10mL Amp resistance, 100rpm 37 DEG C of incubated overnight, the bacterium liquid 2ml getting incubated overnight respectively adds (remaining bacterium liquid is kept in 4 DEG C of refrigerators for subsequent use) in the TB substratum of 18mL Amp resistance, cultivate 2-3h for 37 DEG C, rotating speed 250rpm, when re-activation is 0.8-1.2 to OD600, add IPTG 4.4 μ L, its final concentration is made to be 200 μMs, be placed in shaking table abduction delivering 30 DEG C of 3h again, 16 DEG C of abduction deliverings that spend the night.
2) taken out by the bacterium liquid after abduction delivering, with the centrifugal 2min of 1000rpm, supernatant discarded, adds the mixing of 1mLPBS damping fluid, ultrasonic degradation 3min, then 4 DEG C of centrifugal 15min of 14000rpm, collects supernatant.
2. process supernatant
Get glutathione-Sepharose 4B 20 μ l, after washing 3 times, added by ready supernatant in glutathione-Sepharose 4B with PBS, 4 DEG C of rotations are spent the night combination (or room temperature is in conjunction with 1h).With after the centrifugal 3min of 5000rpm at 4 DEG C, use PBS-0.25% polysorbas20 to wash 2 times, PBS washing once.Glutathione-Sepharose 4B after combining adds 5 μ l 5 × protein sample-loading buffers, adds the centrifugal 2min of process 5min, 10000rpm.
3.SDS-PAGE electrophoresis, pours in glue version by 5% concentrated glue, and glue laminated put down adding distilled water, room temperature places 30min makes it solidify, and is fallen by the distilled water on upper strata dry, then pours into 10% separation gel, plug comb immediately, and room temperature places 30min, and to make it solidify for subsequent use.
4. the Supernatant samples handled well is got respectively 10 μ L loadings, carry out SDS-PAGE electrophoresis.Voltage first 80V electrophoresis 30min, be adjusted to 200V again, after electrophoresis 45min, glue is taken out, be placed in coomassie brilliant blue staining liquid vibration dyeing, after being placed in destainer vibration decolouring again, observations under imaging system, result as shown in Figure 3, PGEX-6P-2-A1S_1523/XL-1blue has at 30 DEG C, the A1S_1523 albumen containing GST label that molecular size range is about 100kDa all can be given expression under 16 DEG C of conditions, and recombinant protein is all in the supernatant of ultrasonic degradation, therefore described recombinant protein is soluble proteins in each inducing temperature condition, all very high with 30 DEG C of expression amounts at 16 DEG C.Two recombinant bacterial strains (1#, 2#) selected are without significant difference.
The preparation of embodiment 3:A1S_1523 proteantigen
1. amplification culture obtains albumen
The pGEX-6P-2-A1S_1523/XL-1blue bacterial classification (1#, 2#) existed in-80 DEG C of refrigerators of going bail for is inoculated in LB ammonia benzyl resistant panel, 37 DEG C of overnight incubation respectively; Picking list colony inoculation in 100ml LB ammonia benzyl resistance culture base, 37 DEG C, 200rpm overnight incubation; The 100ml bacterium liquid of activation is joined 2L and carry out re-activation containing in the LB substratum of Amp resistance, when 37 DEG C of cultivation 3-4h to OD600 are 1.2, adding 420 μ l IPTG (final concentration is 200 μMs) is placed in after 30 DEG C of shaking tables induce 3h, the centrifugal 5min of 6000rpm collects thalline, after adding the resuspended thalline of 80ml PBS again, bacterium liquid is carried out ultrasonic degradation 30min, and the same collected by centrifugation supernatant is combined with 4ml glutathione-Sepharose 4B; Obtain a large amount of A1S_1523 fusion roteins containing GST label.
2. use enzymatic cleavage methods, separates target protein and GST label, obtains A1S_1523 recombinant protein
4ml PBS and 120 μ L PreScission protease (PP enzyme) is added in the glutathione-Sepharose 4B of remainder about 4ml binding purposes albumen, 4 DEG C of vertical rotary enzymes cut through night, after centrifugal absorption supernatant, 2 times are washed respectively with 2ml PBS, after getting 10 μ L denaturing samples process, loading 10 μ L carries out SDS-PAGE protein electrophoresis, observations under imaging system, enzyme cuts rear acquisition A1S_1523 molecular weight of albumen at about 19kDa, be consistent with expection molecular weight of albumen size, No. 1 recombinant bacterial strain expressing protein and preliminary purification electrophoresis result are as shown in Figure 4, after swimming lane 1 represents that enzyme is cut, the three kinds of materials occurred, a: the fusion rotein do not cut completely, b:GST label and enzyme, c: the target protein obtained at supernatant, after swimming lane 2 represents that enzyme is cut, the target protein of first time detergent gel pearl acquisition, swimming lane 3 represents that enzyme cuts front fusion rotein.
3. get No. 1 and the supernatant of No. 2 recombinant bacterial strain expressing proteins after enzyme is cut (containing target protein), replace damping fluid with Sephadex G25 further, target protein is stored in (10 μm of Histidines, pH6.0) in histidine buffering liquid.Result as shown in Figure 5.
4.BCA method measures No. 1 protein concentration, and concentration is 0.8mg/mL.
Embodiment 4: infect the foundation with Acinetobacter bauamnnii (international standard strain 17978) standard quantitative curve
Inoculation is placed in 37 DEG C in MH flat board and hatches 24 hours; On flat board, picking list bacterium colony, is inoculated in MH liquid nutrient medium, be placed in 37 DEG C of constant-temperature tables shake cultivation after 6 hours the centrifugal 10min of 6000rpm collect thalline, with brine thalline 2 times; Again bacterium liquid is carried out 10 times and 1.25 times of dilutions, and under ultraviolet spectrometry system, measure the absorbancy at 600nm place (OD600) of each bacterium liquid, and get each dilution bacterium liquid 100 μ l and coat MH flat board, be placed in 37 DEG C hatch 24 hours after count bacterium colony; According to the OD600 value drawing standard quantitation curves of each flat-plate bacterial colony number and bacterium liquid.
Result: typical curve formula is Y=3.012X+0.0051 (10
9cFU/ml), relation conefficient is 0.9998.
Embodiment 5: the structure of pyemia animal model
1. inoculation is placed in 37 DEG C in MH flat board and hatches 24 hours; On flat board, picking list bacterium colony, is inoculated in MH liquid nutrient medium, is placed in 37 DEG C of constant-temperature tables and shakes cultivation 6 h before harvest thalline, and utilize typical curve formula to carry out quantitatively, then is 2.0 × 10 by bacterium liquid dilution (or concentrated)
9cFU/mL, 2.1 × 10
9cFU/mL, 4 × 10
9cFU/mL different concns group, systemic infection is carried out by the BALB/C mice (100 μ l/ only) that abdominal injection 6-8 week age, body weight are 18-20g again with each group of bacterium liquid, saline control group is set simultaneously, observes 7 days and add up the mortality ratio of each group of mouse;
2, after infecting, timing is adopting colony counting method to detect bacteria planting amount every 24h (observing 7 days): random selecting 3 mouse from each infected group and control group, utilize eyeball excise method, get mouse blood sample 100 μ l and be applied to flat board, be placed in 37 DEG C, counting clone number after 24h; After sacrifice being put into 75% alcohol soaking disinfection after taking blood sample, take out and its four limbs are fixed, dissected, take out spleen, kidney, liver, be placed in the PBS that 1mL is aseptic, in the glass homogenizer of cleaning, carry out homogenate, get 100 μ l homogenates and dilute according to 1:10,1:100,1:1000 ratio; Every extent of dilution is got 100 μ L and is coated gently on solid medium, is placed in 37 DEG C, cultivates 24h, does enumeration.
The results are shown in table 1:
The determination of table 1 Acinetobacter bauamnnii-17978 infective dose and lethal dose
2.0 × 10
8in CFU dosage group 7 days, mouse death rate is 0; 2.2 × 10
8in CFU dosage group 48h, mouse death rate is 20%; 4.2 × 10
8in CFU dosage group 48h, mouse death rate is 90%; Acinetobacter bauamnnii-17978 infective dose is 2.2 × 10 as can be seen here
8cFU, sublethal dose is 4.2 × 10
8cFU.
3. the field planting amount after Acinetobacter bauamnnii-17978 infection BALB/C mice in blood and each internal organs:
After infecting in lung bacterium reach peak value when 24h, maximum field planting amount reaches 2.0 × 10
4cFU/ml, when 48h, in lung, amount of bacteria starts to reduce, in lung during 72h, do not detect bacterium; After infecting in kidney bacterium reach peak value when 24h, maximum field planting amount reaches 1.0 × 10
3cFU/ml, when 48h, in lung, amount of bacteria starts to reduce, in lung during 72h, do not detect bacterium; After infecting, in blood, spleen, heart, liver, the bacterium of field planting does not all detect; Bacteria planting detected result in the blood of control group mice, spleen, kidney, liver is zero.
Above result has carried out the evaluation of animal model for the survival rate of mouse and blood, spleen, kidney, liver major organs bacteria planting amount, and the pathogenetic research that successful development and Acinetobacter bauamnnii for the single subunit vaccine of Acinetobacter bauamnnii and Acinetobacter bauamnnii subunit fusion bacterin infect is laid a good foundation.
Embodiment 6: the detection of immune animal and antibody
1. immune animal
1) first immunisation, by A1S_1523 proteantigen and AlPO
4adjuvant physical mixed, is adjusted to 200 μ g/ml with albumen conserving liquid by antigen concentration, and is placed in 4 DEG C of Rotary adsorption and spends the night and make vaccine; With No. 5 half mould syringe needles, bilateral inguinal is injected, and every BALB/C mice injection volume is 150 μ l, and arranges negative control group (Al (OH) 3 adjuvant group) and blank group (albumen conserving liquid group);
2) second time immunity, within the 14th day, carry out second time immunity, immune component is the same, and injection volume is identical with first immunisation, and immunization route is the same;
3) third time immunity, within the 21st day, carry out third time immunity, immune component is the same, and injection volume is identical with first immunisation, and immunization route is the same;
2. third time the immune rear 7th and 14 days, gather the blood of BALB/C mice, by IgG humoral response level after ELISA detection mouse immune.
1) liquid is prepared
1. the preparation of coating buffer: take NaHCO
31.6g, Na
2cO
32.9g, is dissolved in 1L ddH
2o, is adjusted to 9.6 with PH meter by pH;
2. the preparation of confining liquid: 1g bovine serum V, is dissolved in 100mL antibody diluent (1:100);
3. the preparation of antibody diluent: phosphoric acid salt is dissolved in 1L ddH
2o, then add 500 μ l Tween 20, then with PH meter, pH is adjusted to 7.4;
4. the preparation of washings: take 2.42g Tris and be dissolved in 1L ddH
2o, then add 500 μ l Tween 20, then with PH meter, pH is adjusted to 7.4;
5. nitrite ion (TMB) is sky root Products;
6. stop buffer (2M H
2sO
4) preparation: the 22.2mL vitriol oil is poured into 177.8mL ddH
2in O.
2) ELISA detects the antibody titer that A1S_1523 recombinant protein immune mouse produces
1. being diluted by the A1S_1523 recombinant protein after purifying with coating buffer is 10 μ g/mL;
2. wrap quilt: recombinant protein diluent is added enzyme plate, 200 μ l/ holes, 4 DEG C spend the night after wash 3 times with washings;
3. close: enzyme plate adds confining liquid 100 μ l/ hole, is placed in 37 DEG C of incubators 2 hours, wash 3 times;
4. serum is carried out the doubling dilutions such as 1:1000,1:2000,1:4000,1:8000,1:16000;
5. get the enzyme plate closed, add dilute serum successively, 100 μ l/ holes, be placed in 37 DEG C of incubator 30min, wash 3 times, empty dry;
6. will add the goat anti-mouse igg antibody conserving liquid of HRP mark, dilution 1:5000, makes antibody working fluid;
7. add dilution antibody working fluid, 100 μ l/ holes, are placed in 37 DEG C of incubator 45min, wash three times, empty dry;
8. substrate nitrite ion (TMB) 100 μ l/ hole is added, room temperature lucifuge reaction 5min;
9. stop buffer (2M H is added
2sO
4), be placed in immediately in microplate reader and measure OD value with 450nm wavelength place;
10. result judges: A
samplea
negativezhi≤2.1 are positive (negative control is serum before mouse immune).
Result: the antibody titer detecting the generation of A1S_1523 proteantigen immune mouse reaches 1:128000; After immunity, the antibody positive rate of the 7th day reaches 90%, and after immunity, the antibody positive rate of the 14th day reaches 100%; Illustrate that the A1S_1523 recombinant protein that the present invention builds can make to produce antibody in immune mouse body.
Embodiment 7: by immune mouse determine A1S_1523 recombinant protein immune animal attack poison protection
With the immunization protocol of embodiment 6, for the third time after immune mouse, adopted lethal dose at the 14th day, abdominal injection Acinetobacter bauamnnii-17978 viable bacteria carries out challenge viral dosage, and every BALB/C mice injection bacterium liquid measure is 4 × 10
8cFU, observes 10 days, adds up the survival rate of each group of mouse.Result is shown in table 2.
Table 2
Table 2 shows: be animal immune experiment (each experiment is 10 mouse) result; in table, result display negative control group and the average immune protective rate of blank group are the average immune protective rate that 10%, A1S_1523 recombinant protein is aided with Al (OH) 3 adjuvant group is 50%.
Therefore; A1S_1523 recombinant protein of the present invention has good immunogenicity; and can infect Acinetobacter bauamnnii-17978 and play immune protective effect; body can be induced to produce immunne response, such as can be aided with aluminium adjuvant and prepare subunit vaccine for preventing the infection of Acinetobacter bauamnnii.
The foregoing is only preferred embodiment of the present invention, be not used for limiting practical range of the present invention; If do not depart from the spirit and scope of the present invention, the present invention is modified or equivalent to replace, in the middle of the protection domain that all should be encompassed in the claims in the present invention.
Claims (12)
1. an Acinetobacter bauamnnii A1S_1523 recombinant protein, is characterized in that: comprise A1S_1523 mature peptide, and the aminoacid sequence of described mature peptide is as shown in SEQ ID NO.1.
2. albumen according to claim 1, is characterized in that: the aminoacid sequence of described recombinant protein is as shown in SEQ ID NO.3.
3. recombinant protein according to claim 1, is characterized in that: described recombinant protein is by label protein GST amalgamation and expression, and described label protein is blended in the N end of described A1S_1523 albumen.
4. recombinant protein according to claim 1, it is characterized in that: the aminoacid sequence of described recombinant protein is also included in the aminoterminal of SEQ ID NO.1 and/or carboxy terminal deletion, substitute and/or with the addition of 1-20 amino-acid residue there is immunogenic variant, this variant and described aminoacid sequence have the identity of at least 80%.
5. recombinant protein according to claim 1, is characterized in that: this recombinant protein is by amalgamation and expression and enzyme adds GPLGS five amino acid at aminoterminal according to claim 1 after cutting is formed.
6. the polynucleotide of coding A1S_1523 recombinant protein according to claim 1.
7. the preparation method of A1S_1523 recombinant protein according to claim 1, is characterized in that, comprise the following steps:
1) primer designing PCR is as follows:
Forward primer
5'-CGC
GGATCCGACTATAAAATGATCCAACACA-3'
BamHⅠ
Reverse primer
5'-TTAT
GCGGCCGCTTATTTTTTAGCTGCACTAGCC-3'
NotⅠ
2) step 1 is used) primer that designs to be gone out to encode A1S_1523 albumen goal gene fragment by pcr amplification;
3) step 2) gene fragment clone of gained to expression vector, be then converted into Host Strains;
4) Host Strains after Induction Transformation expresses A1S_1523 recombinant protein;
5) purification of recombinant proteins.
8. expression vector or a host cell, is characterized in that: the polynucleotide or the host cell that comprise recombinant protein described in coding claim 1.
9. carrier according to claim 7 or host cell, is characterized in that: described carrier is pGEX-6P-2 expression vector; Described host cell is intestinal bacteria, and described intestinal bacteria are XL1-Blue.
10. the antibody of anti-A1S_1523 albumen according to claim 1.
11. the application of A1S_1523 recombinant protein according to claim 1 in the medicine that preparation prevents or treatment Acinetobacter bauamnnii infects.
12. A1S_1523 recombinant proteins according to claim 1 are preparing the application in Acinetobacter bauamnnii detection kit.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105582531A (en) * | 2016-01-27 | 2016-05-18 | 中南大学湘雅医院 | Acinetobacter baumannii and subunit protein combined vaccine and preparation method thereof |
| CN109266658A (en) * | 2018-10-17 | 2019-01-25 | 昆明理工大学 | The specific gene and its primer of a kind of Acinetobacter bauamnnii and application |
| CN110079623A (en) * | 2019-05-27 | 2019-08-02 | 深圳市人民医院 | A kind of detection method suitable for Acinetobacter bauamnnii virulence |
| 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 |
| CN110713524A (en) * | 2019-10-31 | 2020-01-21 | 湖北工业大学 | High-sensitivity acinetobacter baumannii antigen Elisa determination kit |
| CN110746496A (en) * | 2019-09-30 | 2020-02-04 | 四川大学华西医院 | PAL recombinant protein of acinetobacter baumannii, coding gene thereof and application thereof |
| 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 |
| CN116445448A (en) * | 2023-06-14 | 2023-07-18 | 四川大学华西医院 | Acinetobacter baumannii PLPFP recombinant protein, preparation method and application |
-
2015
- 2015-04-24 CN CN201510197921.6A patent/CN104877019B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| ACCESSION ID：WP_000550328.1: "hypothetical protein [Acinetobacter baumannii]", 《GENBANK DATABASE》 * |
| 马真等: "鲍曼不动杆菌外膜蛋白CarO的原核表达、纯化和多克隆抗体的制备", 《南方医科大学学报》 * |
| 黄艳飞等: "鲍曼不动杆菌外膜蛋白与耐药性分析", 《中国微生态学杂志》 * |
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| CN109266658A (en) * | 2018-10-17 | 2019-01-25 | 昆明理工大学 | The specific gene and its primer of a kind of Acinetobacter bauamnnii and application |
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| 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 |
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| 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 |
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| CN116445448B (en) * | 2023-06-14 | 2023-09-12 | 四川大学华西医院 | Acinetobacter baumannii PLPFP recombinant protein, preparation method and application |
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