CN104877019B - Acinetobacter bauamnnii assumes the albumen and preparation method and application of albumin A 1S_1523 - Google Patents
Acinetobacter bauamnnii assumes the albumen and preparation method and application of albumin A 1S_1523 Download PDFInfo
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- CN104877019B CN104877019B CN201510197921.6A CN201510197921A CN104877019B CN 104877019 B CN104877019 B CN 104877019B CN 201510197921 A CN201510197921 A CN 201510197921A CN 104877019 B CN104877019 B CN 104877019B
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Classifications
-
- 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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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- 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 present invention relates to a kind of recombinant protein of A1S_1523 and its preparation method and application, which includes A1S_1523 mature peptides, and the amino acid sequence of the recombinant protein is as shown in SEQ ID NO.3.The recombinant protein expression quantity is high, and convenient for isolating and purifying, highly effective and safe can be directly used cooperatively with adjuvant, is used to prepare the subunit vaccine of anti-Acinetobacter bauamnnii infection and relevant detection kit.Animal experiment proves that the genetic engineering recombination monovalent subunit vaccine has the immune protective effect of good anti-Acinetobacter bauamnnii infection.It lays the first stone for further combined vaccine and the research of more subunit's fusion bacterins, while the development and application that prevent vaccine and diagnostic kit is played an important role.
Description
Technical field
The present invention relates to biotechnology, more particularly to Acinetobacter bauamnnii assumes albumin A 1S_1523 albumen and preparation
Methods and applications.
Background technology
Acinetobacter bauamnnii (Acinetobacter baumannii) is a kind of common non-fermentative gram-negative bacilli,
It is widespread in nature, can normally be colonized in human skin surface and cavity, be conditioned pathogen, can mainly lead to lung
Inflammation, bloodstream infection, skin and soft tissue infection, endocarditis and meningitis etc..With its antibody-resistant bacterium recall rate year by year
Increase, Acinetobacter bauamnnii has become a kind of important pathogen in the world, according to U.S. NHSN (National
Healthcare Safety Network) report, between 2009 to 2010 years, all kinds of nosocomial infections monitored are separated to
Acinetobacter bauamnnii bacterial strain in, multidrug resistant (MDR) strain has accounted for 65%.CHINET Bacterial resistance surveillance network datas in China are shown
Show, in 14 teaching hospitals of 10 provinces and cities of China in 2010, the Acinetobacter bauamnnii being clinically separated accounts for all Grain-negative bars
The 16.11% of bacterium, ranks third.In addition, Acinetobacter bauamnnii is also by the important of War injury infections relating in soldier's operation
There are very high recall rate in pathogenic bacteria, U.S. army in Iraq, Kuwait and the Afghan wounded to get into action.
Since Acinetobacter bauamnnii drug resistance enhances year by year, normal antibiotics find its therapeutic effect unobvious
New therapeutic scheme or novel active drug fight the infection of Acinetobacter bauamnnii, particularly Multi-drug resistant Acinetobacter baumannii
Infection become very urgent task.It is developed slowly between new antibiotic, and Acinetobacter bauamnnii drug resistance adapts to
Rapidly, many microbe research scholars begin attempt to exempt from using the full bacterium outer membrane outer membrane vesicles of Acinetobacter bauamnnii or outer membrane protein
Epidemic focus, induction body generate anti-Acinetobacter bauamnnii and are immunized, from the angle entirely different with antibiotic, seek effectively to fight
The new tool of drug resistance Acinetobacter bauamnnii.
At present, multiple researchs are verified:Inactivating full bacterial outer membrane protein outer membrane protein vesica and outer membrane has good exempt from
Epidemic focus and antigenicity, the infection and invasion that can resist Acinetobacter bauamnnii of their immune mouse, but from safety etc.
Consider, outer membrane protein is more safer than full bacterium, and therefore, outer membrane protein may be that the optimal candidate of anti-Acinetobacter bauamnnii resists
One of original.
Invention content
The present invention provides a kind of Acinetobacter bauamnnii and assumes albumin A 1S_1523 recombinant proteins, the recombinant protein expression quantity
Height, convenient for isolating and purifying, highly effective and safe can be directly used cooperatively with adjuvant, be used to prepare anti-Acinetobacter bauamnnii infection
Subunit vaccine and relevant detection kit.
The present invention filters out a kind of hypothesis albumin A 1S_1523 using reverse vaccinology, and the albumen is residual by 196 amino acid
Base forms, and amino acid sequence is as shown in SEQ ID NO.5, and DNA sequence dna is as shown in SEQ ID NO.6.Utilize biological information
It learns and structural-functional analysis is carried out to A1S_1523 albumen, it is found that A1S_1523 is a kind of secretory protein, by 1-21 amino acids groups
Into signal peptide guiding secretion to extracellular (shown in attached drawing 6).The present invention utilizes the mature peptide of A1S_1523 albumen, designs a kind of Asia
Subunit vaccine.
Technical scheme of the present invention is specific as follows：
Acinetobacter bauamnnii A1S_1523 recombinant proteins, comprising A1S_1523 mature peptides, the amino acid sequence of the mature peptide
Row are as shown in SEQ ID NO.1.
The amino acid sequence of the recombinant protein is as shown in SEQ ID NO.3.
For the recombinant protein by label protein GST amalgamation and expressions, the label protein is blended in the A1S_1523 eggs
White N-terminal.
The amino acid sequence of the recombinant protein is additionally included in the aminoterminal of SEQ ID NO.1 and/or carboxy terminal deletion, replaces
Generation, and/or the variant with immunogenicity for being added to 1-20 amino acid residue, the variant have with the amino acid sequence
There is at least 80% homogeneity.
The recombinant protein adds in five amino of GPLGS by aminoterminal described in claim 1 after amalgamation and expression and digestion
Acid is formed.
Encode the polynucleotides of A1S_1523 recombinant proteins.
The preparation method of A1S_1523 recombinant proteins, includes the following steps：
1) primer of design PCR is as follows：
Forward primer
5'-CGCGGATCCGACTATAAAATTGATCCAACACA-3'
BamHⅠ
Reverse primer
5'-TTATGCGGCCGCTTATTTTTTAGCTGCACTAGCC-3'
NotⅠ
2) primer designed using step 1) goes out to encode A1S_1523 albumen target gene fragments by PCR amplification；
3) then the gene fragment clone obtained by step 2) is converted to expression vector to host strain；
4) the host strain expression A1S_1523 recombinant proteins after Induction Transformation；
5) purification of recombinant proteins.
Expression vector or host cell include the polynucleotides or host cell of coding A1S_1523 recombinant proteins.
The carrier is pGEX-6P-2 expression vectors；The host cell is Escherichia coli, and the Escherichia coli are XL1-
Blue。
The antibody of anti-A1S_1523 albumen.
A1S_1523 recombinant proteins of the present invention are in the drug for preparing prevention or treatment Acinetobacter bauamnnii infection
Using.And
Application of the A1S_15230 recombinant proteins in Acinetobacter bauamnnii detection kit is prepared.
The present invention uses technique for gene engineering clonal expression this protective antigens A1S_1523 recombinant proteins, and expression quantity is high,
Convenient for isolating and purifying, and highly effective and safe.A1S_1523 recombinant proteins can directly with adjuvant (such as Al (OH)3Adjuvant, AlPO4Assistant
Agent, MF59, AS03, AS04, incomplete Freund's adjuvant, complete Freund's adjuvant etc.) it is used cooperatively, preferably AlPO4Adjuvant is used for flesh
Meat injecting immune.
The expression of the genetic engineering recombinant protein of the present invention has the advantages that following 6：
1.A1S_1523 albumen and recombinant subunit vaccine field is not used for from A1S_1523 albumen, can effectively excited
Body causes protective immune response, so as to resist Acinetobacter bauamnnii lethal infection；
The expression plasmid of 2.A1S_1523 albumen induced expression in prokaryotic expression system (Escherichia coli), expression quantity is high,
Quality safety is controllable；
3. selecting pGEX-6p-2 expression vectors, A1S_1523 recombinant proteins are protected to greatest extent with fusion protein form expression
Its original space conformation is held；
Just contain in fusion protein expressed by 4. there are one GST labels, this label just becomes the label of protein purification so that
Purification condition is mild, step is simple, does not need to the addition of denaturant, so as to which albumen after purification can keep its space to greatest extent
Conformation and immunogenicity；
5.A1S_1523 expressing fusion protein rates are about 30%, and A1S_1523 fusion protein purity after purification is more than
95%；
6.A1S_1523 fusion protein can induce animal to generate specific antibody.
The subunit vaccine prepared using A1S_1523 fusion proteins of the present invention can by subcutaneous (muscle) injecting pathway into
Row immunity inoculation, excitating organism generate IgG antibody and cellullar immunologic response.And animal experiment proves that, the genetic engineering weight
Group monovalent subunit vaccine has the immune protective effect of good anti-Acinetobacter bauamnnii infection.For further combined vaccine
It lays the first stone with the research of more subunit's fusion bacterins, while for the development of prevention vaccine and diagnostic kit and using with important
Effect.
In order to which the object of the invention, technical solution and advantage is more clearly understood, with reference to the accompanying drawings and embodiments, to this
Invention is further elaborated.It should be appreciated that specific embodiment described herein is only to explain the present invention, and do not have to
It is of the invention in limiting.
Description of the drawings
Fig. 1 is the PCR amplification of A1S_1523 genetic fragments as a result, wherein, swimming lane M：Nucleic acid (DNA) molecular weight standard
(Marker)；Swimming lane 1-2：The pcr amplification product of A1S_1523 bases genetic fragment (540bp)；
Fig. 2 is the digestion qualification result of expression vector pGEX-6p-2-A1S_1523：Wherein, swimming lane M：Nucleic acid (DNA) point
Sub- amount standard (Marker)；Swimming lane 4-6：Qualification results of the recombinant expression plasmid pGEX-6p-2-A1S_1523 after digestion,
Middle swimming lane 4-6 represents the segment 4000bp and 540bp detached after digestion；
Fig. 3 represents inducible protein expression of results under different temperatures：Wherein, swimming lane M：Protein Marker (Marker)；
Swimming lane 1：No. 1 recombination engineering is at 16 DEG C after induced expression, the fusion protein that is obtained in supernatant；Swimming lane 2：No. 1 recombination work
Journey bacterium is at 30 DEG C after induced expression, the fusion protein that is obtained in supernatant.
Fig. 4 represents the fusion protein that No. 1 recombination engineering obtains at 30 DEG C after induced expression, wherein, swimming lane M：Albumen
Molecular weight standard (Marker)；Swimming lane 1：After digestion, fusion egg of the non-specific binding on glutathione-Sepharose 4B
The enzyme (b) and GST labels (b), destination protein (c) of (a), specific binding on glutathione-Sepharose 4B in vain；Swimming lane
2：After digestion, the destination protein of acquisition (shown in arrow)；Swimming lane 3：Before digestion, the fusion protein containing A1S_1523-GST of acquisition
(shown in arrow)；
Fig. 5 represents destination protein after purification, wherein, swimming lane M：Protein Marker (Marker)；Swimming lane 1：Purifying
Afterwards, the A1S_1523 recombinant proteins of No. 1 recombination engineering expression are obtained；Swimming lane 2：After purification, No. 2 recombination engineering expression are obtained
A1S_1523 recombinant proteins；
Fig. 6 utilizes online signal peptide analysis software http://www.cbs.dtu.dk/services/SignalP-4.0/
To A1S_1523 protein signal peptide prediction result figures, the results showed that 1-21 amino acid is signal peptide sequence；
Fig. 7 recombinant expression carriers sequencing after with assume albumen DNA sequence dna comparing result, the results showed that DNA sequence dna with reason
By completely the same.
Specific embodiment
Bacterial strain used in the present invention and various reagents are as follows：
1. bacterial strain
17978 international standard strain of Acinetobacter bauamnnii is provided by American ACT T；
2. reagent
Plasmid pGEX-6p-2 (being purchased from GE companies), pET-22b (being purchased from Novagen companies) and coli strain XL-
1blue (being purchased from general such as spit of fland company) is preserved by applicant microorganism teaching and research room；
PrimeSTAR HS DNA Polymerase, DNA Marker, DNA Ligation Mix, restriction enzyme
BamH I and Not I, albumen Marker are Dalian TakaRa Products；
Plasmid extraction kit and gel reclaims kit are U.S.'s Omega Products；
Bacterial genomes extracts kit, ultra-thin QIAquick Gel Extraction Kit and developing solution are Tiangeng Products；
Glutathione-Sepharose Glutathione Sepharose 4B are produced for GE Healthcare companies of the U.S.
Product.
Embodiment 1：The clone of Acinetobacter bauamnnii A1S_1523 albumen
1. first according to 17978 type strain A1S_1523 amino acid sequences of Acinetobacter bauamnnii, using bioinformatics software
Structural analysis is carried out, analysis result is referring to attached drawing 6, so that it is determined that needing the genetic fragment of A1S_1523 albumen expanded.
2. according to analysis result, PCR method is used using 17978 full-length genome of Acinetobacter bauamnnii as template amplification A1S_
The genetic fragment of 1523 albumen, amplification step are as follows：
1) design PCR primer is as follows, respectively SEQ ID NO.7-8 (underscore shows restriction enzyme site base sequence)
Forward primer PA1S1523B1：SEQ ID NO.7
5'-CGCGGATCCGACTATAAAATTGATCCAACACA-3'
BamHⅠ
Reverse primer PA1S1523N2：SEQ ID NO.8
5'-TTATGCGGCCGCTTATTTTTTAGCTGCACTAGCC-3'
NotⅠ
The nucleotide sequence SEQ ID that the present embodiment will encode A1S_1523 protein amino acid sequences shown in SEQ ID NO.1
Genetic fragment carries out PCR amplification to NO.2 as a purpose.
2) 17978 bacterial strain of Acinetobacter bauamnnii that preservation is taken out in -80 DEG C of freezers is coated on special LB solid mediums
On, 8 hours of culture in LB fluid nutrient mediums are inoculated in 37 DEG C of overnight incubations, then picking single bacterium colony, with reference to bacterial genomes
Extraction agent box extracts full-length genome.
3) using 17978 complete genome DNA of Acinetobacter bauamnnii as template PCR amplifications A1S_1523 protein gene segments
PCR system：
98 DEG C of pcr amplification reaction condition pre-degeneration 40s, 94 DEG C of denaturation 10s, 60 DEG C of annealing 30s, 72 DEG C of extension 1min, 30
A cycle, 72 DEG C of fully extended 10min.PCR amplification is detected as a result, PCR amplification using 1% Ago-Gel after completion of the reaction
The results are shown in Figure 1.
4) A1S_1523PCR products are recycled using gel reclaims kit.
The identification of 3.PCR products and clone, step are as follows：
1) BamH I and Not I digestion pGEX-6P-2 plasmids and A1S_1523PCR products
Endonuclease reaction system：
37 DEG C of digestion 3h.
2) using ultra-thin QIAquick Gel Extraction Kit recycling pGEX-6P-2 plasmids and the PCR product through BamH I and Not I digestions.
3) it connects and converts
Target gene digestion recovery product nucleic acid concentration is measured by ultraviolet specrophotometer：58ng/ μ l, pGEX-6P-2
Digestion recovery product nucleic acid concentration：48ng/ μ l, according to carrier and exogenous sequences molal quantity generally than being 1：2-10, design are following
Coupled reaction system.
Coupled reaction system：
Mixing, 16 DEG C of connection 1h.
4) 3 pipe Escherichia coli XL-1blue competent cells being taken from -80 DEG C of refrigerators, the first pipe adds in pGEX-6P-2 plasmids,
Make positive control；Second pipe adds in DNA connection products；Third pipe is not added with exogenous DNA, makees negative control.Ice bath 30min, 42 DEG C
Thermal shock 90s in metal bath, rapid ice bath 2min.600ul LB blank cultures are added in, mixing is placed in 200rp in 37 DEG C of shaking tables
Shake 1h.
Each pipe centrifuges 5min. with 5000rpm room temperatures, discards 400ul supernatants, then thalline is resuspended, 100 μ l is taken to be coated on Amp
Resistance LB tablets.Tablet is inverted in 37 DEG C of incubators and cultivates for 24 hours.
5) screening, the identification of pGEX-6p-2/A1S_1523 positive recombinant plasmids
1. negative control plates do not have bacterium colony appearance；Positive control tablet covers with bacterium colony, illustrates that competent cell makes just
Really, credible result.Separate good bacterium colony on picking conversion tablet, be inoculated in Amp resistance LB culture mediums, 37 DEG C of shaken cultivations
Overnight；
2. plasmid extraction：It is carried out with reference to plasmid extraction kit specification；
3. Plasmid DNA carries out BamH I and Not I double digestions；
Double digestion reaction system：
37 DEG C of digestion 2h；
4. 1% agarose gel electrophoresis detects double digestion as a result, the results are shown in Figure 2, it is seen that swimming lane 1-4 samples are structure
Build successful pGEX-6p-2/A1S_1523 recombinant plasmids；
5. pGEX-6p-2/A1S_1523 recombinant plasmids are sent to Beijing Huada gene company sequencing, sequencing result comparison result
As shown in Figure 7, it is seen that the sequence of the target gene fragment of recombinant plasmid is correct.
Embodiment 2：Acinetobacter bauamnnii -17978A1S_1523 albumen induces in prokaryotic expression system-Escherichia coli
Expression, purifying and the identification of expression-form
1. destination protein induced expression
1) double digestion is taken to identify correct two recombined engineerings strain pGEX-6P-2-A1S_1523/XL-1blue bacterium solutions
100 μ L are added in the TB culture mediums of 10mL Amp resistances, and 37 DEG C of 100rpm is incubated overnight, and takes the bacterium solution 2ml being incubated overnight respectively
It adds in the TB culture mediums of 18mL Amp resistances (remaining bacterium solution is stored in spare in 4 DEG C of refrigerators), 37 DEG C of culture 2-3h, rotating speed
250rpm when re-activation to OD600 is 0.8-1.2, adds in 4.4 μ L of IPTG, makes its final concentration of 200 μM, then be placed in shaking table
30 DEG C of 3h of induced expression, 16 DEG C of overnight induced expressions.
2) bacterium solution after induced expression is taken out, 2min is centrifuged with 1000rpm, is discarded supernatant, adds in 1mL PBS buffer solution
Mixing, ultrasound cracking 3min, then 4 DEG C of 14000rpm centrifugation 15min, collect supernatant.
2. handle supernatant
20 μ l of glutathione-Sepharose 4B are taken, after washing 3 times with PBS, ready supernatant is added in into gluathione
In peptide-Ago-Gel 4B, 4 DEG C of rotations combine (or room temperature combination 1h) overnight.After centrifuging 3min at 4 DEG C with 5000rpm, make
It is washed 2 times with PBS-0.25% polysorbas20s, PBS washed once.Glutathione-Sepharose 4B after combination adds in 5 μ l
5 × protein sample-loading buffer adds in processing 5min, 10000rpm centrifugation 2min.
5% concentration glue is poured into glue version, equals glue laminated adding in distilled water, be placed at room temperature for by 3.SDS-PAGE electrophoresis
30min makes its solidification, and the distilled water on upper strata is fallen to do, then pour into 10% separation gel, comb is plugged immediately, is placed at room temperature for 30min
Make its solidification spare.
4. the Supernatant samples handled well are taken 10 μ L loadings respectively, SDS-PAGE electrophoresis is carried out.Voltage elder generation 80V electrophoresis
30min, then 200V is adjusted to, after electrophoresis 45min, glue is taken out, is placed in coomassie brilliant blue staining liquid and vibrates dyeing, then be placed in de-
In color liquid after oscillation decoloration, observation is as a result, the results are shown in Figure 3 under imaging system, PGEX-6P-2-A1S_1523/XL-
1blue has the A1S_1523 containing GST labels that can give expression to that molecular size range is about 100kDa under the conditions of 30 DEG C, 16 DEG C
Albumen, and recombinant protein is in the supernatant of ultrasound cracking, therefore the recombinant protein is solvable in each inducing temperature condition
Property albumen is very high with 30 DEG C of expression quantity at 16 DEG C.Two recombination engineerings (1#, 2#) selected are without significant difference.
Embodiment 3：The preparation of A1S_1523 proteantigens
1. amplification culture obtains albumen
The pGEX-6P-2-A1S_1523/XL-1blue strains (1#, 2#) gone bail in -80 DEG C of refrigerators of presence are inoculated in respectively
LB ammonia benzyl resistant panels, 37 DEG C of overnight incubations；Picking single bacterium colony is inoculated in 100ml LB ammonia benzyl resistance culture bases, 37 DEG C,
200rpm overnight incubations；The 100ml bacterium solutions of activation are added in the LB culture mediums of 2L resistances containing Amp and carry out re-activation, 37
DEG C culture 3-4h to OD600 be 1.2 when, add in 420 μ l IPTG (final concentration of 200 μM) be placed in 30 DEG C of shaking tables induce 3h after,
6000rpm centrifugation 5min collect thalline, then add 80ml PBS be resuspended thalline after, by bacterium solution carry out ultrasound cracking 30min, ibid from
The heart is collected supernatant and is combined with 4ml glutathione-Sepharoses 4B；Obtain the largely A1S_1523 fusions containing GST labels
Albumen.
2. using enzymatic cleavage methods, destination protein and GST labels are separated, obtain A1S_1523 recombinant proteins
4ml PBS and 120 μ L are added in into remainder about 4ml the glutathione-Sepharose 4B of binding purpose albumen
PreScission protease (PP enzymes), 4 DEG C of vertical rotary digestions are stayed overnight, and after supernatant is drawn in centrifugation, are washed respectively with 2ml PBS
Wash 2 times, after taking 10 μ L sample denaturation treatments, 10 μ L of loading carry out SDS-PAGE protein electrophoresis, under imaging system observation as a result,
A1S_1523 molecular weight of albumen is obtained after digestion in 19kDa or so, is consistent with expected molecular weight of albumen size, No. 1 recombination work
After journey bacterium expresses albumen and preliminary purification electrophoresis result as shown in figure 4, swimming lane 1 represents digestion, three kinds of substances of appearance, a:It is not complete
The fusion protein of slitting-up；b:GST labels and enzyme；c:In the destination protein that supernatant obtains；After swimming lane 2 represents digestion, for the first time
The destination protein that detergent gel pearl obtains；Swimming lane 3 represents fusion protein before digestion.
3. taking the supernatant (containing destination protein) of No. 1 and No. 2 recombination engineerings expression albumen after digestion, further use
Sephadex G25 replace buffer solution, and destination protein is stored in histidine buffering liquid (10 μm of histidines, pH6.0).As a result
As shown in Figure 5.
4.BCA methods measure No. 1 protein concentration, a concentration of 0.8mg/mL.
Embodiment 4：The foundation of infection Acinetobacter bauamnnii (international standard strain 17978) standard quantitative curve
Inoculation is placed in 37 DEG C in MH tablets to be incubated 24 hours；The picking single bacterium colony on tablet is inoculated in MH liquid
In culture medium, be placed in 37 DEG C of constant-temperature tables shake culture after 6 hours 6000rpm centrifuge 10min and collect thalline, use physiological saline
Washing thalline 2 times；Bacterium solution is carried out 10 times and 1.25 times again to dilute, and measure under ultraviolet spectrometry system each bacterium solution
Absorbance (OD600) at 600nm, and the 100 μ l of bacterium solution of each dilution is taken to be coated on MH tablets, it is small to be placed in 37 DEG C of incubations 24
When after count bacterium colony；Standard quantitative curve is drawn according to the OD600 values of each flat-plate bacterial colony number and bacterium solution.
As a result：Calibration curve formula is Y=3.012X+0.0051 (109CFU/ml), related coefficient 0.9998.
Embodiment 5：The structure of septicopyemia animal model
1. inoculation is placed in 37 DEG C in MH tablets to be incubated 24 hours；The picking single bacterium colony on tablet is inoculated in MH liquid
In body culture medium, it is placed in shake culture in 37 DEG C of constant-temperature tables and thalline is collected after 6 hours, and determined using calibration curve formula
Amount, then it is 2.0 × 10 that bacterium solution is diluted (or concentration)9CFU/mL、2.1×109CFU/mL、4×109CFU/mL various concentration groups,
Again with each group bacterium solution by the way that 6-8 week old is injected intraperitoneally, BALB/C mice that weight is 18-20g (100 μ l/ are only) carry out whole body sense
Dye, while saline control group is set, observe 7 days and count the death rate of each group mouse；
2nd, timing is detected bacteria planting amount using colony counting method in every for 24 hours (observation 7 days) after infecting：From each
3 mouse are randomly selected in infected group and control group, using eyeball excise method, 100 μ l of mouse blood sample is taken to be applied to tablet, are placed in 37
DEG C, count clone's number afterwards for 24 hours；Mouse execution is put into 75% alcohol after soaking disinfection after taking blood sample, is taken out its four limbs
It is fixed, it is dissected, takes out spleen, kidney, liver, be placed in the sterile PBS of 1mL, be homogenized in clean glass homogenizer,
100 μ l homogenates are taken according to 1:10、1:100、1:1000 ratios are diluted；100 μ L is taken gently to be coated on solid per dilution
On culture medium, 37 DEG C are placed in, culture for 24 hours, does bacterium colony counting.
As a result it is shown in table 1：
- 17978 infective dose of 1 Acinetobacter bauamnnii of table and lethal dose determine
2.0×108Mouse death rate is 0 in CFU dosage groups 7 days；2.2×108Mouse death rate is in CFU dosage groups 48h
20%；4.2×108Mouse death rate is 90% in CFU dosage groups 48h；It can be seen that -17978 infectious agent of Acinetobacter bauamnnii
Measure is 2.2 × 108CFU, sublethal dose are 4.2 × 108CFU。
3. the field planting amount after the infection BALB/C mice of Acinetobacter bauamnnii -17978 in blood and each internal organs：
Bacterium reaches peak value when for 24 hours in lung after infection, and maximum field planting amount reaches 2.0 × 104CFU/ml, in 48h
Amount of bacteria starts to reduce in lung, and bacterium is not detected into lung during 72h；Bacterium when for 24 hours reaches peak value in kidney after infection,
Maximum field planting amount reaches 1.0 × 103CFU/ml, in 48h, amount of bacteria starts to reduce in lung, is not detected into lung during 72h thin
Bacterium；The bacterium being colonized in blood, spleen, heart, liver after infection is not detected；Blood, spleen, kidney, the liver of control group mice
In bacteria planting testing result be zero.
Result above has carried out animal for the survival rate and blood, spleen, kidney, liver major organs bacteria planting amount of mouse
The evaluation of model is the successful development of the single subunit vaccine of Acinetobacter bauamnnii and Acinetobacter bauamnnii subunit fusion bacterin
And the research of the pathogenesis of Acinetobacter bauamnnii infection is laid a good foundation.
Embodiment 6：Immune animal and the detection of antibody
1. immune animal
1) first immunisation, by A1S_1523 proteantigens and AlPO4Adjuvant physical mixed preserves liquid antigen is dense with albumen
Degree is adjusted to 200 μ g/ml, is placed in 4 DEG C of Rotary adsorptions and vaccine is made overnight；With No. 5 half mould syringe needles, bilateral inguinal is injected,
Every BALB/C mice injection volume is 150 μ l, and set negative control group (3 adjuvant groups of Al (OH)) and blank control group (albumen
Preserve liquid group)；
2) it is immunized for second, carries out within the 14th day being immunized for second, immune component is same as above, injection volume and the phase of first immunisation
Together, immunization route is same as above；
3) third time is immune, carries out third time within the 21st day and is immunized, immune component is same as above, injection volume and the phase of first immunisation
Together, immunization route is same as above；
7th and 14 day after 2. third time is immune, the blood of BALB/C mice is acquired, with IgG bodies after ELISA detection mouse immunes
Liquid response is horizontal.
1) liquid is prepared
1. the preparation of coating buffer：Weigh NaHCO31.6g, Na2CO32.9g is dissolved in 1L ddH2O is counted with PH and is adjusted to pH
9.6；
2. the preparation of confining liquid：1g cow's serums V are dissolved in 100mL antibody diluents (1:100)；
3. the preparation of antibody diluent：Phosphate is dissolved in 1L ddH2O adds 500 μ l Tween 20, then is counted with PH
PH is adjusted to 7.4；
4. the preparation of cleaning solution：It weighs 2.42g Tris and is dissolved in 1L ddH2O adds 500 μ l Tween 20, then uses PH
PH is adjusted to 7.4 by meter；
5. developing solution (TMB) is Tiangeng Products；
6. terminate liquid (2M H2SO4) preparation：The 22.2mL concentrated sulfuric acids are poured into 177.8mL ddH2In O.
2) ELISA detects the antibody titer that mouse generation is immunized in A1S_1523 recombinant proteins
1. A1S_1523 recombinant proteins after purification are diluted to 10 μ g/mL with coating buffer；
2. it is coated with：By recombinant protein dilution add in ELISA Plate, 200 μ l/ holes, 4 DEG C overnight after washed 3 times with cleaning solution；
3. it closes：ELISA Plate adds 100 μ l/ holes of confining liquid, is placed in 37 DEG C of incubators 2 hours, washs 3 times；
4. serum is carried out 1:1000、1:2000、1:4000、1:8000、1:16000 grade doubling dilutions；
5. taking the ELISA Plate closed, dilute serum is sequentially added, 100 μ l/ holes, 37 DEG C of incubator 30min are placed in, are washed
It washs 3 times, sky is dry；
6. the goat anti-mouse igg antibody that HRP is marked will be added to preserve liquid, dilution 1：5000, antibody working solution is made；
7. adding in dilution antibody working solution, 100 μ l/ holes are placed in 37 DEG C of incubator 45min, wash three times, and sky is dry；
8. adding in substrate developing solution (TMB) 100 μ l/ holes, room temperature is protected from light 5min；
9. add in terminate liquid (2M H2SO4), it is immediately placed in microplate reader to measure OD values at 450nm wavelength；
10. result judges：ASampleAIt is negativeZhi≤2.1 are positive (negative control is serum before mouse immune).
As a result：The antibody titer that mouse generation is immunized in detection A1S_1523 proteantigens reaches 1:128000；7th after immune
It antibody positive rate reaches 90%, and immune antibody positive rate 14th day latter reaches 100%；Illustrate the A1S_ that the present invention is built
1523 recombinant proteins can make to generate antibody in immune Mice Body.
Embodiment 7：Determine that A1S_1523 recombinant proteins were immunized animal attacks malicious protection by the way that mouse is immunized
With the immunization protocol of embodiment 6, after the immune mouse of third time, at the 14th day using lethal dose, Bao is injected intraperitoneally
Graceful -17978 viable bacteria of acinetobacter calcoaceticus carries out challenge viral dosage, and every BALB/C mice injection bacterium solution amount is 4 × 108CFU, observation 10
My god, the survival rate of statistics each group mouse.As a result it is shown in table 2.
Table 2
Table 2 is shown：It is animal immune experiment (experiment is 10 mouse every time) as a result, result shows negative control group in table
Average immune protective rate with blank control group is that 10%, A1S_1523 recombinant proteins are aided with being averaged for 3 adjuvant groups of Al (OH)
Immune protective rate is 50%.
Therefore, A1S_1523 recombinant proteins of the invention have good immunogenicity, and can be to Bao Man not levers
Immune protective is played the role of in the infection of bacterium -17978, and body can be induced to generate immune response, can for example be aided with aluminium adjuvant system
Standby subunit vaccine is used to prevent the infection of Acinetobacter bauamnnii.
The foregoing is merely presently preferred embodiments of the present invention, is not used for limiting the practical range of the present invention；If it does not take off
It from the spirit and scope of the present invention, modifies or equivalently replaces the present invention, should all cover in the claims in the present invention
In protection domain.
Claims (10)
1. a kind of Acinetobacter bauamnnii A1S_1523 recombinant proteins, it is characterised in that：Comprising A1S_1523 mature peptides, the maturation
The amino acid sequence of peptide is as shown in SEQ ID NO.1, and the amino acid sequence of the recombinant protein is as shown in SEQ ID NO.3.
2. the polynucleotides of coding A1S_1523 recombinant proteins described in claim 1.
3. the preparation method of A1S_1523 recombinant proteins described in claim 1, which is characterized in that include the following steps：
1）The primer for designing PCR is as follows：
Forward primer
5'-CGCGGATCCGACTATAAAATTGATCCAACACA -3'
Reverse primer
5'-TTATGCGGCCGCTTATTTTTTAGCTGCACTAGCC -3'；
2）Use step 1）The primer of design goes out to encode by PCR amplification the target gene fragment of the mature peptide；
3）Step 2）Then the gene fragment clone of gained is converted to the expression vector containing GST labels to host strain；
4）Host strain expressed fusion protein after Induction Transformation, the fusion protein are blended in the A1S_ by GST label proteins
The N-terminal of 1523 mature peptides is formed；
5）Purified fusion albumen, PreScission protease digestion fusion proteins, obtains A1S_1523 recombinant proteins.
4. a kind of expression vector, it is characterised in that：Include the polynucleotides of recombinant protein described in coding claim 1.
5. expression vector according to claim 4, it is characterised in that：The expression vector is pGEX-6P-2.
6. a kind of host strain, it is characterised in that：Include the expression vector described in claim 4.
7. host strain according to claim 6, it is characterised in that：The host strain is Escherichia coli XL1-Blue.
8. resist the antibody of A1S_1523 recombinant proteins described in claim 1.
9. A1S_1523 recombinant proteins described in claim 1 are in the drug for preparing prevention or treatment Acinetobacter bauamnnii infection
Application.
10. application of the A1S_1523 recombinant proteins described in claim 1 in Acinetobacter bauamnnii detection kit is prepared.
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| CN105582531B (en) * | 2016-01-27 | 2020-06-12 | 中南大学湘雅医院 | Acinetobacter baumannii combined subunit protein vaccine and preparation method thereof |
| CN109266658B (en) * | 2018-10-17 | 2022-08-19 | 昆明理工大学 | Acinetobacter baumannii specific gene and primer and application thereof |
| CN110079623B (en) * | 2019-05-27 | 2022-11-11 | 深圳市人民医院 | Method for detecting toxicity of acinetobacter baumannii |
| CN110746496B (en) * | 2019-09-30 | 2023-07-07 | 四川大学华西医院 | PAL recombinant protein of Acinetobacter baumannii, encoding gene thereof and application of PAL recombinant protein and encoding gene |
| 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 |
| CN110950939B (en) * | 2019-12-04 | 2022-05-17 | 南京医科大学第二附属医院 | Acinetobacter baumannii Omp22 recombinant multi-antigen epitope polypeptide and application thereof |
| 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 |
-
2015
- 2015-04-24 CN CN201510197921.6A patent/CN104877019B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| hypothetical protein [Acinetobacter baumannii];Accession ID：WP_000550328.1;《GenBank Database》;20150328;ORIGIN部分 * |
| 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;马真等;《南方医科大学学报》;20091231;第29卷(第6期);第1265-1267页 * |
| 鲍曼不动杆菌外膜蛋白与耐药性分析;黄艳飞等;《中国微生态学杂志》;20040630;第16卷(第3期);第144-145页 * |
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