CN105732778A - Pseudomonas aeruginosa recombinant protein OprL as well as preparation method and application thereof - Google Patents
Pseudomonas aeruginosa recombinant protein OprL as well as preparation method and application thereof Download PDFInfo
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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
- C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (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
- A61K39/104—Pseudomonadales, e.g. Pseudomonas
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- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
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Abstract
The invention provides a pseudomonas aeruginosa recombinant protein OprL as well as a preparation method and application thereof. Animal tests show that the recombinant protein can effectively stimulate an organism to generate relatively high humoral immunity response and excellent immune protective effect, so as to facilitate prevention, diagnosis and treatment of pseudomonas aeruginosa. The recombinant protein prepared according to the preparation method is high in expression quantity and convenient to separate and purify.
Description
Technical field
The invention belongs to biotechnological pharmaceutics field, be specifically related to a kind of Pseudomonas aeruginosa recombiant protein OprL, the invention further relates to the preparation method of this recombiant protein and application thereof.
Background technology
Pseudomonas aeruginosa (Pseudomonasaeruginosa, PA) it is commonly called as bacillus pyocyaneus, for the Rhodopseudomonas in non-zymocyte, it is distributed widely in nature, normal human skin, intestinal, respiratory tract, also ubiquity in hospital ward and medical apparatus and instruments, is one of most commonly seen clinically conditioned pathogen.At present in the world, this bacterium has become ICU ward, burn, War injury infection, one of ventilator-associated pneumonia (VAP) pathogen that separation rate is the highest.PA infects and can occur at any position of human body and tissue, such as burn or wound site, middle ear, cornea, urethra and respiratory tract etc., it is possible to causing the systemic infection such as endocarditis, gastroenteritis, pneumonia even septicemia, systemic infection mortality rate is more than 20%.
Additionally, due to reasons such as antibiotic abuses, the resistance problems of PA highlights gradually, occurs in that general tolerant Pseudomonas aeruginosa (PDR-PA) and multi-resistant Pseudomonas aeruginosa (MDR-PA), and the separation rate of drug resistance PA rises year by year.China's continuous monitoring materials of CHINET2005--2012 shows, the resistant rate of Common Antibiotics is maintained at higher level by PA, but slightly on a declining curve.Annual resistant rate respectively 31.0%, 42.8%, 35.8%, 30.5%, 30.5%, 30.8%, 29.1% and 29.1% such as imipenum, to the annual resistant rate of meropenem respectively 32.0%, 34.1%, 28.5%, 24.5%, 25.2%, 25.8%, 25.0% and 27.1%, but wherein general drug resistance (PDR-PA) bacterial strain quantity significantly increases, respectively reached 1.8% and 1.5% (pneumonia caused by Pseudomonas aeruginosa infected specialist's common recognition at 2011 and 2012, China's tuberculosis and breathing magazine, volume 1 phase January 37 in 2014).Constantly soaring due to PA high infection rate clinically and resistant rate, finds new " non-antibiotic therapy " extremely urgent, starts with from immunology angle, develop the selection that safely and effectively vaccine becomes ideal.
Genetic engineering subunit vaccine is by engineered means, and by the gene clone of the virulence factor of pathogen or its mutant protein expression vector extremely, recon is expressed on a large scale in engineering bacteria, prepares into genetic engineering subunit vaccine after purification.This vaccine has that technique is simple, cost is low, workable etc. a little, it has also become the important directions of vaccine research and development.Research and development recombinant vaccine it is crucial that screen good protective antigen molecule.
OprL is the Peptidoglycan associated protein (peptidoglycanassociatedlipoprotein) of Pseudomonas aeruginosa, and it is positioned at the adventitia of Pseudomonas aeruginosa, plays an important role in the physiology and pathological process of antibacterial.OprL albumen is guarded at Pseudomonas aeruginosa camber, has had research to it can be used as diagnosis target spot .Clin.Microbiol.Antimicrob.2004 such as () Xu, J. of Pseudomonas aeruginosa.Studies have found that the existence (Montor finding there is the antibody of anti-OprL in the serum of patient simultaneously; W.R etc., InfectImmun2009), OprL can also produce protectiveness Th17 immune response (Wu by inducing mouse in addition; W etc., AmJRespirCritCareMed2012).For all these reasons, OprL can as good recombinant vaccine candidate antigens.
Summary of the invention
It is an object of the invention to provide a kind of Pseudomonas aeruginosa recombiant protein OprL and preparation method and application; described recombiant protein proves through animal experiment; higher humoral immunoresponse(HI) and good immanoprotection action can be produced by effective stimulus body, be conducive to the prevention of Pseudomonas aeruginosa, diagnose and treat.Adopting recombiant protein prepared by illustrated method, expression is high, be easy to separation purification.
For achieving the above object, the present invention provides following technical scheme:
Pseudomonas aeruginosa recombiant protein OprL comprises Pseudomonas aeruginosa Peptidoglycan albumen (PeptidoglycanAssociatedLipoprotein) fragment, and the nucleotides sequence of described recombiant protein is classified as SEQIDNO:1, and aminoacid sequence is SEQIDNO:2.
A kind of expression vector, comprises the nucleotide sequence encoding above-mentioned recombiant protein.Described expression vector can be connected, by skeleton plasmid, the nucleotide sequence encoding above-mentioned recombiant protein and be formed with modifying；Preferably, described skeleton plasmid is selected from pGex serial carrier, pET serial carrier or pQE serial carrier.
Present invention preferably employs pGEX-6p-2 carrier to build recombinant expression plasmid, express recombiant protein OprL, pGEX is the carrier of the expressed fusion protein built in 1987 by Smith and Johnson, it is mainly characterized by carrier being connected to the glutathione-S-transferase (GST) that molecular weight is 26kDa, just containing a GST label in expressed fusion protein, this label is the labelling of protein purification.Compared with other fusion vectors, pGEX serial carrier has purification condition gentleness, step is simple, do not need the addition of denaturant, so that the albumen after purification can keep its space conformation and immunogenicity to greatest extent.
Comprise the Host Strains of above-mentioned expression vector.Described Host Strains one in E.colistrain XL1 blue, BL21 or HMS174 bacterial strain, it is preferred to E.colistrain XL1 blue bacterial strain.
The preparation method of above-mentioned recombiant protein, comprises the following steps:
1) nucleotide sequence according to coding Pseudomonas aeruginosa recombiant protein OprL synthesizes or designs forward primer and reverse primer, does template pcr amplification with Pseudomonas aeruginosa full-length genome and obtains Pseudomonas aeruginosa recombinant subunit vaccine OprL genes of interest fragment；
2) by step 1) gained gene fragment clone to expression vector, then convert to Host Strains；
3) Host Strains after Induction Transformation expresses OprL recombiant protein.
4) purification of OprL recombiant protein.
Step 1) described in forward primer and the nucleotide sequence of reverse primer such as shown in SEQIDNO:3 and SEQIDNO:4.
Above-mentioned recombiant protein is used for the application diagnosing, prevent or treating in the medicine of Pseudomonas aeruginosa in preparation.
Described medicine is the test kit for diagnosing Pseudomonas aeruginosa.
Described medicine is the subunit vaccine for preventing or treat Pseudomonas aeruginosa.
The present invention adopts this recombination fusion protein of technique for gene engineering clonal expression, and expression is high, it is simple to separation purification, and highly effective and safe.OprL recombiant protein can directly be inoculated with the use of, it is adaptable to injecting immune with adjuvant (such as aluminum hydroxide adjuvant, Aluminium phosphate adjuvant, monostearate aluminium adjuvant, MF59, complete Freund's adjuvant, incomplete Freund's adjuvant, mycobacteria bacillus calmette-guerin vaccine adjuvant etc.).
Owing to have employed technique scheme, present invention have the advantage that:
1) OprL expression of recombinant proteins plasmid abduction delivering in prokaryotic expression system-escherichia coli；
2), when selecting pGEX carrier families, OprL recombiant protein is with fusion protein form expression；Expression vector is connected to the glutathione-S-transferase (GST) that molecular weight is 26kDa, expressed fusion protein just contains a GST label, this label just becomes the labelling of protein purification, the albumen after purification makes the addition that purification condition is gentle, step is simple, do not need denaturant, thus can keep its space conformation and immunogenicity to greatest extent；The OprL recombiant protein purity being purified is more than 95%；
3) OprL recombiant protein can produce specific antibody and have immune protective effect by induced animal.
Utilizing subunit vaccine prepared by OprL recombiant protein of the present invention can carry out immunity inoculation by subcutaneous (muscle) injecting pathway, excitating organism produces high titre IgG antibody.And animal experiment proves that, described genetic engineering recombinant protein vaccine has the good anti-PA immune protective effect infected.Studying for further combined vaccine and many subunits fusion bacterin and lay the first stone, development and application for preventing and treating vaccine and diagnostic kit simultaneously has important effect.
Accompanying drawing explanation
Fig. 1 is the pcr amplification result of OprL genetic fragment；Wherein, swimming lane 1: nucleic acid (DNA) molecular weight standard (Marker), from top to bottom size be respectively as follows: 4500,3000,2000,1200,800,500,200bp；；Swimming lane 2: genes of interest fragment OprL (～450bp)；
Fig. 2 is the double digestion qualification result of recombiant plasmid pGex-6P-2-OprL；Wherein, swimming lane 1: nucleic acid (DNA) molecular weight standard (Marker), from top to bottom size be respectively as follows: 4500,3000,2000,1200,800,500,200bp；Swimming lane 2～6: recombinant expression plasmid pGEX-6p-2-OprL qualification result after enzyme action, the fragment separated after enzyme action is about 4000bp and about 450bp；
Fig. 3 is that albumen OprL induces qualification result；Wherein, swimming lane 1: Protein Marker (Marker), size is respectively as follows: 170Kd, 130Kd, 100Kd, 70Kd, 55Kd, 40Kd, 35Kd, 25Kd, 15Kd, 10Kd from top to bottom；Swimming lane 2&3: the GST filler of the ultrasonic supernatant that zygotic induction is expressed；Swimming lane 4: the supernatant after PP enzyme enzyme action；Swimming lane 5: the GST filler after PP enzyme enzyme action；
Fig. 4 is the albumen OprLSDS-PAGE electrophoresis result after purification, wherein, swimming lane 1: Protein Marker (Marker), size is respectively as follows: 170Kd, 130Kd, 100Kd, 70Kd, 55Kd, 40Kd, 35Kd, 25Kd, 15Kd, 10Kd from top to bottom；Swimming lane 2: the OprL albumen of purification.
Detailed description of the invention
In order to make the purpose of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is described in more detail.Should be appreciated that the specific embodiment herein saying description is only in order to explain the present invention, is not intended to limit the present invention.
Bacterial strain is as follows with various reagent:
1. pseudomonas aeruginosa strains
Pseudomonas aeruginosa international standard strain PA01 by U.S. ATCC provide (BAA-47TM)；
2. (preserving number is CCTCCNO:M2015730 to P. aeruginosa clinical strain XN-1, and Classification And Nomenclature is: Pseudomonas aeruginosa XN-1, Latin title: PseudomonasaeruginosaXN-1, preservation time: 2015.12.9；Depositary institution: China typical culture collection center (CCTCC)；Preservation place: Bayi Road Luo Jia Shan, Wuchang District, Wuhan City, Hubei Province)；
3. reagent
Plasmid pGEX-6p-2 is purchased from GEHealthcareLifeSciences company, and applicant preserves；
Coli strain XL-1blue is purchased from Chao Yan bio tech ltd, Shanghai, and applicant preserves；
PrimeSTARHSDNAPolymerase, DNAMarker, restricted enzyme BamHI and EcoRI, albumen Marker are Dalian TakaRa Products；
It is U.S.'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；
T4DNALigase is Fermentas Products；
Glutathione-Sepharose GlutathioneSepharose4B is U.S.'s GEHealthcare Products.
The clone of embodiment 1:OprL gene and the structure of recombiant plasmid pGEX-6P-2-OprL
1. first OprL full length protein gene order according to Pseudomonas aeruginosa PA01, applying biological information software carries out structural analysis, it is determined that need the OprL genes of interest fragment of amplification.
2. according to analyzing result, adopting PCR method from PA01 genome amplification OprL genes of interest fragment, amplification step is as follows:
1) design PCR primer is as follows, respectively SEQIDNO:3-4 (underscore shows restriction enzyme site base sequence)
| Seq ID | Primer | Sequence (5 '-3 ') | Restriction endonuclease |
| Seq ID 3 | OprL-F | CGCGGATCCTGCTCCTCCAAGGGCG | BamH I |
| Seq ID 4 | OprL-R | CCGGAATTCTTACTTCTTCAGCTCGACG | EcoR I |
2) the pseudomonas aeruginosa strains PA01 taking out preservation in-80 DEG C of freezers coats on LB solid medium, in 37 DEG C of overnight incubation, picking list colony inoculation is cultivated 8 hours in LB liquid bulk culture medium again, with reference to bacterial genomes extraction agent box extracting PA01 genome.
3) with PA01 complete genome DNA for template PCR amplifications OprL genetic fragment
PCR system:
| Template (239.2ng/ μ l) | 2.5μl |
| OprL-F(50μM) | 1.0μl |
| OprL-R(50μM) | 1.0μl |
| PrimeSTAR enzyme | 2.5μl |
| dNTP | 2.0μl |
| Buffer | 15.0μl |
| Sterilizing distilled water | 26.0μl |
| Cumulative volume | 50.0μl |
98 DEG C of denaturation 5min of pcr amplification reaction condition, 98 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C extend 90s, 30 circulations, 72 DEG C of fully extended 6min.Using the agarose gel detection pcr amplification result of 1% after completion of the reaction, pcr amplification result is shown in Figure 1.
4) use gel to reclaim test kit and reclaim OprLPCR product.
The qualification of 3.PCR product and clone, step is as follows:
1) BamHI and EcoRI enzyme action pGEX-6P-2 plasmid and OprLPCR product
Endonuclease reaction system:
| Project | Volume |
| BamH I | 3.0μl |
| EcoR I | 3.0μl |
| 10×K Buffer | 3.0μl |
| 0.1%BSA | 6.0μl |
| Product | 45.0μl |
37 DEG C of enzyme action 2h.
2) ultra-thin recovery test kit is used to reclaim pGEX-6P-2 plasmid and the PCR primer through BamHI and EcoRI enzyme action.
3) connect and convert
Measure OprL enzyme action by ultraviolet spectrophotometer and reclaim product nucleic acid concentration: 21ng/ μ l, pGEX-6P-2 enzyme action reclaims product nucleic acid concentration: 60ng/ μ l.
Coupled reaction system:
| Project | Volume |
| Coupled reaction liquid Soultion I | 5.0μl |
| OprL enzyme action reclaims product | 4.5μl |
| PGEX-6P-2 enzyme action reclaims product | 0.5μl |
Mixing, 16 DEG C connect 2h.
4) taking 3 pipe escherichia coli XL1blue 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 is not added with foreign DNA, makes negative control.Ice bath 50min, thermal shock 90s in 42 DEG C of metal baths, rapid ice bath 2min.Add 600ulLB blank cultures, mixing, be placed in 37 DEG C of shaking tables 220rp jolting 1h.
Each pipe, with the centrifugal 3min. of 5000rpm room temperature, discards 300ul supernatant more resuspended thalline, takes 200 μ l and coat Amp resistance LB flat board.Flat-plate inverted is placed in 37 DEG C of incubators and cultivates 24h.
5) screening of pGEX-6p-2-OprL positive recombiant plasmid, 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 converts and separates good bacterium colony on flat board, is inoculated in Amp resistance LB culture medium, and 37 DEG C of shaken cultivation are overnight；
2. plasmid extraction: carry out with reference to plasmid extraction kit description；
3. plasmid DNA carries out BamHI and EcoRI double digestion；
Double digestion reaction system:
| Project | Volume |
| BamH I | 0.5μl |
| Not I | 0.5μl |
| 10×K Buffer | 0.5μl |
| 0.1%BSA | 1.0μl |
| Recombiant plasmid | 8.0μl |
37 DEG C of enzyme action 2h；
4. the agarose gel electrophoresis detection double digestion result of 1%, result is Fig. 2 such as, it is seen that swimming lane 2 sample is the pGEX-6p-2-OprL recombiant plasmid successfully constructed；
5. pGEX-6p-2-OprL recombiant plasmid is sent to the order-checking of the handsome company in Shanghai, and sequencing result is than GeneBank sequence information comparison, found that the consecutive nucleotides sequence of the two is identical.
Embodiment 2: recombination fusion protein OprL is the qualification of abduction delivering, purification and expression-form in prokaryotic expression system-escherichia coli
1.OprL abduction delivering
Take in the LB culture medium of pGEX-6P-2-OprL/XL-1blue bacterium solution 100 μ L addition 10mLAmp+ resistance of incubated overnight, 180rpm37 DEG C of incubated overnight, take respectively incubated overnight bacterium solution 400 μ L add 20mLAmp+ resistance LB culture medium in (remaining bacterium solution is saved in 4 DEG C of refrigerators standby), cultivate 2～3h for 37 DEG C, rotating speed 200rpm, re-activation to OD600 be 0.8-1.0 time, add IPTG4 μ L, make its final concentration of 200 μMs, then be placed in 30 DEG C of abduction delivering 3h of shaking table abduction delivering.
2) bacterium solution after abduction delivering is taken out, with the centrifugal 5min of 12000rpm, supernatant discarded, add 1mLlysisbuffer (20mMPB, pH7.2,250mMNacl) mixing, ultrasonic degradation 3min (ultrasonic 6 times 30s/ time), 4 DEG C of centrifugal 15min of 14000rpm, cleer and peaceful precipitation in separation again.
2. process supernatant
Taking GlutathioneSepharose4B20 μ l, after washing 3 times with PBS, added in GlutathioneSepharose4B by ready supernatant, room temperature is in conjunction with 1h., after the centrifugal 3min of 14000rpm, to use PBS-0.25% polysorbas20 to wash 2 times at 4 DEG C, PBS washs once.GlutathioneSepharose4B after combining adds 20 μ L2 × protein loading buffer, boils the centrifugal 3min of 5min, 14000rpm.
3. process precipitation
Precipitation adds the 500 resuspended thalline of μ LPBS, takes the 80 resuspended bacterium solution of μ L and add 20 μ L5 × protein loading buffer, boil the centrifugal 3min of 5min, 14000rpm.
4.SDS-PAGE electrophoresis
Pouring in glue version by 5% concentration glue, 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, plugs comb immediately, and it is standby that room temperature placement 30min makes it solidify.The upper cleer and peaceful precipitation handled well is taken respectively 10 μ L loadings, carries out SDS-PAGE electrophoresis.Voltage elder generation 80v electrophoresis 30min, it is adjusted to 180v again, after electrophoresis 1～2h, glue is taken out, it is placed in coomassie brilliant blue staining liquid vibration dyeing, after being placed in destaining solution vibration decolouring again, observed result under imaging system, PGEX-6P-2-OprL/XL-1blue is soluble protein (Fig. 3) at 30 DEG C.
The preparation of embodiment 3:OprL antigen
1. amplification culture obtains albumen
nullThe standby pGEX-6P-2-OprL/XL-1blue bacterium solution 400 μ L in 4 DEG C of refrigerators of existence that goes bail for joins in the 20mL LB culture medium containing Amp resistance and once activates，After 200rpm37 DEG C of cultivation 5～6h，Take the 8mL bacterium solution once activated to join in the 400mL LB culture medium containing Amp resistance and carry out re-activation，When 37 DEG C of cultivation 3～4h to OD600 are 1.0，Add 80 μ LIPTG (final concentration of 200 μMs) be placed in 16 DEG C of shaking tables overnight induction after，The centrifugal 15min of 12000rpm collects thalline，After adding 20mLlysisbuffer (with embodiment 2) resuspended thalline again，Bacterium solution is carried out ultrasonic degradation 3min (200V)，Collect supernatant and 800 μ L to process for GlutathioneSepharose4B (GE company) gel beads (beads) combination in conjunction with gst fusion protein；Carry out PAGE gel electrophoresis again.
2. use enzymatic cleavage methods, destination protein and GST label are separated, obtains OprL destination protein
800 μ LPBS and 120 μ LPreScissionprotease (PP enzymes are added in the remainder about 800 protein-bonded GlutathioneSepharose4B of μ L, GE company), after room temperature vertical rotary enzyme action 5h, after centrifugal absorption supernatant, wash 3 times with 800 μ LPBS respectively, after taking 10 μ L sample degenerative treatments after each, loading 5 μ L carries out protein electrophoresis (method is ibid), observed result under in phase system, OprL molecular weight of albumen～16kDa that enzyme action gets off, it is consistent with expection molecular weight of albumen size, sees Fig. 4.
Embodiment 4: the structure of charrin disease Pneumonia Mice model
1. Pseudomonas aeruginosa bacterium solution preparation
Take frozen P. aeruginosa clinical strain XN-1 (CCTCCM2015730), adopt the recovery of LB nutrient agar panel, 37 DEG C of aerobic overnight incubation.The single colony inoculation 20mLLB fluid medium of picking, after 37 DEG C of aerobic 180rpm shaken cultivation 15h, takes 0.2mL and is inoculated in 20mLLB fluid medium, 37 DEG C of aerobic 230rpm shaken cultivation 6h.The centrifugal thalline of collecting of 6000g, with normal saline resuspension thalline after brine twice.Adopt the OD of spectrophotometric determination bacterium solution600Value, and according to 1OD=6.0 × 109CFU/ml is converted into bacterial concentration.
2. the foundation of mouse anesthesia and collunarium scheme
In the infection experiment of pseudomonas aeruginosa pneumonia model, for ensureing being smoothed out of collunarium process, initially with isoflurane by mouse anesthesia.Anesthesia: carry out sucking induced anesthesia for delivery vehicles with the isoflurane of 5% concentration with oxygen, enters after surgery stage of deep narcosis until mice, and the concentration with 3% maintains anesthesia, and this mode can reach good anaesthetic effect.The control of collunarium dosage is the guarantee of model stability, in order to better control collunarium dosage, prevent bacterium solution in collunarium process from entering oral cavity and producing bubble simultaneously, take following measures: (1) has a physiologic radian due to nasopharynx part, head is faced upward more severe, nasal drop is more easily accessible oral cavity, so collunarium process small mouse head is lain on the back, amplitude not can exceed that the physiologic radian of nasopharynx part；(2) drip medicine along lateral wall of nasal cavity, reduce the generation of bubble in collunarium process as far as possible；(3) get hold of the rhythm of collunarium, drip 4 below μ L amounts in the moment that mice air-breathing starts；(4) in the present embodiment, collunarium amount is 20 μ L, and single-nostril one-time continuous instills.
3. the determination of infective dose
The PAXN-1 bacterium solution that embodiment 1 is obtained by normal saline is adopted to regulate to five kinds of variable concentrations, laboratory animal female BAl BIc/c mice is randomly divided into 5 groups, infect with adopting the mode of collunarium after isoflurane anesthesia mice, every mouse infection dosage is 20 μ L, adopts same dose of normal saline (NS) as blank.Packet and the infection conditions of animal are as shown in table 1.Infection terminate after every 1 day observe dead mouse situation, the observation cycle is 7 days, the observation period terminate after residue animal with CO2Inhalation euthanasia.
Table 1: the determination of pseudomonas aeruginosa pneumonia model infective dose
| Group | Collunarium | Dosage (CFU) | Quantity | Concentration (CFU/ml) | Infusion volume (μ l) |
| 1 | PAXN-1 | 6.00×108 | 10 | 3.00×1010 | 20 |
| 2 | PAXN-1 | 3.00×108 | 10 | 1.50×1010 | 20 |
| 3 | PAXN-1 | 1.50×108 | 10 | 7.50×109 | 20 |
| 4 | PAXN-1 | 7.50×107 | 10 | 3.75×109 | 20 |
| 5 | PAXN-1 | 3.75×107 | 10 | 1.88×109 | 20 |
| 6 | Normal saline | - | 10 | - | 20 |
Adopt variable concentrations (2 times of gradient dilutions) pseudomonas aeruginosa strains PAXN-1 infect BALB/c mouse time, through the observation of 7 days, result as shown in Figure 1: infective dose is 1.5 × 108During CFU, mouse death rate is 40%, and infective dose is 3.0 × 108During CFU, mouse death rate is 80%, and infective dose reaches 6.0 × 108Mouse death rate 100% during CFU.The preferred dose determining mouse infection is 3.0 × 108CFU。
Embodiment 5: the immunity of animal
1) first immunisation, dilutes OprL antigen with PBS, adds the Al (OH) that concentration is 1mg/mL3；With No. 5 half mould syringe needles, bilateral inguinal, vola and dorsal sc are to an injection, and every BALB/C mice injection volume is 100 μ L, and arranges positive controls, negative control group and blank group；
2) second time immunity, carries out second time immunity on the 7th day, and ibid, injection volume proteantigen amount is the 1/2 of first immunisation to immune component, and immunization route is ibid；
3) third time immunity, carries out third time immunity on the 14th day, and ibid, injection volume proteantigen amount is identical with second time immunity, and immunization route is ibid for immune component；
Embodiment 6: the detection of antibody
After third time immunity the 7th and 14 day, gather the blood of BALB/C mice, detect after mouse immune with ELISA, IgG, IgG1, IgG2aHumoral response level.
1. prepare liquid
1) preparation of liquid it is coated: weigh Na2CO31.6g, NaHCO32.9g, is dissolved in 1LddH2O, is adjusted to 9.6 with PH meter by pH；
2) preparation of confining liquid: 1g Ox blood serum V, is dissolved in 100mL antibody diluent (1: 100)；
3) preparation of antibody diluent: phosphate is dissolved in 1LddH2O, adds 500 μ LTween20, then with PH meter, pH is adjusted to 7.4；
4) preparation of cleaning mixture: synantibody diluent
5) nitrite ion (TMB), for sky root Products；
6) stop buffer (2MH2SO4) preparation: 22.2mL concentrated sulphuric acid is poured into 177.8mLddH2In O.
2.ELISA detects the antibody titer that OprL recombiant protein immune mouse produces
1) be coated liquid by after purification OprL recombiant protein dilute be: 1ug/mL, 5ug/mL, 10ug/mL；
2) it is coated: recombiant protein diluent adds ELISA Plate, 100 μ L/ holes, and 4 DEG C are overnight washed 3 times with cleaning mixture afterwards, wrap with preservative film, be placed in 4 DEG C of refrigerators standby after sky is dry；
3) close: ELISA Plate adds confining liquid 200 μ L/ hole, is placed in 37 DEG C of incubators 2 hours, washs 3 times；
4) serum is carried out the doubling dilution such as 1: 100,1: 500,1: 1000,1: 2000,1: 4000,1: 8000；
5) take the ELISA Plate closed, be sequentially added into dilute serum, 100 μ L/ holes, it is placed in 37 DEG C of incubator 1h, washs 3 times, empty dry；
6) by adding the goat anti-mouse igg of HRP labelling, IgG1, IgG2a antibody preservation liquid, dilute 1: 5000, make antibody working solution；
7) add dilution antibody working solution, 100 μ L/ holes, be placed in 37 DEG C of incubator 40min, wash three times, empty dry；
8) substrate nitrite ion (TMB) 100 μ L/ hole is added, room temperature lucifuge reaction 5min；
9) stop buffer (2MH is added2SO4), it is immediately placed in microplate reader and measures OD value with 450nm wavelength place；
10) result judges: ASampleANegativeValue >=2.1 are positive (negative control is serum 1: 1000 times dilution before mouse immune).
Result: the antibody titer that detection OprL proteantigen immune mouse produces reaches 1: 512000；After immunity, the antibody positive rate of the 7th day reaches 100%, illustrates that the OprL recombiant protein that the present invention builds can make to produce in immune mouse body antibody.
The counteracting toxic substances protection of embodiment 7:OprL recombiant protein animal immune is evaluated
After OprL final immunization 10～14 days, adopt the method identical with embodiment 3, by preparation PAXN-1 bacterium solution and regulate concentration to 1.5 × 10 with normal saline10CFU/mL, with adopting the mode of collunarium to infect after isoflurane anesthesia mice, every mouse infection amount is 20 μ L, adopts same dose of normal saline (NS) as blank.Infection terminate after every 1 day observe dead mouse situation, the observation cycle is 7 days, the observation period terminate after residue animal with CO2Inhalation euthanasia.Add up the survival rate of each group of mice.Result is shown in table 2.
Counteracting toxic substances protected effect after table 2OprL recombiant protein immune mouse
Table 2 shows: the average immune protective rate of negative control group and blank group respectively 15% and 20%, recombination fusion protein OprL adds Al (OH)3The average immune protective rate of adjuvant group is 70%.Therefore, the OprL recombiant protein of the present invention has good immunogenicity, it is possible to induction body produces immunne response, and can the infection of PAXN-1 be played a protective role, it is possible to is aided with aluminium adjuvant and prepares subunit vaccine for preventing the infection of Pseudomonas aeruginosa.
Pass through above example, those skilled in the art utilize ordinary skill knowledge can apply the recombiant protein prepared by the present invention and other related reagents apparently, such as it is coated reagent, detection antibody, developer, terminator etc. and prepares related kit, such as detection kit, for whether diagnosis is infected Pseudomonas aeruginosa, is determined prognosis etc..
OprL recombiant protein of the present invention can be used for other any applicable purposes by those skilled in the art.
The foregoing is only presently preferred embodiments of the present invention, be not used for limiting the practical range of the present invention；If without departing from the spirit and scope of the present invention, the present invention is modified or equivalent replacement, all should be encompassed in the middle of the protection domain of the claims in the present invention.
Claims (10)
1. a recombiant protein, it is characterised in that: this recombiant protein comprises Pseudomonas aeruginosa Peptidoglycan protein fragments, and the nucleotides sequence of described recombiant protein is classified as SEQIDNO:1, and aminoacid sequence is SEQIDNO:2.
2. an expression vector, it is characterised in that: comprise the nucleotide sequence of recombiant protein described in coding claim 1.
3. expression vector according to claim 2, it is characterised in that: described expression vector can be connected, by skeleton plasmid, the nucleotide sequence encoding above-mentioned recombiant protein and be formed with modifying；Preferably, described skeleton plasmid is selected from pGex serial carrier, pET serial carrier or pQE serial carrier, it is preferred to pGex-6P-2.
4. comprise the Host Strains of the arbitrary described expression vector of claim 2-3.
5. Host Strains according to claim 4, it is characterised in that: described Host Strains one in E.colistrain XL1 blue, BL21 or HMS174 bacterial strain, it is preferred to E.colistrain XL1 blue bacterial strain.
6. the preparation method of the recombiant protein described in claim 1, it is characterised in that including:
1) nucleotide sequence according to coding Pseudomonas aeruginosa OprL recombiant protein is synthesized into or designs forward primer and reverse primer by full genome, does template pcr amplification with Pseudomonas aeruginosa full-length genome and obtains Pseudomonas aeruginosa recombinant subunit vaccine OprL genes of interest fragment；
2) by step 1) gained gene fragment clone to expression vector, then convert to Host Strains；
3) Host Strains after Induction Transformation expresses OprL recombiant protein.
4) purification of OprL recombiant protein.
7. method according to claim 6, it is characterised in that: step 1) described in forward primer and the nucleotide sequence of reverse primer such as shown in SEQIDNO:3 and SEQIDNO:4.
8. the application in preparing the medicine for diagnosing, prevent or treat Pseudomonas aeruginosa of the recombiant protein described in claim 1.
9. application according to claim 8, it is characterised in that described medicine is the test kit for diagnosing Pseudomonas aeruginosa.
10. application according to claim 8, it is characterised in that described medicine is the subunit vaccine for preventing or treat Pseudomonas aeruginosa.
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| CN109293749A (en) * | 2018-11-20 | 2019-02-01 | 重庆艾力彼生物科技有限公司 | A kind of pseudomonas aeruginosa vaccine recombinant protein rExoU and preparation method thereof |
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Application publication date: 20160706 |