CN104861047B - Single function magnetic nanoparticle based on ferritin - Google Patents
Single function magnetic nanoparticle based on ferritin Download PDFInfo
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Abstract
The invention discloses a kind of magnetic nanoparticle of the single function based on ferritin, it includes single function ferritin shell and magnetic nanoparticle kernel, the single function ferritin shell is the asymmetric globulin with tetrahedral structure, the asymmetric globulin is mainly made up of 1 saltant type Dps subunit and 11 wild type Dps subunits, and the Dps is the DBP of hungry induction.The present invention constructs the single function magnetic nanoparticle based on ferritin using large biological molecule protein as mould material, it is easily transformed, is easy to manipulate, conveniently largely obtains, and it only has One function group, there is great meaning to follow-up Controllable assembly.
Description
Technical field
Present invention relates particularly to a kind of single function magnetic nanoparticle based on ferritin, belongs to nanometer biotechnology neck
Domain.
Background technology
Magnetic nanoparticle is the important component in nano material, because of its special Size Distribution, magnetic Nano
Grain possesses the effect such as surface and interfacial effect, small-size effect, quantum size effect, macroscopic quantum tunneling, so be widely used,
It is related to the every field such as machinery, electronics, optics, magnetics, chemistry and biology.And magnetic nano-particle be applied to nuclear magnetic resonance into
Picture, Magneto separate, a necessary condition of the carrier of drug delivery and tumor thermotherapy etc. are that have good superparamagnetism.
Though non-surface modification with good superparamagnetism, because particle diameter is small, specific surface area is big and the influence of Van der Waals force,
Particle is easy to assemble in itself, and stability is poor, is easily swallowed by the organ that the reticuloendothelial systems such as liver and spleen are enriched, partly declines in vivo
Phase is short, is restricted its application.So one of key for being applied in terms of medical science of development magnetic nanoparticle is transformed into pair
Nano particle carries out modification cladding.
Develop in recent years and provided a series of novel and effective strategy for the modification of magnetic nanoparticle and cladding.
These methods can be by magnetic nanoparticle outside modification amphipathic nature polyalcohol molecule, or wrapping biological macromolecule, so as to increase
Strong biocompatibility, to cytotoxic, and circulation time greatly prolongs in the blood vessel.The large biological molecules such as protein, DNA
It is natural nano material, their various structures can be highly homogeneous with self-replacation, is easy to artificial and manipulates and a large amount of prepare.
DNA has shown unique advantage in Nano-technology Development;Compared with DNA, the structural information that protein carries is more abundant,
Thus it is expected to provide more flexible operating platform for nanometer technology.Wherein, albumen cage structure, for example, viral capsid, ferritin,
Thermal shock albumen has been widely used for the cladding of nano particle or is used as reaction vessel synthesizing magnetic nano particle.
But because magnetic nanoparticle pattern itself and surface nature have Sphere symmetry, in general method can be only formed
Uniform decorative layer, it is impossible to form anisotropic magnetic Nano structure.Recent years, it is a series of effective right to report successively
The asymmetric modification of magnetic nanoparticle, the method for cladding, can by isotropic modified by magnetic nanoparticles itself into it is each to
The opposite sex so that this bottleneck is broken through.But a kind of asymmetric magnetic nanoparticle is obtained, and controllable behaviour on a molecular scale
Their self assembly is indulged, and accurately analyzes their assembling product, is still challenging work.
The content of the invention
In view of deficiency of the prior art, it is an object of the invention to provide a kind of single function magnetic based on ferritin
Nano particle, it can realize the single function of magnetic nanoparticle on a molecular scale so that magnetic nanoparticle can conduct
Construction unit, preferably strategy and development space are provided for follow-up Controllable assembly, and then more complicated, controllable and orientation can be carried out
Assembling.
For achieving the above object, present invention employs following technical scheme:
A kind of single function magnetic nanoparticle based on ferritin, including single function ferritin shell and magnetic Nano
Particle kernel, the single function ferritin shell are the asymmetric globulin with tetrahedral structure, the asymmetric ball egg
In vain mainly by 1 saltant type Dps subunit and 11 wild type Dps subunits(Abbreviation wtDps) composition, the Dps is hungry induction
DNA associated proteins.
Further, the saltant type Dps be wild type Dps N-terminal insert His-tag and one section there is SEQ ID
Sequence shown in No.1 can be obtained by specific biotinylated 15 peptide, and may be simply referred to as HBDps.
Further, the protein-bonded functional groups of DNA of the hungry induction be contained by saltant type Dps subunits can
By specific biotinylated 15 peptide, separation group is His-tag.
Further, the external diameter of the single function ferritin shell is 8-10nm, internal diameter 4.5-5nm.
Further, the average thickness of the single function ferritin shell is 2nm.
Further, the magnetic nanoparticle kernel includes a diameter of 2-3nm ferric oxide nanometer particle.
Compared with prior art, beneficial effects of the present invention include:By using large biological molecule-protein as template
Material, the single function magnetic nanoparticle based on ferritin being constructed, it is easily transformed, is easy to manipulate, conveniently largely obtains,
And be somebody's turn to do the single function magnetic nanoparticle based on ferritin and only have One function group, have to follow-up Controllable assembly great
Meaning.
Brief description of the drawings
Fig. 1 is the SDS-PAGE glue figures in an exemplary embodiments of the invention;
Fig. 2 is that one kind made from the DBP assembling of hungry induction is utilized in one exemplary embodiments of the present invention not
The transmission electron microscope figure of symmetrical protein nano particle;
The transmitted electron that Fig. 3 is the DNA associated proteins absorption AuNPs of unifunctional hungry induction shown in Fig. 2 shows
Micro mirror figure;
Fig. 4 is the positives control of an exemplary embodiments of the invention(The DNA associated proteins of the hungry induction of saltant type)Inhale
Attached AuNPs transmission electron microscope figure;
Fig. 5 is negative control in an exemplary embodiments of the invention(The DNA associated proteins of the hungry induction of wild type)
Adsorb AuNPs transmission electron microscope figure.
Fig. 6 be in an of the invention exemplary embodiments in asymmetric protein nano particle mineralising iron oxide magnetic nano
The transmission electron microscope figure of grain(With phosphotungstic acid negative staining mistake);
Fig. 7 be in an of the invention exemplary embodiments in asymmetric protein nano particle mineralising iron oxide magnetic nano
The transmission electron microscope figure of grain(Unused phosphotungstic acid negative staining mistake);
Fig. 8 is the agarose-gel electrophoresis figure in an exemplary embodiments of the invention;
Fig. 9 is the nuclear magnetic resonance weighted imaging figure in an exemplary embodiments of the invention.
Embodiment
Idea of the invention is that provide a kind of single function magnetic nanoparticle based on ferritin, its cardinal principle
It is:DBP using large biological molecule-starvation induction is operating platform, passes through the DBP to starvation induction
The genetic modification on surface, functional group/separation group is dexterously coupled in structure, then by the ferritin transformed and open country
The ferritin of raw type is sufficiently mixed rear depolymerization and assembled again in specific proportions, realizes that starvation lures with the method for two kinds of protein ratios of control
The Controllable assembly for the DBP led, obtain a kind of nano particle of asymmetric functionalization.Again according to band on asymmetric particle
Some separation groups, further realize the separation and purifying of target product.It is finally separating to obtain the nanometer of asymmetric functionalization
Mineralising magnetic nanoparticle in grain, and preferably use AuNPs(Golden nanometer particle)Its functional group is characterized, confirmation is obtained
Product is the magnetic nanoparticle of single function.
Specifically, the preparation method comprises the following steps:
1)The DBP of starvation induction is subjected to genetic modification, structure carries functional group and separates the egg of group
The carrier of white mutant;
2)The carrier built is transformed into organism and expressed, purifies and identifies to obtain the DNA knots of hungry induction
Hop protein mutant;
3)The ferritin of this mutant and wild type is sufficiently mixed, albumen depolymerization is then carried out under conditions of pH is adjusted
And Hybrid assembling;
4)According to the feature purifying assembling product of separation group;
5)The nano particle of obtained asymmetric functionalization is subjected to mineralising processing, in granular center synthetic iron oxide magnetic
Nano particle.Both the magnetic nanoparticle of purpose product singly-bound had been obtained.
For make the present invention the single function magnetic nanoparticle based on ferritin be more readily understood its substantive distinguishing features and
Its practicality having, an exemplary embodiments and Fig. 1-Fig. 9 are just combined below to structure of the present invention and embodiment
It is described in further detail, but description below in relation to embodiment and explanation do not form any limit to the scope of the present invention
System.
Step 1: structure, expression, the DBP of the hungry induction of purified mutant type.Comprise the following steps that:
In the Loop of the DBP of starvation induction N-terminal insertion His-tag and can be by specific biotinylated 15 peptide
GLNDIFEAQKIEWHE(Shown in SEQ ID No.1), it is built into carrier PET32a-His-Dps(His-Dps);It is true through being sequenced
Determine the correctness of objective gene sequence.PET32a-His-Dps is transferred to E.coli Rosetta with Calcium Chloride Method(DE3)Sense
By state cell, flat board is applied (added with ampicillin and chloramphenicol in flat board)Picking monoclonal connects from flat board after at least 12 h
Enter in 5 mL LB Tube propagation bases, add ampicillin(The μ g/mL of final concentration 100)And chloramphenicol(The μ g/ of final concentration 68
mL), in 37 DEG C of constant temperature, 180 r/min overnight incubations.Transferred according to 1% (v/v) inoculum concentration in 5 mL LB Tube propagation bases
In(Parallel 3 pipe), corresponding antibiotic is added, in 37 DEG C of constant temperature, the h of 180 r/min shaken cultivations 2.5 or so(OD600 is 0.4
Between ~ 0.6), wherein 1 pipe is as blank control(It is not added with derivant IPTG), two manage addition derivant final concentration of 1 in addition
MM IPTG, respectively after 25 DEG C and 37 DEG C are continued the h of Fiber differentiation 10 and 2 h, thalline is collected by centrifugation, is detected with SDS-PAGE
His-Dps expression, can be expressed at a temperature of finding two kinds, while also have inclusion body generation, wherein 37 DEG C of inclusion bodys compared with
It is more.25 DEG C of selection is as inducing temperature when largely preparing albumen.
By E.coli Rosetta(DE3)/ PET32a-His-Dps is inoculated in 5 mL LB Tube propagation bases, is added
Enter corresponding antibiotic in 37 DEG C of constant temperature, 180 r/min overnight incubations.5 mL bacterium solutions were transferred into 500 mL LB tri- in second day
In the bottle of angle, ampicillin is added(The μ g/mL of final concentration 100)And chloramphenicol(The μ g/mL of final concentration 68), in 37 DEG C of constant temperature, 180
After h of r/min shaken cultivations 2.5 or so(OD600 is between 0.4 ~ 0.6), add final concentration of 1 mM's of derivant
IPTG, continue to vibrate the h of Fiber differentiation 10 at 25 DEG C, thalline is collected by centrifugation.Bacterial sediment is cleaned with binding buffer
Once it is resuspended in afterwards in the binding buffer of certain volume and carries out ultrasonication(Condition:Ultrasonic power 400W, work 4s,
Interval 4s, total min of ultrasonication time 60), the bacterium solution after crushing centrifuges 30 min with 12000 r/min, by supernatant loading
In through Ni equilibrated binding buffer2+-- NTA affinity columns, low concentration is then used successively( 20mM、40mM、
60mM、120 mM)Imidazoles wash post and remove foreigh protein removing, finally with elution buffer(500mM imidazoles)Elute purpose egg
In vain.
The HBDps and wtDps prepared exists in the form of spherical nanoparticle.HBDps and wtDps concentration passes through reagent
Box Bradford Protein Assay(Dying method with coomassie brilliant blue)Measure, step are carried out according to kit specification.Simultaneously
Verified with reference to SDS-PAGE.
Step 2: single function Dps assembling and purifying.Comprise the following steps that:
1. by the wtDps that concentration is 8.12mg/mL(18KD)2mL (Count 16.24mg)It is 1.776 mg/mL with concentration
His-Dps(21KD)0.224 mL (Count 0.57mg)Mixing, 4 DEG C vibrate at a slow speed 2h;
2. diluting total protein liquid to final concentration of 1mg/mL, pH value depolymerization and again Hybrid assembling asymmetry albumen are adjusted
Structure.Comprise the following steps that:
Protein liquid is fitted into bag filter by I to be put into 900mL water, with syringe pump with 1.5mL/min speed into water
Add 0.1M HCl, until pH value is transferred into 2.Benefit is filled with water to 1L, and 4 DEG C stand 12 h of dialysis.The principle of the process be Dps pH=
Under conditions of 2, protein monomer can be depolymerized to by the globulin assembled.
II configures 1 L phosphate buffers(0.001M/L), pH=2 are adjusted with concentrated hydrochloric acid, are put into above-mentioned bag filter, are used
Syringe pump adds 2M/L NaOH into water with 0.5mL/min speed, until pH value is transferred into 7, latter 4 DEG C stand 12 h of dialysis.
The principle of the process is Dps in the modulation pH of pH=2=7, can be reassembled into globulin.
3. obtained product contain Hybrid assembling Dps, His-Dps and wtDps globulin and HBDps and wtDps it is mono-
Body.It is to be separated go out asymmetric Hybrid assembling Dps nano particles, it is necessary to following specific steps:
For I toward the mL of 5 × binding of addition buffer 4.2 in assembles concentration, now cumulative volume is 21. mL, will be assembled
Liquid is splined on through Ni equilibrated binding buffer2+- NTA affinity columns, can be adsorbed onto only with HBDps
On chromatographic column, with binding buffer, 15mM, 30mM imidazoles removes the DNA of hungry induction of the non-specific adsorption on post
Associated proteins, single function Dps then is eluted with 100mM imidazoles, then carry multiple His- with the elution of 500-1000mM imidazoles
The globulin of Dps monomers.The principle of the process is:Ni2+A NTA is connected in-NTA chromatography gel matrix
(Nitrilotriacetic acid, nitrilotriacetic acid), can be with Ni ions bindings, the 6-his ammonia of Ni ions and fusion protein
Stronger chelation is produced between base acid, so as to be distinguished with albumen histidine-tagged his-tag and other albumen
Come.Therefore as the when marquis eluted with the imidazole solution of high concentration(Such as 300 mM or higher), imidazoles just with protein his-tag
Imidazole ring competition binding, most fusion protein elutes from gel at last.
4. the single function Dps 0.05M Tris under 100mM imidazoles is eluted, 0.5M NaCl, glycerine 5%, pH=8 are delayed
Fliud flushing is dialysed, and is diluted to more than 10000 times.
Single function Dps is characterized:
There are two methods to verify whether obtained Dps is single function.Specific method is as follows:
1. obtained destination protein is run into SDS-PAGE glue, due to wild type Dps molecular weight is 18KD and mutability Dps points
Son amount is 21KD, and both are in diverse location in SDS-PAGE glue, according to both in the gray value of SDS-PAGE glue, can be sentenced
The amount ratio of both fixed materials, judge whether destination protein is single function with this, as shown in figure 1, according to single function
WtDps and HBDps gray value calculates the amount of both materials in Dps, calculate both ratios are about 30:1, you can card
Bright obtained destination protein is the Dps of single function really.
2. NTA(Nitrilotriacetic acid, nitrilotriacetic acid)The AuNPs of-Ni modifications can be efficiently attached to
On HBDps his-tag, but do not specifically bound but with wild type Dps.With NTA-Ni-AuNPs adsorption experiments pair
Sample is characterized(Fig. 3), while prepare positive control(Fig. 4)And negative control(Fig. 5).In AuNPs and obtain purpose egg
The amount of white material is 2:In the case of 1, destination protein only adsorbs a gold grain substantially, and wild type Dps is unable to ADSORPTION OF GOLD
Grain, and full-mutant Dps can then adsorb the gold grain that 3-6 number does not wait.The result explanation:One is only included in destination protein
The individual His-Dps for having his-tag monomer, remaining 11 be wild type Dps monomer.
Step 3: the synthetic iron oxide magnetic-particle in asymmetric protein nano particle.Specific steps:
1. by the ml of above-mentioned purpose albumen 4.6(330μg/mL)With chuck of the 15mL 0.1M NaCl additions full of N2 protections
In reactor, this reactor is connected with an autotitrator(AG is led in TITRINO, Switzerland ten thousand)And thermometer.With 30mM/L's
The pH value of NaOH regulation reactant mixtures is 8.5.Reactor is fixed on into the aobvious constant temperature electric heating of SHT types stirring to put, agitating and heating
To 65 DEG C.
2. adding the 0.1mL 5mM/L iron ammonium sulfates and 0.075mL 2.5mM/L hydrogen peroxide of Fresh, it is used in combination
The pH value of 30mM/L NaOH regulation reactant mixtures is 8.5, reacts 5min, is repeated 8 times, finally reacts 10min again.
3. product is cooled into room temperature, 12000rpm, 30min are centrifuged.With 0.2 μm of filter filtering supernatant, under filter
Liquid is purpose product:The magnetic nanoparticle of single function.
The sign of magnetic nanoparticle, that is, verify in single function Dps whether include magnetic particle, there are following methods:
First as Fig. 6 will be obvious that of albumen and albumen center with the sample that phosphotungstic acid negative staining is crossed under Electronic Speculum
Grain, without process phosphotungstic acid negative staining as shown in fig. 7, can only see that diameter is in 2-3nm little particle one by one.This proves egg
White inner chamber ore deposit has particle.
Ferric ion, which runs into ferrous cyanogen root, can produce the ferric ferrocyanide precipitation of blueness, so with prussian blue staining method
It can verify in albumen and whether contain iron oxide.As shown in Figure 8:It with the agarose gel of Prussian blue dye, b is to use coomassie that a, which is,
Light blue contaminates the agarose gel that must have same control sample.Wherein a3, b3 are the single function Dps of no mineralising;A2, b2 are ore deposits
The wt-Dps of change, a1, b1 are the single function Dps of mineralising, it can be seen that the ferritin of no mineralising can only be contaminated by Coomassie brilliant blue
Color can not but be dyed to it is Prussian blue, and the wild type ferritin and single function ferritin Jing Guo mineralising can by Mas bright blue and
It is Prussian blue to dye blueness.Ferric oxide nanometer particle is formd really in albumen after illustrating mineralising.
It is finally that NMR signal detection is carried out to sample.As shown in Figure 9:2,3, the 4 of T1, T2 weighted imaging gray-scale map
Hole is plain buffer respectively, the single function Dps of 2 × mineralising(1.5μm/L), the single function Dps of 1 × mineralising(0.75um/l).1
Hole is unrelated sample.As a result show the single function Dps energy more apparent shortening T1 relaxation times of mineralising, while also can substantially shorten T2
In the relaxation time, to sum up it may determine that, what is be combined in asymmetric single function Dps chats is ultra-fine paramagnetic iron oxide
(Ustrasmall superparamagnetic iron oxide, USPIO).
USPIO contrast agent the magnetic marker probe imaging of the mediations such as acceptor application prospect by the extensive attention of people.Institute
Meaning magnetic marker probe, which refers in some way be combined paramagnetic particles with specific receptor, antibody or gene, to be transferred into the cell,
By the accumulation of the paramagnet in the cell is directly proportional to the amount of material connected, therefore, caused by paramagnet
MR signal distributions represent the distribution of acceptor, antibody or gene, and pass through the part of paramagnetic label, under receptor-mediated, production
Raw specific concentration, reach selective reinforcement and then the purpose being imaged.The single function based on ferritin obtained by the present invention
Change magnetic nanoparticle, you can as magnetic marker probe, suitable ligand is added on single function Dps functionalization subunit, you can
Reach selective reinforcement signal and then the purpose being imaged.
It the above is only the embodiment in the numerous concrete application examples of the present invention, protection scope of the present invention do not formed any
Limitation.All technical schemes formed using equivalents or equivalence replacement, all fall within rights protection scope of the present invention.
<110>Suzhou Institute of Nano-tech. and Nano-bionics, Chinese Academy of Sciences
<120>Single function magnetic nanoparticle based on ferritin
<160> 1
<210> 1
<211> 15
<212> PRT
<213>Artificial sequence
<400> 1
Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu
1 5 10 15
Claims (4)
- A kind of 1. single function magnetic nanoparticle based on ferritin, it is characterised in that including single function ferritin shell and Magnetic nanoparticle kernel, the single function ferritin shell are the asymmetric globulin with tetrahedral structure, it is described not Symmetrical globulin is mainly made up of 1 saltant type Dps subunit and 11 wild type Dps subunits, and the Dps is hungry induction DBP, wherein saltant type Dps are N-terminal His-tag and one section of sequence of insertion such as SEQ ID in wild type Dps Can be obtained by specific biotinylated 15 peptide shown in No.1.
- 2. the single function magnetic nanoparticle according to claim 1 based on ferritin, it is characterised in that single work( The external diameter that ferritin shell can be changed is 8-10nm, internal diameter 4.5-5.5nm.
- 3. single function ferritin shell according to claim 1, it is characterised in that the single function ferritin shell Average thickness be 2nm.
- 4. the single function magnetic nanoparticle according to claim 1 based on ferritin, it is characterised in that the magnetic Nano particle kernel includes a diameter of 2-3nm ferric oxide nanometer particle.
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