CN102321590B - Preparation method of asymmetric virus nanoparticles - Google Patents
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Abstract
The invention discloses a preparation method of asymmetric virus nanoparticles. The method comprises the following steps: carrying out gene modification on the virus capsid protein surface, so that the virus capsid protein simultaneously has coupled functional group and separate group; thoroughly mixing the modified virus capsid protein and wild type virus capsid protein while controlling the proportion of the two virus capsid proteins; and meanwhile, adding corresponding inorganic nanoparticles according to the total amount of the virus capsid protein to implement controllable assembly of the virus nanoparticles, thereby obtaining the asymmetric functionalized nanoparticles which are the goal product. The invention adopts biomacromolecule-protein as the nano material, and the biomacromolecule-protein can be easily modified and manually operated, and can be conveniently obtained massively. On the basis of the structural symmetry of the self-assemblable virus capsid protein, the two different protein molecules can be assembled in an oriented mode according to the previous design, and therefore, the assembly body has diversity and controllability; and the invention has the advantage of manageable reaction conditions, and can implement large-scale production.
Description
Technical field
The present invention relates to by a kind of controlled method, handle the self-assembly of biomacromolecule on molecular level, relate in particular to the preparation method of a kind of asymmetric virus nanoparticles (asymmetric VNPs), belong to nanometer biotechnology field.
Background technology
Nanotechnology, refers in the yardstick of 0.1～100 nanometer, research electronics, atom and molecular motion rule and characteristic and the technology that material and material are processed is called as to nanotechnology.Scientists in 1981 invent the important tool-scanning tunnel microscope of research nanometer, and atom, point subworld are from then on visible.Nineteen ninety first international nanotechnology meeting is held in U.S. Baltimore, and nanotechnology form is born.
Nano particle is because of character such as optical, electrical, the magnetic of its uniqueness and mechanics, again because it has the features such as size is little, specific surface area large, surface energy is high, surface atom ratio is large, in fields such as bio-sensing, Materials science, energy and information technologies, get the attention, be to study the earliest and the most ripe nano material, can be used for developing the nano-device with special property and function.But due to surface chemistry isotropy, nano particle, as constructing primitive lack of diversity and stability, is therefore subject to certain limitation in function and application.Therefore one of key issue of development nano material and device is transformed into the assembling of controlling nanometer construction unit.
Development in recent years a series of asymmetric modifying method for nano particle, for the controllability of assemble nanometer particle provides some novel effectively strategies.These methods can, by isotropic nano-particle modified one-tenth anisotropy itself, make common functional nano particle become required construction unit.In recent years, based on anisotropic assembling because its excellent controllability is subject to extensive concern.For the asymmetric nano material of pattern, and have the nanocrystalline of the remarkable different crystal face of character, they are easy to the anisotropy of dependence itself and modify and assembling.A lot of analog results of calculating show, nano particle is carried out to asymmetric modification and can make it from isotropy, become anisotropy, and then can carry out more complicated, controlled and directed assembling.Exactly because but the pattern of nano particle own and surface properties have Sphere symmetry, general method can only form uniform decorative layer.Recent years, reported successively a series of effectively methods to the asymmetric modification of nano particle, this bottleneck is broken through, simultaneously also for controlled assembling provides better strategy and development space.
The biomacromolecule such as protein, DNA is natural nano material, and their structures are various, can self-replacation, and height homogeneous, is easy to artificial manipulation and a large amount of preparation.DNA has shown unique advantage in Nano-technology Development; Compared with DNA, the structural information that protein carries is abundanter, thereby is expected to provide service platform more flexibly for nanotechnology.Wherein, albumen cage structure, as viral capsid, ferritin, thermal shock albumen, has been widely used in studying the model of Macromolecular self-assembly and the synthetic nano-reactor of material.But, on molecular level, by a kind of controlled method, handle their self-assembly, and their assembling product of accurate analysis, remain challenging work.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of asymmetric virus nanoparticles, it can realize the controlled self-assembly of biomacromolecule on molecular level, thereby overcomes deficiency of the prior art.
For achieving the above object, the present invention has adopted following technical scheme:
A preparation method for asymmetric virus nanoparticles, is characterized in that, the method is:
Genetic modification is carried out in viral capsid proteins surface, make viral capsid proteins simultaneously with the functional group and Fen Li group that are coupled, then this improved viral capsid proteins is fully mixed with wild-type virus capsid protein, and control the ratio of this two-strain capsid protein, according to the total amount of viral capsid proteins, add corresponding inorganic nanoparticles simultaneously, realize the controlled assembling of viral sample nano particle, obtain the nano particle of asymmetric functionalization, i.e. target product.
Further say, described viral capsid proteins comprises SV40 capsid protein VP1, and described functional group comprises halfcystine, and described separation group comprises His-tag.
The method comprises the steps:
I. in the VP1 of SV40 capsid protein Loop, introduce Cys, that is, in the 74th amino acids, sport Cys, and insert His-tag at the 139th, carrier construction His-mvp1, and realize and expressing in organism, purified, obtain improved viral capsid proteins His-mvp1;
II. get improved viral capsid proteins His-mvp1 and fully mix with former wild-type SV40 capsid protein wtvp1, by regulating the ratio of these two kinds of capsid proteins, the control of realization to these two kinds of capsid protein component proportionss in assembly virus nanoparticles individuality, according to capsid protein total amount and the amount of nano particle material, than 60:1 ~ 180:1, add inorganic nanoparticles, the common assembling of having dialysed in low salts solution;
III. with metal chelate affinity chromatography method separation and purification assembling product, remove the product of allogenic disease virus capsid protein self-assembly, obtain single asymmetric virus nanoparticles.
The method also comprises the steps:
IV. with sucrose continuous density gradient method to step III obtain asymmetric virus nanoparticles separate, obtain uniform unsymmetrical structure.
Step I is specially:
His-mvp1 expression plasmid is proceeded to by Calcium Chloride Method
e.colirosetta(DE3) competent cell, culturing bacterium abduction delivering recombinant protein His-mvp1 in LB substratum, successively through centrifugal, fragmentation and column chromatography processing, obtain improved viral capsid proteins His-mvp1 thereafter;
In described LB substratum, also contain microbiotic and inductor, described microbiotic comprises penbritin and/or paraxin, and described inductor comprises IPTG.
Step I comprises the steps:
A, His-mvp1 expression plasmid is proceeded to by Calcium Chloride Method
e.colirosetta(DE3) competent cell, be coated with after flat board at least 12 h, in picking mono-clonal access LB substratum, add microbiotic, in 37 ℃ of constant-temperature shaking culture, spend the night, according to 1% inoculum size transfer in LB substratum in thereafter, add microbiotic, in 37 ℃ of constant-temperature shaking culture, and add inductor, at 25 ℃～37 ℃, continue inducing culture, centrifugal collection thalline;
B, thalline that will collect are resuspended in binding buffer and carry out ultrasonication after cleaning, and the bacterium liquid after centrifugal breaking, gets supernatant liquor and carry out column chromatography, obtains improved viral capsid proteins His-mvp1.
Step II is specially: the His-mvp1 of mol ratio 1:50 ~ 1:11 is fully mixed with wtvp1, and add inorganic nanoparticles, the ratio control of inorganic nanoparticles and total protein molecule number is 60:1 ~ 180:1, the common assembling of having dialysed in low salts solution.
The asymmetric virus nanoparticles obtaining in step III has icosahedron structure, and its shell is comprised of 1 His-mVP1 pentamer and 11 wtvp1 pentamers, and center is inorganic nanoparticles.
Step III is specially:
Adopt nickel affinity chromatography method, first with 15-30 mM imidazoles solution, remove the assembling product without improved viral capsid proteins His-mvp1, i.e. the virus nanoparticles of nonfunctional group and Fen Li group; Then get 200 ~ 1000 mM imidazoles eluant solutions and obtain assembling the virus nanoparticles that only contains an improved viral capsid proteins His-mvp1 unit in product, i.e. asymmetric virus nanoparticles.
Virus nanoparticles (virus-based nanoparticles, VNPs) is the hollow bead consisting of one or more viral capsid proteins, and self-assembly produces conventionally, not containing viral nucleic acid, and can not self-replicating.The present invention is by symmetry or the virus surface genetic modification of virus structure, realize the controlled assembling of viral sample nano particle, obtain a kind of nano particle of asymmetric functionalization, thereby likely obtain new physics or chemical property, for virus signature and spike etc. provides new structured material.The success of this strategy is constructed primitive by the nanometer for preparation with controlled assembling performance an effective technological approaches is provided, thereby expedites the emergence of novel nano-device, for physical problems research and downstream application lay a good foundation substantially.
Compare to prior art, the advantage that the present invention has is: select biomacromolecule-protein as nano material, and its easy transformation and artificial manipulation, and conveniently obtain in a large number.By genetic engineering means while integration function group and Fen Li group in virus nanoparticles Knockdown block.Utilization can self-assembly the symmetry of viral capsid proteins structure, after can two kinds of different protein moleculars mixing, by design in advance, carry out NW-TFT, therefore assembly has diversity and controllability; Easily-controlled reaction conditions, and can realize scale operation.
Accompanying drawing explanation
Fig. 1 is that the present invention utilizes the main capsid protein vp1 packing of SV40 quantum dot to assemble altogether the transmission electron microscope photo of a kind of asymmetric virus nanoparticles making;
Fig. 2 is the transmission electron microscope figure of the VNPs of asymmetric shown in Fig. 1 absorption AuNPs;
Fig. 3 is the transmission electron microscope figure of negative control of the present invention (VNPs that wtvp1 packing quantum dot forms) absorption AuNPs;
Fig. 4 is the transmission electron microscope figure of positive control of the present invention (VNPs that His-mvp1 packing quantum dot forms) absorption AuNPs.
Embodiment
The preparation method of the asymmetric virus nanoparticles of one provided by the invention (asymmetric VNPs), utilize biomacromolecule---viral capsid proteins (SV40, CCMV, the capsid protein of the viruses such as TMV) model assembled altogether with novel nano-material, by the genetic modification to viral capsid proteins surface, by functional group and the structurally coupling dexterously of Fen Li group, then after transformed capsid protein fully being mixed in specific proportions with the albumen of wild-type, assemble again, by the method for controlling two kinds of albumen pentamer ratios, realize the controlled assembling of viral sample nano particle, obtain a kind of nano particle of asymmetric functionalization.Again according on asymmetric particle with separation group, further separation and the purifying of realize target product.Finally separation is obtained to the nano particle of asymmetric functionalization, and preferably adopt AuNPs to characterize its functional group.Product prepared by the present invention is asymmetric virus nanoparticles (asymmetric VNPs).
Divided by concrete steps:
1. viral capsid proteins is carried out to genetic modification, build the carrier with the capsid protein mutant of functional group and Fen Li group;
2. the carrier building is transformed in organism and is expressed, purifying evaluation obtain viral capsid proteins mutant;
3. this mutant is fully mixed with the capsid protein of wild-type, then under inorganic nano-particle participates in, through dialysis, assemble;
4. according to the different separation method purifying assembling of the feature selection that separates group product;
5. the nano particle of the asymmetric functionalization obtaining is done to continuous density gradient centrifugal, prepare uniform asymmetric virus nanoparticles (asymmetric VNPs);
For being easier to, the preparation method of asymmetric virus nanoparticles of the present invention (asymmetric VNPs) understands the practicality of its substantive distinguishing features and institute's tool thereof; below just in conjunction with the embodiments 1 and Fig. 1-Fig. 4 technical solution of the present invention is described in further detail, but about description and the explanation of embodiment, protection domain of the present invention is not constituted any limitation below.
embodiment 1
In the Loop structure of the main capsid protein vp1 of SV40, introducing Cys(is that the 74th amino acids sports Cys), at the 139th, insert His-tag, be built into carrier PET32a-His-mvp1(His-mvp1); Through checking order, determine the exactness of goal gene sequence.PET32a-His-mvp1 is proceeded to by Calcium Chloride Method
e.colirosetta(DE3) competent cell, is coated with after dull and stereotyped 12 h picking mono-clonal from flat board and accesses in 5 mL LB test-tube culture mediums, adds penbritin and paraxin, in 37 ℃ of constant temperature, and 180 r/min overnight incubation.According to 1% inoculum size transfer in 5 ml LB test-tube culture mediums (parallel 3 pipes), add corresponding microbiotic, in 37 ℃ of constant temperature, 180 r/min shaking culture 2.5 h left and right (OD600 is between 0.4 ~ 0.6), wherein 1 pipe is as blank (not adding IPTG), it is the IPTG of 1 mM that other two Guan Jun add inductor final concentration, at 25 ℃ and 37 ℃, continue after inducing culture 10 h and 2 h respectively, centrifugal collection thalline, with SDS-PAGE, detect the expression of His-mvp1, find all can express at two kinds of temperature, also there is inclusion body to produce simultaneously, inducing temperature while selecting 25 ℃ of conducts to prepare albumen in a large number.
Will
e.colirosetta(DE3)/PET32a-His-mvp1 is inoculated in 5 mL LB test-tube culture mediums, adds corresponding microbiotic in 37 ℃ of constant temperature, 180 r/min overnight incubation.5 mL bacterium liquid were transferred in 500 mL LB triangular flasks in second day, add penbritin (final concentration 100 ug/mL) and paraxin (final concentration 68 ug/mL), behind 37 ℃ of constant temperature, 180 r/min shaking culture 2.5 h left and right (OD600 is between 0.4 ~ 0.6), adding inductor final concentration is the IPTG of 1 mM, at 25 ℃, continue vibration inducing culture 10 h, centrifugal collection thalline.Bacterial sediment is cleaned in the binding buffer that is once resuspended in afterwards certain volume and carries out ultrasonication (condition: ultrasonic power 400W with binding buffer, 3 s work, intermittently 5 s, total ultrasonication times 60 min), bacterium liquid after fragmentation, with centrifugal 30 min of 12000 r/min, is splined on supernatant liquor the Ni crossing through binding buffer balance
2+---nTA affinity column, then uses the imidazoles of lower concentration (20 mM, 40 mM, 60 mM) to wash post except foreigh protein removing successively, finally uses elution buffer(500 mM imidazoles) wash-out target protein.
The preparation of His-mvp1 and wtvp1 pentamer
His-mvp1 after purifying mainly exists with pentamer form, and also some exists with virus nanoparticles form, therefore need to remove the virus nanoparticles in His-mvp1, prepares pure His-mvp1 pentamer, two steps of this process need:
1. in the His-mvp1 pentamer preparing, adding final concentration is the DTT of 10 mM, then in depolymerization damping fluid, dialysing, (volume is 10 mL His-mvp1 albumen standing dialysis 10 h in 4 ℃ of 1 L depolymerization damping fluid, change once fresh depolymerization damping fluid, continue standing dialysis 10 h);
2. the His-mvp1 albumen ultracentrifugation of dialysing is removed to remaining virus nanoparticles, Ultracentrifugation conditions is 4 ℃, 55000 rpm(Beckman Type 90Ti rotors) centrifugal 1 h, gets supernatant and is His-mvp1 pentamer.
The concentration of His-mvp1 pentamer is by test kit Bradford Protein Assay(coomassie brilliant blue staining method) to measure, step is carried out according to test kit specification sheets.In conjunction with SDS-PAGE, verify simultaneously.
The preparation process of former wild-type SV40 capsid protein wtvp1 pentamer is identical with the preparation method of His-mvp1 pentamer.
Mixed being incorporated under quantum dot participation, low-salt conditions of His-mvp1 and wtvp1 assembled altogether.
Concrete steps are:
1. by concentration, be the wtvp1 17mg(25 mL of 680 ug/mL) be the His-mvp1 1.551 mg(4.7 mL of 330 ug/mL with concentration) pentamer mixes, 4 ℃ of 5 h that vibrate at a slow speed;
2. dilution total protein liquid to final concentration is 200 ug/mL, adding concentration is Quantum dots CdS e 1.67 mL of 3.24 uM, and now cumulative volume is 90.77 mL, after mixing, dialysis assembling altogether (dialysis 20 h, 10 h upgrade once new dialyzate) in assembling damping fluid;
3. the packing product obtaining contains SV40 virus nanoparticles-quantum dot (VNPs that pure wt-vp1 pentamer is assembled into, the VNPs of two kinds of pentamer Hybrid assemblings, the VNPs that pure His-mvp1 is assembled into etc.), quantum dot, capsid protein pentamer, empty virus nanoparticles and the mixture of irregular aggregation.Isolate asymmetric virus nanoparticles, need following concrete steps:
I. add 5 × binding buffer, 22.5 mL, now cumulative volume is 113.27 mL, and assembling liquid is splined on to the Ni crossing through binding buffer balance
2+---nTA affinity column, only with the virus nanoparticles of mutant capsid protein, can be adsorbed onto on chromatography column, with 15 ~ 30 mM imidazoles, remove the non-specific virus nanoparticles being adsorbed on post, then use the imidazoles wash-out of 200 ~ 1000 mM to obtain asymmetric virus nanoparticles.The principle of this process is: in the matrix of his-tag chromatography gel, connected a NTA([=nitrilotriacetic acid] nitrogen base nitrilotriacetic), can with Ni ionic bond, and between the 6Xhis amino acid of Ni ion and fusion rotein, produce stronger sequestering action, thereby will separate with the histidine-tagged albumen of his-tag and other protein region.Therefore as the time marquis (as 300 mM) of the imidazoles eluant solution by high density, just imidazoles is competed combination with the imidazole ring of protein his-tag, fusion rotein elutes from gel the most at last.
II. the VNPs that concentrated 200 ~ 1000 mM imidazoles wash-outs obtain, separates uniform virus nanoparticles by sucrose density gradient centrifugation.
The preparation process of sucrose density gradient:
A) first prepare Packaging buffering liquid (10 mM Tris-HCl, pH 7.2,250 mM NaCl, 1 mM CaCl
2, 5% Glycerol) and 50wt% sucrose solution (preparation of use Packaging buffering liquid), then take the two as mother liquor, be made into mass/mass and compare the sucrose solution as 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%.
B) by after reserved transparent ultracentrifugation pipe the top good sample volume, remaining length is divided into isopyknic 9 parts, uses marking pen marking;
C) ultracentrifugation pipe is fixed on suitable centrifuge tube shelf, slowly joins in centrifuge tube with suction pipe by the order from high density to lower concentration by adherent the sucrose solution of each gradient, suction pipe mouth will contact to avoid disturbance just with sucrose liquid level;
D) after gradient prepares, be statically placed in 4 ℃ of refrigerator overnight, make it to form continuous gradient.
Ultracentrifugation: the concentrated product obtaining is slowly joined in reserved position, ultracentrifugation pipe top, maximum 2 mL of every pipe, 4 ℃ of 38000 rpm centrifugal (Beckman SW40Ti rotor) 4.5 hours in ultracentrifuge, then under ultra violet lamp is auxiliary, position and the situation of sample in continuous gradient after ultracentrifugation taken in photograph, can see that asymmetric virus nanoparticles has formed independently fluorescence band in centrifuge tube, by its taking-up.
Sample desugar: by the PBS dialysis of the sucrose in fluorescence band part without glycerine, be diluted to more than 10000 times.Sample is concentrated with the super filter tube that molecular weight cut-off is 100KD.SDS-PAGE quantitatively and characterize (see figure 1) with transmission electron microscope.
4. the sign of the asymmetric virus nanoparticles of SV40
AuNPs can efficiently be attached on the Cys of His-mvp1 displaying.Sample is characterized to (Fig. 2) with AuNPs adsorption experiment, prepare negative control (Fig. 3) and positive control (Fig. 4) simultaneously.The in the situation that of AuNPs and virus nanoparticles 2:1; asymmetric virus nanoparticles substantially only adsorbs a gold grain; the VNPs that wtvp1 forms can not ADSORPTION OF GOLD particle, and 12 pentamers all show that the VNPs of halfcystine can adsorb the gold grain that 1-3 number does not wait.This presentation of results, only comprises a pentamer (being His-mvp1) of showing cys in asymmetric virus nanoparticles, all the other 11 is wtvp1 pentamer.
Below be only an embodiment in the numerous concrete exemplary applications of the present invention, protection scope of the present invention is not constituted any limitation.All employing equivalents or equivalence are replaced and the technical scheme of formation, within all dropping on rights protection scope of the present invention.
Claims (4)
1. a preparation method for asymmetric virus nanoparticles, is characterized in that, the method comprises the steps:
I. in the VP1 of SV40 capsid protein Loop, introduce Cys, that is, in the 74th amino acids, sport Cys, and insert His-tag at the 139th, carrier construction, and realize and expressing in organism, through Ni
2+---NTA affinity chromatography column purification, obtains improved viral capsid proteins;
II. the improved viral capsid proteins that the former wild-type SV40 capsid protein wtvp1 that is 680 ug/mL by concentration is 330 ug/mL with concentration mixes, adding concentration is the Quantum dots CdS e of 3.24 uM, the volume ratio of former wild-type SV40 capsid protein wtvp1, improved viral capsid proteins and Quantum dots CdS e is 25:4.7:1.67, the common assembling of having dialysed in low salts solution;
III. adopt nickel affinity chromatography method, first with 15-30 mM imidazoles solution, remove the assembling product without improved viral capsid proteins, i.e. the virus nanoparticles of nonfunctional group and Fen Li group; Then get 200 ~ 1000 mM imidazoles eluant solutions and obtain assembling the virus nanoparticles that only contains an improved viral capsid proteins unit in product, it is asymmetric virus nanoparticles, described asymmetric virus nanoparticles has icosahedron structure, its shell is comprised of 1 His-mVP1 pentamer and 11 wtvp1 pentamers, and center is Quantum dots CdS e.
2. the preparation method of asymmetric virus nanoparticles according to claim 1, is characterized in that, the method also comprises the steps:
IV. with sucrose continuous density gradient method to step III obtain asymmetric virus nanoparticles separate, obtain uniform unsymmetrical structure.
3. the preparation method of asymmetric virus nanoparticles according to claim 1, is characterized in that, step I is specially:
Vector expression plasmid is proceeded to by Calcium Chloride Method
e.colirosetta(DE3) competent cell, culturing bacterium abduction delivering recombinant protein in LB substratum, successively through centrifugal, fragmentation and column chromatography processing, obtain improved viral capsid proteins thereafter;
In described LB substratum, also contain microbiotic and inductor, described microbiotic comprises penbritin and/or paraxin, and described inductor comprises IPTG.
4. the preparation method of asymmetric virus nanoparticles according to claim 3, is characterized in that, step I comprises the steps:
A, vector expression plasmid is proceeded to by Calcium Chloride Method
e.colirosetta(DE3) competent cell, be coated with after flat board at least 12 h, in picking mono-clonal access LB substratum, add microbiotic, in 37 ℃ of constant-temperature shaking culture, spend the night, according to 1% inoculum size transfer in LB substratum in thereafter, add microbiotic, in 37 ℃ of constant-temperature shaking culture, and add inductor, at 25 ℃～37 ℃, continue inducing culture, centrifugal collection thalline;
B, thalline that will collect are resuspended in binding buffer and carry out ultrasonication after cleaning, and the bacterium liquid after centrifugal breaking, gets supernatant liquor and carry out column chromatography, obtains improved viral capsid proteins.
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| CN104861047B (en) * | 2014-02-26 | 2018-03-20 | 中国科学院苏州纳米技术与纳米仿生研究所 | Single function magnetic nanoparticle based on ferritin |
| CN106636016B (en) * | 2017-02-24 | 2020-03-17 | 天津大学 | Method for inducing auxiliary virus-like particle self-assembly through positive and negative charges and application |
| CN111505140A (en) * | 2020-04-24 | 2020-08-07 | 厦门大学 | Chemical signal amplification multiplier based on virus capsid protein nanostructure, preparation method and application |
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