CN102321590A - Preparation method of asymmetric virus nanoparticles - Google Patents
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- CN102321590A CN102321590A CN201110214965A CN201110214965A CN102321590A CN 102321590 A CN102321590 A CN 102321590A CN 201110214965 A CN201110214965 A CN 201110214965A CN 201110214965 A CN201110214965 A CN 201110214965A CN 102321590 A CN102321590 A CN 102321590A
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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 on molecular level to handle the self-assembly of biomacromolecule, relate in particular to the preparation method of a kind of asymmetric virus nano particle (asymmetric VNPs), belong to the nanometer biotechnology field with a kind of controlled method.
Background technology
Nanotechnology is meant in the yardstick of 0.1～100 nanometer, research electronics, atom and molecular motion rule and characteristic and material and material are carried out Treatment Technology be called as nanotechnology.Scientists in 1981 invent the important tool-scanning tunnel microscope of research nanometer, atom, divide subworld from then on visible.The international nanosecond science and technology meeting of the nineteen ninety first is held in U.S. Baltimore, and the nanotechnology form is born.
Nano particle is because of character such as its unique light, electricity, magnetic and mechanics; Have characteristics such as size is little, specific surface area is big, surface energy is high, the surface atom ratio is big because of it again; Obtained extensive concern in fields such as bio-sensing, Materials science, energy and information technologies; Be to study the earliest and the most sophisticated nano material, can be used for developing nano-device with special property and function.Yet because the surface chemistry isotropy, therefore nano particle receives certain limitation as constructing primitive lack diversity and stability on function and application.So one of key issue of development nano material and device is transformed into the assembling of control nanometer construction unit.
Development in recent years a series of asymmetric modifying method to nano particle, for assemble nanometer particle controlled property provides some novelty efficient strategy.These methods can make common functional nano particle become required construction unit isotropic nano-particle modified one-tenth anisotropy itself.In recent years, based on anisotropic assembling owing to its excellent controllability receives extensive concern.For the asymmetric nano material of pattern, and the nanocrystalline of the significantly different crystal face of character arranged, they are easy to the anisotropy of dependence itself and modify and assemble.A lot of analog results of calculating show, nano particle is carried out asymmetric modification can make it become anisotropy by isotropy, and then can carry out more complicated, controlled and directed assembling.Exactly because but pattern of nano particle own and surface properties have the ball symmetry, general method can only form uniform decorative layer.Recent years, reported a series of effective methods successively to the asymmetric modification of nano particle, make that this bottleneck is able to break through, also better strategy and development space are provided simultaneously for controlled assembling.
Biomacromolecule such as protein, DNA is natural nano material, and their structures are various, can self-replacation, and the height homogeneous is easy to artificial the manipulation and a large amount of preparations.DNA has shown unique advantage in Nano-technology Development; Compare with DNA, the structural information that protein carries is abundanter, thereby is expected to for nanotechnology service platform more flexibly is provided.Wherein, the albumen cage structure, as viral capsid, ferritin, thermal shock albumen has been widely used in studying the model of macromole self-assembly and material synthetic nano-reactor.But, on molecular level, handle their self-assembly with a kind of controlled method, and the assembling product of accurately analyzing them, remain challenging work.
Summary of the invention
The object of the present invention is to provide a kind of asymmetric virus nano particulate preparation method, it can realize the controlled self-assembly of biomacromolecule on molecular level, thereby overcomes deficiency of the prior art.
For realizing the foregoing invention purpose, the present invention has adopted following technical scheme:
A kind of asymmetric virus nano particulate preparation method is characterized in that this method is:
Genetic modification is carried out on the viral capsid proteins surface; Make viral capsid proteins have the coupled functional group simultaneously and separate group, then should improved viral capsid proteins and wild-type virus capsid protein thorough mixing, and control the ratio of these two kinds of viral capsid proteins; Total amount according to viral capsid proteins adds corresponding inorganic nanoparticles simultaneously; Realize the controlled assembling of virus appearance nano particle, obtain the nano particle of asymmetric functionalization, i.e. title product.
Say that further said viral capsid proteins comprises SV40 capsid protein VP1, said functional group comprises halfcystine, and said separation group comprises His-tag.
This method comprises the steps:
ⅰ. in the VP1 of SV40 capsid protein Loop, introduce Cys, that is, sport Cys in the 74th amino acids; And at the 139th insertion His-tag, carrier construction His-mvp1, and in organism, realize expressing; Purified, obtain improved viral capsid proteins His-mvp1;
ⅱ. get improved viral capsid proteins His-mvp1 and former wild-type SV40 capsid protein wtvp1 thorough mixing; Through regulating the ratio of these two kinds of capsid proteins; Realization is to the control of these two kinds of capsid protein component proportionss in the assembly virus nano individual particles; Add inorganic nanoparticles according to the capsid protein total amount and the amount of nano particle material than 60:1 ~ 180:1, assembling is altogether accomplished in dialysis 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 nano particle.
This method also comprises the steps:
ⅳ. with sucrose continuous density gradient method the asymmetric virus nano particle that step ⅲ obtains is separated, obtain uniform unsymmetrical structure.
Step ⅰ is specially:
The His-mvp1 expression plasmid is changed over to Calcium Chloride Method
E.coliRosetta (DE3) competent cell, culturing bacterium and abduction delivering recombinant protein His-mvp1 in the LB substratum handle through centrifugal, fragmentation and column chromatography thereafter successively, obtain improved viral capsid proteins His-mvp1;
Also contain microbiotic and inductor in the said LB substratum, said microbiotic comprises penbritin and/or paraxin, and said inductor comprises IPTG.
Step ⅰ comprises the steps:
A, the His-mvp1 expression plasmid is changed over to Calcium Chloride Method
E.coliRosetta (DE3) competent cell, be coated with flat board at least 12 h after, the picking mono-clonal inserts in the LB substratum; Add microbiotic, spend the night, transfer in the LB substratum according to 1% inoculum size thereafter in 37 ℃ of constant-temperature shaking culture; Add microbiotic,, and add inductor in 37 ℃ of constant-temperature shaking culture; Continue inducing culture, centrifugal collection thalline at 25 ℃～37 ℃;
B, be resuspended in after the thalline collected cleaned and carry out ultrasonication among the binding buffer, the bacterium liquid after the centrifugal fragmentation is got supernatant and is carried out column chromatography, obtains improved viral capsid proteins His-mvp1.
Step ⅱ is specially: with His-mvp1 and the wtvp1 thorough mixing of mol ratio 1:50 ~ 1:11, and add inorganic nanoparticles, the ratio control of inorganic nanoparticles and total protein molecule number is 60:1 ~ 180:1, and assembling is altogether accomplished in dialysis in low salts solution.
The asymmetric virus nano particle that is obtained among the step ⅲ has the icosahedron structure, and its shell is made up of 1 His-mVP1 pentamer and 11 wtvp1 pentamers, and the center is an inorganic nanoparticles.
Step ⅲ is specially:
Adopt the nickel affinity chromatography method, at first remove the assembling product that does not have improved viral capsid proteins His-mvp1, promptly do not have function group and the virus nano particle that separates group with 15-30 mM imidazoles solution; Getting 200 ~ 1000 mM imidazoles eluant solutions then obtains assembling and only contains a unitary virus nano particle of improved viral capsid proteins His-mvp1, promptly asymmetric virus nano particle in the product.
(virus-based nanoparticles VNPs) is the hollow bead that is made up of one or more viral capsid proteins to the virus nano particle, and self-assembly produces usually, does not contain viral nucleic acid, can not self-replicating.The present invention is through the symmetry or the virus surface genetic modification of virus structure; Realize the controlled assembling of virus appearance nano particle; Obtain a kind of nano particle of asymmetric functionalization, thereby might obtain new physics or chemical property, for virus signature and spike etc. provides new structured material.The success of this strategy will be constructed primitive for the nanometer that preparation has a controlled assembling performance otherwise effective technique approach will be provided, thereby expedite the emergence of novel nano-device, for basic physical problems research and downstream application lay a good foundation.
Compare to prior art, the advantage that the present invention has is: select for use biomacromolecule-protein as nano material, its easy transformation with artificial is handled, and conveniently obtains in a large number.Through the genetic engineering means in virus nano particle Knockdown block simultaneously the integration function group with separate group.But utilize the symmetry of the viral capsid proteins structure of self-assembly, can two kinds of different protein moleculars be mixed the back and carry out the orientation assembling, so assembly has variety and controllability by design in advance; Easily-controlled reaction conditions, and can realize scale operation.
Description of drawings
Fig. 1 is that the present invention utilizes the main capsid protein vp1 packing of SV40 quantum dot to assemble a kind of asymmetric virus nano particulate transmission electron microscope photo that makes altogether;
Fig. 2 is the transmission electron microscope figure of asymmetric VNPs absorption AuNPs shown in Figure 1;
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 a kind of asymmetric virus nano particle provided by the invention (asymmetric VNPs); Utilize biomacromolecule---viral capsid proteins (SV40; CCMV, the capsid protein of viruses such as TMV) model of assembling altogether with novel nano-material is through the genetic modification to the viral capsid proteins surface; With functional group with separate group structurally dexterously the coupling; Assemble again after then the albumen of transformed capsid protein and wild-type being pressed the specified proportion thorough mixing,, obtain a kind of nano particle of asymmetric functionalization with the controlled assembling of the method realization virus kind nano particle of controlling two kinds of albumen pentamer ratios.According to the separation group that has on the asymmetric particle, further realize the separation and the purifying of title product again.At last separation is obtained the nano particle of asymmetric functionalization, and preferably adopt AuNPs that its functional group is characterized.The product of the present invention's preparation is asymmetric virus nano particle (asymmetric VNPs).
Divided by concrete steps:
1. viral capsid proteins is carried out genetic modification, make up the carrier that has functional group and the capsid protein two mutants that separates group;
2. the carrier that builds is transformed in the organism and expresses, purifying and evaluation obtain the viral capsid proteins two mutants;
3. with the capsid protein thorough mixing of this two mutants and wild-type,, assembles inorganic nano-particle under participating in then through dialysis;
4. according to the different separation method purifying assembling of the feature selection of separating group product;
5. it is centrifugal that the nano particle of the asymmetric functionalization that will obtain is done continuous density gradient, prepares uniform asymmetric virus nano particle (asymmetric VNPs);
Be easier to understand the practicality of its substantive distinguishing features and institute's tool thereof for the preparation method who makes asymmetric virus nano particle of the present invention (asymmetric VNPs); Following constipation closes embodiment 1 and Fig. 1-Fig. 4 technical scheme of the present invention is done further to specify, but following description and explanation about embodiment do not constitute any limitation protection domain of the present invention.
Embodiment 1
In the Loop structure of the main capsid protein vp1 of SV40, introduce Cys (promptly the 74th amino acids sports Cys), insert His-tag, be built into carrier PET32a-His-mvp1 (His-mvp1) at the 139th; Confirm the exactness of target gene sequences through order-checking.PET32a-His-mvp1 is changed over to Calcium Chloride Method
E.coliRosetta (DE3) competent cell is coated with behind dull and stereotyped 12 h that the picking mono-clonal inserts the 5 mL LB test tube substratum from flat board, adds penbritin and paraxin, in 37 ℃ of constant temperature, and 180 r/min overnight cultures.Transfer in 5 ml LB test tube substratum (parallel 3 pipes) according to 1% inoculum size, add corresponding microbiotic, in about 37 ℃ of constant temperature, 180 r/min shaking culture, 2.5 h (OD600 is between 0.4 ~ 0.6); Wherein 1 pipe is as blank (not adding IPTG); Other two pipes all add the IPTG that the inductor final concentration is 1 mM, respectively 25 ℃ with 37 ℃ of continuation inducing culture 10 h and 2 h after, centrifugal collection thalline; Detect the expression of His-mvp1 with SDS-PAGE; Find all can express under two kinds of temperature, also have inclusion body to produce the inducing temperature when selecting 25 ℃ of conducts to prepare albumen in a large number simultaneously.
Will
E.coliRosetta (DE3)/PET32a-His-mvp1 is inoculated in the 5 mL LB test tube substratum, adds corresponding microbiotic in 37 ℃ of constant temperature, 180 r/min overnight cultures.5 mL bacterium liquid were transferred in the 500 mL LB triangular flasks in second day; Add penbritin (final concentration 100 ug/mL) and paraxin (final concentration 68 ug/mL); Back (OD600 is between 0.4 ~ 0.6) in about 37 ℃ of constant temperature, 180 r/min shaking culture, 2.5 h; Adding the inductor final concentration is the IPTG of 1 mM, continues vibration inducing culture 10 h, centrifugal collection thalline down at 25 ℃.Be resuspended in after cleaning once with binding buffer bacterial sediment among the binding buffer of certain volume and carry out ultrasonication (condition: ultrasonic power 400W; 3 s work; 5 s intermittently; Total ultrasonication times 60 min), the bacterium liquid after the fragmentation is splined on supernatant the Ni that crosses through binding buffer balance with centrifugal 30 min of 12000 r/min
2+---The NTA affinity column uses the imidazoles of lower concentration (20 mM, 40 mM, 60 mM) to wash post except that foreigh protein removing then successively, uses elution buffer (500 mM imidazoles) wash-out target protein at last.
The preparation of His-mvp1 and wtvp1 pentamer
His-mvp1 behind the purifying mainly exists with the pentamer form, and also some exists with the virus nano particle form, so need remove the virus nano particle among the His-mvp1, prepares pure His-mvp1 pentamer, two steps of this process need:
1. in the His-mvp1 pentamer for preparing, adding final concentration is the DTT of 10 mM; (volume is that 10 mL His-mvp1 albumen leave standstill dialysis 10 h in 4 ℃ of 1 L depolymerization damping fluid in dialysis in the depolymerization damping fluid then; Change once fresh depolymerization damping fluid, continue to leave standstill dialysis 10 h);
2. the His-mvp1 albumen ultracentrifugation that will dialyse is removed remaining virus nano particle, and Ultracentrifugation conditions is 4 ℃, and centrifugal 1 h of 55000 rpm (Beckman Type 90Ti rotor) gets supernatant and is the His-mvp1 pentamer.
The concentration of His-mvp1 pentamer is measured through test kit Bradford Protein Assay (coomassie brilliant blue staining method), and step is carried out according to the test kit specification sheets.Combine the SDS-PAGE checking simultaneously.
The preparation process of former wild-type SV40 capsid protein wtvp1 pentamer is identical with the preparation method of His-mvp1 pentamer.
His-mvp1 and wtvp1 mix and are incorporated in the assembling altogether down of quantum dot participation, low-salt conditions.
Concrete steps are:
1. be that wtvp1 17mg (25 mL) and the concentration of 680 ug/mL is that His-mvp1 1.551 mg (4.7 mL) pentamer of 330 ug/mL mixes 4 ℃ of 5 h that vibrate at a slow speed with concentration;
2. dilution total protein liquid to final concentration is 200 ug/mL; Adding concentration is quantum dot CdSe 1.67 mL of 3.24 uM, and this moment, TV was 90.77 mL, behind the mixing; Dialysis assembling altogether (20 h that dialyse, 10 h upgrade once new dialyzate) in the assembling damping fluid;
3. the packing product that obtains contains SV40 virus nano particle-quantum dot (VNPs that pure wt-vp1 pentamer is assembled into; Two kinds of pentamers mix the VNPs of assembling; The VNPs that pure His-mvp1 is assembled into etc.); Quantum dot, capsid protein pentamer, the empty virus nano particle and the mixture of irregular aggregation.Isolate asymmetric virus nano particle, need following concrete steps:
ⅰ. add 5 * binding buffer, 22.5 mL, this moment, TV was 113.27 mL, and assembling liquid is splined on the Ni that crosses through binding buffer balance
2+---The NTA affinity column; The virus nano particle that only has the two mutants capsid protein can be adsorbed onto on the chromatography column; Remove the non-special virus nano particle that is adsorbed on the post with 15 ~ 30 mM imidazoles, use the imidazoles wash-out of 200 ~ 1000 mM to obtain asymmetric virus nano particle then.The principle of this process is: connected a NTA ([=nitrilotriacetic acid] nitrogen base nitrilotriacetic) on the matrix of his-tag chromatography gel; Can with the Ni ionic bond; And produce stronger sequestering action between the 6Xhis amino acid of Ni ion and fusion rotein, thereby will have the histidine-tagged albumen of his-tag and other albumen makes a distinction.Therefore as the time marquis (like 300 mM) with the imidazoles eluant solution of high density, combine just imidazoles is competed with the imidazole ring of protein his-tag, fusion rotein elutes from gel the most at last.
ⅱ. concentrate the VNPs that 200 ~ 1000 mM imidazoles wash-outs obtain, uniform virus nano particle separation is come out through SDGC.
The preparation process of sucrose density gradient:
A) prepare Packaging buffering liquid (10 mM Tris-HCl, pH 7.2,250 mM NaCl, 1 mM CaCl earlier
2, 5% Glycerol) and 50wt% sucrose solution (with the preparation of Packaging buffering liquid), be that mother liquor is made into mass/mass than the sucrose solution that is 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% with the two then.
B) sample volume has been reserved in transparent ultracentrifugation pipe the top after, remaining length is divided into isopyknic 9 parts, uses the marking pen marking;
C) the ultracentrifugation pipe is fixed on the suitable centrifuge tube shelf, slowly joins in the centrifuge tube the sucrose solution of each gradient is adherent by the order from the high density to the lower concentration with suction pipe, the suction pipe mouth will contact to avoid disturbance with the sucrose liquid level just;
D) after gradient prepares, be statically placed in 4 ℃ of refrigerator overnight, make it to form continuous gradient.
Ultracentrifugation: will concentrate the product that obtains and slowly join in the position of reserving ultracentrifugation pipe top; Maximum 2 mL of every pipe; 4 ℃ of 38000 rpm centrifugal (Beckman SW40Ti rotor) is 4.5 hours in ultracentrifuge, and be auxiliary down at ultra violet lamp then, position and the situation of sample in continuous gradient behind the ultracentrifugation that take of taking a picture; Can see that asymmetric virus nano particle has formed independently fluorescent belt in centrifuge tube, with its taking-up.
Sample desugar:, be diluted to more than 10000 times with the PBS dialysis of the sucrose in the fluorescent belt part with no glycerine.Use molecular weight cut-off sample to be concentrated as the ultrafiltration pipe of 100KD.SDS-PAGE quantitatively and with transmission electron microscope characterizes (see figure 1).
4. the asymmetric virus nano particulate of SV40 characterizes
AuNPs can efficiently be attached on the Cys of His-mvp1 displaying.With the AuNPs adsorption experiment sample is characterized (Fig. 2), prepare negative control (Fig. 3) and positive control (Fig. 4) simultaneously.Under the situation of AuNPs and virus nano particle 2:1; Asymmetric virus nano particle basically only adsorbs a gold grain; The VNPs that wtvp1 forms can not the ADSORPTION OF GOLD particle, and 12 pentamers show that all the VNPs of halfcystine then can adsorb the gold grain that the 1-3 number does not wait.This presentation of results only comprises a pentamer (being His-mvp1) of showing cys in the asymmetric virus nano particle, all the other 11 is the wtvp1 pentamer.
Below only be 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, all drop within the rights protection scope of the present invention.
Claims (9)
1. asymmetric virus nano particulate preparation method is characterized in that this method is:
Genetic modification is carried out on the viral capsid proteins surface; Make viral capsid proteins have the coupled functional group simultaneously and separate group, then should improved viral capsid proteins and wild-type virus capsid protein thorough mixing, and control the ratio of these two kinds of viral capsid proteins; Total amount according to viral capsid proteins adds corresponding inorganic nanoparticles simultaneously; Realize the assembling of virus nano particle controlled, obtain the nano particle of asymmetric functionalization, i.e. title product.
2. asymmetric virus nano particulate preparation method according to claim 1 is characterized in that said viral capsid proteins comprises SV40 capsid protein VP1, and said functional group comprises halfcystine, and said separation group comprises His-tag.
3. asymmetric virus nano particulate preparation method according to claim 1 is characterized in that this method comprises the steps:
ⅰ. in the VP1 of SV40 capsid protein Loop, introduce Cys, that is, sport Cys in the 74th amino acids; And at the 139th insertion His-tag, carrier construction His-mvp1, and in organism, realize expressing; Purified, obtain improved viral capsid proteins His-mvp1;
ⅱ. get improved viral capsid proteins His-mvp1 and former wild-type SV40 capsid protein wtvp1 thorough mixing; Through regulating the ratio of these two kinds of capsid proteins; Realization is to the control of these two kinds of capsid protein component proportionss in the assembly virus nano individual particles; Add inorganic nanoparticles according to capsid protein total amount and inorganic nanoparticles amount of substance than 60:1 ~ 180:1, assembling is altogether accomplished in dialysis 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 nano particle.
4. asymmetric virus nano particulate preparation method according to claim 3 is characterized in that this method also comprises the steps:
ⅳ. with sucrose continuous density gradient method the asymmetric virus nano particle that step ⅲ obtains is separated, obtain uniform unsymmetrical structure.
5. asymmetric virus nano particulate preparation method according to claim 3 is characterized in that step ⅰ is specially:
The His-mvp1 expression plasmid is changed over to Calcium Chloride Method
E.coliRosetta (DE3) competent cell, culturing bacterium and abduction delivering recombinant protein His-mvp1 in the LB substratum handle through centrifugal, fragmentation and column chromatography thereafter successively, obtain improved viral capsid proteins His-mvp1;
Also contain microbiotic and inductor in the said LB substratum, said microbiotic comprises penbritin and/or paraxin, and said inductor comprises IPTG.
6. asymmetric virus nano particulate preparation method according to claim 5 is characterized in that step ⅰ comprises the steps:
A, the His-mvp1 expression plasmid is changed over to Calcium Chloride Method
E.coliRosetta (DE3) competent cell, be coated with flat board at least 12 h after, the picking mono-clonal inserts in the LB substratum; Add microbiotic, spend the night, transfer in the LB substratum according to 1% inoculum size thereafter in 37 ℃ of constant-temperature shaking culture; Add microbiotic,, and add inductor in 37 ℃ of constant-temperature shaking culture; Continue inducing culture, centrifugal collection thalline at 25 ℃～37 ℃;
B, be resuspended in after the thalline collected cleaned and carry out ultrasonication among the binding buffer, the bacterium liquid after the centrifugal fragmentation is got supernatant and is carried out column chromatography, obtains improved viral capsid proteins His-mvp1.
7. asymmetric virus nano particulate preparation method according to claim 3; It is characterized in that; Step ⅱ is specially: with His-mvp1 and the wtvp1 thorough mixing of mol ratio 1:50 ~ 1:11; And the interpolation inorganic nanoparticles, the ratio control of inorganic nanoparticles and the total molecule number of capsid protein is 60:1 ~ 180:1, assembling is altogether accomplished in dialysis in low salts solution.
8. asymmetric virus nano particulate preparation method according to claim 3; It is characterized in that; The asymmetric virus nano particle that is obtained among the step ⅲ has the icosahedron structure; Its shell is made up of 1 His-mVP1 pentamer and 11 wtvp1 pentamers, and the center is an inorganic nanoparticles.
9. asymmetric virus nano particulate preparation method according to claim 3 is characterized in that step ⅲ is specially:
Adopt the nickel affinity chromatography method, at first remove the assembling product that does not have improved viral capsid proteins His-mvp1, promptly do not have function group and the virus nano particle that separates group with 15-30 mM imidazoles solution; Getting 200 ~ 1000 mM imidazoles eluant solutions then obtains assembling and only contains a unitary virus nano particle of improved viral capsid proteins His-mvp1, promptly asymmetric virus nano particle in the product.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102787164A (en) * | 2012-06-12 | 2012-11-21 | 中国科学院苏州纳米技术与纳米仿生研究所 | Colloidal gold dimer, and synthesis method of colloidal gold and colloidal gold dimer |
| CN104861047A (en) * | 2014-02-26 | 2015-08-26 | 中国科学院苏州纳米技术与纳米仿生研究所 | Ferritin-based monofunctionalized magnetic nanometer particle |
| CN106636016A (en) * | 2017-02-24 | 2017-05-10 | 天津大学 | Method for assisting self-assembly of virus-like particles by introducing 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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2011
- 2011-07-29 CN CN201110214965.7A patent/CN102321590B/en not_active Expired - Fee Related
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102787164A (en) * | 2012-06-12 | 2012-11-21 | 中国科学院苏州纳米技术与纳米仿生研究所 | Colloidal gold dimer, and synthesis method of colloidal gold and colloidal gold dimer |
| CN104861047A (en) * | 2014-02-26 | 2015-08-26 | 中国科学院苏州纳米技术与纳米仿生研究所 | Ferritin-based monofunctionalized magnetic nanometer particle |
| 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 |
| CN106636016A (en) * | 2017-02-24 | 2017-05-10 | 天津大学 | Method for assisting self-assembly of virus-like particles by introducing positive and negative charges and application |
| 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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| CN102321590B (en) | 2014-04-16 |
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