CN105861418A - Application of simian virus 40 capsid protein VP1 assembled virus-like particles in mediating microparticles into cells - Google Patents

Abstract

The present invention discloses application of simian virus 40 capsid protein VP1 assembled virus-like particles in mediating microparticles into cells. Chemical modification is carried out to combine simian virus 40 capsid protein VP1 assembled virus-like particles with inorganic microparticles, so as to coat the microparticles; and the microparticles can be brought into mammalian cells to achieve intracellular delivery of the inorganic microparticles. By wrapping of nanoparticles with different functions and combination with microparticles of different properties, a method for introducing the microparticles into cells is established and a multi-level multi-functional nano-architecture is achieved. The invention has potential application value in targeting diagnosis, targeted delivery, high capacity drug deliver and hyperthermia.

Description

The virus-like particle that simian virus 40 capsid protein VP1 assembles enters the application in cell at mediation micron particle

Technical field

The present invention relates to cell transduction technical field, be more particularly to the virus-like particle that simian virus 40 capsid protein VP1 assembles The application in cell is entered at mediation micron particle.

Background technology

Biological nano granule had both had nano-scale and architectural characteristic, had again the bioactive functions of uniqueness, should in nanotechnology By aspect, there are great potentiality.Current most research in nanotechnology also focuses in manufactured chemical's nanostructured, is less frequently utilized sky Right biological nanostructures, and biological nano granule has convenient for production, the advantages such as structure is homogeneous, and biocompatibility is strong, the suitableeest Share in life and health medicine aspect.Such as virus, as natural nano grade biological, has the homogeneous structure of rule and uniqueness Biological effect.The hollow bead that the capsid protein of some viruses can be self-assembled into, does not have viral nucleic acid, it is impossible to replicate, And form is similar with natural viral.This cage modle granule can be as the various foreign particle of carrier package, including organic granular, nothing Machine granule and little molecule, thus as carrier system transport " goods " to cell or in vivo.

Viral capsid is the rules layer level structure formed by a lot of same protein.Nano material based on virus is nano science One infusive research field, has become as material science, biochemistry, viral physicochemical crossing research heat at present Point.The key element of this kind of nano material be namely based on viral protein capside (shell) and the self assembly of organic or inorganic goods or Controllable assembly, it is thus achieved that the virus nanoparticles (VNP) of functionalization.VNP can carry polymer, enzyme and other organic goods Thing, equally can also coated metal ion.Inorganic particle is loaded in the nano material of viral capsid and is designed with two possible strategy: 1, inorganic particle is packaged in the inside of capsid；2, inorganic material is combined in the outside of viral capsid.Both of which is capsid Interiorly or exteriorly, or certain part directly formation of virus protein, pack or combine inorganic goods.Postpone phage Research, also explores the formation of plant and animal virus VNP packing inorganic goods.Plant virus includes BMV, CPMV, TMV, CCMV, RCNMV, except bar-shaped TMV, other viruses are all regular dodecahedron structures.And animal virus What intermediate package crossed inorganic goods has SV40, Alphavirus and HIV-1 etc..

From the viewpoint of material science, viral capsid can play the part of different roles in VNP.If inorganic particle is in disease The outside of poison capsid is formed, and viral capsid decorates upper inorganic nanoparticles as masterplate or scaffold, then can obtain surface and take The capsule structure of the hollow with inorganic material, tubular structure etc..If inorganic particle nucleation in viral capsid, virus clothing Shell can be as a nano-reactor, and its inwall can promote, be catalyzed, control the growth of nano-particle.The third may be recognized For when viral capsid is self-assembly of around inorganic nanoparticles, make viral capsid certainly due to the nucleation of nano-particle Assemble.VNP has multiple, and we pay close attention to VNP as the cell transport agent of inorganic nanoparticles and imaging, The biomedical wide application prospect such as medicine delivery, thermotherapy and nano material design.Such as, bio-imaging is based on inorganic particulate One of grain most promising application of VNP.In experiment, VNP packs or combines nanometer gold and can be visualized spike in vitro. VNP packaging or incorporating quantum point then can be by Fluirescence observation for external or experiment in vivo.Additionally, outside CPMV The complex of portion's incorporating quantum point, the local concentration of quantum dot is the highest, and the quantum dot ratio free with in solution, fluorescent quantum Productivity improves 15%.A large amount of ferric oxide nanometer particles are combined in CPMV surface and have the local magnetic field strength of reinforcement, have uncommon Hope for nuclear magnetic resonance.But not must not proved the most completely of above-mentioned major part example.Virion can be used for medicine and delivers, Many VLP and VNP loading organic goods have been used successfully to the in vitro and in vivo of medicine delivery and have applied, and inorganic goods Also simply just it is taken into account.In packaging inorganic particle, drug molecule can be adsorbed at functional modification (particularly by we Amphiphile, amphiphilic molecule) nano grain surface, and discharge VNP depolymerization when.Inorganic particle can simultaneously imaging and Magnetic loads.When inorganic particle is combined in viral capsid outer surface, the inside of VNP then can load drug molecule, but The most such experiment is not also reported.In terms of tumor thermotherapy, various outer cover packaging magnetic nanoparticles have got more and more Obtain studied, both can carry magnetic nanoparticle, it is also possible to control the response of its treatment according to organic shell character.

The perfect outer surface of viral capsid can promote the combination of specific molecular, and this can make its some cancerous cell selectively targeted, The VNP of packaging magnetic-particle just can be used for thermotherapy.Certainly, virion can be additionally used in and builds novel nano-device Material.Self-assembling in dielectric metamaterials as single dispersing is packed the VNP of nanometer gold, cobalt oxide etc., manufacture can improve The gold of battery capacity-cobalt oxide heterozygosis nano wire, the nano-electrode of processability extremely excellence or battery.

Cell membrane can allow little molecule to pass through, cell absorb nutrition and and microenvironment between all communications utilize various mechanism to send out Raw during permeates cell membranes.Oxygen, carbon dioxide, water and little hydrophobic or nonpolar molecule can pass through Concentraton gradient Difference free diffusing passes through cell membrane.Such as ion, the little molecule such as aminoacid is by the active transport system such as memebrane protein pump or ion channel Pass through cell membrane.And the hydrophilic biomacromolecule of nanoscale usually relies on endocytosis and enters cell, this mode utilizes and derives from carefully Goods internalization is entered cell by the transport vesicle of after birth.Cellular uptake and mechanism for nano-particle have had the most research. The nano-particle such as designed, such as metal cluster, CNT, fullerene and quantum dot, it is possible to permeates cell membranes, and energy Enough enter endotheliocyte, pulmonary epithelial cells, enterocyte, pulmonary alveolar macrophage, other macrophage and neuronal cell Etc. various cells.But, different experiments room is inconsistent, even for the mechanism that cellular uptake nano-particle is proposed sometimes Conflict completely.And, every kind of nano-particle all shows the preference for different cell internalizing approach.To nano-particle carefully Therefore absorption and the systematization understanding of transport in born of the same parents become most important thing.

The size and shape of nano-particle is also the key parameter arranging their characteristic, so also determining the process of cellular uptake. Such as, HeLa cellular uptake nanometer gold is as the change of nanometer gold size and changes.The nanometer gold that internalization herceptin is modified (from 2nm to 100nm) is largely determined by its size: the size range of endocytosis full blast is 25-50nm, This is in receptor-mediated endocytic processes, and the orientation between cross-linking due to the multivalence of membrane receptor and film packaging process balances and causes 's.Intracellular gathering is found to strengthen its magnetic ability, and has more preferable magnetic resonance imaging contrast's degree.Nanometer The shape of grain is the factor that another one is important, and it directly affects the approach of absorption.Or illustrate with gold nano grain, have reality Test the geometry aspect that have studied gold nanorods (NRs) for the impact of cellular uptake, find the geometry of NRs for Cellular uptake has significant effect: the short nanometer rods with similar face electric charge is easier to into cell than long nanometer rods.Equivalently-sized Spherical nanoparticle be easier to into cell than rod-like nano granule, it may be possible to rod-like nano granule needs the longer film bending time.

Ferric oxide particles can be used for nuclear magnetic resonance, tumor thermotherapy etc., is the most promising functional material of one.By size, Ferric oxide particles substantially can be divided into superparamagnetic iron oxide particle (superparamagnetic iron oxide, SPIO), microminiature Superparamagnetic iron oxide particle (ultra-small superparamagneticironoxide, USPIO) and monocrystalline iron oxides nanometer Grain (monocrystalline ironoxide nanoparticles.MION).2004, Shapiro etc. found the super suitable of micron dimension Magnetic iron oxide particle (micron-sizedsuperparamagnetic iron oxide, MPIO), can produce more with labeled cell Strong signal contrast, reaches the spike imaging of individual cells, which results in the research interest of numerous scientist.In recent years, based on The research of MPIO and application obtain significant achievement and breakthrough.But because the superparamagnetic iron oxide particle MPIO of micron dimension Particle is big, and coating inertia is strong, is more difficult to import cell.MPIO and some transfection mediums are combined into cell, but efficiency is very Low, and transfection medium used may suppress the proliferation and differentiation of cell.And viral capsid is as importing carrier, it is expected to become one Plant new efficient, safe transduction method.

Summary of the invention

The virus-like particle that object of the present invention is to provide simian virus 40 capsid protein VP1 assembling is mediating inorganic micron Grain enters the application in cell.The virus-like particle making simian virus 40 capsid protein VP1 assemble by chemical modification is micro-with inorganic Rice grain combines so that it is is coated with by micron particle, can bring in mammalian cell by micron particle, it is achieved inorganic microparticle Cell deliver.

In order to achieve the above object, the present invention takes techniques below measure:

The virus-like particle that simian virus 40 capsid protein VP1 assembles enters the application in cell at mediation micron particle, applies Journey is as follows:

1) virus-like particle making simian virus 40 capsid protein VP1 assemble by chemical modification is combined with inorganic microparticle, makes Micron particle is coated with by it, it is thus achieved that compound micron particle.

2) by step 1) the compound micron particle that obtains hatches with mammalian cell, and micron particle can be by virus-like particle Mediation enters cell.

The virus-like particle that described simian virus 40 capsid protein VP1 assembles, the known method of available this area or reference literary composition Offer (Small, 2009,5 (6): 718-726) to obtain the virus-like particle that simian virus 40 capsid protein VP1 assembles, this virus sample Grain can pack the conventional tracer grain of this area such as quantum dot or nanometer gold as required.

According to above-mentioned application, utilize the virus-like particle that simian virus 40 capsid protein VP1 assembles at mediation micron dimension magnetic oxygen Change ferrum granule MPIO and enter the application in cell, specific as follows:

(1), build VP1 albumen expression plasmid (PET-VP1), escherichia coli (E.coli Rosetta) express, purification VP1, Its pentamer form is characterized by ultramicroscope.

(2), pack quantum dot at VP1 albumen, then be purified by sucrose density gradient, obtain the viral sample of packaging quantum dot Granule (SVQDs), is characterized by ultramicroscope.

(3), SVQDs is mixed with biotinylation reagent, remove unnecessary unconjugated biotin, obtain Biotin-SVQDs. MPIO with Biotin-SVQDs that Streptavidin is modified is mixed, is purified into the magnetic wrapped up by SVQDs by magnetic frame Pearl (MPIOs-VLP-QDs).

(4) taken pictures by fluorescence microscopy systematic observation, obtain individual particle micro-image.

(5) being mixed on ice with cultured Vero cell by MPIOs-VLP-QDs and hatch, it is unnecessary to wash MPIOs-VLP-QDs, places into incubator and continues to cultivate.

(6) positioned by 4 degree of incubated cell different time granules of fluorescence microscope MPIOs-VLP-QDs respectively.

(7) outside culture dish, place a Magnet to continue to cultivate, use fluorescence microscope MPIOs-VLP-QDs in cell Location, " after the cell trypsinization infected suspends, observe the magnetic field control to cell movement direction simultaneously.

The present invention compared with prior art, has the following advantages and effect:

The present invention utilizes the major capsid protein VP1 self-assembly systems of simian virus 40, packs quantum dot, and then passes through biotin Interact with Streptavidin, viral capsid self assembly particle encapsulation is combined in magnetic micrometer particle surface, by virus clothing Shell enters the ability of cell and micron particles brings into mammalian cell, thus develops one and can carry micron particle and enter suckling The method of zooblast.Viral capsid proteins itself has good biocompatibility, easily degrades.Can wrap up in its capsid Inorganic nanoparticles, function micron particle by chemistry or genetically modified organisms functional molecular, then can be loaded by capsid, Just can build multi-layer, multi-functional micron functional particulate, the targeting diagnosis of disease, targeting transport, high power capacity drug carrier, The aspects such as thermotherapy have potential using value.

This system is not only able to self and loads nano-particle, and can be combined with micron particle and deliver into mammalian cell. Enter the magnetic micrometer granule after cell to be controlled it by external magnetic field, including controlling the arrangement of intracellular magnetic-particle, And the direction of motion of suspension cell.The method is the granule import system of a kind of good biocompatibility, multi-layer, multifunction.

Accompanying drawing explanation

Fig. 1 VP1 albumen and pentamer schematic diagram thereof.

Wherein a is VP1 protein SDS-PAGE electrophoretogram, and b is VP1 albumen western blot qualification figure, and c is VP1 pentamer Electronic Speculum schematic diagram.

Fig. 2 is SVQDs Electronic Speculum schematic diagram.

Fig. 3 is MPIOs-VLP-QDs schematic diagram.

Wherein a is that MPIOs-VLP-QDs simulates schematic diagram, and b is MPIOs-VLP-QDs fluorescence microscope schematic diagram.

Fig. 4 be MPIOs-VLP-QDs with cell incubation different time after fluorescence localization schematic diagram.

Wherein a is MPIOs-VLP-QDs and the little fluorescence localization figure constantly of cell incubation 0, b be with cell incubation 16 hours after Fluorescence localization figure.

Fig. 5 is the fluorogram that externally-applied magnetic field controls intracellular MPIOs-VLP-QDs granule orientation.

Fig. 6 is the fluorogram that externally-applied magnetic field controls the suspension cell direction of motion of intension MPIOs-VLP-QDs granule.

Detailed description of the invention

The embodiment of the present invention mediates as a example by MPIO entrance cell by the virus-like particle that simian virus 40 capsid protein VP1 assembles, It is further illustrated its operating process, but the scope of the present invention is not limited to following embodiment.Technical scheme of the present invention, as Not special standby explanation, is the conventional scheme of this area.

Embodiment 1:

Prepared by packaging quantum dot virus-like particle

(1), build VP1 albumen expression plasmid (PET-VP1), escherichia coli (E.coli Rosetta) express, purification VP1, Its pentamer form is characterized by ultramicroscope.

It is template amplification VP1 with pSV21SphI-N1 (J Virol Methods, 1997,64 (1): 1-9), the double enzyme of BglII-XhoI After cutting, it is connected into the pQE-30 carrier (purchase of Qiagen company) of BamHI-SalI double digestion, is built into pQEVP1；With pQEVP1 For template, amplification N end merges the VP1 encoding gene having Histag, by NdeI-XhoI site insert pET32a (+) Carrier (carrier contains two NdeI sites, the abundant enzyme action of NdeI), is built into pET32a-hisVP1.All enzyme action and PCR Operating procedure is all carried out by Related product description.PQEVP1 and pET32a-hisVP1 determines genes of interest sequence through order-checking The correctness of row.

By pET32a-hisVP1 CaCl₂Method is transformed into E.coli Rosetta (DE3) competent cell, picking list from flat board Clone accesses in 5mL LB Tube propagation base, adds ampicillin and chloromycetin, cultivates in 37 DEG C of constant temperature, 200r/min Overnight.Next day, transfer in 5mL LB Tube propagation base (parallel 4 pipes) by 1% inoculum concentration, add corresponding antibiotic, In 37 DEG C of constant temperature, 200r/min shaken cultivation to OD600 between 0.4～0.6, wherein 1 pipe is as blank, separately Outer 3 pipes all add IPTG to final concentration 1mM, respectively 20 DEG C, 25 DEG C and 37 DEG C continue inducing culture 16h, 8h and After 3h, collect thalline, with the expression of SDS-PAGE detection VP1, find that at three temperature, VP1 all can express, and There is inclusion body to produce, select 25 DEG C as inducing temperature when preparing albumen in a large number.

E.coli Rosetta (DE3) (pET32a-hisVP1) is inoculated in 5mL LB Tube propagation base, adds ammonia benzyl penicillium sp Element and chloromycetin, in 37 DEG C of constant temperature, 200r/min overnight incubation.Next day, transfer in 500mL LB triangular flask, add Corresponding antibiotic, in 37 DEG C of constant temperature, 200r/min shaken cultivation to OD600 between 0.4～0.6, adds IPTG to end Concentration 1mM, after 25 DEG C are continued inducing culture 8h, collects thalline, carries out ultrasonic breaking by resuspended after bacterial sediment cleaning once Broken, it is centrifuged 30min with 10000r/min, supernatant is splined on Ni²⁺-NTA affinity chromatograph column purification destination protein (figure 1a), Western blot carries out verifying (Fig. 1 b), and VP1 pentamer is carried out Electronic Speculum sign (Fig. 1 c).

(2), in VP1 albumen, add quantum dot (605nm launches light), carry out dialysis (10mMTris-HCl pH in Packaging buffering liquid 7.2,250mMNaCl, 1mM CaCl₂, 5% glycerol), then be purified by sucrose density gradient, obtain packaging quantum dot Virus-like particle (SVQDs), carry out characterizing (Fig. 2) by ultramicroscope.

Embodiment 2:

The preparation of MPIOs-VLP-QDs granule

(1), by SVQDs and biotinylation reagent (EZ-Link Sulfo-NHS-LC-LC-Biotin, Thermo company, article No.: Pierce Part no.21343) carry out mixing (mol ratio 1:3), room temperature stands 30 minutes.Use 100KD super filter tube and Mixture is diluted 104 times by PBS, to remove unnecessary unconjugated biotin.By magnetic-particle (MPIO, MyOne^TMStreptavidin C1, Thermo Fisher company, article No.: 65001) with magnetic frame absorption on EP tube wall after, Suck supernatant, add PBS, after so washing 3 times, by magnetic-particle (MPIO) with Biotin-SVQDs in molar ratio 1:1000, or make Biotin-SVQDs definitely excess mixing, vortex oscillator is slowly shaken, under room temperature, reacts 30 Minute.After reaction, sample is placed on magnetic frame absorption, magnetic bead 3 minutes, magnetic nanoparticle 30min, sucks supernatant, And wash twice with PBS, obtain SVQDs parcel and combine the granule (MPIOs-VLP-QDs) of magnetic bead.By final magnetic bead Be suspended in 500 μ L PBS, be placed on 4 DEG C standby.

(2) being taken pictures by fluorescence microscopy systematic observation, use 60 times of oily lens heads, optical filter is set to red channel and light field passage, Obtain MPIOs-VLP-QDs individual particle micro-image (Fig. 3).

Embodiment 3:

Cell imports MPIOs-VLP-QDs granule and controlled manipulation

(1) Cultivation of Vero, takes out the MPIOs-VLP-QDs of 200 μ L 5mg/mL, is added separately to Vero cell 80-90% It is paved with the glass bottom capsule of rate.Capsule is placed on 4 DEG C of refrigerators and hatches 1.5h, use DMEM culture medium to be washed by capsule afterwards Three times, add 5 μ L Hoechst 33258 and 500 μ LOpti-MEM culture medium incubated at room 5 minutes, by capsule DMEM Culture medium is washed three times, adds 1.5mL fresh DMEM medium, is placed in 37 DEG C of CO2 gas incubator and continues cultivation 24 h。

(2) after 4 DEG C of incubated cell 1.5h of MPIOs-VLP-QDs, using fluorescence microscope, 60X object lens are observed in capsule Sample, the nucleus of use DAPI filter set imaging Hochest dye, TRITC filter set imaging magnetic coupling granule, Light field shooting cellular morphology, result display MPIOs-VLP-QDs can be adsorbed in cell surface (Fig. 4 a).Cell continues to put After 37 DEG C of CO2 gas incubator continue to cultivate 16h, micro-imaging finds that MPIOs-VLP-QDs enters cell and is positioned at Close on nuclear region (Fig. 4 b).

If in cell cultivation process, add a magnetic field in capsule side, after MPIOs-VLP-QDs " infects " 24h, carefully The magnetic-particle of intracellular and fluorescence thereof all align (Fig. 5) by magnetic direction.MPIOs-VLP-QDs " is infected " After cell becomes round with 200 microlitre trypsinizations, add 2mL DMEM culture medium, with liquid-transfering gun, cell blown afloat suspension, At capsule on one side plus Magnet, shoot dynamic process with light field under the microscope, it can be seen that containing magnetic-particle The cell of MPIOs-VLP-QDs, can be under the control of externally-applied magnetic field, by magnetic direction directed movement (Fig. 6).

Claims (4)

1. the virus-like particle that simian virus 40 capsid protein VP1 assembles enters the application in cell at mediation micron particle.

Application the most according to claim 1, described micron particle is inorganic microparticle.

Application the most according to claim 1, it is characterised in that: the virus-like particle that simian virus 40 capsid protein VP1 assembles enters the application in cell at mediation micron dimension magnetic iron oxide particle MPIO.

Application the most according to claim 3, its step includes:

(1), build VP1 albumen expression plasmid, escherichia coli expression, purification VP1, characterize its pentamer form by ultramicroscope；

(2), pack quantum dot at VP1 albumen, then be purified by sucrose density gradient, obtain the virus-like particle SVQDs of packaging quantum dot, characterized by ultramicroscope；

(3), SVQDs is mixed with biotinylation reagent, remove unnecessary unconjugated biotin, obtain Biotin-SVQDs；

MPIO with Biotin-SVQDs that Streptavidin is modified is mixed, is purified into the magnetic bead MPIOs-VLP-QDs wrapped up by SVQDs by magnetic frame；

(4) taken pictures by fluorescence microscopy systematic observation, obtain individual particle micro-image；

(5) MPIOs-VLP-QDs is mixed on ice with cultured Vero cell hatch, wash unnecessary MPIOs-VLP-QDs, place into incubator and continue to cultivate；

(6) positioned by the granule under fluorescence microscope MPIOs-VLP-QDs incubated cell difference incubation time respectively；

(7) outside culture dish, place a Magnet to continue to cultivate, use fluorescence microscope MPIOs-VLP-QDs location in cell, after the cell trypsinization infected being suspended, observe the magnetic field control to cell movement direction simultaneously.