CN100351279C - Preparation of monodispersion high molecular nanometer sphere using envelop virus as template - Google Patents

Abstract

The present invention relates to a method for preparing monodisperse macromolecule nano spheres by hollow case viruses, which solves the problem that the prior art is difficult to prepare consistent nano materials on size and shapes. Hollow cowpea chlorosis piebald viruses of icosahedral plants are used as templates, and 2-allyl amido-2-methyl-1-propane sulfonic acid which is a monomer and N, N'-methylene acrylamide which is used as a crosslinking agent are filled in interior cavities of the viruses. A polymerization reaction is carried out under the space limitation of the viruses for preparing crosslinking type poly 2-allyl amido-2-methyl-1-propane sulfonic acid macromolecular compounds. After the macromolecular compounds are disengaged from the hollow viruses, the products have monodispersity on scales, the size of the products is equal to the diameters of the interior cavities of the viruses, and the shapes of the products are complementary with the spherical cavities of the viruses. The method provides an approach for designing nano macromolecule granules of specified size and shapes by different virus molecules.

Description

With ghost virus is that template prepares the mono-disperse polymer nanometer ball

[technical field]: the invention belongs to biomaterial and nano-high molecule synthetic technology cross-application field, particularly make polymer nano material for " container " with natural microbe.It can obtain that traditional preparation method of nano material is difficult to obtain singly disperses product, and is expected to produce a large amount of regular particle assembling formed synergistic effect of getting up.

[background technology]: sol-gel method, complex alkoxide method, microemulsion method, vapour deposition process, plasma method, situ aggregation method, molion graft process etc. are adopted in the preparation of nano material more.All these methods are all because the fluctuation of microenvironment, and material is consistent on size dimension and outer shape fully.Nanoparticle presents a distribution on particle diameter, though its median size is in the nanometer range, and maximum have bigger difference with minimum grain size, and product is actually the mixture of various yardstick particles.In addition, the nano material of method for preparing is spherical basically, and perhaps the interference because of microenvironment produces irregular globoid.The big multipotency of existing method for preparing polymer micro provides micron-sized particle.Though people can make nano level polymer microballoon, generally be difficult to control the homogeneous degree of particle diameter.The ultra-fine grain of preparation different quantities level needs different operational meanss usually, perhaps needs extremely accurate and harsh reaction conditions just can finish.

At nature, exist the material that some have the microstructure of rule.For example, C ₆₀The cage structure of similar football, the tubular structure of carbon nanotube, the Cup structure of cyclodextrin and calixarene, the ring texture of crown ether and cyclic peptide etc.But their each compounds all have only a kind of fixed size and dimension, and some can not be gone back some and not be difficult to catch and the encapsulation organic molecule because of there being suitable duct as polymkeric substance synthetic template because of size is too little.

Virus, as a kind of half natural life entity microorganism, its size just is in the nano level scope between 10～300nm.Virion is made of the nucleic acid (RNA or DNA) and the protein enclosure at center.If remove inner nucleic acid, different viral big and small protein " cage " can extensively provide size different nanometer reaction vessel.The investigator can prepare the nanoparticle of specifying size by selecting different virions.In addition, virus of the same race has identical yardstick and shape, this means that with ghost virion of the same race be template, synthesizes size and the identical nano material of profile.

Find to store up the mineralising reaction that ferritin can be used for space constraint as, people such as Meldrum and (see Meldrum, F.C.Heywood, B.R.﹠amp; Mann, S.Science, 1992,257:522.).Douglas and Young have carried out encapsulation and mineralising with virion to inorganic mineral tungstate and vanadate, and will gather methyl allylphenol sulfonic acid and introduce in the ghost virus, thereby the mineralising that has hindered virion (is seen Douglas, T.﹠amp; Young, M.Nature, 1998,393:152).But these two kinds of methods only are that ready-made material is packed in the viral cavity, and chemical transformation does not take place.

[summary of the invention]: the present invention seeks to solve the problem that nano material that prior art can not make preparation is consistent on size dimension and outer shape fully, providing a kind of is the method that template prepares the mono-disperse polymer nanometer ball with ghost virus.The plant ghost virus that this method is known with structure is template, carries out high molecular synthesizing in its cavity, the polymer nano-particle of preparation specified size and shape and homogeneous grain diameter.

The present invention is that template prepares the mono-disperse polymer nanometer ball with ghost virus, this nanometer ball consist of 2-acrylic-amino-2-methyl isophthalic acid-propane sulfonic acid and N, N '-methylene-bisacrylamide multipolymer; Multipolymer has crosslinking structure; Outer shape is spherical; Size is in the nano level scope; Its concrete preparation process is as follows:

---viral self-assembly and purifying:

Suspend in the Tris-hydrochloride buffer ghost cowpea chlorotic mottle virus coat protein of 0.5～5.0mg/ml with suspension dialysis 10～20h, assembles the ghost virion under 4 ℃; The assembling mixture be written into 10～40% saccharose gradients, and with the ultracentrifugation rotor 38, centrifugal 4～6h under the 000rpm; Slice gradient, the layer part of collecting the 280nm ultraviolet absorption peak;

---monomeric encapsulation:

Under 5 ℃, in the Tris-of 10～20ml hydrochloride buffer (0.05M), 25～50mg ghost cowpea chlorotic mottle virus is dispersed in 2-acrylic-amino-2-methyl isophthalic acid-propane sulfonic acid of 15～24mg, the N of 13～21mg, hatches 3～5h in Potassium Persulphate-sodium sulfite solution of N '-methylene-bisacrylamide and 2～4mg; Reduce the pH value of solution then with phthalic acid-hydrochloride buffer, and wash with a large amount of phthalic acids-hydrochloride buffer; Sample is written into foregoing 10～40% saccharose gradients subsequently, carries out ultracentrifugation and separates; Collect the gradient cut of 280nm absorption peak;

---synthesizing of polymkeric substance:

At N ₂Protection down, temperature under 5 ℃, the monomer polymerization that virus is inner and crosslinked and solidify; Be warming up to 60～80 ℃, make the polymkeric substance slaking; The urea that in system, adds 8～12mol/l; Sample is written into foregoing 10～40% saccharose gradients subsequently, carries out the ultracentrifugation fractionation, and washing and enriched product obtain the mono-disperse polymer nanometer ball.

Advantage of the present invention and positively effect: because viral yardstick is general all in nano level scope, RNA or DNA that virus is inner remove, will stay next cavity in the remaining protein enclosure, this cavity has nano level volume and specific shape, if make the polymerization in this cavity of certain monomer, formed polymkeric substance coincide with viral cavity on yardstick, in shape with the cavity complementation; Reaction environment just can be prepared the high molecular nanometer particles that is complementary with virus accurately.Virus is diversified simultaneously, and they have very big difference on size and dimension, adopts the inventive method can prepare the high molecular nanometer particles of different size and shape.The present invention is with the template of the symmetric plant ghost virus of icosahedron as the preparation macromolecule nanometer ball, and in alkaline environment, it is inner that the hole that the capsid surface has 60 diameters to be about 2nm leads to virus; In acidic medium, then can closure get up in these ducts, can be at the inner polymerization system that forms relative closure of virus.Monomer molecule will couple together with covalent linkage in viral inside, forms a complete reticulated structure.These capsids can derive from the virus of removing the nucleic acid gene group, also can derive from the assembly of the expressed capsid protein matter of genetically engineered.Adopt the inventive method can obtain being complementary with ghost virus cavity size and shape, and the polymer nano-particle of homogeneous grain diameter.

[description of drawings]:

Fig. 1 is to be the synthetic synoptic diagram of template macromolecule nanometer ball with ghost virus;

Fig. 2 is the transmission electron microscope image of polymer nanocomposite ball.

[embodiment]:

Embodiment 1

1. viral self-assembly and purifying

The ghost cowpea chlorotic mottle virus coat protein of suspension 0.5mg/ml in Tris-hydrochloride buffer (0.05M), the 15h that under 4 ℃ suspension dialysed assembles the ghost virion.The assembling mixture be written into 10% saccharose gradient, and with the ultracentrifugation rotor 38, centrifugal 4h under the 000rpm.Slice gradient, the layer part of collecting the 280nm ultraviolet absorption peak.

2. monomeric encapsulation

Under 5 ℃, in the Tris-of 10ml hydrochloride buffer (0.05M), 25mg ghost cowpea chlorotic mottle virus is dispersed in 2-acrylic-amino-2-methyl isophthalic acid-propane sulfonic acid of 15mg, the N of 13mg, hatches 3h in Potassium Persulphate-sodium sulfite solution of N '-methylene-bisacrylamide and 2mg.Use the pH value of phthalic acid-hydrochloride buffer (0.05M) reduction solution then, and wash with a large amount of phthalic acids-hydrochloride buffer.Sample is written into foregoing 10% saccharose gradient subsequently, carries out ultracentrifugation and separates.Collect the gradient cut of 280nm absorption peak.

3. polymkeric substance is synthetic

At N ₂Protection down, temperature under 5 ℃, the monomer polymerization that virus is inner and crosslinked and solidify.Be warming up to 60 ℃, make the polymkeric substance slaking.The urea that in system, adds 8mol/l.Sample is written into foregoing 10% saccharose gradient subsequently, carries out the ultracentrifugation fractionation.Washing also concentrates, and obtains the macromolecule nanometer ball product.As shown in Figure 2, with monomer dyeing, be of the encapsulation of the transmission electron microscope observing virus of unit to polymkeric substance with 100nm.

Embodiment 2

1. viral self-assembly and purifying:

The ghost cowpea chlorotic mottle virus coat protein of suspension 1.0mg/ml in the Tris-hydrochloride buffer, the 10h that under 4 ℃ suspension dialysed assembles the ghost virion; The assembling mixture be written into 20% saccharose gradient, and with the ultracentrifugation rotor 38, centrifugal 5h under the 000rpm; Slice gradient, the layer part of collecting the 280nm ultraviolet absorption peak;

2. monomeric encapsulation:

Under 5 ℃, in the Tris-of 15ml hydrochloride buffer (0.05M), 35mg ghost cowpea chlorotic mottle virus is dispersed in 2-acrylic-amino-2-methyl isophthalic acid-propane sulfonic acid of 20mg, the N of 18mg, hatches 4h in Potassium Persulphate-sodium sulfite solution of N '-methylene-bisacrylamide and 3mg; Reduce the pH value of solution then with phthalic acid-hydrochloride buffer, and wash with a large amount of phthalic acids-hydrochloride buffer; Sample is written into foregoing 30% saccharose gradient subsequently, carries out ultracentrifugation and separates; Collect the gradient cut of 280nm absorption peak;

3. polymkeric substance is synthetic:

At N ₂Protection down, temperature under 5 ℃, the monomer polymerization that virus is inner and crosslinked and solidify; Be warming up to 70 ℃, make the polymkeric substance slaking; The urea that in system, adds 10mol/l; Sample is written into foregoing 20% saccharose gradient subsequently, carries out the ultracentrifugation fractionation, and washing and enriched product obtain the mono-disperse polymer nanometer ball.

Embodiment 3

1. viral self-assembly and purifying:

The ghost cowpea chlorotic mottle virus coat protein of suspension 5.0mg/ml in the Tris-hydrochloride buffer, the 20h that under 4 ℃ suspension dialysed assembles the ghost virion; The assembling mixture be written into 40% saccharose gradient, and with the ultracentrifugation rotor 38, centrifugal 6h under the 000rpm; Slice gradient, the layer part of collecting the 280nm ultraviolet absorption peak;

2. monomeric encapsulation:

Under 5 ℃, in the Tris-of 20ml hydrochloride buffer (0.05M), 50mg ghost cowpea chlorotic mottle virus is dispersed in 2-acrylic-amino-2-methyl isophthalic acid-propane sulfonic acid of 24mg, the N of 21mg, hatches 5h in Potassium Persulphate-sodium sulfite solution of N '-methylene-bisacrylamide and 4mg; Reduce the pH value of solution then with phthalic acid-hydrochloride buffer, and wash with a large amount of phthalic acids-hydrochloride buffer; Sample is written into foregoing 40% saccharose gradient subsequently, carries out ultracentrifugation and separates; Collect the gradient cut of 280nm absorption peak;

3. polymkeric substance is synthetic:

At N ₂Protection down, temperature under 5 ℃, the monomer polymerization that virus is inner and crosslinked and solidify; Be warming up to 80 ℃, make the polymkeric substance slaking; The urea that in system, adds 12mol/l; Sample is written into foregoing 40% saccharose gradient subsequently, carries out the ultracentrifugation fractionation, and washing and enriched product obtain the mono-disperse polymer nanometer ball.

Claims (1)

1, a kind of is the method that template prepares the mono-disperse polymer nanometer ball with ghost virus, and what it is characterized in that this nanometer ball consists of 2-acrylic-amino-2-methyl isophthalic acid-propane sulfonic acid and N, N '-methylene-bisacrylamide multipolymer; Multipolymer has crosslinking structure; Outer shape is spherical; Size is in the nano level scope; Its concrete preparation process is as follows:

---viral self-assembly and purifying:

Suspend in the Tris-of the 0.05M hydrochloride buffer ghost cowpea chlorotic mottle virus coat protein of 0.5～5.0mg/ml with suspension dialysis 10～20h, assembles the ghost virion under 4 ℃; The assembling mixture be written into 10～40% saccharose gradients, and with the ultracentrifugation rotor 38, centrifugal 4～6h under the 000rpm; Slice gradient, the layer part of collecting the 280nm ultraviolet absorption peak;

---monomeric encapsulation:

Under 5 ℃, in the Tris-hydrochloride buffer of the 0.05M of 10～20ml, 25～50mg self-assembly and sublimed ghost cowpea chlorotic mottle virus are dispersed in 2-acrylic-amino-2-methyl isophthalic acid-propane sulfonic acid of 15～24mg, the N of 13～21mg, hatch 3～5h in Potassium Persulphate-sodium sulfite solution of N '-methylene-bisacrylamide and 2～4mg; Reduce the pH value of solution then with phthalic acid-hydrochloride buffer of 0.05M, and wash with a large amount of phthalic acids-hydrochloride buffer; Sample is written into foregoing 10～40% saccharose gradients subsequently, carries out ultracentrifugation and separates; Collect the gradient cut of 280nm absorption peak;

---synthesizing of polymkeric substance:

At N ₂Protection down, temperature under 5 ℃, the monomer polymerization that virus is inner and crosslinked and solidify; Be warming up to 60～80 ℃, make the polymkeric substance slaking; The urea that in system, adds 8～12mol/l; Sample is written into foregoing 10～40% saccharose gradients subsequently, carries out the ultracentrifugation fractionation, and washing and enriched product obtain the mono-disperse polymer nanometer ball.

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