CN105367630A - Method for preparing two-dimensional nanometer structure from tobacco mosaic virus capsid protein mutant - Google Patents
Method for preparing two-dimensional nanometer structure from tobacco mosaic virus capsid protein mutant Download PDFInfo
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Abstract
According to the present invention, a tobacco mosaic virus capsid protein mutant is used, an amino acid sequence represented by SEQ ID No.1 is subjected to point mutation to obtain the sequence of the tobacco mosaic virus capsid protein mutant, the point mutation is that the site 103 is mutated into cysteine, and the tobacco mosaic virus capsid protein mutant is used to prepare a two-dimensional nanometer structure; according to the preparation method, a tubular virus particle template is formed by using the mutant or recombinant protein containing the mutant, the specific nucleation is achieved by using the efficient chemical adsorption effect of the mercapto functional groups distributed on the inner surface of the pore channel and the metal ions, and through the subsequent growth regulation, the selective growth of the metal having the superfine diameter in the nano-tubes is achieved, and the two-dimensional nanometer structure preparation based on the tubular virus particle template mineralization is achieved; and the preparation method has characteristics of simple step and mild reaction conditions, and the prepared two-dimensional nanometer structure has characteristics of good biocompatibility and easy surface modification.
Description
Technical field
The present invention relates to field of nano biotechnology, be specifically related to a kind of tobacco mosaic disease virus capsid protein mutant, particularly relate to the method for this tobacco mosaic disease virus capsid protein Mutant Preparation two-dimensional nanostructure of application.
Background technology
Protein is made up of 20 multiple amino acids, and structure-rich is various.The protein structure had been found that at present just has ball, bar, layer and polyhedron etc.The template that these structures provide desirable for preparing various nano material.Viral capsid proteins is the wherein important representative of a class, and the organic nano material as a kind of structure explication is widely used in field of nanometer technology.First, virus has size advantage, and most viral sizes concentrates between 10nm ~ 100nm, just in time meets nano material dimensional requirement.Secondly, viral granule-morphology of the same race is homogeneous, and monodispersity is good.3rd, virus is often only made up of nucleic acid and protein enclosure, and its protein coat can be used as nano container and carries out drug carrier or as microreactor.Its protein enclosure of different virus is often different, and structural difference provides more stencil-chosen.
In conjunction with gene clone technology, conveniently can realize transforming viral protein specific site or modifying, as introduced functional group, inserting or knock out specific amino acids or polypeptide, thus obtain the protein template possessing ideal functionality.
Biomineralization refers to that organism produces the process of inorganic mineral by the regulation and control of biomacromolecule.Namely under certain physical and chemical condition, by functional group and the interaction of mineral ion in interface of biomacromolecule, control crystallization, the growth of inorganic mineral phase from molecular level, thus make biomineralization have special hierarchy and assembling mode.The important component part of nanometer biotechnology development in recent years that to take viral capsid proteins as template mineralising inorganic nano material be, but make one's way in life at present and mainly combine in spherical viruses templated synthesis function nano particle.
Two-dimensional nanostructure, particularly ultra-fine two-dimensional nanostructure, often have excellent physico-chemical property.Often synthesis step is complicated, reaction conditions is gentle not to utilize viral protein template of the prior art to form two-dimensional nanostructure, and the two-dimensional nanostructure biocompatibility formed is poor.
Summary of the invention
The object of the invention is to the above-mentioned defect for prior art, the method that a kind of tobacco mosaic disease virus capsid protein mutant assembling of application forms virion Template preparation two-dimensional nanostructure is proposed, solve two-dimensional nanostructure synthesis step complexity in prior art, reaction conditions is gentle not, and the technical problem that the two-dimensional nanostructure biocompatibility formed is poor.
Invention applies a kind of tobacco mosaic disease virus capsid protein mutant, described mutant sequence is obtained through point mutation by the aminoacid sequence shown in SEQIDNo.1, and described point mutation is: the 103rd sports halfcystine.
Preferably, described tobacco mosaic disease virus capsid protein mutant sequence is the aminoacid sequence shown in SEQIDNo.2.
In addition, present invention also offers the encoding gene of above-mentioned tobacco mosaic disease virus capsid protein mutant.
Preferably, described coding gene sequence is for shown in SEQIDNo.3.
The invention provides a kind of preparation method of the two-dimensional nanostructure based on tubulose virion, application said mutation body Nanostructure fabrication, the method comprises the steps:
S1, in the capsid protein of tobacco mosaic virus (TMV), introduce halfcystine:
The capsid protein single amino acid sequence the 103rd of tobacco mosaic virus (TMV) is sported halfcystine, carrier construction, and realizes expressing in organism, through anion chromatography column purification, obtain improved viral capsid proteins monomer;
The generation of S2, tubulose virus granular formwork:
Improved for step S1 gained viral capsid proteins monomer is placed in phosphate buffered saline buffer dialyse, carry out sucrose continuous density gradient subsequently centrifugal, again centrifugal rear collected precipitation is placed in phosphate buffered saline buffer to dialyse, finally carries out ultrafiltration and concentration and obtain tubulose virus granular formwork;
S3, tubulose virus two-dimensional nanostructure generates:
Step S2 gained tubulose virus granular formwork, precious metal ion solution are mixed, then adds the first reductive agent and mix, to form tubulose virus kernel; Continuation adds precious metal ion several times and the second reductive agent mixes, to form tubulose virus two-dimensional nanostructure;
Wherein, precious metal ion is selected from the one in gold ion, palladium ion or platinum ion.
Preferably, step S1 is specially: the capsid protein single amino acid sequence the 103rd of tobacco mosaic virus (TMV) is sported halfcystine, carrier construction, vector expression plasmid is proceeded to E.coliBL21 (DE3) competent cell, culturing bacterium in LB substratum abduction delivering recombinant protein, thereafter successively through centrifugal, broken, ammonium sulfate precipitation and column chromatographic isolation and purification, improved viral capsid proteins is obtained.
Preferably, step S1 comprises the steps:
A, vector expression plasmid Calcium Chloride Method is proceeded to E.coliBL21 (DE3) competent cell, be coated with flat board at least after 12h, in picking mono-clonal access LB substratum, add microbiotic, spend the night in 37 DEG C of constant-temperature shaking culture, thereafter transfer in LB substratum according to 1% inoculum size, add microbiotic, in 37 DEG C of constant-temperature shaking culture, and add inductor, continue inducing culture at 30 DEG C, collected by centrifugation thalline, wherein, described microbiotic comprises penbritin, and described inductor comprises isopropylthiogalactoside;
B. the thalline collected is resuspended in lysis buffer and carries out ultrasonication, centrifugal in 4 DEG C, get centrifugal rear gained supernatant liquor and carry out ammonium sulfate precipitation and anion chromatography successively, obtain improved viral capsid proteins.
Preferably, step S2 is specially: improved for step S1 gained viral capsid proteins is placed in phosphate buffered saline buffer and dialyses, at least 5 days, carry out sucrose continuous density gradient subsequently centrifugal, again centrifugal rear collected precipitation is placed in phosphate buffered saline buffer carry out dialysing and undertaken quantitatively by SDS-PAGE, be concentrated into 0.8mg/mL finally by 30KD ultra-filtration centrifuge tube, obtain tubulose virus granular formwork.
Preferably, in step S3, precious metal ion is selected from the one in hydrochloro-auric acid, the acid of chlorine palladium or Platinic chloride.
Preferably, the first reductive agent described in step S3 is sodium borohydride, and described second reductive agent is vitamins C or dimethyamine borane.
Compared with prior art, beneficial effect of the present invention comprises: the tobacco mosaic disease virus capsid protein mutant that the present invention is formed by genetic modification introduces halfcystine in original capsid protein, disulfide linkage is formed between subunit, change the non covalent bond self-assembly system that capsid protein is originally fragile, the external self-assembly ability of this mutant improves greatly, and this mutant can form stable tubulose virus granular formwork; The preparation method of the two-dimensional nanostructure based on tubulose virion of the present invention, the recombinant protein adopting said mutation body or comprise this mutant forms tubulose virus granular formwork, the mineralising achieved based on tubulose virus granular formwork prepares two-dimensional nanostructure, this preparation method's step is simple, reaction conditions is gentle, prepared two-dimensional nanostructure has good biocompatibility, and surface is easy to modify.
Accompanying drawing explanation
Fig. 1 is the Electronic Speculum figure of the tubulose virus granular formwork of the embodiment of the present invention 2;
Fig. 2 is the Electronic Speculum figure of the two-dimensional nanostructure of the embodiment of the present invention 3,
Wherein, in Fig. 2, a represents and continues to add several times at tubulose virus kernel the Electronic Speculum figure that precious metal ion and the second reductive agent carry out mixing to be formed to add indegree be 7 the formation two-dimensional nanostructure of precious metal ion and the second reductive agent in tubulose virus two-dimensional nanostructure process, and the indegree that adds of b, c, d is respectively 10,14 and 24;
Fig. 3 is the Electronic Speculum figure of the two-dimensional nanostructure of the embodiment of the present invention 4,
Wherein, in Fig. 3, a is the Local map of Electronic Speculum figure, and b is partial enlarged drawing;
Fig. 4 is the Electronic Speculum figure of the two-dimensional nanostructure of the embodiment of the present invention 5;
Fig. 5 is the Electronic Speculum figure of the two-dimensional nanostructure of the embodiment of the present invention 6.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
Unless otherwise defined, all scientific and technical terminologies that the present invention uses have the identical meanings that those of ordinary skill in the art understand.About definition and the term of this area, professional can with reference to CurrentProtocolsinMolecularBiology (Ausubel).The abbreviation of amino-acid residue is standard 3 letter referring to one of 20 conventional L-amino acid used in this area and/or 1 alphanumeric codes.
Although the numerical value shown in broad scope of the present invention natively must containing certain error, it is caused by the standard deviation existed in their each measurement.In addition, all scopes disclosed by the invention are interpreted as containing any and all subranges wherein comprised.The scope of " 1 to 10 " such as recorded is interpreted as comprising any and all subranges (comprising end points) between minimum value 1 and maximum value 10; That is, all with minimum value 1 or subrange initial more greatly, such as 1 to 6.1, and with the subrange of maximum value 10 or less termination, such as 5.5 to 10.In addition, to be anyly called that the reference of " being incorporated to herein " is interpreted as being incorporated to its entirety.
It should be noted that as used in this description, singulative comprises the plural form of its referent in addition, be limited to a referent unless clear and clear and definite.Term "or" can be exchanged with term "and/or" and be used, unless context separately has clear indicating.
The capsid protein of ripe tobacco mosaic virus (TMV) (TMV) consists of the mode of self assembly the protein monomers that about 2130 have a same acid sequence, it is non covalent bond self-assembly system, its nanotube-shaped geometric shape, highly active surfaces externally and internally can by as a kind of template of constructing low-dimensional materials, wherein, each monomer is made up of (as shown in SEQIDNo.1) 158 amino acid, modification transformation is carried out to above-mentioned monomer, halfcystine is introduced in its aminoacid sequence, obtain improved monomer, TMV capsid protein mutant mentioned by the present invention is above-mentioned improved monomer, by disulfide-bonded between each improved monomer, contribute to improving external self-assembly ability, the improved TMV capsid protein that multiple improved monomer (TMV capsid protein mutant) self-assembly is formed is more suitable for the growth of nano material than wild-type TMV capsid protein, that is, the improved TMV capsid protein that multiple improved monomer self-assembly is formed, can be used as a kind of tubulose virus granular formwork, this tubulose virus granular formwork is a kind of more superior more stable protein template.
In the solution of the present invention, the Threonine (Thr) of the 103rd in aminoacid sequence shown in wild-type TMV capsid protein monomer SEQIDNo.1 is sported halfcystine (Cys), improved aminoacid sequence is for shown in SEQIDNo.2, first the encoding gene of improved aminoacid sequence is synthesized according to subject amino acid sequence (shown in SEQIDNo.2), such as: nucleotides sequence is classified as the encoding gene shown in SEQIDNo.3, above-mentioned encoding gene is inserted in expression vector, then be converted in organism (as intestinal bacteria) and express, obtain improved monomer.
After the improved monomer of above-mentioned gained carries out purifying, assemble in phosphate buffered saline buffer, carry out sucrose continuous density gradient subsequently centrifugal, again centrifugal rear collected precipitation is placed in phosphate buffered saline buffer to dialyse, finally carry out ultrafiltration and concentration and obtain the improved TMV capsid protein that improved monomer assembles, this albumen carries out nanostructure growth as tubulose virus granular formwork, and after this albumen forms tubulose, its inside pipe wall diameter can reach 4nm.
The preparation method of the two-dimensional nanostructure based on tubulose virion of the present invention, there is the improved TMV capsid protein of narrow and small cavity internal diameter for tubulose virus granular formwork, first by sulfydryl in pipe and the coordination containing precious metal element group, at its narrow and small cavity internal adsorption containing precious metal element group, finally add the first reductive agent that reducing power is stronger, achieve the growth of precious metal element group in this template pipe, obtain the tubulose virus kernel building two-dimensional nanostructure further; Then, in tubulose virus kernel, continue gradation adds containing precious metal element group and more weak the second reductive agent of reducing power, the continued growth on tubulose virus kernel by coordination and electrostatic adsorption of precious metal element group, form two-dimensional nanostructure, as nano particle, nano chain, nanometer rod etc.
Particularly, the preparation method of the above-mentioned two-dimensional nanostructure based on tubulose virion, mainly comprises following three steps:
First be step S1, halfcystine is introduced: the capsid protein single amino acid sequence the 103rd of tobacco mosaic virus (TMV) is sported halfcystine in the capsid protein of tobacco mosaic virus (TMV), carrier construction, and realize expressing in organism, through anion chromatography column purification, obtain improved viral capsid proteins monomer.
Particularly, in this step, the capsid protein single amino acid sequence the 103rd of tobacco mosaic virus (TMV) is sported halfcystine, in this, as TMV capsid protein mutant (improved monomer), when the cDNA complete genome sequence of known said mutation body aminoacid sequence and wild-type TMV capsid protein monomer, those of ordinary skill in the art can obtain the encoding gene of said mutation body easily, thus carrier construction, and realize expressing in organism, those of ordinary skill in the art can select different organisms to carry out the expression of said mutation body (improved monomer) according to specific needs with condition, as escherichia expression system, yeast expression system or animal somatic cell expression system, such as, select escherichia expression system, the prokaryotic expression carrier of structure is proceeded to E.coliBL21 (DE3) competent cell, culturing bacterium in LB substratum abduction delivering recombinant protein, thereafter successively through centrifugal, broken, ammonium sulfate precipitation and column chromatographic isolation and purification, obtain improved viral capsid proteins, be specially: vector expression plasmid Calcium Chloride Method is proceeded to E.coliBL21 (DE3) competent cell, be coated with flat board at least after 12h, in picking mono-clonal access LB substratum, add microbiotic, spend the night in 37 DEG C of constant-temperature shaking culture, thereafter transfer in LB substratum according to 1% inoculum size, add microbiotic, in 37 DEG C of constant-temperature shaking culture, and add inductor, inducing culture is continued at 30 DEG C, collected by centrifugation thalline, wherein, described microbiotic comprises penbritin, described inductor comprises isopropylthiogalactoside, the thalline collected is resuspended in lysis buffer and carries out ultrasonication, centrifugal in 4 DEG C, get centrifugal rear gained supernatant liquor and carry out ammonium sulfate precipitation and anion chromatography successively, obtain improved viral capsid proteins.
Next, tubulose virion template is generated in step S2, improved for step S1 gained viral capsid proteins monomer is placed in phosphate buffered saline buffer dialyse, carry out sucrose continuous density gradient subsequently centrifugal, again centrifugal rear collected precipitation is placed in phosphate buffered saline buffer to dialyse, finally carries out ultrafiltration and concentration and obtain tubulose virus granular formwork.
Particularly, improved for step S1 gained viral capsid proteins is placed in phosphate buffered saline buffer dialyse, at least 5 days, carry out sucrose continuous density gradient subsequently centrifugal, again centrifugal rear collected precipitation is placed in phosphate buffered saline buffer carry out dialysing and undertaken quantitatively by SDS-PAGE, be concentrated into 0.8mg/mL finally by 30KD ultra-filtration centrifuge tube, obtain tubulose virus granular formwork.
Generate tubulose virus two-dimensional nanostructure subsequently in step s3, step S2 gained tubulose virus granular formwork, precious metal ion solution are mixed, then add the first reductive agent and mix, to form tubulose virus kernel; Continuation adds precious metal ion several times and the second reductive agent mixes, and to form tubulose virus two-dimensional nanostructure, wherein, precious metal ion is selected from the one in gold ion, palladium ion or platinum ion.
Above-mentioned is precious metal ion containing precious metal element group, as gold ion, platinum ion, palladium ion, is specially hydrochloro-auric acid, the acid of chlorine palladium and Platinic chloride; Preferably but be not limited to sodium borohydride, and institute adds the first reductive agent to first reductive agent is excessive, and the second reductive agent preferably but be not limited to vitamins C.
Different precious metal ions is used to carry out growing and adopt different treatment processs in above-mentioned preparation method of the present invention, different two-dimensional nanostructures can be formed, such as, as a preferred version of the present invention, hydrochloro-auric acid is added in step S3, and after formation tubulose virus kernel, add hydrochloro-auric acid several times and the second reductive agent mixes, add the second reductive agent again after the 5-10min of interval after adding hydrochloro-auric acid at every turn, two-dimensional nanostructure is chain, namely gold nano chain (or gold nano string, Fig. 2 shown in) is defined; And for example, as a preferred version of the present invention, hydrochloro-auric acid is added in step S3, and after formation tubulose virus kernel, add hydrochloro-auric acid several times and the second reductive agent mixes, add hydrochloro-auric acid at every turn and add the second reductive agent at once, two-dimensional nanostructure is bar-shaped, namely defines gold nanorods (shown in Fig. 3); Add Platinic chloride or the acid of chlorine palladium in step S3, two-dimensional nanostructure is particulate state (shown in Fig. 4 and Fig. 5).
The concentration of agents useful for same in above-mentioned steps; as the concentration of concrete reagent used in TMV capsid protein mutant, tubulose virus granular formwork, tubulose virus kernel, hydrochloro-auric acid and purification step is the selection that those of ordinary skill in the art carry out as the case may be under the spirit of above-mentioned preparation method, all within protection scope of the present invention.
Set forth the present invention further below by way of specific embodiment, these embodiments are only presented for purposes of illustration, do not limit the scope of the invention.Indicate apart from concrete, the condition in the following example and working method are all carried out according to this area routine operation.
In following specific embodiment mentioned T103C-TMVcp represent TMV capsid protein single amino acid sequence the 103rd Threonine is become halfcystine after the mutant that formed, be improved monomer, mentioned plasmid vector PET32a-T103C-TMVcp represents to be proceeded to by the encoding gene of T103C-TMVcp after in plasmid vector PET32a and formed.In following specific embodiment mentioned unit " mM " expression " mmol/L " (mmole often rises).
embodiment 1:
The structure of TMV capsid protein mutant expression plasmid and the expression and purification in intestinal bacteria thereof
Step 1: the cDNA synthesizing ribonucleotide sequence according to wild-type TMV capsid protein monomer is the mutant code gene shown in SEQIDNo.3, is proceeded to in plasmid vector PET32a, builds plasmid vector PET32a-T103C-TMVcp;
Step 2: after the exactness of DNA sequencing determination mutant gene sequence, plasmid vector PET32a-T103C-TMVcp Calcium Chloride Method is proceeded in E.coliBL21 competent cell, access in 5mLLB test-tube culture medium from picking mono-clonal flat board after being coated with dull and stereotyped 12h, add penbritin (final concentration 100 μ g/mL), in 37 DEG C of constant temperature, 180r/min overnight incubation; According to the inoculum size of 1%, 5mL bacterium liquid is transferred in 500mLLB triangular flask, add penbritin (final concentration 100 μ g/mL), after 37 DEG C of constant temperature, 180r/min about shaking culture 2.5h (OD600 is between 0.4 ~ 0.6), add IPTG inductor (final concentration is the IPTG of 1mM), vibration inducing culture 10h is continued, collected by centrifugation thalline at 30 DEG C; Bacterial sediment Tris-HCl buffer solution for cleaning is once resuspended in afterwards in the lysis buffer of certain volume and carries out ultrasonication (condition: ultrasonic power 400W, work 4s, interval 4s, total ultrasonication time 60min), bacterium liquid after fragmentation, with the centrifugal 30min of 12000r/min, gets supernatant liquor;
The purifying of step 3:T103C-TMVcp; First, slowly adding solid ammonium sulfate to final concentration is 35% saturation ratio.Stop adding ammonium sulfate, 4 DEG C of centrifugal 15min of standing 2h, 12000rpm, collecting precipitation.Precipitation pH9.5,50mM sodium carbonate-bicarbonate damping fluid is resuspended, and add 20mMDTT.Dissolving of slowly vibrating at low ambient temperatures is spent the night.12000rpm4 DEG C of centrifugal 15min, gets supernatant; Then, supernatant liquor is dialysed in supreme sample damping fluid, through 0.45 μm of filter filtering and impurity removing matter, carry out loading operation after being balanced by HiTraPQHP prepacked column sample-loading buffer simultaneously, load in AKTAPrimeplusFPLC system by being adsorbed with target protein HiTraPQHP prepacked column after loading, carry out wash-out by continuous gradient elution mode, gradient 0-800mMNaCl, elution flow rate is 0.8mL/min.Collect elution peak on ice, 4 DEG C of dialysis are to sodium carbonate-bicarbonate damping fluid (50mM, pH9.5; 20mMDTT), with SDS-PAGE testing goal purity of protein, high purity protein liquid is put-80 DEG C frozen.
The present embodiment gained albumen T103C-TMVcp is improved TMV capsid protein monomer, and aminoacid sequence the 103rd is sported the mutant of halfcystine by Threonine, its aminoacid sequence is as shown in SEQIDNo.2.
embodiment 2:
The generation of tubulose virus granular formwork
Dialysed by the high purity T103C-TMVcp of embodiment 1 gained in assembling damping fluid assembling damping fluid (pH7,400mM phosphate buffered saline buffer), incubated at room 5 days, carries out self-assembly; By sucrose density gradient centrifugation by the tubulose virus granular formwork after separation and purification self-assembly.
Wherein, the preparation process of sucrose density gradient:
A) PB damping fluid (pH7.0 is first prepared, 150mM phosphoric acid salt) and 50wt% sucrose solution (using PB buffer), then with both for mother liquor is made into mass/mass than the sucrose solution being 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%;
B), after sample volume having been reserved in transparent ultracentrifugation pipe the top, remaining length is divided into isopyknic 9 parts, uses marking pen marking;
C) ultracentrifugation pipe is fixed on suitable centrifuge tube shelf, slowly joins adherent for the sucrose solution of each gradient in centrifuge tube by the order from high density to lower concentration with suction pipe;
D) after gradient prepares, be statically placed in 4 DEG C of refrigerator overnight, make it to form continuous gradient.
Ultracentrifugation: slowly joined by the assembling product obtained in position reserved above ultracentrifugation pipe, 38000rpm centrifugal (Beckman SW40Ti rotor) 3.5 hours, then takes out by the packing of original line mark difference.
Sample desugar: simultaneously dialysed by all packing samples in PB damping fluid (pH7.0,100mM phosphoric acid salt), Electronic Speculum characterizes respectively.
Finally carry out ultrafiltration and concentration to self-assembly product, obtain tubulose virus granular formwork, be stored in-20 DEG C of refrigerators, as shown in Figure 1, which form tubular structure as seen, inside pipe wall diameter can reach 4nm to the electromicroscopic photograph of tubulose virus granular formwork.
embodiment 3:
Tubulose virus two-dimensional nanostructure generates
Based on the tubulose of embodiment 2 gained virus granular formwork, grow, concrete steps are:
2.5 μ L hydrochloro-auric acids (50mM) are added in 150 μ LT103-TMVcp (0.8mg/ml), incubated at room 3 hours, add and now join borane reducing agent sodium hydride (20 μ L, 10mM), left at room temperature over night after 800rpm magnetic agitation 5min; Collected by centrifugation supernatant, adds 2 μ L hydrochloro-auric acids (50mM) and 10 μ L vitamins Cs (20mM), hatches centrifuging and taking supernatant after 2 hours; Every secondary growth adds after 2 μ L hydrochloro-auric acids (5mM) hatch 10min, then adds 5 μ L vitamins Cs (2mM) and hatch 15min, and carry out 7,10,14 and 24 circulations so respectively, gained two-dimensional nanostructure as shown in Figure 2.
embodiment 4:
Tubulose virus two-dimensional nanostructure generates
Based on the tubulose of embodiment 2 gained virus granular formwork, grow, concrete steps are:
2.5 μ L hydrochloro-auric acids (50mM) are added, more than incubated at room 30min in 150 μ LT103-TMVcp (0.8mg/ml).Ultrafiltration removes excessive hydrochloro-auric acid.Add 5 μ L sodium borohydrides.Hatch 5min, again remove unnecessary sodium borohydride by super filter tube.Add the hydrochloro-auric acid (5mM) of 2 μ L and the vitamins C (5mM) of 2 μ L, 15min is hatched at interval, and cycling deposition 10 times, gained two-dimensional nanostructure as shown in Figure 3 at every turn.
embodiment 5:
Tubulose virus two-dimensional nanostructure generates
Based on the tubulose of embodiment 2 gained virus granular formwork, grow, concrete steps are:
2.5 μ L Platinic chlorides (50mM) are added, more than incubated at room 30min in 150 μ LT103-TMVcp (0.8mg/ml).Ultrafiltration removes excessive Platinic chloride.Add 5 μ L sodium borohydrides.Hatch 5min, again remove unnecessary sodium borohydride by super filter tube.Add the Platinic chloride (10mM) of 10 μ L and the dimethyamine borane (10mM) of 10 μ L, 15min is hatched at interval, and cycling deposition 3 times, gained two-dimensional nanostructure as shown in Figure 4 at every turn.
embodiment 6:
Tubulose virus two-dimensional nanostructure generates
Based on the tubulose of embodiment 2 gained virus granular formwork, grow, concrete steps are:
2.5 μ L chlorine palladium acid (50mM) are added, more than incubated at room 30min in 150 μ LT103-TMVcp (0.8mg/ml).Ultrafiltration removes excessive chlorine palladium acid.Add 5 μ L sodium borohydrides.Hatch 5min, again remove unnecessary sodium borohydride by super filter tube.The vitamins C (50mM) of the chlorine palladium acid (50mM) and 2 μ L that add 2 μ L hatches growth, and gained two-dimensional nanostructure as shown in Figure 5.
The above the specific embodiment of the present invention, does not form limiting the scope of the present invention.Various other that any technical conceive according to the present invention is made change and distortion accordingly, all should be included in the protection domain of the claims in the present invention.
Claims (10)
1. a tobacco mosaic disease virus capsid protein mutant, is characterized in that, described mutant sequence is obtained through point mutation by the aminoacid sequence shown in SEQIDNo.1, and described point mutation is: the 103rd sports halfcystine.
2. comprise a recombinant protein for tobacco mosaic disease virus capsid protein mutant sequence, it is characterized in that, described tobacco mosaic disease virus capsid protein mutant sequence is the aminoacid sequence shown in SEQIDNo.2.
3. the encoding gene of tobacco mosaic disease virus capsid protein mutant described in claim 1.
4. encoding gene according to claim 3, is characterized in that, described coding gene sequence is for shown in SEQIDNo.3.
5. based on a preparation method for the two-dimensional nanostructure of tubulose virion, it is characterized in that, the method comprises the steps:
S1, in the capsid protein of tobacco mosaic virus (TMV), introduce halfcystine:
The capsid protein single amino acid sequence the 103rd of tobacco mosaic virus (TMV) is sported halfcystine, carrier construction, and realizes expressing in organism, through anion chromatography column purification, obtain improved viral capsid proteins monomer;
The generation of S2, tubulose virus granular formwork:
Improved for step S1 gained viral capsid proteins monomer is placed in phosphate buffered saline buffer dialyse, carry out sucrose continuous density gradient subsequently centrifugal, again centrifugal rear collected precipitation is placed in phosphate buffered saline buffer to dialyse, finally carries out ultrafiltration and concentration and obtain tubulose virus granular formwork;
S3, tubulose virus two-dimensional nanostructure generates:
Step S2 gained tubulose virus granular formwork, precious metal ion solution are mixed, then adds the first reductive agent and mix, to form tubulose virus kernel; Continuation adds precious metal ion several times and the second reductive agent mixes, to form tubulose virus two-dimensional nanostructure;
Wherein, precious metal ion is selected from the one in gold ion, palladium ion or platinum ion.
6. preparation method according to claim 5, it is characterized in that, step S1 is specially: the capsid protein single amino acid sequence the 103rd of tobacco mosaic virus (TMV) is sported halfcystine, carrier construction, vector expression plasmid is proceeded to E.coliBL21 (DE3) competent cell, culturing bacterium in LB substratum abduction delivering recombinant protein, thereafter successively through centrifugal, broken, ammonium sulfate precipitation and column chromatographic isolation and purification, obtain improved viral capsid proteins.
7. preparation method according to claim 6, is characterized in that, step S1 comprises the steps:
A, vector expression plasmid Calcium Chloride Method is proceeded to E.coliBL21 (DE3) competent cell, be coated with flat board at least after 12h, in picking mono-clonal access LB substratum, add microbiotic, spend the night in 37 DEG C of constant-temperature shaking culture, thereafter transfer in LB substratum according to 1% inoculum size, add microbiotic, in 37 DEG C of constant-temperature shaking culture, and add inductor, continue inducing culture at 30 DEG C, collected by centrifugation thalline, wherein, described microbiotic comprises penbritin, and described inductor comprises isopropylthiogalactoside;
B. the thalline collected is resuspended in lysis buffer and carries out ultrasonication, centrifugal in 4 DEG C, get centrifugal rear gained supernatant liquor and carry out ammonium sulfate precipitation and anion chromatography successively, obtain improved viral capsid proteins.
8. preparation method according to claim 5, it is characterized in that, step S2 is specially: improved for step S1 gained viral capsid proteins is placed in phosphate buffered saline buffer and dialyses, at least 5 days, carry out sucrose continuous density gradient subsequently centrifugal, again centrifugal rear collected precipitation is placed in phosphate buffered saline buffer carry out dialysing and undertaken quantitatively, being concentrated into 0.8mg/mL finally by 30KD ultra-filtration centrifuge tube by SDS-PAGE, obtains tubulose virus granular formwork.
9. preparation method according to claim 5, is characterized in that, in step S3, precious metal ion is selected from the one in hydrochloro-auric acid, the acid of chlorine palladium or Platinic chloride.
10. preparation method according to claim 5, is characterized in that, the first reductive agent described in step S3 is sodium borohydride, and described second reductive agent is vitamins C or dimethyamine borane.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106822030A (en) * | 2017-02-27 | 2017-06-13 | 中国科学院理化技术研究所 | Preparation method of tobacco mosaic virus capsid protein capsule |
| CN109988755A (en) * | 2018-01-03 | 2019-07-09 | 中国科学院苏州纳米技术与纳米仿生研究所 | RhuA mutant and its preparing the application in Two-dimensional Composites |
| CN111349149A (en) * | 2020-03-17 | 2020-06-30 | 中国科学院苏州纳米技术与纳米仿生研究所 | Tobacco mosaic virus capsid protein mutant and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102049226A (en) * | 2010-11-16 | 2011-05-11 | 中国烟草总公司郑州烟草研究院 | Method for preparing TMV template-based nano-gold material |
| CN102886049A (en) * | 2011-07-19 | 2013-01-23 | 南开大学 | Nanobiomaterial, its preparation method and nucleic acid compound |
| CN102899346A (en) * | 2012-09-12 | 2013-01-30 | 国家烟草质量监督检验中心 | Tobacco mosaic virus coat protein template structure modification method |
| CN103613647A (en) * | 2013-06-05 | 2014-03-05 | 浙江大学 | Tobacco mosaic virus capsid protein cysteine mutant and applications thereof |
| CN103706800A (en) * | 2014-01-16 | 2014-04-09 | 国家烟草质量监督检验中心 | Method for growing nanogold on gene-modified tobacco mosaic virus nuclecapsid protein template |
-
2014
- 2014-08-11 CN CN201410391846.2A patent/CN105367630A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102049226A (en) * | 2010-11-16 | 2011-05-11 | 中国烟草总公司郑州烟草研究院 | Method for preparing TMV template-based nano-gold material |
| CN102886049A (en) * | 2011-07-19 | 2013-01-23 | 南开大学 | Nanobiomaterial, its preparation method and nucleic acid compound |
| CN102899346A (en) * | 2012-09-12 | 2013-01-30 | 国家烟草质量监督检验中心 | Tobacco mosaic virus coat protein template structure modification method |
| CN103613647A (en) * | 2013-06-05 | 2014-03-05 | 浙江大学 | Tobacco mosaic virus capsid protein cysteine mutant and applications thereof |
| CN103706800A (en) * | 2014-01-16 | 2014-04-09 | 国家烟草质量监督检验中心 | Method for growing nanogold on gene-modified tobacco mosaic virus nuclecapsid protein template |
Non-Patent Citations (3)
| Title |
|---|
| KEITH M. BROMLEY等: "Preparation of high quality nanowires by tobacco mosaic virus templating of gold nanoparticles", 《JOURNAL OF MATERIALS CHEMISTRY》 * |
| KUN ZHOU等: "Disulfide Bond: Dramatically Enhanced Assembly Capability and Structural Stability of Tobacco Mosaic Virus Nanorods", 《BIOMACROMOLECULES》 * |
| MOROZOV,S.YU.等: "Coat protein [Tobacco mosaic virus]", 《GENBANK DATABASE》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106822030A (en) * | 2017-02-27 | 2017-06-13 | 中国科学院理化技术研究所 | Preparation method of tobacco mosaic virus capsid protein capsule |
| CN106822030B (en) * | 2017-02-27 | 2019-09-27 | 中国科学院理化技术研究所 | Preparation method of tobacco mosaic virus capsid protein capsule |
| CN109988755A (en) * | 2018-01-03 | 2019-07-09 | 中国科学院苏州纳米技术与纳米仿生研究所 | RhuA mutant and its preparing the application in Two-dimensional Composites |
| CN111349149A (en) * | 2020-03-17 | 2020-06-30 | 中国科学院苏州纳米技术与纳米仿生研究所 | Tobacco mosaic virus capsid protein mutant and application thereof |
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