CN111349149A - Tobacco mosaic virus capsid protein mutant and application thereof - Google Patents

Tobacco mosaic virus capsid protein mutant and application thereof Download PDF

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CN111349149A
CN111349149A CN202010184704.4A CN202010184704A CN111349149A CN 111349149 A CN111349149 A CN 111349149A CN 202010184704 A CN202010184704 A CN 202010184704A CN 111349149 A CN111349149 A CN 111349149A
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王强斌
董津辰
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Suzhou Institute of Nano Tech and Nano Bionics of CAS
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Abstract

The invention discloses a tobacco mosaic virus capsid protein mutant and application thereof. The tobacco mosaic virus capsid protein mutant is obtained by point mutation of an amino acid sequence shown in SEQ ID No.1, wherein the point mutation is as follows: the 103 th mutation is cysteine, and the carboxyl terminal is inserted with RGD1 sequence. The tobacco mosaic virus capsid protein is reformed to be self-assembled into disc-shaped nano particles with consistent size and shape height, the disc-shaped nano particles can cause organism immunity less, have enhanced tumor tissue penetrating capability, can be widely used as nano drug carriers, and can be applied to detection and/or treatment reagents of diseases such as tumors and the like by functional modification, such as fluorescent substances, targeting substances and the like, so that the tobacco mosaic virus capsid protein has wide application prospect.

Description

Tobacco mosaic virus capsid protein mutant and application thereof
Technical Field
The invention relates to a tobacco mosaic virus capsid protein (TMVcp), in particular to a tobacco mosaic virus capsid protein mutant and application thereof, for example, a method for assembling the tobacco mosaic virus capsid protein mutant into a disk-shaped nano material and applying the disk-shaped nano material as a drug carrier, belonging to the technical field of nano biology.
Background
The application of the nano material in the field of biotechnology is concerned by people, and a great amount of reports are already made on aspects of biological imaging, drug transportation, screening diagnosis and the like. Proteins are used as biological supramolecular nanomaterials, which have been functionalized for in vivo drug delivery due to their characteristics of consistent size and shape, biodegradability and compatibility, and multifunctional sites for chemical and biological modification.
The tobacco mosaic virus capsid protein subunit (TMVcp) is one of the simplest natural protein assembly motifs, and the application of Tobacco Mosaic Virus (TMV) in biomedicine in recent years is probably divided into disease diagnosis and disease treatment. As a good nano-carrier, the virus protein specific site is modified or reformed by combining with a gene cloning technology, so that the virus protein specific site is connected with a specific ligand or a drug molecule for in vivo specific tissue targeting and drug delivery treatment. However, intact TMV particles contain genetic material and a length of 300nm is likely to elicit an immune response, which is not conducive to therapy or imaging.
Disclosure of Invention
The invention mainly aims to provide a tobacco mosaic virus capsid protein mutant and application thereof, so as to overcome the defects in the prior art.
In order to solve the above problems, the technical scheme provided by the invention is as follows:
the embodiment of the invention also provides a tobacco mosaic protein capsid protein mutant which comprises a polypeptide obtained by point mutation of an amino acid sequence shown by SEQ ID No.1, wherein the point mutation is as follows: the 103 th position is mutated into cysteine, and the carboxyl terminal of the polypeptide is inserted with an RGD1 sequence shown in SEQ ID No. 7.
The embodiment of the invention also provides a coding gene of the tobacco mosaic virus capsid protein mutant.
The embodiment of the invention also provides a recombinant expression vector containing the coding gene.
The embodiment of the invention also provides a transgenic cell line containing the coding gene.
The embodiment of the invention also provides a host bacterium transformed/transfected with the recombinant expression vector. Further, the host bacterium includes Escherichia coli.
The embodiment of the invention also provides a method for constructing the tobacco mosaic virus capsid protein mutant, which comprises the following steps:
providing a site-directed mutagenesis reagent;
taking DNA of natural TMV as a template, uniformly mixing the DNA with the site-directed mutagenesis reagent, carrying out PCR directed mutagenesis, mutating threonine at the 103 th site of the amino acid sequence of the natural TMV monomer into cysteine, inserting an RGD1 sequence into the carboxyl terminal, and then carrying out T4 enzyme linkage to obtain a recombinant expression vector carrying the gene;
and transferring the recombinant expression vector into a host cell for expression to obtain the TMV mutant.
In some embodiments, the method specifically comprises: with CaCl2Transferring the recombinant expression vector into competent escherichia coli, selecting monoclonal to inoculate in a liquid culture medium containing antibiotics, culturing for 6-10h at 37 ℃, expanding the culture volume to the liquid culture medium containing the antibiotics, culturing for 2-3h at 37 ℃, adding isopropyl thiogalactoside, and continuing to perform induction culture for 14-16h at 27 ℃ to obtain the TMV mutant.
The embodiment of the invention also provides a disk-shaped nanoparticle, which is formed by self-assembly of tobacco mosaic virus capsid protein mutants with the sequence shown as SEQ ID No.3 or SEQ ID No.5 and has a double-layer disk-shaped structure, wherein the disk-shaped nanoparticle has the diameter of 18-20nm and the height of 4-5nm, and the disk-shaped nanoparticle has a hollow through hole with the diameter of 4-5 nm.
Furthermore, the disc-shaped nanoparticles can be modified with fluorescent substances, targeting substances capable of being specifically combined with tumor cells and the like.
Furthermore, the disc-shaped nanoparticles can be modified with fluorescent substances, targeting substances capable of being specifically combined with tumor cells and the like.
The embodiment of the invention also provides a preparation method of the disc-shaped nano particles, which comprises the following steps:
constructing tobacco mosaic virus capsid protein mutant with sequence shown as SEQ ID No.3 or SEQ ID No. 5;
dissolving the tobacco flower leaf virus capsid protein mutant in potassium phosphate/β -mercaptoethanol buffer solution with the pH value of 9.0-9.5, wherein the potassium phosphate/β -mercaptoethanol buffer solution comprises 50-100mmol/L potassium phosphate and 5-10mmol/L β -mercaptoethanol, dialyzing in tris (hydroxymethyl) aminomethane buffer solution with the pH value of 7.0-7.5 and the concentration of 50-100mmol/L, the molecular weight cut-off of the dialysis bag is 8000-14000, and then concentrating to 4-5mg/mL by 30-100KD ultrafiltration centrifugation, so that the tobacco flower leaf virus capsid protein mutant self-assembles to form double-layer disc-shaped nanoparticles, the diameter of the disc-shaped nanoparticles is 18-20nm, the height of the disc-shaped nanoparticles is 4-5nm, and the disc-shaped nanoparticles have hollow through holes with the diameter of 4-5 nm.
The embodiment of the invention also provides application of the tobacco floral leaf protein capsid protein mutant or the disc-shaped nano-particles in preparation of a drug carrier.
The embodiment of the invention also provides a functional product, which comprises the tobacco floral leaf protein capsid protein mutant or the disc-shaped nanoparticle, and the product can be at least used for detecting and/or treating tumors.
Compared with the prior art, the tobacco mosaic virus capsid protein is transformed into the tobacco mosaic virus capsid protein mutant, so that the tobacco mosaic virus capsid protein mutant can be self-assembled to form disc-shaped nano particles with consistent size and shape, the disc-shaped nano particles can cause organism immunity less, have enhanced tumor tissue penetrating capability, can be widely used as nano drug carriers, and can be applied to detection reagents, treatment drugs and the like for diseases such as tumors and the like by performing functional modification on the disc-shaped nano particles, such as modification of fluorescent substances, targeting substances and the like, so that the tobacco mosaic virus capsid protein mutant has wide application prospect.
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FIGS. 1a to 1b are electron micrographs of the TMV-T103C and TMV-T103C-RGD1 templates obtained in example 2, respectively.
FIGS. 2 a-2 b are electron micrographs of the fluorescent dye-bearing TMV-T103C and TMV-T103C-RGD1 templates obtained in example 3, respectively.
FIGS. 3 a-3 d are fluorescence micrographs of TMV-T103C and TMV-T103C-RGD1 template-labeled U87MG cells, with and without fluorescent dye, respectively, obtained in example 4.
Detailed Description
The technical solution of the present invention will be explained in more detail with reference to the drawings and examples, but the present invention is not limited thereto.
The embodiment of the invention also provides a tobacco mosaic protein capsid protein mutant which comprises a polypeptide obtained by point mutation of an amino acid sequence shown by SEQ ID No.1, wherein the point mutation is as follows: the 103 th position is mutated into cysteine, and the carboxyl terminal of the polypeptide is inserted with an RGD1 sequence shown in SEQ ID No. 7.
Further, the amino acid sequence of the tobacco mosaic protein capsid protein mutant is shown in SEQ ID No. 5.
The embodiment of the invention also provides a coding gene of the tobacco mosaic virus capsid protein mutant.
Furthermore, the sequence of the coding gene is shown as SEQ ID No. 6.
The capsid protein of the native Tobacco Mosaic Virus (TMV) is a non-covalent bond system consisting of roughly 2130 protein monomers with the same amino acid sequence, each of which consists of 158 amino acids (as shown in SEQ id No. 1) by self-assembly. In the above embodiment of the invention, the monomer is modified, cysteine is introduced into the amino acid sequence of the monomer, and cysteine and the carboxyl terminal of the monomer are introduced to insert the targeting peptide, so as to obtain two modified monomers, namely the two tobacco mosaic virus capsid protein mutants, each modified monomer can be combined through a disulfide bond, which is beneficial to improving the in vitro self-assembly capability, and the stability is remarkably improved.
The embodiment of the invention also provides a disc-shaped nanoparticle which is formed by self-assembly of the tobacco mosaic virus capsid protein mutant and has a double-layer disc-shaped structure, wherein the diameter of the disc-shaped nanoparticle is 18-20nm, the height of the disc-shaped nanoparticle is 4-5nm, and the disc-shaped nanoparticle is provided with a hollow through hole with the diameter of 4-5 nm.
Further, the disc-shaped nanoparticles are disc-shaped.
Further, the disc-shaped nanoparticles may be modified with a fluorescent substance (e.g., cy5, etc., without being limited thereto). Preferably, the fluorescent substance is bound to the disc-shaped nanoparticle through a covalent bond, such as a peptide bond.
The disc-shaped nanoparticles formed by the mutant TMV capsid protein provided by the embodiment of the invention are nano-sized, the small size can weaken the immune clearance effect of the body on the disc-shaped nanoparticles, the penetrating capability of the disc-shaped nanoparticles on tumor tissues can be enhanced, and the disc-shaped nanoparticles can be utilized as drug delivery carriers. Furthermore, the discoid nano-particles can be endowed with more functions, such as targeting effect, by performing functional treatment on the discoid nano-particles, such as fluorescent modification or targeted modification on the discoid nano-particles, so that the phagocytosis of tumor cells on the discoid nano-particle drug carriers can be explored, and the discoid nano-particles have important morphological and biological therapeutic significance.
The embodiment of the invention also provides a preparation method of the disc-shaped nano particles, which comprises the following steps:
constructing a tobacco mosaic virus capsid protein mutant with a sequence shown as SEQ ID No.3 or SEQ ID No.5 (preferably the latter);
dissolving the tobacco mosaic virus capsid protein mutant in potassium phosphate/β -mercaptoethanol buffer solution with the pH value of 9.0-9.5, wherein the potassium phosphate/β -mercaptoethanol buffer solution comprises 50-100mmol/L potassium phosphate and 5-10mmol/L β -mercaptoethanol, then dialyzing in tris (hydroxymethyl) aminomethane buffer solution with the pH value of 7.0-7.5 and the concentration of 50-100mmol/L, the cut-off molecular weight of a dialysis bag is 8000-14000, and then carrying out ultrafiltration centrifugal concentration with 30-100KD to enable the tobacco mosaic virus capsid protein mutant to self-assemble into double-layer disc-shaped nano particles, wherein the disc-shaped nano particles have the diameter of 18-20nm and the height of 4-5nm, and have hollow through holes with the diameter of 4-5 nm.
In some embodiments, the preparation method may further comprise: and connecting a fluorescent substance to the disc-shaped nanoparticles by a chemical modification mode.
For example, in some more specific embodiments, the discotic nanoparticles with stable and uniform dispersion can be mixed with a reducing agent to react and expose the thiol group of cysteine, and then the discotic nanoparticles are mixed with a fluorescent dye with a cross-linking group to bind the fluorescent dye to the discotic nanoparticles, so as to obtain discotic nanoparticles with fluorescent dye (which can also be considered as a discotic protein nanocarrier).
In some embodiments, the preparation method may further comprise: at least the fluorescent substance is attached to the wall of the hollow through-hole of the discotic nanoparticle.
In some embodiments, the modified TMV capsid protein mutant targeting tumor cell surface integrin is a discoid nano virus particle as a template, and the fluorescent dye is firstly bonded on the discoid nano material through the cross-linking reaction of the sulfydryl in the hollow through hole and the fluorescent dye containing maleimide, and then the above material is added into PBS buffer solution, and the tumor cell is incubated to phagocytose the nano material, so as to realize enhanced tumor phagocytosis effect.
The types of the above chemical modification, the process steps and the process conditions are well known to those skilled in the art, and are not described herein.
In a more specific embodiment of the invention, threonine (Thr) at position 103 in the amino acid sequence shown in the wild-type TMV capsid protein monomer SEQ ID No.1 can be mutated to cysteine (Cys) to obtain the modified TMV-T103C (i.e., a TMVcp mutant), the amino acid sequence of which is shown in SEQ ID No. 3. According to the amino acid sequence of the modified TMV-T103C, the encoding gene PET32a-TMV-T103C can be obtained, and the sequence of the encoding gene is shown as SEQ ID No. 4.
In a more specific embodiment of the invention, RGD1(GRDGSRG) can be inserted into the carboxyl terminal end of the amino acid sequence of TMV-T103C shown in SEQ ID No.3 to obtain the modified TMV-T103C-RGD1 (i.e. another TMVcp mutant), and the amino acid sequence of the modified TMV-T103 8925 is shown in SEQ ID No. 5. Similarly, the encoding gene PET32a-TMV-T103C-RGD1 can be obtained according to the amino acid sequence of the modified TMV-T103C-RGD1, and the sequence of the encoding gene is shown as SEQ ID No. 6.
Furthermore, the TMVcp mutant obtained after the expression is finished can be resuspended by a lysis buffer solution, placed on ice for ultrasonic disruption, centrifuged to obtain a supernatant, and then filtered, purified and processed.
Wherein, the purification treatment includes, but is not limited to, ultra-strong anion chromatography column purification, ammonium sulfate purification, and the like.
Further, after the TMVcp mutant is purified, it can be assembled as a monomer in a phosphate buffer, and then column elution purification is performed, and then the product is placed in a phosphate buffer for dialysis, and finally ultrafiltration concentration is performed to obtain the disk-shaped nanoparticles formed by self-assembly of the modified TMV capsid protein, wherein the diameter of the disk-shaped nanoparticles is about 18-20nm, the height of the disk-shaped nanoparticles is about 4-5nm, and the disk-shaped nanoparticles have hollow through holes with the diameter of about 4-5 nm.
The embodiment of the invention also provides application of the tobacco floral leaf protein capsid protein mutant or the disc-shaped nano-particles in preparation of a drug carrier.
The embodiment of the invention also provides a functional product, which comprises the tobacco floral leaf protein capsid protein mutant or the disc-shaped nanoparticle, and the product can be at least used for detecting and/or treating tumors.
The embodiment of the invention obtains the tobacco floral leaf protein capsid protein mutant by gene modification, and the mutant is self-assembled to form a nano disc shape, and when the mutant is used as a protein nano carrier, the mutant has better stability and targeting effect under the condition of maintaining the original property. By performing fluorescent modification on the discoid nanoparticles, the tumor cell phagocytosis effect and cell distribution of the discoid nanoparticles can be tracked. The discoidal nanoparticles, as a morphology which is just being studied, have far-reaching significance in exploring phagocytosis thereof by cells.
The stably dispersed discoid nano-particles with the targeting effect have enhanced phagocytosis on tumor cells, can carry medicaments and crosslink various functional ligands and the like as the bioaffinity and various modification sites of proteins, overcomes the ubiquitous toxic and side effects and non-degradability of nano-materials, and provides a selectable strategy for treating tumors.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. With regard to the definitions and terminology in this field, the expert can refer to Current Protocols in molecular biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.
Notwithstanding that the numerical values set forth in the broad scope of the invention inherently contain certain errors necessarily resulting from the standard deviation found in their respective measurements. Moreover, all areas of applicability of the present disclosure should be understood to encompass any and all subranges subsumed therein. For example, a stated range of "1 to 10" should be understood to include subranges beginning with a minimum value of 1 or more, e.g., 1 to 6.1, and subranges ending with a maximum value of 10 or less, e.g., 5.5 to 10. Additionally, any reference that is said to be "incorporated herein" is to be understood as being incorporated in its entirety.
It should also be noted that, as used in this specification, the singular forms "a," "an," and "the" include plural referents unless the context clearly and clearly dictates otherwise. The term "or" may be used interchangeably with the term "and/or" unless context dictates otherwise.
The present invention is further illustrated by the following specific examples and figures, which are provided for illustrative purposes only and do not limit the scope of the present invention. The conditions and procedures in the following examples were carried out according to conventional procedures in the art, unless otherwise specified.
The TMV-T103C variant mentioned in the following examples represents a mutant formed by mutating the 103 th position of the TMV capsid protein monomer amino acid sequence to cysteine, the TMV-T103C-RGD1 variant mentioned represents a mutant formed by mutating the 103 th position of the TMV capsid protein monomer amino acid sequence to cysteine and inserting RGD1(SEQ ID No.7) sequence into the carboxyl terminal, namely a modified monomer, the mentioned recombinant expression vector PET32a-TMV-T103C is formed by connecting the coding gene of-TMV-T103C to the plasmid vector PET32a, and the mentioned recombinant expression vector PET32a-TMV-T103C-RGD1 is formed by connecting the coding gene of TMV-T103C-RGD1 to the plasmid vector PET32 a. The unit "mM" mentioned in the specific examples below means "mmol/L" (millimoles per liter).
Example 1 construction of a mutant expression plasmid of the capsid protein of tobacco mosaic Virus, prokaryotic expression, and isolation and purification
Step 1: according to the coding gene shown in the cDNA nucleotide sequence SEQ ID No.2 of the wild tobacco mosaic virus capsid protein monomer, the coding gene is used as a PCR template, and the following two pairs of primers synthesized by the designed gene are used as mutagens:
A1：GCCAATCCGTGTACCGCG
A2：CGCGGTAGACGGATTGGC
B1：GAGATATACATATGAGCTATAGCAT
B2：GGAATTCTCAGGTCGCCGGGCCG。
adding 38 μ L sterilized ddH2O, 5 μ L10 × Taq Buffer, 4 μ L dNTP, 1 μ L PCR template, 1 μ L A1, 1 μ L B2, 0.4 μ L Taq enzyme into a 200 μ L PCR tube 1, adding 38 μ L sterilized ddH2O, 5 μ L10 × Taq Buffer, 4 μ L dNTP, 1 μ L PCR template, 1 μ L A2, 1 μ L B1, 0.4 μ L Taq enzyme into another 200 μ L PCR tube 2, blowing up and amplifying in PCR instrument, recovering PCR product after electrophoresis for 30min with 1% agarose, concentrating to 80-100 μ L, adding 38 μ L sterilized ddH2O, 5 μ L10 TP 10, 1 μ L Taq DNA 464, PCR product as PCR gene sequence, PCR product after PCR amplification for 30min, concentrating to 80-100 μ L PCR tube, adding 38 μ L sterilized ddH2O, 5 μ L Taq DNA 468, PCR product for 30min PCR product, PCR product after PCR amplification for 5 μ L PCR amplification, PCR product for 5 μ L PCR amplification for 3 min, PCR product for detecting DNA sequence, PCR product for 5 μ L4632, PCR product for detecting, PCR product for detecting for 5 μ L4632, PCR product for detecting, PCR product for detecting, PCR product for detecting:
C1：GAGATATACATATGAGCTATAGCAT
C2：GGAATTCTCAGCCCGGGCTATCGCCGCGGCCGGTCGCCGG。
adding 38 mu L of sterilized ddH2O, 5 mu L of 10 × Taq Buffer, 4 mu L of dNTP, 1 mu L of PCR template, 1 mu L of C1, 1 mu L of C2 and 0.4 mu L of Taq enzyme into another 200 mu L PCR tube, respectively blowing and beating, then putting into a PCR instrument for amplification, after electrophoresis for 30 minutes by 1% agarose, recovering a PCR product by using the kit, concentrating to 80-100 ng/mu L to obtain a PCR product, after the PCR product and a PET32a vector are respectively subjected to enzyme digestion and purification by NdeI enzyme and EcoRI enzyme, connecting T4 enzyme, successfully constructing a recombinant vector PET32a-TMV-T103C-RGD1, and verifying the gene sequence by DNA sequencing to be identical to SEQ ID No. 6.
Step 2: DNA sequencing to determine the complete correctness of the mutant gene sequence in the transferred vector, and using CaCl for recombinant vectors PET32a-TMV-T103C and PET32a-TMV-T103C-RGD12Transferring into E.coli BL21 competent cells, respectively, coating LB plate in sterile environment, culturing overnight at 37 deg.C, selecting monoclonal strain from the plate, inoculating into 5mL LB test tube culture medium, adding ampicillin (final concentration 50 μ g/mL), culturing at 37 deg.C at 200r/min for 12-16 h; transferring 5mL of bacterial liquid into 500mL of LB culture medium according to the inoculation amount of 0.5%, adding ampicillin (the final concentration is 50 mu g/mL), carrying out shake culture at constant temperature of 37 ℃ and 200r/min for about 2-3h (the OD600 is between 0.4 and 0.6), adding an isopropyl thiogalactoside inducer (IPTG with the final concentration of 1 mM), continuing shake-induced culture at 27 ℃ for 14h-16h, and centrifuging 8000rmp for 5min to collect thalli; collecting the collected mycelia with tris (hydroxymethyl) aminoThe supernatant is collected by washing once with methane buffer, then resuspending in 40mL lysis buffer, performing ice-bath ultrasonication, and centrifuging at 12000r/min for 30 min.
And step 3: purification of TMV-T103C and TMV-T103C-RGD 1; and D, filtering the supernatant obtained in the step A2 by using a 0.22-micrometer syringe filter to remove impurities, balancing the Hi Trap Q HP prepacked column by using a peristaltic pump to perform loading buffer solution, then loading by using the peristaltic pump, then loading the Hi Trap Q HP prepacked column adsorbed with the target protein into an AKTA Prime plus FPLC system, performing gradient elution, wherein the elution gradient is 0-1M NaCl, the elution flow rate is 3mL/min, respectively collecting elution peaks on ice, then slowly adding 1M solid ammonium sulfate for purification, standing at 4 ℃ for thirty minutes, and centrifuging at 12000rpm to collect precipitates. The pellet was resuspended in lysis buffer (100mM sodium carbonate-sodium bicarbonate, pH 9.5). Slowly oscillating overnight at low temperature, centrifuging at 12000rpm at 4 deg.C for 15min, collecting supernatant, detecting the purity of target protein by SDS-PAGE, and freezing at-80 deg.C.
The proteins TMV-T103C and TMV-T103C-RGD1 obtained in this example are mutant TMV protein monomers. Wherein, the 103 th position of the amino acid sequence of TMV-T103C is mutated into cysteine, and the amino acid sequence is shown as SEQ ID No. 3. The TMV-T103C-RGD1 has the point mutation, and an RGD1 sequence is added at the carboxyl terminal, and the amino acid sequence of the RGD1 sequence is shown as SEQ ID No. 5.
Example 2 generation of TMV nanodisc nanocarriers (i.e., the aforementioned discotic nanoparticles)
The high purity TMV-T103C and TMV-T103C-RGD1 protein monomers obtained in example 1 were dialyzed from a potassium phosphate/β -mercaptoethanol buffer solution (containing 50-100mmol/L potassium phosphate and 5-10mmol/L β -mercaptoethanol) having a pH of 9.0-9.5, respectively, to a concentration of 50mM from tris (hydroxymethyl) aminomethane buffer solution, a cut-off molecular weight of dialysis bag was 8000-14000, ammonium sulfate and carbonate were removed by dialysis 3-5 times, the concentration was measured by a nucleic acid protein analyzer, and then concentrated to 4-5mg/mL by a 100KD ultrafiltration centrifuge tube, the concentration was measured by an enzyme-linked immunosorbent assay instrument, impurities were removed again by a needle filter, and electron microscopy was performed, and the self-assembled product was stored in a 4 ℃ refrigerator, and photographs of nano discs (hereinafter, each of which is referred to as TMV-T103C and TMV-T103C-RGD1 protein monomers, respectively, were formed into a disc-shaped nano-disc (hereinafter, each of which is approximately 18nm in diameter and approximately 18nm, as shown in FIG. 1a photograph.
Example 3 crosslinking of TMV nanodisk nanocarriers with fluorescent dyes
Based on the nano-disc assembly formed by the TMV-T103C and TMV-T103C-RGD1 proteins obtained in the example 2, the nano-disc assembly is respectively crosslinked with Mal-cy5, and the specific steps are as follows:
the TMV-T103C nanodisk assembly was dialyzed into sodium dihydrogen phosphate-disodium hydrogen phosphate (pH 7.0, 50mM) buffer, the concentration was quantified to 0.5mg/mL, tris (2-carboxyethyl) phosphine (TCEP) was added to 1mL system at a final concentration of 5mM, mixed well and allowed to stand for half an hour, then Mal-cy5 was added at a final concentration of 0.02mg/mL and allowed to stand at room temperature overnight in the absence of light. TMV-T103C-RGD nanodisk assembly as described above. And then, removing unreacted Mal-cy5 dye by ultrafiltration, and characterizing by an electron microscope to obtain TMV-T103C-cy5 nanodisks and TMV-T103C-RGD1-cy5 nanodisk structures as shown in FIGS. 2 a-2 b respectively.
Example 4 uptake of TMV nanodisk nanocarriers by tumor cells
10 in 12-well plate6Density culture of human glioblastoma cells (U87MG) for 12h, adjusting the absorption values of TMV-T103C-cy5 nanodiscs and TMV-T103C-RGD1-cy5 nanodiscs at 560nm wavelength to be consistent, diluting the same in proportion with DMEM low-sugar medium, replacing serum-containing medium in U87MG cells with serum-free medium containing PBS solution, serum-free medium containing TMV-T103C-RGD1 nanodisc, serum-free medium containing TMV-T103C-cy5 nanodisc and serum-free PBS medium containing TMV-T103C-RGD1-cy5 nanodiscs, incubating for 15min, washing 3 times with PBS solution, and finally replacing with PBS solution, observing fluorescence imaging of cy5 in U87MG cells under a fluorescence microscope, the fluorescence imaging graphs are respectively shown in fig. 3 a-3 d.
The embodiment of the invention adopts the stable nanodisc assembly formed by the TMV-T103C and TMV-T103C-RGD1 mutant proteins as a protein carrier, and utilizes the interaction between functional sulfydryl exposed on the surface of the stable nanodisc assembly and maleimide to crosslink the two protein assemblies and the cy5 dye, so that the nanodisc assembly formed by the stable TMV-T103C and TMV-T103C-RGD1 mutant proteins is used as the protein carrier, and the phagocytosis of tumor cells on the carrier is increased.
The preparation process provided by the embodiment of the invention has simple steps and mild conditions, and solves the technical problems of complicated steps, poor dispersibility, poor stability and the like when the drug nano-carrier is constructed in the prior art; and the obtained nano protein carrier material has good dispersibility, stability and biological safety, and has potential value in the directions of biological imaging and drug carriers.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
It should be understood that the above-mentioned embodiments are merely illustrative of the technical concepts and features of the present invention, which are intended to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and therefore, the protection scope of the present invention is not limited thereby. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Figure BDA0002413745480000101
Figure BDA0002413745480000111
Figure BDA0002413745480000121
Figure BDA0002413745480000131
Sequence listing
<110> Suzhou nanotechnology and nano-bionic institute of Chinese academy of sciences
<120> tobacco mosaic virus capsid protein mutant and application thereof
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20 25 30
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35 40 45
Ser Glu Val Trp Lys Pro Ser Pro Gln Val Thr Val Arg Phe Pro Asp
50 55 60
Ser Asp Phe Lys Val Tyr Arg Tyr Asn Ala Val Leu Asp Pro Leu Val
65 70 75 80
Thr Ala Leu Leu Gly Ala Phe Asp Thr Arg Asn Arg Ile Ile Glu Val
85 90 95
Glu Asn Gln Ala Asn Pro Thr Thr Ala Glu Thr Leu Asp Ala Thr Arg
100 105 110
Arg Val Asp Asp Ala Thr Val Ala Ile Arg Ser Ala Ile Asn Asn Leu
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Ile Val Glu Leu Ile Arg Gly Thr Gly Ser Tyr Asn Arg Ser Ser Phe
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<210>2
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atgagctata gcattaccac cccgagccag tttgtgtttc tgagcagcgc ctgggcggat 60
ccgattgaac tgattaacct gtgcaccaat gcgctgggca accaatttca gacccagcag 120
gcgcgcaccg ttgtgcagcg tcagttcagc gaagtttgga aaccgagccc gcaggttacc 180
gtgcgctttc cggatagcga ttttaaagtg tatcgctata acgccgtgct ggatccgctg 240
gtgaccgcgc tgctgggcgc ctttgatacc cgtaatcgta tcattgaagt ggaaaaccag 300
gccaatccga ctaccgcgga aaccctggat gcgacccgtc gtgtggatga tgccaccgtg 360
gcgattcgca gcgccatcaa taacctgatt gtggaactga ttcgtggcac cggcagctat 420
aaccgtagca gctttgaaag cagcagcggc ctggtgtgga cgagcggccc ggcgacctga 480
<210>3
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<213> Artificial sequence (Artificial sequence)
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Ser Tyr Ser Ile Thr Thr Pro Ser Gln Phe Val Phe Leu Ser Ser Ala
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Arg Val Asp Asp Ala Thr Val Ala Ile Arg Ser Ala Ile Asn Asn Leu
115 120 125
Ile Val Glu Leu Ile Arg Gly Thr Gly Ser Tyr Asn Arg Ser Ser Phe
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atgagctata gcattaccac cccgagccag tttgtgtttc tgagcagcgc ctgggcggat 60
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gcgcgcaccg ttgtgcagcg tcagttcagc gaagtttgga aaccgagccc gcaggttacc 180
gtgcgctttc cggatagcga ttttaaagtg tatcgctata acgccgtgct ggatccgctg 240
gtgaccgcgc tgctgggcgc ctttgatacc cgtaatcgta tcattgaagt ggaaaaccag 300
gccaatccgt gtaccgcgga aaccctggat gcgacccgtc gtgtggatga tgccaccgtg 360
gcgattcgca gcgccatcaa taacctgatt gtggaactga ttcgtggcac cggcagctat 420
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20 25 30
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35 40 45
Ser Glu Val Trp Lys Pro Ser Pro Gln Val Thr Val Arg Phe Pro Asp
50 55 60
Ser Asp Phe Lys Val Tyr Arg Tyr Asn Ala Val Leu Asp Pro Leu Val
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gcgcgcaccg ttgtgcagcg tcagttcagc gaagtttgga aaccgagccc gcaggttacc 180
gtgcgctttc cggatagcga ttttaaagtg tatcgctata acgccgtgct ggatccgctg 240
gtgaccgcgc tgctgggcgc ctttgatacc cgtaatcgta tcattgaagt ggaaaaccag 300
gccaatccgt gtaccgcgga aaccctggat gcgacccgtc gtgtggatga tgccaccgtg 360
gcgattcgca gcgccatcaa taacctgatt gtggaactga ttcgtggcac cggcagctat 420
aaccgtagca gctttgaaag cagcagcggc ctggtgtgga cgagcggccc ggcgaccggc 480
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1 5

Claims (12)

1. A tobacco mosaic virus capsid protein mutant is characterized by comprising a polypeptide obtained by point mutation of an amino acid sequence shown in SEQ ID No.1, wherein the point mutation is as follows: the 103 th position is mutated into cysteine, and the carboxyl terminal of the polypeptide is inserted with RGD1 sequence shown in SEQ ID No. 7.
2. The tobacco floral leaf protein capsid protein mutant according to claim 1, characterized in that: the amino acid sequence of the polypeptide is shown as SEQ ID No. 5.
3. The gene encoding the mutant tobamovirus capsid protein according to claim 1 or 2, said gene having the sequence shown in SEQ ID No. 6.
4. A recombinant expression vector comprising the coding gene of claim 3.
5. A transgenic cell line comprising the gene encoding claim 3.
6. A disc-like nanoparticle characterized by: the tobacco mosaic virus capsid protein mutant is formed by self-assembly of tobacco mosaic virus capsid protein mutants with sequences shown as SEQ ID No.3 or SEQ ID No.5, and has a double-layer disc-shaped structure, the diameter of the disc-shaped nanoparticle is 18-20nm, the height of the disc-shaped nanoparticle is 4-5nm, and the disc-shaped nanoparticle is provided with a hollow through hole with the diameter of 4-5 nm.
7. The discotic nanoparticle of claim 6, wherein: the disc-shaped nanoparticles are also modified with a fluorescent substance, and preferably, the fluorescent substance is combined with the disc-shaped nanoparticles through a covalent bond.
8. The discotic nanoparticle of claim 6, wherein: the discoid nanoparticles are also combined with a targeting substance capable of being specifically combined with tumor cells, and preferably, the targeting substance is combined with the discoid nanoparticles through a covalent bond; more preferably, the covalent bond is a peptide bond.
9. A method for preparing a disc-shaped nanoparticle, comprising:
constructing a tobacco mosaic virus capsid protein mutant with a sequence shown as SEQ ID No.3 or SEQ ID No. 5;
dissolving the tobacco flower leaf virus capsid protein mutant in potassium phosphate/β -mercaptoethanol buffer solution with the pH value of 9.0-9.5, wherein the potassium phosphate/β -mercaptoethanol buffer solution comprises 50-100mmol/L potassium phosphate and 5-10mmol/L β -mercaptoethanol, then dialyzing in tris (hydroxymethyl) aminomethane buffer solution with the pH value of 7.0-7.5 and the concentration of 50-100mmol/L, the molecular weight cut-off of the dialysis bag is 8000-14000, and then carrying out ultrafiltration centrifugal concentration with 30-100KD to 4-5mg/mL, so that the tobacco flower leaf virus capsid protein mutant self-assembles to form double-layer disc-shaped nanoparticles, the diameter of the disc-shaped nanoparticles is 18-20nm, the height of the disc-shaped nanoparticles is 4-5nm, and the disc-shaped nanoparticles have hollow through holes with the diameter of 4-5 nm.
10. The method of claim 9, further comprising: and (3) connecting a fluorescent substance to the disc-shaped nanoparticles by a chemical modification mode, preferably, connecting the fluorescent substance to the wall of the hollow through hole of the disc-shaped nanoparticles.
11. Use of a mutant tobacco mosaic protein coat protein according to claim 1 or 2 or a discoid nanoparticle according to claim 6, 7 or 8 for the preparation of a pharmaceutical carrier.
12. A functional product, characterized in that it comprises a tobacco floral leaf protein coat protein mutant according to claim 1 or 2 or a discoid nanoparticle according to claim 6, 7 or 8, and in that it is at least useful for the detection and/or treatment of tumors.
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