CN111363013B - Construction method of multi-component nanoparticle cluster - Google Patents
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Abstract
The invention provides a construction method of the multi-component nanoparticle cluster, which comprises the following steps: respectively providing N zinc finger proteins, N protein tags and M nanoparticles; crosslinking one kind of the nanoparticles with at least one kind of the protein tags, and crosslinking only one kind of the nanoparticles with one kind of the protein tags to obtain N kinds of nanoparticle/protein tag complexes; crosslinking one zinc finger protein with one ligand according to the unique pairing relation to obtain N zinc finger protein/ligand complexes; providing a nucleotide sequence, mixing and incubating the nucleotide sequence with the N types of nano-particle/protein tag complexes and the N types of zinc finger protein/ligand complexes to form a nucleotide sequence-zinc finger protein-protein tag-nano-particle connecting system, and collecting the obtained nano-particle cluster after separation, wherein bases in the nucleotide sequence are set according to the preset arrangement sequence of zinc finger proteins in the nano-particle cluster.
Description
Technical Field
The invention belongs to the technical field of nano particle clusters, and particularly relates to a construction method of a multi-component nano particle cluster.
Background
The nanoparticle cluster is a specific composition or arrangement formed by nanoparticles through special interaction, can better integrate and enhance the excellent optical, electric and magnetic properties of the nanoparticles, and further shows properties superior to those of the nanoparticles when simply aggregated. In addition, the good structural stability and low blood dispersibility of the nano particle cluster can effectively improve the signal to noise ratio, and the nano particle cluster has wide application value in the fields of biochemical sensing, biological markers, medical images and the like.
At present, the nanoparticle cluster is mostly prepared by using small molecules as media, for example, Kumacheva et al modifies sulfhydrylation polystyrene molecules at the wide part of a gold nanorod, and forms a linear structure in which the gold nanorod is connected end to end through the behavior of spontaneously reducing the surface energy in a poor solvent, and the gold nanorod and the palladium nanorod are connected in a staggered manner. Sailor and Karathanasis et al respectively use self-assembly of dextran and chemical reaction between thiol-amino groups to construct a worm-like magnetic nanoparticle cluster, which has good drug-loading capability in chemotherapy for tumors. However, when the functional group of the small molecule medium method reacts, randomness exists in different positions, so that the size or the shape of the nanoparticle cluster structure is difficult to accurately control, and uncertainty in practical application is increased.
In recent years, researchers have provided a new idea of guiding nanoparticles to be arranged into linear particle clusters by using template nucleic acids, taking advantage of the evolutionary structure of organisms in the nature. The Chengdian courier subject group at the university of Fudan utilizes amino-modified nanogold to be combined with a DNA phosphate skeleton with negative charges to form a nanogold particle cluster; hughes et al bind biotin molecules to DNA via thymine to form streptavidin-quantum dot complexes aligned along the DNA nanotubes; furthermore, Liedl, Gang et al, through base complementation, combine nanoparticles modified with single-stranded nucleic acids with their complementary single-stranded nucleic acids to form nanoparticle clusters. Researchers at Korea scientific and technical institute and Shenzhen advanced technology research institute, Zhongkoku institute have inserted biotin (biotin) -converted zinc finger proteins into double-stranded nucleic acids to obtain nucleic acid templates having biotin activity. The nucleic acid template can be combined with magnetic nanoparticles modified with Streptavidin (SA) in a high specificity manner, and the magnetic nanoparticles are effectively guided to be arranged orderly along a nucleic acid skeleton. However, because the nucleic acid chain and the nanoparticle are based on 4:1 combination between the nucleic acid chain and the nanoparticle, and a single nanoparticle often has a plurality of streptavidin, a cross-linking phenomenon that one nanoparticle is connected with a plurality of template nucleic acids or a plurality of nanoparticles are connected with a plurality of template nucleic acids is easy to occur, a network structure of nucleic acid wound nanoparticles with difficult size control is generated, and the yield of target nanoparticle clusters is greatly influenced. Currently, the target form of nanoparticle clusters can be obtained only by increasing the feeding times of nanoparticles and centrifuging the product at high speed for many times. The cross-linking easily causes the agglomeration of nano-particles, and reduces the stability of the nano-particles, and in biomedical imaging analysis, the irregular complex cross-linking structure influences the distribution of signals, so that the signals are wrongly analyzed. Furthermore, a single combination approach can only achieve a single component of nanoparticle clusters.
Disclosure of Invention
The invention aims to provide a construction method of a multi-component nanoparticle cluster, and aims to solve the problem that only a single-component nanoparticle cluster can be constructed in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a construction method of a nanoparticle cluster, wherein the nanoparticle cluster is a multi-component nanoparticle cluster, and the construction method of the multi-component nanoparticle cluster comprises the following steps:
providing N zinc finger proteins, M nanoparticles, N protein tags and N ligands for identifying different said protein tags, respectively, wherein one said ligand uniquely identifies a corresponding said protein tag; n is a positive integer greater than or equal to 2, M is a positive integer greater than or equal to 2, and M is less than or equal to N;
crosslinking one kind of the nanoparticles with at least one kind of the protein tags, and crosslinking only one kind of the nanoparticles with one kind of the protein tags to obtain N kinds of nanoparticle/protein tag complexes; crosslinking one zinc finger protein with one ligand according to the unique pairing relation to obtain N zinc finger protein/ligand complexes;
providing a nucleotide sequence, mixing and incubating the nucleotide sequence with the N types of nano-particle/protein tag complexes and the N types of zinc finger protein/ligand complexes to form a nucleotide sequence-zinc finger protein-protein tag-nano-particle connecting system, and collecting the obtained nano-particle cluster after separation, wherein bases in the nucleotide sequence are set according to the preset arrangement sequence of zinc finger proteins in the nano-particle cluster.
According to the construction method of the nanoparticle cluster, different zinc finger proteins are adopted to mediate ordered arrangement of different nanoparticles on a nucleotide sequence, so that construction of multi-component and diverse nanoparticle clusters is achieved. Specifically, different zinc finger proteins are connected with different ligands, different protein tags are used for marking different nanoparticles, finally, specific types of nanoparticles are connected with specific zinc finger proteins through one-to-one correspondence between the ligands and the protein tags, and finally, the nanoparticles are connected to nucleotide sequences with bases arranged according to the arrangement sequence of the zinc finger proteins in a preset nanoparticle cluster, so that the construction of multi-component and diverse nanoparticle clusters is realized. In addition, the protein tags and the ligands, the ligands and the zinc finger proteins, and the zinc finger proteins and the nucleic acids can be combined according to the ratio of 1:1, so that the multi-component nanoparticle cluster constructed by the method can effectively improve the controllability of the components of the particle cluster, and is convenient for subsequent purification.
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FIG. 1 is a schematic diagram of the construction of a multi-component nanoparticle cluster provided by an embodiment of the present invention;
FIG. 2 is a UV spectrum of a single particle quantum dot, a quantum dot composite, a quantum dot/protein tag composite, and a quantum dot nanoparticle cluster according to an embodiment of the present invention;
FIG. 3 is a fluorescence spectrum of a single-particle quantum dot, a quantum dot complex, a quantum dot/protein tag complex, and a quantum dot nanoparticle cluster according to an embodiment of the present invention;
fig. 4 is a TEM image of a nanoparticle cluster provided by an embodiment of the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In the description of the present invention, it is to be understood that the terms "first", "second" and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
With reference to fig. 1 to 4, an embodiment of the present invention provides a method for constructing a nanoparticle cluster, where the nanoparticle cluster is a multicomponent nanoparticle cluster, and the method for constructing the multicomponent nanoparticle cluster includes the following steps:
s01, respectively providing N zinc finger proteins, M nanoparticles, N protein tags and N ligands for identifying different protein tags, wherein one ligand uniquely identifies one corresponding protein tag; n is a positive integer greater than or equal to 2, M is a positive integer greater than or equal to 2, and M is less than or equal to N;
s02, crosslinking one type of nano-particles and at least one type of protein tag, wherein only one type of nano-particles are crosslinked by one type of protein tag, so that N types of nano-particle/protein tag complexes are obtained; crosslinking one zinc finger protein with one ligand according to the unique pairing relation to obtain N zinc finger protein/ligand complexes;
s03, providing a nucleotide sequence, mixing and incubating the nucleotide sequence with the N nano-particle/protein tag complexes and the N zinc finger protein/ligand complexes to form a nucleotide sequence-zinc finger protein-protein tag-nano-particle connecting system, and collecting the obtained nano-particle cluster after separation, wherein bases in the nucleotide sequence are set according to a preset arrangement sequence of zinc finger proteins in the nano-particle cluster.
According to the construction method of the nanoparticle cluster provided by the embodiment of the invention, different zinc finger proteins are adopted to mediate ordered arrangement of different nanoparticles on a nucleotide sequence, so that construction of multi-component and diverse nanoparticle clusters is realized. Specifically, different zinc finger proteins are connected with different ligands, different protein tags are used for marking different nanoparticles, finally, specific types of nanoparticles are connected with specific zinc finger proteins through one-to-one correspondence between the ligands and the protein tags, and finally, the nanoparticles are connected to nucleotide sequences with bases arranged according to the arrangement sequence of the zinc finger proteins in a preset nanoparticle cluster, so that the construction of multi-component and diverse nanoparticle clusters is realized. In addition, the protein tags and the ligands, the ligands and the zinc finger proteins, and the zinc finger proteins and the nucleic acids can be combined according to the ratio of 1:1, so that the multi-component nanoparticle cluster constructed by the method can effectively improve the controllability of the components of the particle cluster, and is convenient for subsequent purification.
Specifically, in step S01, M types of nanoparticles are provided in combination with the type of multicomponent nanoparticle cluster to be formed. The M kinds of nanoparticles can be M kinds of nanoparticles of the same kind but different specific models, or M kinds of nanoparticles of different kinds. Wherein M is a positive integer greater than or equal to 2.
N protein tags and N ligands for identifying different protein tags are provided, and one ligand uniquely identifies one corresponding protein tag, namely, the protein tags and the ligands have one-to-one correspondence. For example, when the protein tag is SNAPtag, the corresponding ligand is BG-Maleimide; when the protein tag is Halotag, the corresponding ligand is HaloTag-Maleimide, wherein the Maleimide is Maleimide.
N zinc finger proteins are provided, which realize connection with the label protein connected with the nanoparticles through connecting specific ligands. The N zinc finger proteins are finally linked to the nucleotide sequences provided in step S03. Therefore, the base arrangement of the nucleotide sequence can be reasonably edited, so that the zinc finger protein is connected to the nucleotide sequence according to a certain sequence, the arrangement of the multi-component nanoparticles in the nanoparticle cluster is further realized, and the multi-component nanoparticle cluster is further obtained.
Wherein N is a positive integer greater than or equal to 2, and M is less than or equal to N. Thus, one nanoparticle may be labeled with one or more zinc finger proteins (protein tags and ligands) (while one zinc finger protein (or protein tag and ligand) labels only one nanoparticle), achieving the alignment of multicomponent nanoparticles in the nanoparticle cluster. Of course, in order to improve the convenience of operation, it is preferable that M be equal to N. Namely, a zinc finger protein is adopted to be connected with a specific ligand, and then the protein tag of the nanoparticle is connected through the specific ligand in a crosslinking way. Namely, the protein tags and the ligands, the ligands and the zinc finger proteins, and the zinc finger proteins and the nucleic acids can be combined according to the proportion of 1:1, so that the constructed multi-component nanoparticle cluster can effectively improve the controllability of the components of the particle cluster, and is convenient for subsequent purification.
In the embodiment of the invention, the zinc finger protein, the nanoparticle, the protein tag and the ligand are selected only by the condition that the unique corresponding relationship exists between the protein tag and the ligand; while one ligand is connected to only one zinc finger protein between the zinc finger proteins, the types of the ligands connected to the zinc finger proteins are not strictly limited, for example, zinc finger protein zif268 is connected with only one ligand, the ligand can be BG-Maleimide, HaloTag-Maleimide or other, but once selected, all zinc finger proteins zif268 are connected with only one corresponding ligand; similarly, between the nanoparticle and the protein tag, only one type of the protein tag needs to be uniquely marked with only one type of nanoparticle.
As a preferred embodiment, N, M are both 2, the two zinc finger proteins are the first zinc finger protein zif268 and the second zinc finger protein SNAP; the two N protein labels are SNAPtag and Halotag respectively; the two ligands are BG-Maleimide and HaloTag-Maleimide respectively, wherein the BG-Maleimide is used for identifying SNAPtag, and the HaloTag-Maleimide is used for identifying Halotag. At this time, a schematic diagram of a construction method of the multicomponent nanoparticle cluster is shown in fig. 1.
In some embodiments, the zinc finger protein is prepared by:
s011, providing primers, and performing PCR amplification by taking pET30a-C-zif268 and pSNAP-tag as templates to obtain a first gene DNA fragment encoding zif268 protein and a second gene DNA fragment encoding SNAP protein, wherein histidine tags are introduced into the C ends of the first gene DNA fragment and the second gene DNA fragment; NdeI enzyme cutting sites and XhoI enzyme cutting sites are respectively designed at the 5 'end and the 3' end of the first gene DNA fragment; NdeI and XhoI enzyme cutting sites are respectively designed at the 5 'end and the 3' end of the second gene DNA fragment;
s012, providing a vector pET21a, and carrying out enzyme digestion treatment by using Nde I and Xho I to obtain a linear vector; and (2) incubating the first gene DNA fragment and the second gene DNA fragment with a linearized pET21a vector by using a homologous recombinase to obtain a recombinant vector, transforming the recombinant vector into a competent cell, culturing and expressing the competent cell, and extracting the competent cell by using an AKTA protein purification instrument and a Ni column to obtain a first zinc finger protein zif268 and a second zinc finger protein SNAP.
Specifically, in step S011, PCR amplification was performed using pET30a-C-zif268 and pSNAP-tag as templates, respectively, to obtain a first gene DNA fragment encoding zif268 protein and a second gene DNA fragment encoding SNAP protein. In order to effectively purify the protein expressed by the obtained target gene, a histidine tag (6 × His-tag) is introduced into the C ends of the first gene mRNA fragment and the second gene DNA fragment which are amplified when a primer sequence is designed. In addition, when designing the primers, in order to conveniently recombine the zinc finger protein into the vector, the 5 'end and the 3' end of the amplified first gene DNA fragment are respectively designed with NdeI enzyme cutting sites and XhoI enzyme cutting sites; the 5 'end and the 3' end of the amplified second gene DNA fragment are respectively designed with NdeI restriction enzyme site and XhoI restriction enzyme site.
In some preferred embodiments, the primers used to amplify the first gene DNA fragment are:
an upstream primer: 5 'TAAGAAGGAGATATACATATGGCTAGCACCATGGATATCAAGCTT 3'
A downstream primer: 5 'GTGGTGGTGGTGGTGCTCGAGATTAACCTCGAGCCC 3'.
In some preferred embodiments, the primers used to amplify the second gene DNA fragment are:
an upstream primer: 5 'TAAGAAGGAGATATACATATGCATATGATGTGCAAAACCGG 3'
A downstream primer: 5 'GTGGTGGTGGTGGTGCTCGAGGAATTCCTTCTCACCGGTGTGGAT 3'.
After PCR amplification, new target fragments zif268(AZP4) and SNAP are obtained through electrophoresis and gel recovery, wherein both ends of the target fragments zif268(AZP4) and SNAP contain 15 bases overlapped with the vector pET21a, and both ends of the C terminal are provided with a histidine tag.
Notably, the amplification of the first gene DNA fragment and the second gene DNA fragment encoding SNAP protein can integrate the target gene fragments into vectors respectively and express them respectively; it is also possible to integrate two target gene fragments into one recombinant vector and express both proteins simultaneously.
In step S012, vector pET21a was provided and digested with Nde I and Xho I to obtain a linear vector. And (3) incubating the first gene DNA fragment and the second gene DNA fragment with a linearized pET21a vector by using homologous recombinase (TaKaRa) to obtain a recombinant vector. In some embodiments, preferably, the incubation is performed at 50 ℃ for 15 min.
Further, the recombinant vector is transformed into competent cells, followed by culturing. Before culturing, the transformation effect can be further verified and verified. In some embodiments, the verification method is: after the recombinant vector was transformed into competent cells DH 5. alpha. by heat treatment at 42 ℃ for 45s, the transformed cells were further recovered by culture at 37 ℃ for 1 hour and then plated on LB plates (containing ampicillin) for selection, and cultured overnight at 37 ℃. And selecting a single colony for colony PCR and double enzyme digestion identification positive cloning the next day, culturing the screened positive strain, extracting plasmid, and finally performing gene sequencing identification. The plasmid with successful sequencing was transformed into competent cells BL21(DE3) by heat treatment at 42 ℃ for 45s, recovered by culture at 37 ℃ and plated on LB plates (containing ampicillin) for screening, and cultured overnight at 37 ℃. On the next day, single colonies were picked for colony PCR and double restriction enzyme to identify positive clones.
In some embodiments, culturing the transformed cells can be performed by: adding fresh LB culture medium (containing ampicillin) into glycerol bacteria (transformed cells) for culturing overnight, adding into the culture medium for amplification culture the next day, adding inducer IPTG when OD600 is 0.4-0.6 (entering into logarithmic phase of growth), inducing expression, and collecting bacteria. In particular, since the vector pET21a contained the ampicillin resistance gene, only the strain transformed with recombinant pET21a was ampicillin resistant, and thus by adding ampicillin to the medium, the positive strain was allowed to grow normally while inhibiting the growth of the other strains. In the embodiment of the invention, IPTG is added as an inducer in the culture process, has a similar structure with a lactose operon which is a promoter of pET21a, and is not easy to be metabolized by cells (lactose can be utilized by the cells), so that the continuous expression of protein can be realized. Preferably, the working concentration of ampicillin is 0.1mg/ml and the working concentration of IPTG is 0-1 mM.
In a specific embodiment, the culturing of the transformed cells can be performed by the following method: from the glycerol strain (transformed cells) of the stock keeping, add 1:100 into 4mL of fresh LB medium (containing ampicillin), culture under 37 deg.C, 200rpm overnight, add 1:100 into 400mL of medium the next day, expand culture under 37 deg.C, 200rpm, monitor its OD600 0.4-0.6 (enter into growth log phase), add 0.5mg/mL inducer IPTG, induced expression at 25 deg.C for 7h, at 10000rpm, 5min centrifugation, collect the thalli.
Reselecting the collected thallus, preferably re-suspending the thallus by using PBS, then crushing the thallus by using ultrasound (power 600w × 37%) to release soluble protein in the thallus, clarifying the solution, centrifuging and taking supernatant, wherein the preferable centrifugation conditions are as follows: centrifuging at 11000rpm and 4 deg.C for 15 min.
Further, the protein obtained after centrifugation was purified. In order to obtain high-purity protein, an AKTA protein purification instrument and a Ni column are adopted for extraction, and a first zinc finger protein zif268 and a second zinc finger protein SNAP are obtained. The nickel sulfate filler in the Ni column is specifically combined with the histidine fusion protein to remove the impurity protein, and then the nickel sulfate filler is combined with the Ni column filler in a competitive way by using an imidazole solution, so that the histidine fusion protein is eluted from the column, and the high-purity expression proteins of the first zinc finger protein zif268 and the second zinc finger protein SNAP can be obtained.
In a preferred embodiment, the conditions for purification of the first zinc finger protein zif268 and the second zinc finger protein SNAP are:
the column was washed with water, the ethanol was washed off, and then the column was equilibrated with PBS. After the UV line (indicating protein) and cout line (indicating salt concentration) on the instrument were brought to levels, the sample was loaded (supernatant collected in the previous step). After the sample loading is finished, the column is washed by PBS, proteins (impurity proteins) which are not combined with the column are washed, gradient concentration elution is carried out by imidazole when the UV rays are horizontal, the UV rays are monitored in real time during elution, and the eluent is collected when the UV rays are changed.
More preferably, the imidazole eluent is subjected to gradient elution at a concentration of 25mM, 75mM, 100mM, 125mM, 150mM, and 200mM in this order to obtain a high-purity protein.
After purification is complete, the collected protein may be verified. In some embodiments, the collected eluate is fractionated by SDS-PAGE and stained with Coomassie Brilliant blue. Observing the band corresponding to the molecular weight position, selecting eluent with high expression amount and less impurity protein, removing imidazole by using 10kD ultrafiltration tube, replacing with PBS as storage system, and storing at-80 deg.C.
In some embodiments, the preparation method of the tag protein SNAPtag is as follows:
the constructed pET21a-SNAP is transformed into an escherichia coli expression host cell BL21(DE3), the overnight culture is carried out for plate screening, a single colony is selected and inoculated into an LB liquid culture medium containing ampicillin to culture an activated strain, the activated strain liquid is inoculated into a fresh LB liquid culture medium containing ampicillin to be cultured until OD600 reaches 0.4-0.6, namely the strain is cultured until the logarithmic growth phase of the strain, and then an inducer IPTG (final concentration is 1mM) is added to induce the SNAP to be efficiently and soluble expressed. Further, the protein obtained by expression is extracted and separated. Here, the action and preferred concentration of the ampicillin with the inducer IPTG is as described above and will not be described in further detail for the sake of brevity.
Specifically, the cells were centrifuged to discard the supernatant, and then PBS buffer (ph7.4) was added to sufficiently suspend the cells, the cells were disrupted on ice by ultrasonic waves, and insoluble proteins were removed by centrifugation to obtain a supernatant soluble crude protein solution. Finally, the nickel sulfate filler in the Ni column and the histidine fusion protein can be specifically combined to remove the impurity protein, and then the imidazole solution is competitively combined to the Ni column filler to elute the histidine fusion protein from the column. Because the protein expression quantity and the purity are high, an obvious protein band can be obtained when the concentration of an eluent is 75-125 mM, and the pure target protein SNAPtag is obtained after imidazole and small molecular hybrid protein are removed by dialyzing the protein.
In a specific preferred embodiment, the preparation method of the tag protein SNAPtag comprises the following steps:
the constructed pET21a-SNAP is transformed into an escherichia coli expression host cell BL21(DE3) by heat shock at 42 ℃ for 45s, plates are coated on an LB solid culture medium (added with ampicillin for screening) at 37 ℃ for overnight culture, plates are screened, a single colony is selected and inoculated into an LB liquid culture medium containing ampicillin for culturing an overnight activated strain at 37 ℃ and 200rpm, the activated strain liquid is inoculated into 200mL of fresh LB liquid culture medium containing ampicillin according to the proportion of 1:100 for culturing until the OD600 reaches 0.4-0.6, namely the strain is cultured to the logarithmic growth phase, and then an inducer IPTG (final concentration is 1mM) is added for inducing SNAP high-efficiency soluble expression under the condition of overnight culture at 16 ℃ and 200 rpm. Centrifuging at 4 deg.C and 10000g for 10min to collect thallus, discarding supernatant LB culture medium, adding PBS buffer (pH7.4) to fully suspend thallus, crushing thallus on ice with ultrasonic wave, centrifuging at 4 deg.C and 10000rpm for 20min to remove insoluble protein, and obtaining supernatant soluble crude protein solution. Finally, purifying by Ni column chromatography to obtain the pure target protein SNAPtag.
In some embodiments, the preparation method of the tag protein Halotag is as follows:
the constructed pH6HTN His6HaloTag T7 expression vector is transformed into an escherichia coli expression host cell KRX, then a single colony on a plate is picked to be cultured in an LB culture medium (with glucose and ampicillin added), and the activated bacterial liquid is inoculated into a fresh LB culture medium (containing ampicillin). Furthermore, glucose and rhamnose can be added as promoters to promote the efficient and rapid soluble expression of Halo. Further, the protein obtained by expression is extracted and separated.
Specifically, the supernatant of the cells was discarded, and then PBS buffer (pH7.4) was added to the cells to sufficiently suspend the cells, and the cells were disrupted by ultrasonic waves, and insoluble proteins were removed by centrifugation to obtain soluble proteins in the supernatant. Removing hybrid protein by utilizing the principle that filler nickel sulfate in a Ni column can be specifically combined with histidine fusion protein, then competitively combining the filler with imidazole solution to the Ni column, eluting the histidine fusion protein from the column, and dialyzing the protein to remove imidazole and micromolecular hybrid protein to obtain the purer target tag protein Halotag.
In a specific preferred embodiment, the preparation method of the tag protein Halotag comprises the following steps:
the constructed pH6HTN His6HaloTag T7 expression vector is thermally shocked for 20s at 42 ℃ and transformed into an escherichia coli expression host cell KRX, then picking single colony on the plate to LB culture medium (adding 0.2% -0.4% glucose and 100mg/ml ampicillin), shake culturing at 37 deg.C and 200rpm for 6-8h, inoculating the activated bacteria liquid into a fresh LB culture medium (containing 100mg/ml ampicillin) according to the proportion of 1:100, adding 0.05% of glucose and 0.05% of rhamnose, culturing overnight at the temperature of 16 ℃ and the speed of 200rpm to promote Halo efficient and rapid soluble expression, centrifuging for 10min at the temperature of 4 ℃ and the speed of 10000g to collect thalli, discarding a supernatant LB culture medium, adding PBS (phosphate buffer solution) (pH7.4) to fully suspend the thalli, crushing the thalli by using ultrasonic waves, and centrifuging to remove insoluble protein to obtain supernatant soluble protein. And purifying by using a Ni column to obtain the pure target tag protein Halotag.
In step S02, one kind of the nanoparticles is cross-linked with at least one kind of the protein tags, and one kind of the protein tags is cross-linked with only one kind of the nanoparticles, so as to obtain N kinds of nanoparticle/protein tag complexes. Preferably, one of said nanoparticles is cross-linked with one of said protein tags, and one protein tag is cross-linked with only one nanoparticle, i.e. M ═ N.
Specifically, the method comprises the following steps: providing stock solutions of EDC and NHS, preparing a stock solution of a protein label and a nano-particle solution, mixing the stock solutions of EDC and NHS, the stock solution of the protein label and the nano-particle solution, and carrying out a crosslinking reaction. The stock solutions of EDC and NHS may be a stock solution of EDC and a stock solution of NHS, or a mixed stock solution of EDC and NHS. And mixing the stock solutions of the EDC and the NHS, the stock solution of the protein tag and the nano-particle solution, wherein the EDC and the NHS can modify the nano-particles and even the protein tag, so that the nano-particles can be crosslinked on the surface of the protein tag, and preparation is made for the protein tag to be connected with the zinc finger protein through a specific ligand.
Preferably, in the step of mixing the EDC and NHS stock solution, the protein tag stock solution and the nanoparticle solution, the nanoparticles, the protein tag, the EDC and the NHS are mixed according to the molar ratio of 1 (1-100) to (100-10000), so that better coupling reaction efficiency is obtained. Specifically, in the step of mixing the EDC and NHS stock solution, the protein tag stock solution, and the nanoparticle solution, the respective substances are mixed in a molar ratio of 1:5:1000:1000 of the nanoparticles, the protein tags, the EDC, and the NHS.
Mixing the stock solutions of EDC and NHS, the stock solution of the protein label and the nano-particle solution, reacting at room temperature (10-35 ℃), preferably reacting for 2 hours, and purifying by using a 100KD ultrafiltration tube.
When the protein tags are SNAPtag and Halotag, the preparation method of the stock solution of the protein tags is preferably as follows: the tagged proteins were dissolved in PBS buffer at pH7.4, respectively, to prepare a stock solution of protein tags at a concentration of 2. mu.M.
And crosslinking one zinc finger protein with one ligand according to the unique pairing relationship to obtain N zinc finger protein/ligand complexes, thereby realizing the connection between the zinc finger protein and the tag protein through the specific ligand crosslinked on the zinc finger protein. A step of cross-linking one of said zinc finger proteins with a ligand in a unique pairing relationship, comprising: providing stock solution of TCEP, preparing stock solution of ligand solution and zinc finger protein, mixing the stock solution of TCEP, the ligand solution and the stock solution of zinc finger protein, and carrying out cross-linking reaction. The TCEP can be used for carrying out cremation on the zinc finger protein and the ligand, so that the zinc finger protein and the ligand are crosslinked, and preparation is made for connecting the protein tag with the zinc finger protein through a specific ligand.
And mixing the stock solution of the TCEP, the ligand solution and the stock solution of the zinc finger protein, wherein the molar weight of the TCEP is more than 5 times of the molar weight of the zinc finger protein, and the molar weight of the ligand is more than 1 time of the molar weight of the zinc finger protein, so that the crosslinking can be performed. Preferably, in the step of mixing the stock solution of TCEP, the ligand solution and the stock solution of zinc finger protein, the ratio of zinc finger protein: TCEP: the molar ratio of the ligand is 1 (5-100) to (1-200), and all the substances are mixed, so that better crosslinking reaction efficiency is obtained. Specifically, in the step of mixing the stock solution of TCEP, the ligand solution, and the stock solution of zinc finger protein, the ratio of zinc finger protein: TCEP: the molar ratio of the ligands was 1:10:20, and the substances were mixed.
The step of mixing the stock solutions of TCEP, ligand solution and zinc finger protein is performed at room temperature, preferably overnight, and the reaction is followed by purification using a 10K ultrafiltration tube.
When the zinc finger protein is zif268 and SNAP, the preparation method of the zinc finger protein stock solution is preferably as follows: the zinc finger proteins were dissolved in PBS buffer at pH7.4, respectively, to prepare stock solutions of zinc finger proteins at a concentration of 50 μ M.
When the tag proteins are SNAPtag and Halotag, the ligands correspondingly connected with the two zinc finger proteins are BG-Maleimide and HaloTag-Maleimide respectively.
In the step S03, providing a nucleotide sequence, mixing and incubating the nucleotide sequence with the N nanoparticle/protein tag complexes and the N zinc finger protein/ligand complexes to form a nucleotide sequence-zinc finger protein-protein tag-nanoparticle connection system, and separating and collecting the obtained nanoparticle clusters, wherein bases in the nucleotide sequence are set according to a preset arrangement order of zinc finger proteins in the nanoparticle clusters.
In one embodiment, the nucleotide sequence may be mixed with the N nanoparticle/protein tag complexes and the N zinc finger protein/ligand complexes, incubated, and the resulting nanoparticle clusters may be collected after isolation. Further preferably, the incubation conditions are: incubating at 4-45 deg.C, specifically 37 deg.C, for more than 15min, specifically 0.5 h.
In a preferred embodiment, in the step of mixing and incubating the nucleotide sequence with the N kinds of nanoparticle/protein tag complexes and N kinds of zinc finger protein/ligand complexes, after mixing and reacting the N kinds of nanoparticle/protein tag complexes with the N kinds of zinc finger protein/ligand complexes, collecting zinc finger protein-protein tag-nanoparticle complexes; and then mixing and incubating the zinc finger protein-protein tag-nanoparticle complex with the nucleotide sequence to prepare a nanoparticle cluster.
Further preferably, the step of incubating the zinc finger protein-protein tag-nanoparticle complex in admixture with the nucleotide sequence places the zinc finger protein-protein tag-nanoparticle complex and the nucleotide sequence in NaCl and ZnSO at a temperature of 4-45 ℃, particularly 37 ℃4The buffer solution (2) is incubated for more than 15min, specifically for 0.5 h.
Finally, the nanoparticle clusters are collected by centrifugal separation, and the centrifugal condition is preferably 5000rpm for 12 h.
In a preferred embodiment, the first zinc finger protein zif268 and the second zinc finger protein SNAP are used as the two zinc finger proteins; the two N protein labels are SNAPtag and Halotag respectively; the two ligands are BG-Maleimide and HaloTag-Maleimide respectively, and the nano-particles are quantum dots. When a multi-component nanoparticle cluster is constructed, ultraviolet spectrograms of single-particle quantum dots, quantum dot complexes, quantum dot/protein label complexes and quantum dot nanoparticle clusters are shown in FIG. 2; the fluorescence spectra of the single-particle quantum dot, the quantum dot complex, the quantum dot/protein tag complex and the quantum dot nanoparticle cluster are shown in FIG. 3; the TEM image of the resulting nanoparticle cluster is shown in fig. 4.
As can be seen from fig. 2, the nanoparticle cluster obtained in the example of the present invention has a characteristic uv absorption peak (around 520 nm) of all quantum dots, and the formation of the nanoparticle cluster and its uv properties can be confirmed; as can be seen from fig. 3, the nanoparticle cluster obtained in the example of the present invention has a characteristic peak (around 530 nm) of fluorescence emission of the quantum dots, and the formation of the nanoparticle cluster and the fluorescence property thereof can be confirmed; fig. 4 is a transmission electron microscope representation, in which a "necklace-like" structure is formed in a red region in an electron microscope, and the formation of a particle cluster is confirmed for a designed nanoparticle cluster.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A method for constructing a nanoparticle cluster, wherein the nanoparticle cluster is a multicomponent nanoparticle cluster, and the method for constructing the multicomponent nanoparticle cluster comprises the following steps:
providing N zinc finger proteins, M nanoparticles, N protein tags and N ligands for identifying different said protein tags, respectively, wherein one said ligand uniquely identifies a corresponding said protein tag; n is a positive integer greater than or equal to 2, M is a positive integer greater than or equal to 2, and M is less than or equal to N;
crosslinking one kind of the nanoparticles with at least one kind of the protein tags, and crosslinking only one kind of the nanoparticles with one kind of the protein tags to obtain N kinds of nanoparticle/protein tag complexes; crosslinking one zinc finger protein with one ligand according to the unique pairing relation to obtain N zinc finger protein/ligand complexes;
providing a nucleotide sequence, mixing and incubating the nucleotide sequence with the N nano-particle/protein tag complexes and the N zinc finger protein/ligand complexes, forming a nucleotide sequence-zinc finger protein-protein tag-nano-particle connection system through one-to-one correspondence between ligands and protein tags, and collecting the obtained nano-particle cluster after separation, wherein bases in the nucleotide sequence are set according to the arrangement sequence of zinc finger proteins in a preset nano-particle cluster.
2. The method of claim 1, wherein the step of cross-linking one of said nanoparticles with at least one of said protein tags and one protein tag with only one nanoparticle comprises:
providing stock solutions of EDC and NHS, preparing a stock solution of a protein label and a nano-particle solution, mixing the stock solutions of EDC and NHS, the stock solution of the protein label and the nano-particle solution, and carrying out a crosslinking reaction.
3. The method of claim 2, wherein in the step of mixing the EDC and NHS stock solution, the protein tag stock solution and the nanoparticle solution, the materials are mixed in a molar ratio of 1 (1-100): 100-.
4. The method of claim 1, wherein the step of cross-linking a zinc finger protein with a ligand in a unique pair-wise relationship comprises:
providing stock solution of TCEP, preparing stock solution of ligand solution and zinc finger protein, mixing the stock solution of TCEP, the ligand solution and the stock solution of zinc finger protein, and carrying out cross-linking reaction.
5. The method of claim 4, wherein in the step of mixing the stock solution of TCEP, the ligand solution and the stock solution of zinc finger proteins, the ratio of zinc finger proteins: TCEP: the molar ratio of the ligand is 1 (5-100) to 1-200, and all the substances are mixed.
6. The method for constructing a nanoparticle cluster according to claim 1, wherein in the step of mixing and incubating the nucleotide sequence with the N nanoparticle/protein tag complexes and the N zinc finger protein/ligand complexes, the zinc finger protein-protein tag-nanoparticle complex is collected after mixing and reacting the N nanoparticle/protein tag complexes with the N zinc finger protein/ligand complexes; and then mixing and incubating the zinc finger protein-protein tag-nanoparticle complex with the nucleotide sequence to prepare a nanoparticle cluster.
7. The method for constructing a nanoparticle cluster according to claim 6, wherein the step of incubating the zinc finger protein-protein tag-nanoparticle complex in a mixture with the nucleotide sequence is performed at a temperature of 4-45 ℃Placing the zinc finger protein-protein tag-nanoparticle complex and the nucleotide sequence in NaCl and ZnSO4The cells were incubated in the buffer solution (2) for 15min or more.
8. The method of any one of claims 1 to 7, wherein N, M are both 2, and the two zinc finger proteins are a first zinc finger protein zif268 and a second zinc finger protein SNAP; the two protein labels are SNAPtag and Halotag respectively; the two ligands are BG-Maleimide and HaloLigase-Maleimide respectively, wherein BG-Maleimide is used for identifying SNAPtag, and HaloLigase-Maleimide is used for identifying Halotag.
9. The method for constructing a nanoparticle cluster according to claim 8, wherein the zinc finger protein is prepared by:
providing primers, and respectively using pET30a-C-zif268 and pSNAP-tag as templates, carrying out PCR amplification to obtain a first gene DNA fragment encoding zif268 protein and a second gene DNA fragment encoding SNAP protein, wherein histidine tags are introduced into the C ends of the first gene DNA fragment and the second gene DNA fragment; NdeI enzyme cutting sites and XhoI enzyme cutting sites are respectively designed at the 5 'end and the 3' end of the first gene DNA fragment; NdeI and XhoI enzyme cutting sites are respectively designed at the 5 'end and the 3' end of the second gene DNA fragment;
providing a vector pET21a, and carrying out enzyme digestion treatment by using Nde I and Xho I to obtain a linear vector; and (2) incubating the first gene DNA fragment and the second gene DNA fragment with a linearized pET21a vector by using a homologous recombinase to obtain a recombinant vector, transforming the recombinant vector into a competent cell, culturing and expressing the competent cell, and extracting the competent cell by using an AKTA protein purification instrument and a Ni column to obtain a first zinc finger protein zif268 and a second zinc finger protein SNAP.
10. The method for constructing a nanoparticle cluster according to claim 9, wherein the primers for amplifying the first gene DNA fragment are:
an upstream primer: 5 'TAAGAAGGAGATATACATATGGCTAGCACCATGGATATCAAGCTT 3'
A downstream primer: 5 'GTGGTGGTGGTGGTGCTCGAGATTAACCTCGAGCCC 3'
The primers used to amplify the second gene DNA fragment were:
an upstream primer: 5 'TAAGAAGGAGATATACATATGCATATGATGTGCAAAACCGG 3'
A downstream primer: 5 'GTGGTGGTGGTGGTGCTCGAGGAATTCCTTCTCACCGGTGTGGAT 3'.
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