CN112553232A - Controllable biosynthesis method of multifunctional self-assembled nanoparticles - Google Patents
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Abstract
The invention provides a controllable biosynthesis method of multifunctional self-assembled nanoparticles, wherein the multifunctional self-assembled nanoparticles comprise two or more different functional ligands, and the synthesis of different functional ligands in different proportions can be completed in an organism. The controllable biosynthesis method of the asymmetric self-assembly nano-particles regulated and controlled by the promoter greatly simplifies the steps of preparing multifunctional nano-particles, realizes the one-step synthesis of the multifunctional nano-particles and completely avoids the conventional preparation process of in vitro disassembly and assembly. In the synthesis method provided by the invention, the whole self-assembly process is carried out in a host body, only the conditions suitable for the growth of the host need to be provided, and a harsh environment is not needed.
Description
Technical Field
The invention relates to the technical field of biological nano material synthesis, in particular to a controllable biological synthesis method of multifunctional self-assembled nano particles.
Background
Protein nanoparticles, which are typically self-assembled from a specific number of protein subunits (self-assembling elements) to form 10-100nm cage-like nanostructures, are common protein nanoparticles, such as viruses, ferritin, and heat shock proteins. Compared with other nanoparticles, the protein nanoparticles have the characteristics of easy genetic modification, diversified functional ligand selection, good biological safety, good monodispersity and the like.
By means of protein engineering technology, the molecular orientation and the quantity of functional ligands (the self-assembly elements of the protein nanoparticles have well-oriented molecular conformations and structural composition quantity, and the corresponding molecular orientation and consistent quantity of the functional ligands genetically modified thereon) can be precisely controlled at the nanoscale. Under the assistance of an in-vitro self-assembly control technology, more kinds of functional ligands can be accurately modified on the surface of the porous material, and the assembly quantity of different functional ligands is controlled to enable the porous material to generate asymmetric self-assembly, so that more complex advanced functions are obtained.
The current multifunctional self-assembly nanoparticles are mainly established on the basis of depolymerization-reassembly of self-assembly protein nanoparticles and realized by an in vitro self-assembly control technology, for example, Trevor Douglas topic group reports that two bifunctional DPS (DNA binding protein) nanoparticles are obtained on the basis of a mask method and a topological selection modification method in 2009 in JACS and Nano Lett, respectively, and functional ligands on the surfaces of the nanoparticles have a definite quantitative ratio; plum peak and the like use capsid protein VP1 of genetically modified monkey Virus40 (Simian Virus 40) to continuously prepare a complex Janus nanoparticle system with single functionalization and adjustable surface functional ligand quantity through an in-vitro self-assembly control technology of disassembly-reassembly, so as to obtain a nano optical assembly.
The dissembling-self-assembling preparation of the asymmetric nano-particles with multiple functions and controllable functional ligand quantity ratio is a feasible technical route for developing complex functional nano-particles. However, this technology still presents a major technical hurdle in preparing multifunctional nanoparticles with multiple immunoaffinity activities: 1. the types of self-assembly protein nanoparticles capable of being disassembled under mild conditions are few, most self-assembly protein nanoparticles occur when the pH value is lower than 2, and the self-assembly protein nanoparticles can generate large influence on proteins and even cause irreversible denaturation of the proteins; 2. genetic modification of the protein coat mostly occurs in the surface loop region, which is difficult to modify macromolecular functional ligands; 3. the disassembly and assembly of protein nanoparticles which are easy to modify macromolecular foreign proteins usually require harsh conditions, and the recovery rate of the disassembly and assembly is low or the recovery structure is incomplete. This is the biggest obstacle to the research of preparing the asymmetric nanoparticles of the controlled multifunctional protein. Therefore, the biggest problem in preparing the controlled multifunctional self-assembled nanoparticles is to avoid the steps of disassembly-reassembly.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention establishes the technology of controllably and asymmetrically self-assembling a promoter regulation and control metabolic network in an organism, avoids the steps of disassembly and reassembly in the organism in the conventional process of synthesizing the multifunctional self-assembled nano particles, not only can realize the construction of the multifunctional self-assembled nano particles, but also can simultaneously control the type and the quantity of surface functional ligands.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
in one aspect, the present invention provides a method for the controlled biosynthesis of multifunctional self-assembled nanoparticles comprising two or more different functional ligands, which allow the synthesis of different ratios of different functional ligands in vivo.
Illustratively, the controllable biosynthesis method of the multifunctional self-assembled nanoparticle utilizes a gene recombination method to realize the synthesis of different ratios of different functional ligands by regulating and controlling promoters of different functional ligands. Referring to the schematic diagram in fig. 1, two different functional ligands are differentially transcribed under the control of a promoter, and then the multifunctional self-assembled nanoparticles are obtained through asymmetric self-assembly.
Illustratively, the expression of a functional ligand regulated by a promoter can be regulated by replacing the promoter and/or modifying the promoter and/or inserting regulatory sequences into the promoter.
In one embodiment of the invention, different promoters are used to regulate the expression of different functional ligands.
The promoter of the present invention is not particularly limited, and may be selected depending on the functional ligand and the expression level of the desired functional ligand.
Illustratively, the promoter may be one or more of T7, tac1, lacUV5, tac2, trc, and promoters of different strengths obtained by mutating T7, tac1, lacUV5, tac2, trc, and the like.
In one embodiment of the invention, the promoters used are T7 and tac 1.
The functional ligand is not particularly limited in the present invention, and different functional ligands can be selected according to the need.
Illustratively, the functional ligand is an enzyme, antigen, antibody, fluorescent protein, biotin-binding protein, protein G, protein a, various protein purification tags, nucleic acid-binding protein, and the like.
In a specific embodiment of the present invention, the functional ligands are Green Fluorescent Protein (GFP) and protein G (SPG, protein G).
In a specific embodiment of the present invention, the functional ligands are Pyruvate Phosphokinase (PPDK) and Luciferase (Luciferase).
Illustratively, the asymmetric self-assembling nanoparticles are virus-like particles, e.g., one or more of Ferritin (Ferritin), Streptavidin (SA), starvation induced DNA binding proteins (Dps), phage capsid protein (P22), simian vacuolating virus40 (SV vacuolating virus40, SV40), Cowpea Chlorotic Mottle Virus (CCMV), and the like.
In one embodiment of the present invention, the virus-like particle is a nanoparticle formed by self-assembly of hepatitis b core antigen.
In another aspect, the invention provides asymmetric self-assembled nanoparticles, which are prepared by the above controllable biosynthesis method.
The functional ligands for preparing the asymmetric self-assembly nano-particles are different, and the obtained nano-particles have different functions. For example, when the functional ligand is an enzyme, the nanoparticle has a function of catalyzing the reaction; when the functional ligand is an antibody, the nanoparticle has an immune function; when the functional ligand is fluorescent protein, the nanoparticle has the function of a molecular label and the like.
In a specific embodiment of the invention, the multifunctional self-assembled nanoparticle comprises hepatitis b core antigen, green fluorescent protein and protein G. The hepatitis B core antigen can form nanoparticles.
In a specific embodiment of the invention, the multifunctional self-assembled nanoparticle comprises hepatitis b core antigen, pyruvate phosphokinase, and luciferase. The hepatitis B core antigen can form nanoparticles.
The controllable biosynthesis method of the multifunctional self-assembled nanoparticles provided by the invention specifically comprises the following steps:
through gene cloning technology, different functional ligands are cloned into expression vectors respectively, and the expression vectors are transferred into the same host for expression.
For example, by gene cloning, different functional ligand genes (containing promoters) are fused with protein genes capable of forming nanoparticles respectively, and then the fusion products are transferred into an expression vector. The expression vectors may be the same expression vector or different expression vectors. If the expression vector is the same, the ligand genes with different functions respectively have promoters carried by the genes; if different expression vectors are used, they are compatible in the same host.
Or, through gene cloning, the protein gene and the functional gene capable of forming the nano-particles are firstly split, and the split gene segments are then fused and transferred into an expression vector for expression.
The expression vector and the host are not particularly limited in the present invention. The invention does not limit the type and category of the expression vector and the host, and can select the vector and the host which are used for genetic modification in the field, and concretely, the expression vector can be pET series, pMAL series, pQE series, pETDuet, pACYCdue, pCDFDuet or pRSFduet and the like; the expression host is Escherichia coli host cell BL21(DE3), Rosetta (DE3), M15, etc.
In one embodiment of the present invention, the fusion protein is prepared by transferring the fusion proteins with different functions into two different expression vectors, respectively, but transferring the fusion proteins into the same host for expression. The method specifically comprises the following steps:
selecting a 149 amino acid length fragment (HBcAg149) at the N end of a hepatitis B core antigen to form nanoparticles, Green Fluorescent Protein (GFP) and protein G (SPG) as functional ligands to form asymmetric self-assembly nanoparticles, adopting a T7 promoter to regulate the expression of GFP protein, adopting a tac1 promoter to regulate the expression of protein G, selecting pETDuet and pCDFDuet as expression vectors, and taking Escherichia coli E.coli as a host strain, wherein the preparation process comprises the following steps:
firstly, splitting an HBcAg149 (hepatitis B core antigen) fragment into an N-terminal fragment (core-N) and a C-terminal fragment (core-C) by a gene manipulation means; the GFP structure is divided into two parts of beta 1-10(1-651bp) and beta 11(652-726bp), wherein the beta 1-10 of the GFP is fused to the C terminal of the core-N through a gene fusion technology, SPG is fused to the N terminal of the GFP-beta 11, then the SPG and the SPG are fused to the N terminal of the core-C together, and the fusion protein is transferred into an expression vector pETDuet;
or splitting the HBcAg149 fragment into an N-terminal fragment (core-N) and a C-terminal fragment (core-C) by a gene manipulation means; the SPG protein was directly fused to the C-terminus of core-N or to the N-terminus of core-C, and the fusion protein was transferred into the expression vector pETDuet.
Secondly, splitting the HBcAg149 fragment into an N-terminal fragment (core-N) and a C-terminal fragment (core-C); the GFP structure is divided into two parts of beta 1-10(1-651bp) and beta 11(652-726bp), wherein the beta 1-10 of GFP is fused to the C terminal of core-N by a gene fusion technology, the GFP-beta 11 is fused to the N terminal of core-C, and the fusion protein is transferred into an expression vector pCDFDuet.
And thirdly, simultaneously transferring the expression vector pETDuet in the first step and the expression vector pCDFDuet in the second step into Escherichia coli E.coli for induced expression, thus obtaining the asymmetric self-assembled nanoparticles.
In one embodiment of the present invention, the fusion protein is prepared by transferring the fusion proteins with different functions into two different expression vectors, respectively, but transferring the fusion proteins into the same host for expression. The method specifically comprises the following steps:
firstly, the HBcAg149 (hepatitis B core antigen) fragment is split into an N-terminal fragment (core-N) and a C-terminal fragment (core-C) by a gene manipulation means.
Fusing pyruvate phosphokinase to the N end of core-C, transferring the fused gene into an expression vector pETDuet, and constructing a plasmid with the name: pET-PPDK-strep.
And thirdly, luciferase is fused to the N terminal of core-C, and the fused gene is transferred into an expression vector pCDFDuet to construct a plasmid with the name: pCDF-Luc-strep.
Illustratively, the present invention has at least one of the following advantages:
the controllable biosynthesis method of the multifunctional self-assembled nanoparticles provided by the invention greatly simplifies the preparation steps of multifunctional nanoparticles, realizes the one-step synthesis of the multifunctional nanoparticles, and completely avoids the conventional preparation process of in-vitro disassembly and assembly. In the synthesis method provided by the invention, the whole self-assembly process is carried out in a host body, only the conditions suitable for the growth of the host need to be provided, and a harsh environment is not needed.
Drawings
FIG. 1 is a schematic diagram of the multifunctional self-assembled nanoparticles provided by the present invention.
FIG. 2 is a schematic diagram showing the structure of expression vector pETDuet provided in example 1 of the present invention.
FIG. 3 is a schematic structural diagram of the expression vector pCDFDuet provided in example 1 of the present invention.
FIG. 4 is a schematic structural diagram, an ultracentrifugation distribution diagram and a transmission electron microscope diagram of the multifunctional self-assembled nanoparticles provided in example 1 of the present invention.
FIG. 5 is a western blot of two functional proteins of the multifunctional self-assembled nanoparticle obtained in example 1 of the present invention.
Fig. 6 is a schematic diagram showing the function of the multifunctional self-assembled nanoparticle provided in example 2 of the present invention.
FIG. 7 is a schematic diagram of the process of using the multifunctional self-assembled nanoparticles provided in example 2 of the present invention in ELISA detection.
FIG. 8a is a graph comparing the results of the multifunctional self-assembled nanoparticles provided in example 2 of the present invention with GFP when used in ELISA assay. In these, GFP-IgG was present on the left side of the adjacent histogram, and HBcAg-GFP was present on the right side.
Fig. 8b is a graph comparing the results of multifunctional self-assembled nanoparticles provided in example 2 of the present invention when used in ELISA assay with Alexa 488.
FIG. 9 is a schematic diagram of the process of using the multifunctional self-assembled nanoparticles provided in example 3 of the present invention in indirect ELISA detection.
FIG. 10 is a graph showing the results of the multifunctional self-assembled nanoparticles provided in example 3 of the present invention in indirect ELISA assay.
FIG. 11 is an immunoblot of two functional proteins of the bi-enzyme multifunctional self-assembled nanoparticle obtained in example 4 of the present invention.
FIG. 12 is a graph showing the results of the cascade catalytic reaction of the double-enzyme self-assembled nanoparticles in example 4 of the present invention.
FIG. 13 shows the data of the promoter library constructed by the present invention, different promoters are used to express the same green fluorescent protein, and light spots with different fluorescent intensities regulated by different promoters are obtained. Wherein, the light spot 1 is tac1 promoter, and the light spot 2 is T7 promoter.
Detailed Description
The controllable biosynthesis method of the multifunctional self-assembled nanoparticle of the present invention is further illustrated below with reference to the specific examples, wherein the nanoparticle is formed by hepatitis B virus core antigen, and Green Fluorescent Protein (GFP) and protein G (SPG) are taken as functional ligands.
The Virus-like Particles (VLPs) have a hollow shell or envelope-shaped particle structure without Virus genome, are hollow Particles which are assembled by structural proteins of viruses and have a length of 15-400 nm, and have a structure similar to that of natural Virus Particles. VLPs do not contain viral genomes, cannot replicate autonomously, are morphologically similar to real virions, and can be presented to immune cells in the same way as viral infection, effectively inducing the body to produce an immunoprotective response. VLPs are assembled from structural proteins of viruses and can self-assemble into VLPs in cells. In the case of non-enveloped viruses, the VLPs component is derived from the capsid protein (or mutated, modified versions thereof) of the virus. Viral capsid proteins generally have natural self-assembly ability, VLPs can be isolated directly from cells or culture medium expressing viral capsid proteins, or VLPs can be assembled in vitro after purification of capsid protein subunits. VLPs are produced by the principle of expressing one (or more) structural protein(s) of a virus in vitro with high efficiency, allowing it to self-assemble into hollow particles that are morphologically similar to the native virus.
Hepatitis B virus core antigen (HBcAg) is the capsid protein of hepatitis B virus, and is wrapped around the viral genome and polymerase. Molecular weight of about 21kD, capable of self-assembly into 30(T ═ 3) or 34nm (T ═ 4) icosahedral particles, forming capsid-like particles (CLPs) of whole viruses. In terms of structure, 180 or 240 identical polypeptide subunits (hbcags) are arranged in an array with a large number of α -helical structures. The four α -helical bundles form spikes on the surface of the particles of CLPs.
The total length of the hepatitis B core antigen is 183-185 amino acids (depending on the different subtypes of hepatitis B virus), which mainly comprises two parts: 1. 140 amino acids from its N-terminus are the major sites for its self-assembly into capsid-like particles; 2. the C-terminal region is rich in basic amino acids and serves the main function of binding to nucleic acids. The hepatitis B core antigen selected by the invention is a full-length 183aa mutant type: the 149 amino acids at the N-terminus were selected as monomers, and the region of the C-terminal nucleic acid binding function was removed. Compared with the full-length hepatitis B core antigen mutant with deletion of the C terminal, the mutant is easier to heterologously express in escherichia coli.
Example 1 preparation of multifunctional nanoparticles
The number of the hepatitis B core antigen gene selected in the example is Accession No. CAA24706 in Genebank, 149 amino acid fragments (HBcAg149) at the N end of the gene are selected, the 149 amino acid fragments are split into an N-end fragment (core-N) and a C-end fragment (core-C) at C/e1 loop by a gene manipulation means, and the split core-N and core-C are transferred to the same expression vector and use the same promoter. The two amino acid fragments are translated separately by two Ribosome Binding Sites (RBS). Two small subunits translated separately using two RBSs can self-assemble into capsid-like nanoparticles in the same way as the intact monomer translated by the same RBS in multiple copies. The advantage of splitting HBcAg149 is that the foreign protein can be fused at the C end of core-N, the N end of core-C or even two foreign proteins can be fused at the C end of N segment fragment and the N end of C segment fragment simultaneously without influencing self-assembly to form capsid-like nanoparticles.
The two foreign proteins fused in this example are green fluorescent Protein (GFP, Access No.: AF004665.1, 726bp in Genebank) and Protein G (Protein G, SPG, Access No.: DQ016035.1, 171bp in Genebank), respectively, and the specific fusion method is as follows:
the GFP structure is divided into two parts of beta 1-10(1-651bp) and beta 11(652-726bp), wherein the beta 1-10 of the GFP is fused to the C terminal of the core-N through a gene fusion technology, the SPG is fused to the N terminal of the GFP-beta 11, and then the two parts are fused to the N terminal of the core-C together. The specific fusion sequence is shown in FIG. 2. The fused gene is transferred into an expression vector pETDuet, and the name of a constructed plasmid can be as follows according to the difference of promoters: pET-T7-SPG-strep or pET-tac-SPG-strep.
② the GFP structure is divided into two parts of beta 1-10(1-651bp) and beta 11(652-726bp), wherein the beta 1-10 of the GFP is fused to the C terminal of core-N by the gene fusion technology, and the GFP-beta 11 is fused to the N terminal of core-C. The specific fusion sequence is shown in FIG. 3. The fused gene is transferred into an expression vector pCDFDuet, and according to the difference of promoters, a plasmid name is constructed as follows: pCDF-tac-his or pCDF-T7-his.
The two different expression plasmids fused with different functional subunit genes are simultaneously transformed into bacteria, and can be self-assembled into the multifunctional fluorescent nanoparticles in one step in the bacteria after induction expression (as shown in the leftmost diagram of figure 4).
Coli e.coli is selected as the host bacterium, competent cells are transformed by a chemical method, the expression vector petdeut and the expression vector pCDFDuet are co-transformed into the e.coli expression strain e.coli, and then the e.coli expression strain e.coli is coated on a plate with corresponding antibiotic resistance to carry out single colony screening.
Protein induction expression process: coli E.coli grows to the exponential growth region, IPTG is added and placed in a shaking table for induction expression. After induction expression, 500 mu L of bacterial liquid is reserved for SDS-PAGE detection, and the two constructed fusion proteins can be seen in obvious green after induction expression.
The two types of nanoparticles formed by fusion and self-assembly have green fluorescence, so that the self-assembly result and efficiency can be easily judged, and convenience is brought to particle purification. And the nano particles are easier to observe by using an electron microscope, and the virus capsid-like particles can be easily characterized.
The steps of nanoparticle purification are as follows:
(1) inducing and expressing the bacteria solution which is green, collecting the bacteria, carrying out ultrasonic crushing, filtering with a filter membrane to remove impurities, and precipitating with ammonium sulfate to obtain crude protein;
(2) centrifuging the crude protein obtained in the step (1) by using a sucrose density gradient, and collecting a sucrose gradient layer with green fluorescence;
(3) dialyzing the solution of the green fluorescence obtained in the step (2), and concentrating to obtain a concentrated solution;
(4) purifying the concentrated solution obtained in the step (3) by using a molecular sieve to obtain the nano-particles.
This example uses two promoters of different strengths, the T7 promoter and the tac1 promoter, the T7 promoter having greater transcriptional regulatory strength than the tac1 promoter. The expression amount of GFP protein was controlled by the T7 promoter, and the expression amount of protein G was controlled by the tac1 promoter.
The GFP protein gene was also inserted into pETDuet plasmid in this example. In the pETDuet plasmid, the amount of GFP protein expressed is the same as the amount of protein G expressed, whereas in the pCDFDuet plasmid, only expression of GFP protein regulated by T7 results in expression of the amount of GFP protein in a specific ratio to the amount of protein G, with a theoretical value of about 17: 1 in quantitative ratio. FIG. 13 is data of promoter library constructed by the present invention, different promoters are used to express the same green fluorescent protein, light spots with different fluorescent intensities regulated by different promoters are obtained, and the ratio of tac1 to T7 promoter intensities obtained by comparing the optical densities corresponding to different promoters is about 1: 17.7, it can be assumed that the theoretical value of the amount of GFP protein and the amount of protein G in this example is about 17: 1.
the gray scale values in FIG. 5 reflect that the expression levels of GFP and protein G are about 15: about 1. With the self-assembly of the monomers formed by core-N and core-C to form nanoparticles, GFP protein is displayed on the surface of the nanoparticles in proportion to protein G, and thus multifunctional self-assembled nanoparticles are formed, the schematic structure of which is shown in the leftmost diagram of FIG. 4. The middle and right-most images in FIG. 4 are the distribution diagram of the ultracentrifugation and transmission electron microscope images of the multifunctional self-assembled nanoparticles obtained in this example, respectively.
In this example, the GFP protein gene was similarly inserted into the petdue plasmid, but in the specific implementation, only protein G was expressed in the petdue plasmid, as desired.
In this example, two different expression vectors, pETDuet and pCDFDuet, were selected, and one of them was also selected. For example, only expression vector pETDuet was selected. One part of the expression vector pETDuet is introduced with core-N, core-C and GFP protein gene controlled by a T7 promoter, the other part of the expression vector pETDuet is introduced with core-N, core-C and SPG protein gene controlled by a tac1 promoter, and the expression vectors pETDuet carrying different fusion protein genes are all transferred into E.coli Rosetta for expression.
The multifunctional self-assembled nanoparticle obtained in this example is characterized by its expression level of two functional proteins regulated by a promoter using an immunoblot assay, as shown in fig. 5. As can be seen from FIG. 5, the expression level of pET-T7-SPG-strep is higher than that of pET-tac1-SPG-strep and that of pCDF-tac1-his, respectively, than that of pCDF-T7-his, respectively.
The promoter in this embodiment can be replaced by a promoter with any strength to arbitrarily regulate the expression of different functional proteins, or the strength of the promoter is changed by modifying codons of the promoter, inserting a regulatory sequence at the upstream of the promoter, or other ways, so as to change the expression of a functional ligand regulated by the promoter. Because two different functional ligands are regulated by promoters with different strengths, differential expression is carried out in escherichia coli, and after expression, the two different functional ligands are mixed in a specific proportion in cells of the escherichia coli, so that asymmetric self-assembly is further carried out, and the asymmetric self-assembly multifunctional nanoparticles with controllable quantities of different functional ligands can be obtained in one step.
In the embodiment of the invention, the preparation of the nanoparticles can be visualized by introducing the fluorescent protein, the self-assembly result and efficiency can be observed by naked eyes, the fluorescent protein can bring great convenience to the purification of the nanoparticles, the splitting and recombination of the fluorescent protein introduces more stable intermolecular interaction related to the self-assembly for the splitting and recombination of hepatitis B core antigen, on one hand, the macromolecular protein is displayed by the protein shell loop, on the other hand, the self-assembly is easier, and the obtained nanoparticles are more stable.
The protein G introduced from the surface of the self-assembled nanoparticle can be conveniently combined with the Fc end of the antibody, so that the multifunctional asymmetric nanoparticle can be directionally marked on the Fc end of the antibody as a fluorescent probe of the antibody (as shown in FIG. 6).
The green fluorescent protein and protein G in this example can be substituted for other functional proteins as desired. For example, enzymes, antigens, antibodies, fluorescent proteins, biotin binding proteins, protein a, various protein purification tags, nucleic acid binding proteins, and the like.
Example 2 use of asymmetric fluorescent nanoparticles for direct ELISA detection
The asymmetric fluorescent nanoparticles prepared in example 1 are used as fluorescent probes for direct ELISA detection to detect Human Immunodeficiency Virus (HIV), and the detection target is p24 protein of HIV, and the specific experimental steps are as follows:
1. antigen coating: HIV-p24 protein was diluted using carbonate buffer to a range of concentrations: 1000. mu.g/ml, 200. mu.g/ml, 40. mu.g/ml, 8. mu.g/ml, 1.6. mu.g/ml, 0.32. mu.g/ml, 0.064. mu.g/ml and a negative control of 0. mu.g/ml (containing carbonate buffer only), 100. mu.l/well of each concentration gradient was added to the microplate and coated overnight at 4 ℃ and 9 replicate wells were made for each concentration gradient;
2. sealing an enzyme label plate: taking out the enzyme label plate coated overnight, throwing away the residual antigen, washing for 4 times by using PBST buffer solution, adding 300ul of 5% skimmed milk powder into each hole, and sealing for 2 hours at 37 ℃;
3. three probe preparations: while sealing, taking 5 mul of p24 monoclonal antibody and 3ml of asymmetric fluorescent nano-particles with the concentration of 0.2mg/ml, and incubating for 30min at 37 ℃ to prepare an HBcAg-GFP probe; corresponding 5. mu.l of commercial reagent Alexa 488-labeled p24 monoclonal antibody was diluted to 3ml with PBS as an Alexa488-IgG probe; meanwhile, 5. mu.l of p24 monoclonal antibody was incubated with 3ml of 0.1mg/ml SPG-GFP protein at 37 ℃ for 30min to prepare a GFP-IgG probe.
4. And (3) probe incubation: after the sealing is finished, throwing away the skimmed milk powder, washing for 4 times by using PBST buffer solution, adding 100ul of the prepared probes into each hole, repeating the steps of 3 times for each probe in each antigen concentration gradient, and incubating for 1 hour at 37 ℃;
5. data reading: and after the probe incubation is finished, throwing away redundant probes in the ELISA plate, washing for 5 times by using PBST buffer solution, and detecting the fluorescence intensity of each hole by using light with the wavelength of 488nm and the emission wavelength of 520nm on an ELISA reader. The detection principle is shown in FIG. 7, and the experimental results are shown in FIGS. 8a and 8b (the abscissa is. mu.g/ml, and the ordinate is fluorescence intensity).
From fig. 8a and fig. 8b, it can be seen that the detection limit of the asymmetric nanoparticle as a probe is 320ng/ml, which is 25 times higher than that of the commercial reagent Alexa488 as a probe, and 125 times higher than that of the monomeric fluorescent protein as a probe, and the fluorescence intensity of the detection signal of the asymmetric nanoparticle as a probe is significantly higher than that of Alexa488 and much higher than that of the monomeric fluorescent protein. Therefore, the detection sensitivity and detection signals of the asymmetric fluorescent nanoparticles as fluorescent probes are superior to those of commercial reagents Alexa488 and monomeric green fluorescent protein as probes.
Example 3 use of asymmetric fluorescent nanoparticles for indirect ELISA detection
The asymmetric fluorescent nanoparticles prepared in example 1 are used as a fluorescent probe for indirect ELISA detection to detect an HIV antibody (HIV antibody) and a p24 protein antibody of HIV as a detection target, and the specific experimental steps are as follows:
1. antigen coating: diluting HIV-p24 protein to 5 μ g/ml with carbonate buffer solution, adding to 9 rows (8 wells/row) of ELISA plate with each well at 100 μ l, and coating overnight at 4 deg.C;
2. sealing an enzyme label plate: taking out the enzyme label plate coated overnight, throwing away the residual antigen, washing for 4 times by using PBST buffer solution, adding 300ul of 5% skimmed milk powder into each hole, and sealing for 2 hours at 37 ℃;
3. primary antibody incubation: after blocking, the skim milk powder was thrown away, washed 4 times with PBST buffer, and 1mg/ml of p24 mab was diluted with PBS buffer to a range of concentrations: 10. mu.g/ml, 1. mu.g/ml, 100ng/ml, 10ng/ml, 1ng/ml, 0.1ng/ml, 0.01ng/ml and a negative control of 0ng/ml (only PBS buffer contained), 9 wells per concentration gradient were added to the microplate at 100. mu.l/well and incubated at 37 ℃ for 1 hour.
4. Three probe preparations: while the primary antibody was incubated, 5. mu.l of unlabeled goat anti-mouse IgG was incubated with 3ml of 0.2mg/ml asymmetric fluorescent nanoparticle at 37 ℃ for 30min to prepare a HBcAg probe secondary antibody, and 5. mu.l of commercially available Alexa 488-labeled goat anti-mouse IgG was diluted to 3ml with PBS as an Alexa488-IgG probe while 5. mu.l of unlabeled goat anti-mouse IgG was incubated with 3ml of 0.1mg/ml SPG-GFP protein at 37 ℃ for 30min to prepare a GFP-SPG-IgG secondary antibody probe.
4. And (3) probe incubation: after primary antibody incubation is finished, throwing away redundant primary antibody liquid, washing for 5 times by using PBST buffer solution, adding 100ul of prepared probes into each hole, repeating 3 rows of probes, and incubating for 1 hour at 37 ℃;
5. data reading: and after the probe incubation is finished, throwing away redundant probes in the ELISA plate, washing for 5 times by using PBST buffer solution, and detecting the fluorescence intensity of each hole by using light with the wavelength of 488nm and the emission wavelength of 520nm on an ELISA reader. The detection principle is shown in fig. 9, and the experimental result is shown in fig. 10.
From fig. 10, it can be seen that the detection limit of the asymmetric nanoparticle as a probe is 1ng/ml, which is increased by about 100 times compared with that of a commercial reagent Alexa488 as a probe, and is increased by more than 1000 times compared with monomeric fluorescent protein, and the fluorescence intensity of the detection signal of the asymmetric nanoparticle as a probe is significantly higher than that of Alexa488 and also much higher than that of monomeric fluorescent protein, and the background signals are basically the same. Therefore, the detection sensitivity and detection signals of the asymmetric fluorescent nanoparticles as fluorescent probes are still obviously superior to those of commercial reagents Alexa488 and monomeric green fluorescent protein as probes.
Example 4
The method for manipulating the hepatitis B core antigen gene selected in this example is the same as in example 1.
The two foreign proteins fused in this example are Pyruvate phosphokinase (PPDK, Access No.: AB025020.1, 2637bp) and Luciferase (Luciferase, Access No.: AF311601.1, 1653bp) respectively, and the specific fusion method is as follows:
pyruvate phosphokinase is fused to the N-terminus of core-C described in example 1, and the fused gene is transferred into an expression vector pETDuet, constructing a plasmid name: pET-PPDK-strep.
② luciferase is fused to the N-terminal of core-C described in example 1, the fused gene is transferred into expression vector pCDFDuet, and plasmid name is constructed: pCDF-Luc-strep.
In this embodiment, two different expression plasmids fusing different functional subunit genes are simultaneously transformed into bacteria, and after induction co-expression, nanoparticles of a double-enzyme system can be synthesized in the bacteria by one-step self-assembly.
The T7 promoter and tac promoter were also used in this example to regulate two functional protein genes. Different enzymes are regulated by different promoters to obtain the self-assembled nanoparticles with controllable quantity of the double enzymes, and the self-assembled nanoparticles can be combined to form the following 3 double-enzyme catalytic particles with different quantity ratios:
pET-T7-PPDK-strep and pCDF-T7-Luc-strep, wherein the two carriers are independently or jointly transferred into escherichia coli, and PPDK and Luciferase are equivalently displayed on the surface of the HBcAg protein nanoparticle.
pET-tac-PPDK-strep and pCDF-T7-Luc-strep, wherein the two carriers are transferred into Escherichia coli, PPDK is regulated by tac, while Luciferase is regulated by T7, and the difference of the two enzymes is displayed on the surface of HBcAg protein nano-particles.
And thirdly, pET-T7-PPDK-strep and pCDF-tac-Luc-strep, wherein the two carriers are transferred into escherichia coli together, PPDK is regulated and controlled by T7, while Luciferase is regulated and controlled by tac, and double enzyme differential quantity is displayed on the surface of the HBcAg protein nano-particle.
The double-enzyme self-assembly nanoparticles obtained in this example were characterized by the molecular number of two functional proteins regulated by a promoter using an immunoblot assay, as shown in fig. 11. The two vectors respectively show one band when being independently transferred into escherichia coli, and simultaneously show two bands when being jointly transferred into the escherichia coli, and the band widths and the colors of the two bands are not greatly different, which represents equivalent expression. pET-tac-PPDK-strep and pCDF-T7-Luc-strep, as well as pET-T7-PPDK-strep and pCDF-tac-Luc-strep are co-transferred into Escherichia coli, two bands are displayed simultaneously, but the bandwidth and color of the two bands are greatly different, which indicates differential expression.
The three double-enzyme self-assembly nanoparticles constructed in the embodiment and using the promoter to regulate the quantity ratio of two enzymes verify the cascade catalysis effect.
The specific experimental method is as follows:
the system of the double-enzyme cascade catalytic reaction is as follows:
0.083mM AMP (adenosine monophosphate); 3.3mM PEP (phosphoenolpyruvate); 3.3mM DTT (dithiothreitol); 83mM (NH)4)2SO4；10mM MgSO4(ii) a 83mM bis-Tris propane (1, 3-bis [ Tris (hydroxymethyl) methylamino)]Propane, pH 6.8); 0.4mM D-luciferin, 50. mu.L of double-enzyme catalytic particles (200. mu.g/mL) are mixed uniformly, 20. mu.L of PPi (2mM) are added for full mixing, and a luminescence signal is continuously detected by a microplate reader at 37 ℃.
The results are shown in FIG. 12, where the curves correspond to pET-T7-PPDK-strep + pCDF-T7-Luc-strep, pET-T7-PPDK-strep + pCDF-tac-Luc-strep, pET-tac-PPDK-strep + pCDF-T7-Luc-strep, respectively, from top to bottom. Different promoters are used for controlling different vectors to regulate the expression ratio of the two enzymes, and a stronger luminescent signal and longer-lasting luminescent time are displayed after cascade catalysis, so that the enzyme catalysis rate of the cascade catalysis can be regulated by controlling the ratio of the two enzymes in a two-enzyme system.
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 the like that are within the spirit and principle of the present invention are included in the present invention.
Claims (10)
1. A controllable biosynthesis method of multifunctional self-assembled nanoparticles, wherein the asymmetric self-assembled nanoparticles comprise two or more different functional ligands, and the synthesis of different ratios of the different functional ligands can be completed in vivo.
2. A controlled biosynthesis process as claimed in claim 1, wherein the synthesis of different ratios between different functional ligands is achieved by controlling the promoters of different functional ligands.
3. The controllable biosynthesis method according to claim 2, wherein the expression of a functional ligand regulated by the promoter is regulated by replacing the promoter and/or modifying the promoter and/or inserting a regulatory sequence into the promoter.
4. The controlled biosynthesis process of claim 3, wherein said promoters include, but are not limited to, T7, tac1, lacUV5, tac2, trc, and one or more of the promoters with different strengths obtained by mutating T7, tac1, lacUV5, tac2, trc, preferably, said promoters are T7 and tac 1.
5. The controlled biosynthesis method of any one of claims 1-4, wherein said functional ligand includes, but is not limited to, one or more of an enzyme, an antigen, an antibody, a fluorescent protein, a biotin-binding protein, protein G, protein A, a plurality of protein tags (e.g., spy tag, HALO tag, transmembrane peptide), a nucleic acid-binding protein;
preferably, the functional ligands are green fluorescent protein and protein G;
preferably, the functional ligands are pyruvate phosphate kinase and luciferase.
6. The controllable biosynthesis method according to any one of claims 1-4, wherein the multifunctional self-assembled nanoparticle is a virus-like particle.
7. The method of controlled biosynthesis according to claim 6, wherein said virus-like particle comprises but is not limited to nanoparticles formed by self-assembly of one or more of hepatitis B core antigen, ferritin, streptavidin, starvation induced DNA binding protein, phage capsid protein, Simian vacuolation Virus40, cowpea chlorotic mottle virus, preferably said virus-like particle is a nanoparticle formed by self-assembly of hepatitis B core antigen.
8. Asymmetric self-assembled nanoparticles made by the controllable biosynthetic method described in claims 1-7.
9. The asymmetric self-assembled nanoparticle of claim 8, comprising hepatitis b core antigen, green fluorescent protein, protein G, pyruvate phosphate kinase, luciferase.
10. The method for controlled biosynthesis according to claims 1 to 7 or the method for preparing asymmetric self-assembled nanoparticles according to claims 8 or 9, comprising the following steps:
through gene cloning technology, different functional ligands are cloned into expression vectors respectively, and the expression vectors are transferred into the same host for expression.
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| CN116083467A (en) * | 2022-10-24 | 2023-05-09 | 西南大学 | Attenuated salmonella and phage P22 double-vector, construction method and application thereof |
| CN116396398A (en) * | 2023-01-16 | 2023-07-07 | 四川大学 | A method to realize the controllable self-assembly of ferritin nanoparticles and a nanoparticle antigen display platform based on this method |
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