CN116139105A - Protein nucleic acid nano-particle and preparation method thereof - Google Patents

Abstract

The invention discloses a protein nucleic acid nanoparticle and a preparation method thereof. The invention belongs to the field of biological medicine, and in particular relates to a protein nucleic acid nanoparticle and a preparation method thereof. The protein in the nanoparticle can be assembled with deoxyribonucleotide or ribonucleotide with different lengths, single chains or double chains and different sequences to form the nanoparticle with the size of about 30nm by only adding a certain amount of positive amino acid into the sequence and reducing the salt concentration in the buffer solution. The three-dimensional structure of the amino terminal of the protein in the nanoparticle can be completely identified, thereby playing a corresponding function. At the same time, the nanoparticle may be taken up by the cell.

Description

Protein nucleic acid nano-particle and preparation method thereof

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a protein nucleic acid nanoparticle and a preparation method thereof.

Background

In the biomedical field, protein therapy is the most common technical means, and plays a very important role in the fields of vaccines, antibody therapy and the like. The nano particles have the characteristics of better stability, higher density of carried antigen epitopes, convenience in transportation into cells and the like, and have very wide application prospects in protein treatment. However, the design limitations of proteins carried by nanoparticles formed by current protein drugs are high, and the introduction of metal ions and other non-biological source materials is often required, which may lead to large side effects.

Meanwhile, nucleic acid medicaments are greatly lightened in the fields of cancer treatment, vaccines, gene editing and the like due to the characteristics of short research and development period, wider targets, capability of radically treating genetic diseases and the like. In order to be able to deliver nucleic acids safely and accurately into specific cells, it is often necessary to use nucleic acid delivery vehicles to achieve this process, however existing nucleic acid delivery vehicles such as lipid nanoparticles and the like have large side effects, and the development of new nucleic acid delivery vehicles is urgent.

Disclosure of Invention

The invention aims to solve the technical problems of avoiding the limitations of the existing protein nano-particles and avoiding the possibility of causing larger side effects due to the introduction of metal ions and other non-biological source materials.

In order to solve the above problems, the present invention provides a protein nucleic acid nanoparticle.

The protein nucleic acid nanoparticle provided by the invention is a nanoparticle prepared from a nucleic acid molecule and a protein molecule; the protein molecule contains a peptide fragment, the length of the peptide fragment is greater than or equal to 50 amino acid residues, the peptide fragment contains greater than or equal to 12 positively charged amino acid residues, and the positively charged amino acid residues are arginine residues.

Further, the diameter of the protein nucleic acid nanoparticle is 10nm-100nm.

Further, the nucleic acid molecule may be 21bp to 4733bp in size. The size of the protein molecule may be 20768Da-50462 Da.

Still further, the nucleic acid molecule is at least one selected from the group consisting of 4 nucleic acid molecules of single-stranded deoxyribonucleotides, double-stranded deoxyribonucleotides, single-stranded ribonucleotides, and double-stranded ribonucleotides.

The protein nucleic acid nanoparticle described above, which is prepared according to any one of the methods described below.

The invention provides a method for preparing the protein nucleic acid nano-particles, which comprises the steps of reacting the nucleic acid molecules with the protein molecules in a buffer solution with high ionic strength, and reacting the obtained reaction product in a buffer solution with low ionic strength to obtain the protein nucleic acid nano-particles; the high-ionic strength buffer solution contains 500mM NaCl,20mM Tris-HCl, pH 8.0 or 2000mM NaCl,20mM Tris-HCl and pH 8.0, the content of NaCl in the high-ionic strength buffer solution is more than or equal to 500mM, the low-ionic strength buffer solution contains 100mM NaCl,20mM Tris-HCl and pH 8.0, and the content of NaCl in the low-ionic strength buffer solution is less than or equal to 100mM.

The NaCl content is 500mM or more and 500mM to 2000mM, and the NaCl content is 100mM or less and 0mM to 100mM.

In the above method, the mass ratio of the nucleic acid molecule to the protein molecule (based on the mass of PRM1 in the protein) is 1:0.3-3.3.

The nucleic acid molecules may be present in a buffer of high ionic strength in an amount of 20 ng/. Mu.L to 4. Mu.g/. Mu.L.

In the above method, the 500mM NaCl,20mM Tris-HCl, pH 8.0 or 2000mM NaCl,20mM Tris-HCl, pH 8.0 high ionic strength buffer solution is composed of a solute and a solvent, wherein the solvent is water, and the solute is NaCl or KCl or ammonium sulfate or sodium hydrogen phosphate or disodium hydrogen phosphate or sodium dihydrogen phosphate or potassium dihydrogen phosphate or dipotassium hydrogen phosphate or potassium dihydrogen phosphate, tris-HCl or HEPES (4-hydroxyethyl piperazine ethane sulfonic acid) or boric acid or calcium hydroxide or MOPS (3-morpholinopropionic acid) or CAPS (3- (cyclohexylamine) -1-propane sulfonic acid) or CHES (2-cyclohexylamine ethane sulfonic acid) or Tricine (tri (hydroxymethyl) methylglycine) or triethanolamine or barbital sodium or sodium carbonate.

The low-ionic strength buffer solution consists of a solute and a solvent, wherein the solvent is water, and the solute is NaCl or KCl or ammonium sulfate or sodium hydrogen phosphate or disodium hydrogen phosphate or sodium dihydrogen phosphate or potassium hydrogen phosphate or dipotassium hydrogen phosphate or potassium dihydrogen phosphate, tris-HCl or HEPES (4-hydroxyethyl piperazine ethane sulfonic acid) or boric acid or calcium hydroxide or MOPS (3-morpholinopropane sulfonic acid) or CAPS (3- (cyclohexylamine) -1-propane sulfonic acid) or CHES (2-cyclohexylamine ethane sulfonic acid) or Tricine (tri (hydroxymethyl) methylglycine) or triethanolamine or barbital sodium or sodium carbonate.

In the above method, said reacting in a buffer of low ionic strength comprises reacting said reaction product in said buffer of high ionic strength having a NaCl content of 500-2000mM, and then in said buffer of low ionic strength having a NaCl content of 0-100 mM.

In the above method, the protein nucleic acid nanoparticle is a nanoparticle made of a nucleic acid molecule and a protein molecule; the nucleic acid molecule is at least one of 4 nucleic acid molecules selected from the group consisting of single-stranded deoxyribonucleotides, double-stranded deoxyribonucleotides, single-stranded ribonucleotides and double-stranded ribonucleotides.

In the above method, the protein molecule comprises a peptide fragment having a length of 50 amino acid residues or more and 12 positively charged arginine residues or more.

The protein may be any one of the following A), B), C), (D) or (E):

the protein of A) may be any of the following:

a1 A protein having an amino acid sequence of SEQ ID No. 1;

a2 Fusion proteins obtained by fusion of the protein tags at the amino terminus of the proteins indicated in A1),

a3 Protein which is obtained by substituting and/or deleting and/or adding amino acid residues on the protein of A1) or A2), has more than 80% of identity with the protein of A1) or A2) and has the same function;

The protein of B) may be any of the following:

b1 A protein having an amino acid sequence of SEQ ID No. 2;

b2 Fusion protein obtained by fusing protein tags at the amino terminus of the protein shown in B1),

b3 A protein which is obtained by substituting and/or deleting and/or adding amino acid residues of the protein of B1) or B2), has more than 80% of identity with the protein of B1) or B2) and has the same function;

the protein of C) may be any of the following:

c1 A protein having an amino acid sequence of SEQ ID No. 3;

c2 Fusion protein obtained by fusing protein tags at the amino terminus of the protein shown in C1),

c3 A protein which is obtained by substituting and/or deleting and/or adding amino acid residues of the protein of C1) or C2), has more than 80% of identity with the protein of C1) or C2) and has the same function;

the D) protein may be any of the following:

d1 A protein having an amino acid sequence of SEQ ID No. 4;

d2 Fusion protein obtained by fusing protein tags at the amino terminus of the protein shown in D1),

d3 Protein obtained by substituting and/or deleting and/or adding amino acid residues of the protein of D1) or D2) and having more than 80% of identity with the protein of D1) or D2) and the same function;

The E) protein may be any of the following:

e1 A protein having an amino acid sequence of SEQ ID No. 5;

e2 Fusion protein obtained by fusing protein tags at the amino terminus of the protein shown in E1),

e3 Protein obtained by substituting and/or deleting and/or adding amino acid residues of the protein of E1) or E2) and having 80% or more identity with the protein of E1) or E2) and the same function.

The invention also provides application of the protein nucleic acid nano-particles in preparation of products for identifying tumor cells.

The invention also provides application of the protein nucleic acid nanoparticle in preparation of products for delivering proteins or nucleic acids or proteins and nucleic acids into cells.

The preparation method of the nucleic acid protein nano-particles can assemble proteins and nucleic acids by a corresponding method, and finally form nano-particles with uniform size and diameter of about 30 nm. The protein and nucleic acid in the particle have great engineering capability. Meanwhile, as the protein and the nucleic acid are produced from biological sources, the materials used in the assembly process are mainly inorganic substances with relatively wide abundance in organisms such as sodium chloride, and the particles have relatively good biocompatibility and can deliver the protein and the nucleic acid into cells, so that the particles are applied to the fields of vaccines, antibodies and the like which relate to treatment or prevention by using the protein, and the fields of nucleic acid vaccines, gene editing, cancer treatment and the like which relate to treatment or prevention by using the nucleic acid.

Drawings

Fig. 1 shows a particle obtained by the assembly method 1. Wherein (a) a chromatogram of the molecular sieve; (B) results of SDS-PAGE stained with coomassie brilliant blue; (C) results after GelRed staining of agarose gel; l: load, i.e., sample not purified by molecular sieve, M: a marker; (D) And (3) detecting a negative staining result of the Peak 1 sample by using an electron microscope, wherein particles are arranged in a green ring, and the proportion is as follows: 100nm.

Fig. 2 is a particle obtained by assembly mode 2. Wherein (A) is based on the results of GelRed staining of Native agarose gel samples after assembly by modified dialysis with different protein to nucleic acid ratios, M: a marker; (B) The particles were assembled by modified dialysis using a nucleic acid to protein ratio of 1:3, with particles in the green circle and a scale of 100nm. The ratio used is the mass ratio of DNA to protein at the time of assembly.

FIG. 3 is a particle formed by the assembly of different amino-terminal proteins with different nucleic acids.

FIG. 4 is a particle formed with nucleic acid after mutation of the carboxy terminal protein.

FIG. 5 shows the result of the cleavage of the particles by different types of enzymes. Wherein (A) SDS-PAGE of ULP1 digested particles is stained with Coomassie brilliant blue, M: a marker; (b) Results of the uraa-PAGE of MNase digested particles after GelRed staining, C: control, i.e. 59nt ssdna alone, m: a marker; (C) Negative staining electron microscope detection results of the particles after ULP1 enzyme digestion; (D) negative staining electron microscopy of the particles; (E) And (3) detecting the result of the negative staining electron microscope after the particles are subjected to Mnase digestion. Green circles in the detection result of the negative electron microscope are particles, and the proportion scale is: 100nm.

FIG. 6 is a cell uptake assay for HEK 293T cells. Nucleic acid-protein nanoparticles formed from SUMO-PRM1 were used. (A) The results observed after 2 hours after addition of the particles, after staining with Hoechst dye; (B) The results observed after 4 hours after addition of the particles, after staining with Hoechst dye; (C) The results observed after 6 hours after addition of the particles, after staining with Hoechst dye; (D) The results observed after 6 hours of staining with Hoechst dye after addition of equal amounts of DNA alone; (E) Directly mixing the protein and DNA in the same proportion used in assembly under low salt condition, adding the cells for 6 hours, and then staining with Hoechst dye. Merge: fusion of images of bright field, 488nm wavelength excitation and 346nm wavelength excitation light. 488: images at 488nm wavelength excitation. Hoechest: an image at 346nm wavelength excitation. The scale bar in the figure is 50. Mu.m.

FIG. 7 shows the cell uptake assay of Hela cells. A. Adding nucleic acid-protein nanoparticles formed by SUMO-PRM1 into Hela cells, allowing the mixture to act for 2 hours, and observing the mixture after the mixture is dyed by Hoechst dye; B. the nucleic acid-protein nanoparticles formed by GE11-SUMO-PRM1 were added to Hela cells and observed 2 hours after staining with Hoechst dye; C. adding nucleic acid-protein nanoparticles formed by SUMO-PRM1 into Hela cells, allowing the mixture to act for 4 hours, and observing the mixture after the mixture is dyed by Hoechst dye; D. adding nucleic acid-protein nanoparticles formed by GE11-SUMO-PRM1 into Hela cells, allowing the mixture to act for 4 hours, and observing the mixture after the mixture is dyed by Hoechst dye; E. after adding nucleic acid-protein nanoparticles formed from SUMO-PRM1 to Hela cells, the reaction was observed after 6 hours of staining with Hoechst dye; F. after adding nucleic acid-protein nanoparticles formed by GE11-SUMO-PRM1 to Hela cells, the reaction was observed after 6 hours of staining with Hoechst dye; G. the addition of a separate equivalent amount of DNA to HeLa cells was followed by 6 hours incubation with HeLa cells and then observation after staining with Hoechst dye. Merge: fusion of images of bright field, 488nm wavelength excitation and 346nm wavelength excitation light. 488: images at 488nm wavelength excitation. Hoechest: an image at 346nm wavelength excitation. The scale bar in the figure is 50. Mu.m.

FIG. 8 is a cell uptake assay for HEK 293T cells. A. The nucleic acid-protein nanoparticles were added to HEK 293T cells and observed 8 hours later. B. The nucleic acid-protein nanoparticles were added to HEK 293T cells and observed 28 hours later.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The quantitative experiments in the following examples were performed in triplicate unless otherwise indicated.

Vector pET-28a of the present invention has been described in the following literature: zhang L, serra-Cardona A, zhou H, et al Multi site Substrate Recognition in Asf1-Dependent Acetylation of Histone H K56 by Rtt109.Cell,2018,174 (4): 818-830. The public is available from the national academy of sciences biophysics, and the biomaterial is only used for repeated experiments of the invention and is not available for other uses.

In the present invention the plasmids pBluescriptII, pET-22b, BL21-CondonPlus-RIL E.coli competence and DH 5. Alpha. E.coli competence have been described in: yang D, fang Q, wang M, et al, N.alpha. -activated Sir3 stabilizes the conformation of a nucleosome-binding loop in the BAH domain. Nature Structure & Molecular Biology,2013,20 (9): 1116-1118. The public is available from the national academy of sciences biophysics, and the biomaterial is only used for repeated experiments of the invention and is not available for other uses.

HEK 293T cells and plasmid pGEX-6P-1 according to the invention are described in: liu C P, xiong C, wang M, et al Structure of the variant histone H3.3.3-H4 heterodimer in complex with its chaperone DAXX. Nat Structure Mol Biol 2012,19 (12): 1287-1292. The public is available from the national academy of sciences biophysics, and the biomaterial is only used for repeated experiments of the invention and is not available for other uses.

The plasmid pEGFP-N1 of the invention has been described in: liu C P, jin W, hu J, et al distingt history H3-H4 binding modes of sNASP reveal the basis for cooperation and competition of histone chaperones.genes Dev,2021,35 (23-24): 1610-1624. The public is available from the national academy of sciences biophysics, and the biomaterial is only used for repeated experiments of the invention and is not available for other uses.

In the present invention, hela cells have been described in: fang J, liu Y, wei Y, et al Structure transitions of centromeric chromatin regulate the cell cycle-dependent recruitment of CENP-N.genes Dev,2015,29 (10): 1058-1073. The public is available from the national academy of sciences biophysics, and the biomaterial is only used for repeated experiments of the invention and is not available for other uses.

In the present invention pri-miRNA has been described in: jin W, wang J, liu C-P, et al structural Basis for pri-miRNA Recognition by Drosha. Molecular Cell,2020,78 (3): 423-433. The public is available from the national academy of sciences biophysics, and the biomaterial is only used for repeated experiments of the invention and is not available for other uses.

ULP1 according to the invention has been described in: hou P, huang C, liu C-P, et al Structure Insights into Stimulation of Ash L's H K36 Methyltransferase Activity through Mrg15binding. Structure,2019,27 (5): 837-845. The public is available from the national academy of sciences biophysics, and the biomaterial is only used for repeated experiments of the invention and is not available for other uses.

The proteins selected herein are: SUMO-PRM1, GST-PRM1 and MBP-PRM1. The amino acid sequence of the specific protein is shown in Table 1, and the nucleotide sequence of the protein is shown in Table 3.

The proteins SUMO-PRM1, GST-PRM1, MBP-PRM1, SUMO-PRM1 (mut) and GE11-SUMO-PRM1 were prepared as follows:

1. expression of proteins using prokaryotic expression systems

pET-28a gene expression vectors with SUMO-PRM1 gene (specific sequence shown in SEQ 13), pET-22b gene expression vectors with MBP-PRM1 gene (specific sequence shown in SEQ 14), pGEX-6P-1 gene expression vectors with GST-PRM1 gene (specific sequence shown in SEQ 15), pET-28a gene expression vectors with SUMO-PRM1 (mut) gene (specific sequence shown in SEQ 16) and pET-28a gene expression vectors with GE11-SUMO-PRM1 gene (specific sequence shown in SEQ 17) are used respectively.

1) Conversion: 50ng of expression plasmid (SUMO-PRM 1 gene expression vector, SUMO-PRM1 (mut) gene expression vector, GE11-SUMO-PRM1 gene expression vector, GST-PRM1 gene expression vector or MBP-PRM1 gene expression vector) was added to 50. Mu.L BL21-CondonPlus-RIL E.coli competence prepared in advance, incubated on ice for 30min, heat-shocked at 42℃for 90s, added with 800. Mu.L of medium, placed on a shaking table at 37℃for 180rpm for 1h, 200. Mu.L of bacterial liquid was transferred to a solid incubator with corresponding antibiotics (SUMO-PRM 1 gene expression vector, SUMO-PRM1 (mut) gene expression vector or GE11-SUMO-PRM1 gene expression vector used for 50. Mu.g/mL and chloramphenicol 37. Mu.g/mL, GST-PRM1 gene expression vector or MBP-PRM1 gene expression vector used for ampicillin 50. Mu.g/mL and chloramphenicol 37. Mu.g/mL) for 37. Mu.g/mL, and 37. Mu.g/mL for 37℃for 12h.

2) Culturing bacterial liquid: a monoclonal colony was picked up, and 5mL of LB medium having carried thereon the corresponding antibiotic (SUMO-PRM 1 gene expression vector, SUMO-PRM1 (mut) gene expression vector or GE11-SUMO-PRM1 gene expression vector was used as kanamycin 50. Mu.g/mL and chloramphenicol 37. Mu.g/mL; GST-PRM1 gene expression vector or MBP-PRM1 gene expression vector was used as ampicillin 50. Mu.g/mL and chloramphenicol 37. Mu.g/mL), was added to a shaking table at 37℃for 8 hours at 180rpm, followed by culturing according to 1:1000 were added to 200mL of LB medium, which had been provided with the corresponding antibiotics, and incubated at 180rpm for 12h in a shaker at 37 ℃.

3) And (3) bacterial liquid expansion culture: culturing bacterial liquid according to the following formula 1:40 is added into 800mL LB culture medium with corresponding antibiotics, and cultured for about 4h at 160rpm in a shaking table at 37 ℃ until the OD of the bacterial liquid is reached ₆₀₀ The value is about 0.6, at this point IPTG was added to a final concentration of 0.5mM, and the mixture was placed in a shaking table at 37℃and incubated at 160rpm for a further 4h.

4) Collecting bacterial liquid: each bottle of bacterial liquid is transferred into a 1L bacteria collection centrifugal bottle. Using a floor centrifuge, centrifugation was performed at 4000rpm for 15min at 4℃to collect the cells and the supernatant was discarded.

5) The cells were resuspended at a rate of 200mL of cell lysate per 10L of cell lysate.

2. Purification of proteins

1) Bacteria breaking

Crushing the bacterial liquid obtained by re-suspension by using an ultrasonic crusher or a high-pressure crusher, centrifuging the bacterial liquid obtained by crushing by using a high-speed centrifuge at 4 ℃ and 20000g for 60min, retaining the supernatant and discarding the precipitate.

2) Affinity chromatography

The supernatant after disruption centrifugation was incubated with the corresponding chromatography medium at 4℃for 30min for Ni-excel beads (for purification of SUMO-PRM1, SUMO-PRM1 (mut) and GE11-SUMO-PRM 1) and for 3h for GST beads (for purification of GST-PRM 1) and Amylose Resin beads (for purification of MBP-PRM 1).

The supernatant was allowed to flow through using a gravity column, after which the chromatographic medium was washed with 10 column volumes of cell lysate to remove non-specifically bound heteroproteins.

Eluting the chromatographic medium by using the corresponding eluent, and collecting the eluent.

3) Ion exchange chromatography

The ion column used was a heparin column (purchased from GE Healthcare company under the trade designation 17040703).

a. Ion column was washed using 10 column volumes of ion column buffer a.

b. The affinity chromatography buffer is reduced in salt to the salt concentration of the ion column buffer A, and the ion column is flowed through.

c. The ion column was again washed with ion column buffer a.

d. The AKTA system program is set to the flow rate that the concentration of the ion column buffer solution B in the ion column flowing through liquid is increased from 0 to 100 percent in 50min and 2mL/min, and the ion column is eluted.

e. Corresponding samples were collected according to the chromatogram of the AKTA system, combined with coomassie brilliant blue staining results of SDS-PAGE.

f. The column volumes were washed 10 times again using ion column buffer B.

g. The resulting water was purified using Millipore, and after suction filtration the system and ion column were washed.

4) Size exclusion chromatography

a. The HiLoad 16/600Superdex 75pg molecular sieve (GE Healthcare Co., ltd., cat. No. 28989333) was washed for 1.5 column volumes using molecular sieve buffer A until the salt concentration of the molecular sieve effluent was the same as the salt concentration of the molecular sieve buffer.

b. The sample collected by the ion column is concentrated, centrifuged for 30min at 20000g at 4 ℃, and the sample is loaded into the molecular sieve using loading loop.

3. According to the chromatogram of the AKTA system, the coomassie brilliant blue staining result of SDS-PAGE is combined, and corresponding samples are collected to obtain proteins SUMO-PRM1, GST-PRM1 and MBP-PRM1 respectively.

The selected nucleic acid is: 59nt ssDNA, 59bp DNA, pEGFP-N1 linearization product, 6X 187bp DNA, siRNA, pri-miRNA, EGFP mRNA, and 94bp DNA.

The preparation of DNA fragments in large quantities is as follows (94 bp DNA and 6X 187bp DNA are taken as examples):

1. mass replication of plasmids

94bp DNA (nucleotide sequence shown in SEQ ID No. 10) and 6×187bp DNA (nucleotide sequence shown in SEQ ID No. 11) were inserted into the multiple cloning site of pBluescriptII, respectively, to obtain a recombinant vector containing 94bp DNA, and a recombinant vector containing 6×187bp DNA was obtained.

1) Conversion: 50ng of any one of the recombinant vectors is taken, 50 mu L of DH 5. Alpha. E.coli competent prepared in advance is added, the mixture is incubated on ice for 30min, heat shock is carried out at 42 ℃ for 90s, 800 mu L of culture medium is added, the mixture is placed on a shaking table at 37 ℃ for culturing for 1h at 180rpm, 200 mu L of bacterial liquid is transferred to LB solid culture medium added with corresponding antibiotics (50 mu g/mL ampicillin for pBluescriptII plasmid and 50 mu g/mL kanamycin for pEGFP-N1) and the culture is carried out for 12h at 37 ℃.

2) Culturing bacterial liquid according to the following formula 1:40 to 800mL of TB medium to which the corresponding antibiotic (50. Mu.g/mL ampicillin for pBluescriptII plasmid, 50. Mu.g/mL kanamycin for pEGFP-N1) and phosphate buffer had been added, and culturing at 160rpm in a shaker at 37℃for about 6 hours until the OD of the bacterial solution had been reached ₆₀₀ The value was about 0.8, and the shaking table temperature was raised to 42℃and the culture was continued at 160rpm for 12 hours.

3) Bacterial liquid is collected, and each bottle of bacterial liquid is transferred into a 1L bacterial collection centrifugal bottle. Using a floor centrifuge, centrifugation was performed at 4000rpm for 15min at 4℃to collect the cells and the supernatant was discarded.

2. Extraction of plasmid by alkaline lysis

1) Solution configuration

S1: 15mL Tris-HCl, pH 8.0,5.93g D-glucose, 12mL EDTA, pH 8.0 were weighed, water was added to volume 500mL and ready to use.

S2: 6.4g NaOH and 8g SDS are weighed, water is added to a volume of 800mL, and the mixture is prepared immediately.

S3: 588.8g of potassium acetate and 229.3mL of glacial acetic acid are weighed, water is added to fix the volume to 2L, and suction filtration is performed.

40% PEG6000 solution: 20g PEG6000 is weighed into 40mL of water, heated in a water bath at 65 ℃ and dissolved, and then the volume is fixed to 50mL for standby.

TE：10mM Tris-HCl pH 8.0，1mM EDTA。

TE 10/0.1：10mM Tris-HCl pH 8.0，0.1mM EDTA。

TE 10/50：10mM Tris-HCl pH 8.0，50mM EDTA。

3M sodium acetate solution: 40.8g of CH are weighed ₃ COONa·H ₂ O, adding about 40mL of water, stirring for dissolution, adding glacial acetic acid to adjust the pH value to 5.2, adding water to fix the volume to 100mL, and placing at 4 ℃ for standby.

Phenol chloroform extract: tris saturated phenol was mixed with chloroform according to 1:1, the water phase is reserved and kept stand for standby.

Chloroform isoamyl alcohol extract: isoamyl alcohol and chloroform were mixed according to 1:24, and standing for standby.

2) The bacterial cells were resuspended in 360mL of plasmid extraction buffer S1 at a ratio of 10L of bacterial solution in a shaking table at 37℃at 160rpm for about 30min to no macroscopic clumps.

3) According to S1: s2=1: 2, fresh plasmid extraction buffer S2 was added in the volume ratio, gently inverted to mix well. The mixture was left at room temperature for 5min.

4) According to S1: s3=1: 3.5, adding precooled plasmid extraction buffer solution S3 by sticking to the wall, shaking until no layering exists, and forming the impurity into egg-shaped.

5) Placing on ice for 10min. The supernatant was carefully aspirated using a floor standing centrifuge 4000rpm, centrifuged at 4℃for 30min, filtered through 4 layers of gauze to remove suspended impurities, and transferred to a clean beaker.

6) Isopropanol precipitation of DNA: to the collected supernatant, 0.52 times of isopropyl alcohol was added, and the mixture was stirred and mixed well, followed by standing at room temperature. Until there is a significant precipitation and delamination from the liquid phase. The mixture was centrifuged at 4000rpm at 4℃for 30 minutes using a floor centrifuge, and the supernatant was discarded to recover the nucleic acid precipitate. The tube wall and bottom sediment of the centrifuge tube were gently rinsed with a small amount of 70% ethanol and left to evaporate at room temperature. 50mL of TE 10/50 was then added to dissolve the precipitate.

7) Rnase digestion: an appropriate amount of RNase was added to the recovered plasmid, and digested overnight at 37℃to completely chop RNA in the plasmid.

8) Phenol chloroform removal of protein: phenol chloroform was prepared in advance and allowed to stand until significant phase separation occurred. After the RNase digestion is finished, 1/5 volume of phenol-chloroform is added, the mixture is stirred and mixed uniformly, 20 ℃ and 20000g are centrifuged for 20min, the upper water phase is carefully sucked out for retention, and the process is repeated until the interface between the two phases is clear and no white precipitate is separated out.

9) Removing phenol from chloroform isoamyl alcohol: then adding 2/5 volume of chloroform isoamyl alcohol, shaking and uniformly mixing, centrifuging for 20min at 20 ℃ and 20000g, and carefully sucking out the upper water phase to remain.

10 PEG-minus-RNA: to the aspirated aqueous phase, 1/5 volume of 4M NaCl,2/5 volume of 40% PEG6000 were added, and after mixing, incubated at 37℃for 5min and on ice for 30min. Centrifuging at 4deg.C for 15min at 20000g, discarding supernatant (containing RNA), washing the tube wall and precipitate with a small amount of 70% ethanol, removing ethanol without disturbing the precipitate, drying with the aid of a blower, dissolving the precipitate in 30mL TE10/0.1, and standing in a 37℃incubator for several hours until the precipitate is completely dissolved.

11 Removing PEG: adding 1/5 volume of chloroform isoamyl alcohol, shaking and mixing, centrifuging at 4 ℃ and 20000g for 10min, and repeating until no obvious precipitation is generated after centrifugation.

12 Ethanol precipitation of plasmid collection: 1/10 volumes of 3M sodium acetate solution (pH 5.2) and 2.5 volumes of ice-cold absolute ethanol were added and placed at-20℃for at least 1 h. Centrifuging at 4deg.C and 20000g for 15min, precipitating to obtain plasmid DNA, washing the container wall and the surface of the precipitate with a small amount of 70% ethanol, pouring out ethanol, drying with blower, and dissolving in 30mL TE10/0.1.

3. Restriction enzyme cutting to obtain DNA fragment

1) And (3) enzyme cutting: the corresponding enzyme was added at a rate of 30U per 1 mg plasmid (pEGFP-N1 plasmid linearization product using AseI, others using EcorV) while DTT was added to a final concentration of 1 mM.

2) Isolation of fragments (plasmid linearization products do not require this step): 0.192 times the volume of 4M NaCl was added, and the corresponding volume of 40% PEG6000 was incubated at 37℃for 5 min, 1 h was incubated on ice (ice for a period of time not too long to prevent some of the desired fragment from being deposited), centrifuged at 4℃for 20 min at 20000 g, and precipitated as plasmid DNA. Specific PEG volumes require small attempts to be made in advance using the same ratio until the supernatant has no large fragments and little fragments are precipitated.

3) Ethanol precipitation and collection of the product: 1/10 volumes of ice-cold 3M NaAc (pH 5.2) and 2.5 volumes of ice-cold absolute ethanol were added and left at-20℃for at least 1 h. Centrifuge at 4℃and 20000 g for 15 min. The precipitated plasmid DNA was washed with a small amount of 70% ethanol, the wall of the vessel and the surface of the precipitate were dried by a blower after the ethanol was removed, and the resultant was dissolved in 30 mL of TE 10/0.1. The pEGFP-N1 linearized product, 6X 187 bp DNA and 94 bp DNA were obtained, respectively.

4. 59 nt ssDNA is custom made by Biotechnology (Shanghai) Inc., order number 111930348.

5. 59 bp DNA was obtained by annealing 59 nt ssDNA by a PCR apparatus (BIO-RAD Co., ltd., cat# 1851148). And (3) annealing: 59 nt ssDNA was dissolved in water at a concentration of 1 mg/mL, and after heating the sample to 100deg.C for 5 minutes, the temperature was set to drop by 0.5℃every 30 seconds until the temperature dropped to 20deg.C.

6. The nucleotide sequences of the siRNAs are shown in Table 2, custom made products by the biological engineering (Shanghai) Co., ltd., order number: r12444.

The preparation method of Tris-HCl used in the invention comprises the following steps:

firstly, preparing Tris-HCl with the concentration of 1M and mother solution with the pH of 8.0: 121.1 g Tris (available from sigma-aldrich company, cat# v 900483) was weighed, dissolved in 800 mL water, fully dissolved, adjusted to pH 8.0 with hydrochloric acid, and fixed to a volume of 1L. When in use, the mother solution is diluted to the corresponding concentration.

The solvent of the buffer solution used in the particle assembly in the invention is water, and the solute can be NaCl or KCl or ammonium sulfate or sodium hydrogen phosphate or disodium hydrogen phosphate or sodium dihydrogen phosphate or potassium hydrogen phosphate or potassium dihydrogen phosphate, tris-HCl or HEPES (4-hydroxyethyl piperazine ethane sulfonic acid) or boric acid or calcium hydroxide or MOPS (3-morpholinopropanesulfonic acid) or CAPS (3- (cyclohexylamine) -1-propane sulfonic acid) or CHES (2-cyclohexylamine ethane sulfonic acid) or Tricine (tri (hydroxymethyl) methyl glycine) or triethanolamine or barbital sodium or sodium carbonate. The solute used in the following examples was NaCl, tris-HCl.

Example 1, nucleic acid protein nanoparticle assembly mode:

the preparation method of the buffer A comprises the following steps: 2000 mM NaCl,20 mM Tris-HCl, pH 8.0;

The preparation method of the buffer B comprises the following steps: 500mM NaCl,20mM Tris-HCl, pH 8.0;

the preparation method of the buffer solution C comprises the following steps: 100mM NaCl,20mM Tris-HCl, pH 8.0

1. Assembly mode 1

The following illustrates the preparation of protein nucleic acid nanoparticles (Peak 1 samples) using a protein (amino acid sequence: SEQ ID No. 1) named SUMO-PRM1 as a protein molecule and a 94bp double-stranded DNA molecule (nucleotide sequence: SEQ ID No.10, abbreviated as 94bp DNA) as a nucleic acid molecule:

(1) precooling the required buffer A, buffer B and buffer C at 4 ℃;

(2) SUMO-PRM1 and 94bp DNA were added to buffer A at 4℃to give reaction solution A. The volume of the reaction solution A was 0.5mL, the content of SUMO-PRM1 was 4mg/mL, and the content of 94bp DNA was 4mg/mL. The reaction solution A was placed in a dialysis tube (Millipore Sigma Co., ltd., cat. No. 71509-3) having a molecular weight cut-off of 6-8kDa, the dialysis tube was placed in a buffer solution A, placed on a magnetic stirrer (Liberer Co., ltd., cat. No. 81-2), and dialyzed at 4℃for 3 hours;

(3) taking out the dialysis tube, putting the dialysis tube into the buffer solution B, putting the buffer solution B on a magnetic stirrer, and dialyzing for 3 hours at 4 ℃;

(4) taking out the dialysis tube, putting the dialysis tube into a buffer solution C, putting the buffer solution C on a magnetic stirrer, dialyzing the solution for 3 hours at 4 ℃ to obtain protein nucleic acid nano-particles, and naming the protein nucleic acid nano-particles as protein nucleic acid nano-particles SUMO-PRM1-94 bp DNA. The samples were taken out for subsequent experiments.

The assembled sample was subjected to ultrafiltration using a 30kDa ultrafiltration tube (Millipore Sigma Co., ltd.; product No. UFC 903096) at 3000g rotation speed and at 4℃until the volume was about 0.4mL, the sample was taken out, centrifuged at 20000g rotation speed and 4℃for 15 minutes, and then subjected to gel filtration chromatography on Superose6 (GE Healthcare Co., ltd., product No. 29-0915-96), the eluate was 100mM NaCl,20mM Tris-HCl solution, the loading volume was 0.4mL, and the elution rate was 0.5mL/min. Panel A shows a chromatogram of the protein purifier (GE Healthcare, model AKTA Pure25M 1) used. And (3) carrying out polyacrylamide protein electrophoresis on the eluted samples at the positions shown in Peak1 and Peak 2 in the graph A to obtain a graph B, carrying out agarose gel electrophoresis under a denaturation condition to obtain a graph C, and carrying out negative staining treatment on the eluted sample at the position shown in Peak1 and observing through an electron microscope to obtain a graph D.

As can be seen from the chromatogram of the molecular sieve in FIG. 1, a first sample Peak appears at about 0.5 column volumes (about 12 mL), which is designated Peak1, and a second sample Peak appears at about 0.625 column volumes (15 mL), which is designated Peak 2.

SDS-PAGE examined the nucleic acid-protein nanoparticles obtained in the above procedure, and as shown in FIG. 1B, both Peak1 and Peak 2 contained SUMO-PRM1, with the proteins concentrated mainly in Peak 1.

The result of agarose Gel staining with Gel-Red is shown in FIG. 1C, and 94bp DNA was contained in both Peak 1 and Peak 2.

Electron microscopy (D in fig. 1) after negative staining of the Peak 1 sample revealed that the Peak 1 sample was a particle of uniform size with a diameter of about 30 nm.

2. Assembly mode 2

SUMO-PRM1 (SEQ ID No. 1) and 94bp DNA (SEQ ID No. 10) gave protein nucleic acid nanoparticles by the following steps:

(1) the desired buffer C was pre-chilled at 4 ℃.

(2) Protein SUMO-PRM1 and nucleic acid (shown as SEQ ID No. 10) were mixed in the following proportions under buffer A, the mass ratio of nucleic acid to protein SUMO-PRM1 being 1:1, 1:2 and 1:3. The mixture was then added to a dialysis tube (MilliporeSigma Co., ltd., product No. 71509-3), which was placed in buffer C and dialyzed against a magnetic stirrer for 3 hours. The samples were taken out for subsequent experiments.

And (3) performing Gel-Red staining on the particles obtained in the assembly mode 2, and performing agarose Gel electrophoresis under non-denaturing conditions, wherein the result is shown as A in fig. 2, the degree of upward migration of the assembled product on the agarose Gel is correspondingly improved along with the increase of the protein proportion during assembly, which indicates that the protein is combined with the nucleic acid, and the combination strength is also improved along with the increase of the protein proportion during assembly. As can be seen from FIG. 2B, the particles assembled by modified dialysis using nucleic acid and protein in a mass ratio of 1:3 exhibited particles with a diameter of about 30nm under electron microscopy.

The electron microscope results show that both assembly mode 1 and assembly mode 2 can obtain particles with the diameter of about 30nm under the electron microscope. The uniformity of the particles obtained in assembly mode 1 is better. But the steps of assembly mode 2 are simpler and require less time.

Example 2 investigation of the Effect of different proteins and nucleic acids on the formation of protein nucleic acid nanoparticles

(1) Comparison of proteins with nucleic acids

Different kinds of proteins (specific protein sequences are shown in Table 1) were assembled with different kinds of nucleic acids (specific sequences are shown in Table 2).

Wherein the selected proteins are SUMO-PRM1, GST-PRM1 and MBP-PRM1. The nucleotide sequences corresponding to the proteins are shown in Table 3.

The selected nucleic acids were 59nt ssDNA, 59bp DNA, pEGFP-N1 linearization product, 6X 187bp DNA, siRNA, pri-miRNA, EGFP mRNA, 94bp DNA, respectively. The specific nucleic acid sequences are shown in Table 2.

The amino acid sequence of the protein used in Table 1

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Table 2 sequences of nucleic acids used

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Note that: EGFP mRNA was 996nt in length from APExBio corporation and was designated as R1001. The siRNA synthesized by conventional chemistry is 21-25nt with a 2nt overhang at the 3' end of the double stranded small RNA (usually dTdT or UU). The engineered double-stranded siRNA (i.e., the siRNA used in this patent) has the following characteristics: the sense strand 21 base is a target sequence of 19 bases plus a overhang TT of 2 bases at the 3' end, and the sequence is 5'-CAAGCUGACCCUGAAGUUCTT-3'; the antisense strand 21 bases are 19 bases complementary to the sense strand, and the 3' -end is added with a overhang TT of 2 bases, and the sequence is 5'-GAACUUCAGGGUCAGCUUGTT-3'.

Table 3 nucleotide sequences corresponding to the proteins used

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These proteins involved in assembly are characterized by arginine at 12 amino acid residues of 50 amino acids, whereas nucleic acids are not very different.

The specific assembly mode and the proportion (mass ratio) of the nucleic acid protein nano-particles are as follows:

1) 59nt ssDNA: SUMO-prm1=1:3, wherein the mass ratio of SUMO to PRM1 in SUMO-PRM1 is 2:1, a step of;

2) 59bp DNA: SUMO-prm1=1:3, wherein the mass ratio of SUMO to PRM1 in SUMO-PRM1 is 2:1, a step of;

3) 6X 187bp DNA: SUMO-prm1=1:6, wherein the mass ratio of SUMO to PRM1 in SUMO-PRM1 is 2:1, a step of;

4) pEGFP-N1 linearization product: SUMO-prm1=1:5, wherein the mass ratio of SUMO to PRM1 in SUMO-PRM1 is 2:1, a step of;

5) siRNA: SUMO-prm1=1:10, wherein the mass ratio of SUMO to PRM1 in SUMO-PRM1 is 2:1, a step of;

6) pri-miRNA: SUMO-prm1=1:2, wherein the mass ratio of SUMO to PRM1 in SUMO-PRM1 is 2:1, a step of;

7) EGFP mRNA: SUMO-prm1=1:4, wherein the mass ratio of SUMO to PRM1 in SUMO-PRM1 is 2:1, a step of;

8) 59nt ssDNA: GST-prm1=1:6, wherein the mass ratio of GST to PRM1 in GST-PRM1 is 3:1, a step of;

9) 59bp DNA: GST-prm1=1:6, wherein the mass ratio of GST to PRM1 in GST-PRM1 is 3:1, a step of;

10 94bp DNA: GST-prm1=1:6, wherein the mass ratio of GST to PRM1 in GST-PRM1 is 3:1, a step of;

11 pEGFP-N1 linearization product: GST-prm1=1:4, wherein the mass ratio of GST to PRM1 in GST-PRM1 is 3:1, a step of;

12 59nt ssDNA: MBP-prm1=1:15, wherein the mass ratio of MBP to PRM1 in MBP-PRM1 is 6:1, a step of;

13 59bp DNA: MBP-prm1=1:15, wherein the mass ratio of MBP to PRM1 in MBP-PRM1 is 6:1, a step of;

14 94bp DNA: MBP-prm1=1:15, wherein the mass ratio of MBP to PRM1 in MBP-PRM1 is 6:1, a step of;

15 pEGFP-N1 linearization product: MBP-prm1=1:15, wherein the mass ratio of MBP to PRM1 in MBP-PRM1 is 6:1.

as a result, as shown in FIG. 3, 59nt ssDNA was able to form particles with a diameter of about 30nm with SUMO-PRM1 by assembly 2; the 59bp DNA can form particles with the diameter of about 30nm with SUMO-PRM1 through an assembly mode 2; 6X 187bp DNA was able to form particles of about 30nm in diameter with SUMO-PRM1 by assembly mode 2; the pEGFP-N1 linearized product was able to form particles of about 30nm in diameter with SUMO-PRM1 by assembly mode 2; the siRNA can form particles with the diameter of about 30nm with SUMO-PRM1 through an assembly mode 2; the per-miRNA can form particles with the diameter of about 30nm with SUMO-PRM1 through an assembly mode 2; EGFP mRNA was able to form particles with a diameter of about 30nm with SUMO-PRM1 by assembly mode 2; 59nt ssDNA forms particles of about 30nm in diameter with GST-PRM1 by assembly mode 2; the 59bp DNA can form particles with the diameter of about 30nm with GST-PRM1 through an assembly mode 2; the 94bp DNA can form particles with the diameter of about 30nm with GST-PRM1 through an assembly mode 2; the pEGFP-N1 linearization product was able to form particles of about 30nm in diameter with GST-PRM1 by assembly mode 2; 59nt ssDNA forms particles with a diameter of about 30nm with MBP-PRM1 by assembly mode 2; the 59bp DNA can form particles with the diameter of about 30nm with MBP-PRM1 through an assembly mode 2; the 94bp DNA can form particles with the diameter of about 30nm with MBP-PRM1 through an assembly mode 2; the pEGFP-N1 linearized product was able to form particles of about 30nm in diameter with MBP-PRM1 by assembly mode 2.

In summary, SUMO-PRM1 was able to form particles of about 30nm in diameter by assembly 2 with DNA or RNA of different lengths or sequences, single-stranded or double-stranded. Whereas SUMO-PRM1, GST-PRM1 and MBP-PRM1 are capable of forming particles of about 30nm in diameter with DNA of different lengths or sequences, either single-stranded or double-stranded.

It follows that, after fusion of the N-terminus of PRM1 with other proteins, particles with a diameter of about 30nm can be formed by assembly mode 2 with nucleic acids of different lengths and sequences.

(2) Effect of mutation of half of the arginine residues of the PRM1 sequence in SUMO-PRM1 to alanine on nucleic acid-protein nanoparticle formation

Mutating half of arginine residue of PRM1 sequence in SUMO-PRM1 into alanine (specific sequence is SEQ ID No. 4), and the mass ratio of protein to pEGFP plasmid linearization product is SUMO-PRM1 (mut): nucleic acid = 4:1, wherein SUMO: PRM1 (mut) mass ratio is 2:1, according to the assembly mode 2.

The assembly results are shown in FIG. 4, and the protein after mutation can still form particles with a diameter of about 30nm with nucleic acid on the basis of using assembly mode 2.

From the above, it can be seen that if more than 12 arginine residues are present in any 50 amino acid sequence in a protein, the protein can form nanoparticles of uniform size under the assembly method herein.

The protein in the nanoparticle can be assembled with deoxyribonucleotides or ribonucleotides with different lengths, single chains or double chains and different sequences to form the nanoparticle with the size of about 30nm by a method of reducing the salt concentration in a buffer solution only by adding a certain amount of positive amino acid such as arginine into the sequence of the protein. The three-dimensional structure of the amino terminal of the protein in the nanoparticle can be completely identified, thereby playing a corresponding function.

Example 3 use of protein nucleic acid nanoparticles

The protein nucleic acid nanoparticles obtained in example 1 (SUMO-PRM 1 and 59nt ssDNA) were treated with MNase (Micrococcus nuclease, thermoFisher Co., ltd., cat. No. 88216) and ULP 1. The method comprises the following specific steps: the particles were assembled according to assembly mode 2, and diluted with a buffer solution of pH 8.0 by adding 100mM NaCl,20mM Tris-HCl to give a final concentration of 20 ng/. Mu.L (the result obtained by measuring single-stranded DNA using a Nanodrop ultra-micro ultraviolet spectrophotometer, model of which is the Nanodrop 2000 spectrophotometer). A: after incubation on ice for 3h, 1. Mu.L of ULP1 was mixed in a proportion of 1. Mu.L of ULP1 (4 mg/mL) per 50. Mu.L of sample, and SDS-PAGE was performed with 1. Mu.L of ULP 1-added particles and 5. Mu.L of non-added particles. B: adding MNase into low-salt buffer solution containing particles and low-salt buffer solution containing 20 ng/mu L59 nt ssDNA according to the proportion of adding 1 mu L MNase into each 50 mu L sample, incubating for 3 hours on ice, and taking the particle buffer solution added with MNase, the buffer solution with particles without adding MNase and the buffer solution with 59nt ssDNA for urea gum detection.

Efficient cleavage of SUMO by ULP1 enzyme depends on its recognition of the SUMO three-dimensional sequence, as can be seen in fig. 5, under which conditions the proteins in the particles are efficiently cleaved by ULP1, while under which conditions 59nt ssDNA alone is completely degraded by MNase, whereas no serious degradation of 59nt ssDNA assembled to form nanoparticles occurs.

Therefore, the protein nucleic acid nano-particle has a certain protection effect on nucleic acid, and simultaneously, the three-dimensional structure of the amino end of the protein can be identified by other proteins and can play a corresponding role, thus indicating the corresponding capability of the particle in protein delivery.

EXAMPLE 4 results of cellular experiments with protein nucleic acid nanoparticles

1. Cell uptake experiments in HEK 293T cells (the nucleic acid used was one with a fluorescent label)

The DNA molecule used in the experiment is DNA with Alexa fluorine 488 fluorescence modification at the 5' end (custom made product of biological engineering (Shanghai) Co., ltd., order number: 111989851, specific sequence shown in SEQ ID No. 8), and the protein used is SUMO-PRM1, specific sequence shown in SEQ ID No.1. The mass ratio of protein SUMO-PRM1 to 59nt ssDNA was =4: 1, according to the assembly mode 2. The experimental groupings were as follows:

(A) After adding nucleic acid-protein nanoparticles to HEK 293T cells for 2 hours, staining with Hoechst dye (purchased from Biyun Tian Biotechnology Co., product number: C1022, see product description for specific methods of use), and observing;

(B) After adding nucleic acid-protein nanoparticles to HEK 293T cells for 4 hours, the cells were visualized after staining with Hoechst dye;

(C) 6 hours after adding the nucleic acid-protein nanoparticles to HEK 293T cells, the mixture was observed after staining with Hoechst dye;

(D) Equal amounts of DNA were added separately to HEK 293T cells and observed 6 hours after staining with Hoechst dye;

(E) The protein and DNA were directly mixed in the ratio used for assembly, and after 6 hours of addition to the cells, they were observed.

Observations were made under conditions of Merge, bright field, 488nm wavelength excitation and 346nm wavelength excitation. The results are shown in FIG. 6, and the results of (A) (B) (C) indicate that the nucleic acid is taken up by the cells two hours after the addition of the particles, and that the nucleic acid taken up by the cells is more and more as time goes by, compared with the direct addition of the equivalent amount of DNA of (D). Also comparing with the results of (E) it can be concluded that uptake of nucleic acids by cells is dependent on assembly of 30nm particles. The particles were shown to be capable of uptake by HEK 293T cells, thereby delivering nucleic acids into the cells.

2. Cell uptake assay of Hela cells

The DNA molecule used in this experiment was DNA with a fluorescent modification of Alexa fluorine 488 at the 5' end (custom made product of Biotechnology (Shanghai) Co., ltd., order number 111989851, specific sequence shown in SEQ ID No. 8). The protein used is SUMO-PRM1, the specific sequence of the GE11-SUMO-PRM1 is shown in SEQ ID No.1, and the protein sequence of the GE11-SUMO-PRM1 is shown in SEQ ID No.5. The 59nt ssDNA sequence used is shown in SEQ ID No.8. The mass ratio of GE11-SUMO-PRM1 to 59nt ssDNA is GE11-SUMO-PRM1: nucleic acid = 5:1, SUMO-PRM1 to 59nt ssDNA mass ratio of SUMO-PRM1: nucleic acid = 4:1, according to the assembly mode 2. The experimental groupings were as follows:

(A) Adding nucleic acid-protein nanoparticles formed by SUMO-PRM1 into Hela cells, allowing the mixture to act for 2 hours, and observing the mixture after the mixture is dyed by Hoechst dye;

(B) The nucleic acid-protein nanoparticles formed by GE11-SUMO-PRM1 were added to Hela cells and observed 2 hours after staining with Hoechst dye;

(C) Adding nucleic acid-protein nanoparticles formed by SUMO-PRM1 into Hela cells, allowing the mixture to act for 4 hours, and observing the mixture after the mixture is dyed by Hoechst dye;

(D) Adding nucleic acid-protein nanoparticles formed by GE11-SUMO-PRM1 into Hela cells, allowing the mixture to act for 4 hours, and observing the mixture after the mixture is dyed by Hoechst dye;

(E) After adding nucleic acid-protein nanoparticles formed from SUMO-PRM1 to Hela cells, the reaction was observed after 6 hours of staining with Hoechst dye;

(F) After adding nucleic acid-protein nanoparticles formed by GE11-SUMO-PRM1 to Hela cells, the reaction was observed after 6 hours of staining with Hoechst dye;

(G) The addition of a separate equivalent amount of DNA to HeLa cells was observed after 6 hours incubation with HeLa cells stained with Hoechst dye.

Observations were made under conditions of Merge, bright field, 488nm wavelength excitation and 346nm wavelength excitation. The results are shown in FIG. 7, and the results of (A) (B) (C) (D) (E) indicate that nucleic acid is taken up by cells two hours after the addition of the particles, and that more nucleic acid is taken up by cells over time, as compared with the direct addition of the equivalent amount of DNA of (G). It can thus be concluded that the particles are capable of transporting nucleic acids into Hela cells. Meanwhile, compared with SUMO-PRM1, the particle formed by GE11-SUMO-PRM1 and nucleic acid is promoted by the amount of cell uptake at the same time, which indicates that absorption of particles by Hela cells can be promoted after fusion of GE11 at the N end of PRM1, and GE11 small peptide can realize targeting of the Hela cells in vivo.

In conclusion, through adding a peptide segment sequence with targeting ability at the N end of the protein during recombinant expression, the uptake ability of the targeting cell to the particle can be enhanced, so that the directional recognition of the particle to the target cell is completed.

3. Cell uptake experiments in HEK 293T cells (the nucleic acid used was that with a fluorescent label and the protein used was that with a fluorescent label)

The DNA molecule used in this experiment was 59 nt ssDNA with Alexa fluorine 488 fluorescence modification at the 5' end, (custom made product by Shanghai Co., ltd., order number: 111989851, specific sequence shown in SEQ ID No. 8). SUMO-PRM1 used was prepared by Alexa Fluor ^TM 546 C5 (available from ThermoFisher company under the product number A10258, specific methods of use see product description) was modified. The protein sequence is shown in SEQ ID No.1.

The mass ratio of the protein SUMO-PRM1 to 59 nt ssDNA is 4:1, according to the assembly mode 2. The experimental groupings were as follows:

(A) Observations were made 8 hours after addition of the nucleic acid-protein nanoparticles to HEK 293T cells.

(B) Observations were made 28 hours after nucleic acid-protein nanoparticles were added to HEK 293T cells.

Observation was performed under conditions of Merge, bright field, 488nm wavelength and 564nm wavelength excitation light. As a result, as shown in fig. 8, after 8 hours from the addition of the nanoparticles, the cells were already able to complete the uptake of the particles, and the uptake of the particles by the cells was enhanced over time; the observation of both 488nm wavelength and 564nm wavelength excitation light shows that the particles are capable of transporting not only nucleic acids into cells, but also proteins into cells.

Taken together, it can be concluded that the protein nucleic acid nanoparticle is capable of transporting proteins and nucleic acids into different cells.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims (10)

1. A protein nucleic acid nanoparticle, the protein nucleic acid nanoparticle being a nanoparticle made of a nucleic acid molecule and a protein molecule; the protein molecule contains a peptide fragment, the length of the peptide fragment is greater than or equal to 50 amino acid residues, the peptide fragment contains greater than or equal to 12 positively charged amino acid residues, and the positively charged amino acid residues are arginine residues.

2. The protein nucleic acid nanoparticle of claim 1, wherein: the diameter of the protein nucleic acid nano-particles is 10nm-100nm.

3. The protein nucleic acid nanoparticle of claim 1 or 2, wherein: the nucleic acid molecule is at least one of 4 nucleic acid molecules selected from the group consisting of single-stranded deoxyribonucleotides, double-stranded deoxyribonucleotides, single-stranded ribonucleotides and double-stranded ribonucleotides.

4. A proteinaceous nucleic acid nanoparticle according to claim 1, 2 or 3, characterized in that: the protein nucleic acid nanoparticle prepared according to the method of any one of claims 5-8.

5. A method for preparing the protein nucleic acid nanoparticle of claim 1 or 2, comprising reacting the nucleic acid molecule of claim 1 or 2 with the protein molecule in a buffer of high ionic strength, and reacting the resulting reaction product in a buffer of low ionic strength to obtain the protein nucleic acid nanoparticle; the high-ionic strength buffer solution contains NaCl and Tris-HCl, the content of NaCl in the high-ionic strength buffer solution is more than or equal to 500mM, the low-ionic strength buffer solution contains NaCl and Tris-HCl, and the content of NaCl in the low-ionic strength buffer solution is less than or equal to 100mM.

6. The method according to claim 4, wherein: the mass ratio of the nucleic acid molecule to the protein molecule (calculated as PRM1 in the protein) is 1:0.3-3.3.

7. The method according to claim 4, wherein: the high-ionic strength buffer solution consists of a solute and a solvent, wherein the solvent is water, and the solute is NaCl or KCl or ammonium sulfate or sodium hydrogen phosphate or disodium hydrogen phosphate or sodium dihydrogen phosphate or potassium hydrogen phosphate or dipotassium hydrogen phosphate or potassium dihydrogen phosphate, tris-HCl or HEPES (4-hydroxyethyl piperazine ethane sulfonic acid) or boric acid or calcium hydroxide or MOPS (3-morpholinopropane sulfonic acid) or CAPS (3- (cyclohexylamine) -1-propane sulfonic acid) or CHES (2-cyclohexylamine ethane sulfonic acid) or Tricine (tri (hydroxymethyl) methylglycine) or triethanolamine or barbital sodium or sodium carbonate; the low-ionic strength buffer solution consists of a solute and a solvent, wherein the solvent is water, and the solute is NaCl or KCl or ammonium sulfate or sodium hydrogen phosphate or disodium hydrogen phosphate or sodium dihydrogen phosphate or potassium hydrogen phosphate or dipotassium hydrogen phosphate or potassium dihydrogen phosphate, tris-HCl or HEPES (4-hydroxyethyl piperazine ethane sulfonic acid) or boric acid or calcium hydroxide or MOPS (3-morpholinopropane sulfonic acid) or CAPS (3- (cyclohexylamine) -1-propane sulfonic acid) or CHES (2-cyclohexylamine ethane sulfonic acid) or Tricine (tri (hydroxymethyl) methylglycine) or triethanolamine or barbital sodium or sodium carbonate.

8. The method according to claim 4, wherein: the reaction in a low ionic strength buffer comprises reacting the reaction product in the high ionic strength buffer with NaCl content of 500-2000mM, and then in the low ionic strength buffer with NaCl content of 0-100 mM.

9. Use of a proteinaceous nucleic acid nanoparticle according to any one of claims 1 to 4 for the preparation of a product for the recognition of tumor cells.

10. Use of a proteinaceous nucleic acid nanoparticle according to any one of claims 1 to 4 for the preparation of a product for delivering a protein or nucleic acid or protein and nucleic acid into a cell.

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