A polypeptide having transfection activity and use thereof

Abstract

This invention discloses a transfection-active peptide and its applications, relating to the field of peptide technology. The invention synthesizes a novel peptide that, like liposomes, can encapsulate nucleic acid fragments in vitro and activate membrane receptors for transport into the cytoplasm. Compared to liposomes, this peptide exhibits lower toxicity and greater stability in vivo.

Description

Technical Field

[0001] This invention relates to the field of polypeptide technology, specifically to a polypeptide with transfection activity and its applications. Background Technology

[0002] Transfection is the process by which cells acquire a new phenotype by actively or passively introducing exogenous nucleic acid fragments under certain conditions. Transfection is widely used in scientific research, disease treatment, and vaccine coating. Currently, the mainstream transfection reagents are liposome reagents such as PEI and LIPO2000.

[0003] PEI is a synthetically produced organic macromolecular cationic polymer, while Lipo2000 and Lipo3000 are cationic liposome transfection reagents. Negatively charged nucleic acid molecules bind to positively charged liposomes to form nucleic acid-cationic liposome complexes, which are then adsorbed onto the negatively charged cell membrane surface and introduced into the cell through endocytosis by activating membrane receptors.

[0004] Numerous studies have reported that liposomes themselves participate in cellular physiological activities, causing upregulation or downregulation of gene expression; for example, they participate in the regulation of the PKC (protein kinase C) pathway; inhibit ATPase activity; interact with the mitochondrial membrane; and cause off-target effects when transfected with siRNA. The magnitude of cytotoxicity often indicates the magnitude of its impact on cellular physiological activities, and these effects of liposomes are the root cause of cytotoxicity.

[0005] Traditional liposome transfection reagents (such as Lipo2000) are easily degraded after entering the body and accumulate in lung tissue, which may cause inflammatory reactions or toxicity problems. Therefore, they cannot be used directly for nucleic acid delivery in live animals. Summary of the Invention

[0006] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a polypeptide with transfection activity and its application.

[0007] The technical solution of the present invention is as follows:

[0008] A transfection-active polypeptide has an amino acid residue in the middle backbone linked to a branched side chain, and the core structure of each branched side chain is -Arg-His-His-Lys-His-His-Lys-His-His-His-Lys-His-His.

[0009] Preferably, the amino acid amino residues of the intermediate main chain are linked to a branched side chain, and the core structure of each branched side chain is -Arg-His-His-His-His-His-His-His-His-His-His-His-His (abbreviated as RHHKHHKHHHKHH, as shown in SEQ ID NO.1 of the amino acid sequence listing), abbreviated as X, and its structure is as follows:

[0010] .

[0011] Preferably, the preparation method includes the following steps:

[0012] Weigh the resin, add DMF to swell for more than half an hour, remove the DMF, and perform Fmoc deprotection reaction with deprotection reagent for 10 minutes on a shaker. Remove the deprotection reagent, wash with DMF 3 times, and then proceed to the amino acid condensation reaction. Take the corresponding amino acid and condensation reagent, dissolve them in the reaction solution, add them to the reactor, stir the reaction, and obtain a peptide resin after amino acid condensation. Repeat the above Fmoc deprotection and amino acid condensation reaction steps on the obtained peptide resin until the last amino acid has reacted completely to obtain the target peptide.

[0013] Preferably, the resin is Cl-Resin chloride ion exchange resin;

[0014] The deprotecting agent is prepared by dissolving a 20 wt% piperidine solution in N,N-dimethylformamide (DMF).

[0015] The condensation reagent is a mixed solution of N,N-diisopropylcarbodiimide and 1-hydroxybenzotriazole.

[0016] The present invention also discloses the application of any of the above-described polypeptides in the preparation of cell transfection agents.

[0017] Preferably, the formulation is an injection or a topical application.

[0018] The beneficial effects of this invention are: this invention synthesizes a novel peptide that, like liposomes, can encapsulate nucleic acid fragments in vitro and transport them into the cytoplasm; this polypeptide is a biological macromolecule, which is more stable and less toxic than liposomes.

[0019] This invention first attracts and adheres to the cell membrane surface through positive and negative charges, and then enters the cell by binding to the cell surface NPR receptor NRP-1, which has certain biological chemotactic tumor enrichment characteristics. NRP-1 often shows high expression in a variety of malignant tumors, and its high expression is usually closely related to tumor progression, angiogenesis, metastasis and poor prognosis. Attached Figure Description

[0020] Figure 1 Electrophoretic analysis of the encapsulation effect of the peptide xsh007 on got1 and siAR plasmids;

[0021] Figure 2 The effect of transfecting 2 mg of plasmid per six-well plate for single-well overexpression of the target molecule in vitro;

[0022] Figure 3 To demonstrate the knockdown effect of siRNF41 transfection in vitro;

[0023] Figure 4 To reduce the knockdown effect of siAR transfection in vitro;

[0024] Figure 5 This is the chromatogram of polypeptide xsh007. Detailed Implementation

[0025] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0026] A transfection-active polypeptide has an amino acid residue in its central backbone linked to a branched side chain. The core structure of each side chain is -Arg-His-His-Lys-His-His-His-His-His-His-His-His. This invention synthesized one sample with a core structure of three lysine residues. The amino groups of its side chains are linked to three branched side chains, and each amino residue in the main chain is also linked to a side chain. Its structure can be represented as X-His-His-His, with each His side chain amino group linked to an X, where X is -Arg-His-His-His-His-His-His-His-His-His-His-His-His (abbreviated as RHHKHHKHHHKHH, as shown in SEQ ID NO.1 of the amino acid sequence listing). The structure is as follows:

[0027] .

[0028] The following specific experiments further illustrate the technical solution of the present invention.

[0029] It should be noted that:

[0030] AR and RNF41 are gene names;

[0031] got1 is plasmid DNA;

[0032] si is a transfection siRNA used to knock down gene expression; siAR is a siRNA used to target and knock down AR expression; siRNF41 is a siRNA used to target and knock down RNF41.

[0033] siRNA is a small interfering RNA transfected to knock down gene expression, while plasmid is a circular DNA molecule used to overexpress a specific sequence.

[0034] Example 1

[0035] Weigh the resin, add DMF to swell for more than half an hour, remove the DMF, and perform Fmoc deprotection reaction with deprotection reagent for 10 minutes on a shaker. Remove the deprotection reagent, wash with DMF 3 times, and then proceed to the amino acid condensation reaction. Take the corresponding amino acid and condensation reagent according to the designed amino acid sequence structure, dissolve them in the reaction solution, put them into the reactor, stir the reaction, and obtain a peptide resin after amino acid condensation. Repeat the above Fmoc deprotection and amino acid condensation reaction steps on the obtained peptide resin until the last amino acid has reacted, and obtain the target peptide (peptide xsh007).

[0036] The resin is: Cl-Resin chloride ion exchange resin;

[0037] The deprotecting agent is prepared by dissolving piperidine in N,N-dimethylformamide (DMF) to obtain a 20 wt% piperidine solution;

[0038] The condensation reagent is a mixed solution of N,N-diisopropylcarbodiimide and 1-hydroxybenzotriazole in a volume ratio of 3:1.

[0039] Application: In vitro transfection, as detailed below:

[0040] After the cells are seeded to 30% in a six-well plate, replace with 1.8 ml of OPTI medium and starve the cells for at least half an hour.

[0041] Prepare solutions A and B. Solution A: 50 nM siRNA at a final concentration, diluted to 100 μl with OPTI transfection medium. Solution B: 0.1 mg peptide, diluted to 100 μl with OPTI medium. After standing for five minutes, mix solutions A and B, and let stand for another half hour. Add the mixture to a culture dish and incubate for 6 hours. Then change the medium to complete culture medium, i.e., 10% fetal bovine serum for this cell culture.

[0042] Experiment 1: The encapsulation effect of this polypeptide on nucleic acid fragments was detected by agarose gel electrophoresis.

[0043] Two micrograms of peptide were added to each group along with siRNA in the corresponding proportion, and the volume was adjusted to 100 microliters with enzyme-free water. After standing for half an hour, the samples were subjected to agarose gel electrophoresis. The results are shown below. Figure 1 .

[0044] Experiment 2: Effect of transfection of the target molecule with 2 mg of plasmid per six-well plate for single-well overexpression.

[0045] Procedure: After seeding 30% of 293t cells into a six-well plate, replace with 1.8 ml of serum-free culture medium and starve the cells for at least half an hour;

[0046] Prepare solutions A and B. Solution A: 2 mg plasmid diluted to 100 μl with serum-free medium. Solution B: 10 mg peptide diluted to 100 μl with serum-free medium. After standing for five minutes, mix solutions A and B, let stand for another half hour, and then add to a culture dish for 6 hours of incubation. Change the medium to complete medium, i.e., 10% FBSDMEM or high-glucose complete medium (containing 10% FBS).

[0047] Protein was extracted 48 hours later and plasmid tags were detected by Western blot.

[0048] NC is the reference group consisting only of peptides and empty vector plasmids, excluding the influence of peptides on protein detection.

[0049] stub1, got1, foxa1, RNF41, and SMAD3 are the corresponding molecular plasmid groups for the peptide transfection tags.

[0050] See results Figure 2 This indicates that each molecular plasmid was successfully expressed, and xsh007 can be transfected with multiple plasmids.

[0051] Experiment 3: Effect of in vitro transfection of this molecule with siRNF41 knockdown

[0052] Procedure: After seeding 30% of PC3 cells in a six-well plate, replace with 1.8 ml of OPTI to starve the cells for at least half an hour;

[0053] Prepare solutions A and B. Solution A: 5 mg siRNF41, diluted to 100 μl with OPTI. Solution B: 10 mg peptide, diluted to 100 μl with OPTI. After standing for five minutes, mix solutions A and B, and let stand for another half hour. Add the mixture to a culture dish and change the medium after 6 hours.

[0054] Protein was extracted 48 hours later and the RNF41 knockdown was detected by wb.

[0055] The internal control ACT is a reference protein Actin used to correct the total amount of protein loaded onto the sample and ensure consistency in the total amount.

[0056] NC stands for the control group containing only peptides, thus excluding the influence of peptides on molecular expression.

[0057] See results Figure 3It can be seen that xsh007 does not interfere with RNF41 expression, and the knockdown effect still depends on the siRNA concentration. At a concentration of 50 nM, there is no significant difference in transfection efficiency compared with the commercially available lipo2000.

[0058] Experiment 4: In vitro transfection and siAR knockdown effect of this molecule

[0059] Procedure: After seeding 22RV1 cells to 30% in a six-well plate, replace with 1.8 ml of OPTI and starve the cells for at least half an hour;

[0060] Prepare solutions A and B. Solution A: 5 mg siAR, diluted to 100 μl with OPTI. Solution B: 10 mg peptide, diluted to 100 μl with OPTI. After standing for five minutes, mix solutions A and B, and let stand for another half hour. Add the mixture to a culture dish and change the medium after 6 hours to complete the culture medium.

[0061] Protein was extracted 48 hours later and the AR knockdown was detected by wb.

[0062] The internal control ACT is a reference protein ACTIN used to correct the total amount of protein loaded onto the sample and ensure consistency in the total amount.

[0063] NC stands for the control group containing only peptides, thus excluding the effect of peptides on AR expression.

[0064] S1 and S2 are siRNAs with different sequences and different knockdown effects, respectively.

[0065] See results Figure 4 It can be seen that the xsh007 transfection effect remains stable under gene replacement and multi-target siRNA transfection.

[0066] Experiment 5: Particle potential of siRNA encapsulated in vitro at a specific ratio.

[0067] Prepare solutions A and B. Solution A: 50 nM siRNA with enzyme-free water to a final volume of 100 μl. Solution B: 0.1 mg peptide with enzyme-free water to a final volume of 100 μl. After standing for five minutes, mix solutions A and B, and then let stand for another half hour before taking samples to detect particle potential.

[0068] The results are shown in Table 1:

[0069] Table 1. Particle size potential of siRNA encapsulated in vitro at various ratios

[0070]

[0071] Table 1 shows that when the mass ratio of siRNA to xsh007 is 1:2, the particle potential is positive, making it easier to bind to the negatively charged cell membrane and improve the transfection effect.

[0072] Experiment 5: Reversed-phase high-performance liquid chromatography analysis

[0073] Sample: xsh007; Instrument: Reversed-phase high-performance liquid chromatography (HPLC);

[0074] Purification method: Gradient elution;

[0075] Sample number: C226B915G0-1;

[0076] Elution buffer A: 0.065% trifluoroacetic acid aqueous solution (v / v);

[0077] Elution buffer B: 0.05% trifluoroacetic acid in acetonitrile solution (v / v);

[0078] Flow rate: 1 ml / min;

[0079] Wavelength: 220nm;

[0080] Gradient elution is shown in Table 2;

[0081] The test results are shown in Table 3 and Figure 5 .

[0082] Table 2 Elution gradient

[0083]

[0084] Table 3 Peak values

[0085]

[0086] From Table 3 and Figure 5 It can be seen that the xsh007 peptide has extremely high purity, thus eliminating the interference of impurities on the experimental results.

[0087] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various other corresponding changes and modifications based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of the present invention.

Claims

1. The application of a transfection-active polypeptide in the preparation of transfection agents, characterized in that, The structure of the polypeptide is as follows: ； Where, X is -Arg-His-His-Lys-His-His-Lys-His-His-His-Lys-His-His.

2. The application according to claim 1, characterized in that, The transfection agent is an injection or a topical application.

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