Polypeptide with transfection activity and application thereof

Abstract

The invention discloses a polypeptide with transfection activity and application thereof, and relates to the technical field of polypeptides, a novel peptide is synthesized, and the novel peptide can wrap nucleic acid fragments in vitro and activate a membrane receptor to be transferred into cytoplasm as a liposome. Compared with lipidosome, the polypeptide has lower toxicity and is more stable in vivo.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polypeptides, in particular to a polypeptide with transfection activity and application thereof. BACKGROUND

[0002] Transfection is a process in which a cell is actively or passively introduced with an exogenous nucleic acid fragment under certain conditions to obtain a new phenotype. Transfection is widely used in scientific research, disease treatment, vaccine coating and other fields. Currently, the mainstream transfection reagents are PEI, LIPO2000 and other liposome reagents.

[0003] PEI is an artificially synthesized organic macromolecular cationic polymer, while Lipo2000 and Lipo3000 are cationic liposome transfection reagents. A negatively charged nucleic acid molecule is combined with a positively charged liposome to form a nucleic acid-cationic liposome complex, which is then adsorbed to the surface of a negatively charged cell membrane and is introduced into the cell by activating the membrane receptor endocytosis.

[0004] Numerous documents have reported that liposomes themselves can participate in cell physiological activities, causing up-regulation or down-regulation of gene expression; for example, participating in PKC (protein kinase C) pathway regulation; for example, inhibiting the activity of ATPase; for example, interacting with mitochondrial membranes; transfection of siRNA causing off-target effects, etc. The size of cytotoxicity often means the size of the impact on cell physiological activities, and these effects of liposomes are the root cause of cytotoxicity.

[0005] Traditional liposome transfection reagents (such as Lipo2000) are easily decomposed after entering the living body and accumulate in the lung tissue, which may cause inflammatory reactions or toxicity problems, and therefore cannot be directly used for nucleic acid delivery in living animals. SUMMARY

[0006] The present application aims to at least solve one of the problems in the prior art, and provides a polypeptide with transfection activity and application thereof.

[0007] The technical solution of the present application is as follows: A polypeptide with transfection activity, in which the amino acid amino residues of the middle main chain are 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.

[0008] Preferably, the amino acid amino residue of the intermediate main chain links one branch side chain, and the core structure of each branch side chain is -Arg-His-His-Lys-His-His-Lys-His-His-His-Lys-His-His (abbreviated as RHHKHHKHHHKHH, as shown in the amino acid sequence table SEQ ID NO. 1), abbreviated as X, and the structure is as follows: .

[0009] Preferably, the preparation method comprises the following steps: The resin is weighed, swelled in DMF for more than half an hour, the Fmoc deprotection reaction is carried out with a deprotection reagent, 10 minutes on a shaker, the deprotection reagent is removed, and the deprotection reagent is washed with DMF for 3 times, and then the amino acid condensation reaction is carried out, the corresponding amino acid and condensation reagent are taken, dissolved in the reaction liquid, and then put into the reactor, and the reaction is stirred to obtain a peptide resin after amino acid condensation. The obtained peptide resin is repeatedly subjected to the above Fmoc deprotection and amino acid condensation reaction steps until the last amino acid reaction is completed, and the target peptide is obtained.

[0010] Preferably, the resin is Cl-Resin, a chloride ion exchange resin; The deprotection reagent is prepared by dissolving a 20wt% piperidine solution in N,N-dimethylformamide (DMF); The condensation reagent is a mixed solution of N,N-diisopropyl carbodiimide and 1-hydroxybenzotriazole; The application further discloses a use of the polypeptide in the preparation of a cell transfection preparation.

[0011] Preferably, the preparation is an injection or a smearing agent.

[0012] The application has the beneficial effects that the application synthesizes a new type of peptide, which can wrap nucleic acid fragments in vitro and transport into the cytoplasm, like liposomes; the polypeptide is a biological macromolecule, is more stable and has smaller toxicity than liposomes.

[0013] The application has certain biological tumor enrichment properties by attracting the cell membrane surface through positive and negative charges and then entering the cell through the combination of the cell surface NPR receptor NRP-1; NRP-1 often presents a high expression state in various malignant tumors, and the high expression is usually closely related to tumor progression, angiogenesis, metastasis and poor prognosis. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Electrophoretic detection of the wrapping effect of polypeptide xsh007 wrapping got1 and siAR plasmids; Figure 2To transfect plasmid 2mg per six-hole plate single hole to overexpress the target molecule in vitro; Figure 3 To transfect siRNF41 to knock down in vitro; Figure 4 To transfect siAR to knock down in vitro; Figure 5 The chromatogram of the polypeptide xsh007. DETAILED DESCRIPTION

[0015] The embodiments of the present application are described in detail below. The embodiments described below are exemplary and are used to explain the present application, and cannot be understood as a limitation of the present application. If the specific technology or condition is not indicated in the embodiments, the technology or condition described in the literature in the art or according to the product instruction is used. If the reagent or instrument used is not indicated by the manufacturer, it is a conventional product that can be obtained by purchase.

[0016] A polypeptide with transfection activity, the amino acid amino residues of the intermediate main chain link a branch side chain, and the core structure of each side chain is -Arg-His-His-Lys-His-His-Lys-His-His-His-Lys-His-His. One sample of the present application is synthesized, the core structure of which is three lysine residues, the amino groups of the side chain of which link three branch side chains, and one amino residue of the main chain also links a side chain, the structure of which can be expressed as: X-His-His-His, and each His side chain amino links an X, X is -Arg-His-His-Lys-His-His-Lys-His-His-His-Lys-His-His (abbreviated as RHHKHHKHHHKHH, as shown in the amino acid sequence table SEQ ID NO. 1), and the structure is as follows: .

[0017] The following specific tests further illustrate the technical solutions of the present application.

[0018] It should be noted that: AR and RNF41 are gene names, respectively; got1 is plasmid DNA; si is siRNA used for transfection to knock down gene expression; siAR is siRNA used to target and knock down AR expression; siRNF41 is siRNA used to target and knock down RNF41; siRNA is small interfering RNA used for transfection to knock down gene expression, and plasmid is a circular DNA molecule used for overexpression of a certain sequence.

[0019] Example 1 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).

[0020] The resin is: Cl-Resin chloride ion exchange resin; The deprotecting agent is prepared by dissolving piperidine in N,N-dimethylformamide (DMF) to obtain a 20 wt% piperidine solution; The condensation reagent is a mixed solution of N,N-diisopropylcarbodiimide and 1-hydroxybenzotriazole in a volume ratio of 3:1. Application: In vitro transfection, as detailed below: 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.

[0021] 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.

[0022] Experiment 1: The encapsulation effect of this polypeptide on nucleic acid fragments was detected by agarose gel electrophoresis. 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 .

[0023] Experiment 2: Effect of transfection of the target molecule with 2 mg of plasmid per six-well plate for single-well overexpression. 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; 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).

[0024] 48h later, extract protein to detect plasmid tag by WB.

[0025] NC is the reference group with only polypeptide and empty vector plasmid, excluding the influence of polypeptide on protein detection.

[0026] stub1, got1, foxa1, RNF41, SMAD3 are respectively corresponding to each molecular plasmid group of polypeptide transfection tag.

[0027] The results are shown in Figure 2 It can be seen that each molecular plasmid is successfully expressed, and xsh007 can transfect various plasmids.

[0028] Test three: in vitro transfection of siRNF41 by the molecule Operation steps: replace the 1.8ml of OPTI of the 30% plated PC3 cells in a six-well plate with starved cells for at least half an hour; Prepare A liquid and B liquid, A liquid: 5mg siRNF41 is made up to 100ul with OPTI, B liquid: 10mg polypeptide is made up to 100ul with OPTI, mix A liquid and B liquid after standing for five minutes, and then stand for half an hour before adding to the culture dish. Replace with complete medium after 6h.

[0029] 48h later, extract protein to detect RNF41 knockdown by WB.

[0030] Internal reference ACT is a reference protein Actin used to correct the total amount of loaded protein to ensure consistent total amount; NC is the reference group with only polypeptide, excluding the influence of polypeptide on molecular expression; The results are shown in Figure 3 It can be seen that xsh007 has no interference with RNF41 expression, and the knockdown effect still depends on siRNA concentration, and there is no obvious difference in transfection efficiency with lipo2000 on the market at a concentration of 50nM.

[0031] Test four: in vitro transfection of siAR by the molecule Operation steps: replace the 1.8ml of OPTI of the 30% plated 22RV1 cells in a six-well plate with starved cells for at least half an hour; Prepare A liquid and B liquid, A liquid: 5mg siAR is made up to 100ul with OPTI, B liquid: 10mg polypeptide is made up to 100ul with OPTI, mix A liquid and B liquid after standing for five minutes, and then stand for half an hour before adding to the culture dish. Replace with complete medium after 6h.

[0032] 48h later, extract protein to detect AR knockdown by WB. Internal reference ACT is a reference protein ACTIN used to correct the total amount of loaded protein to ensure consistent total amount; NC is the reference group of only polypeptide, excluding the influence of polypeptide on AR expression; S1, S2 are siRNAs with different sequences and different knockdown effects; The results are shown in Figure 4 It can be seen that the transfection effect of xsh007 is still stable under the replacement of genes and multi-target siRNA transfection.

[0033] Test five: particle potential of siRNA wrapped in proportion in vitro.

[0034] Prepare A liquid and B liquid. A liquid: final concentration of 50nM siRNA, constant volume to 100ul with enzyme-free water. B liquid: 0.1mg polypeptide, constant volume to 100ul with enzyme-free water. After standing for five minutes, mix A liquid and B liquid, and then stand for half an hour before sampling and testing particle potential.

[0035] The results are shown in Table 1: Table 1: Particle size and potential of siRNA wrapped in proportion in vitro

[0036] From Table 1, it can be seen that when the mass ratio of siRNA:xsh007 is 1:2, the particle potential is positive, which is more easily combined with the negatively charged cell membrane to improve the transfection effect.

[0037] Test five: reverse phase high performance liquid chromatography analysis Sample: xsh007; Instrument: reverse phase high performance liquid chromatography (HPLC); Purification method: gradient elution; Sample number: C226B915G0-1; Eluent A: 0.065% trifluoroacetic acid aqueous solution (v / v); Eluent B: 0.05% trifluoroacetic acid in acetonitrile solution (v / v); Flow rate: 1ml / min; Wavelength: 220nm; Gradient elution is shown in Table 2; The detection results are shown in Table 3 and Figure 5 .

[0038] Table 2: Elution gradient

[0039] Table 3: Peak value

[0040] From Table 3 and Figure 5 It can be seen that the purity of xsh007 polypeptide is very high, excluding the interference of impurities on the experimental results.

[0041] The above described embodiments only express the preferred embodiments of the present application, which are described in a more specific and detailed manner, but should not be understood as a limitation to the patent scope of the present application. It should be noted that for ordinary skilled in the art, other various corresponding changes and modifications can be made according to the above described technical solutions and concepts, and all of these changes and modifications should belong to the protection scope of the claims of the present application.

Claims

1. A polypeptide having transfection activity, characterized in that, The amino acid amino residue of the middle main chain links one branch side chain, and the core structure of each branch side chain is -Arg-His-His-Lys-His-His-Lys-His-His-His-Lys-His-His.

2. The polypeptide having transfection activity according to claim 1, characterized in that, The core structure is three lysine residues, the amino groups of the side chains of which correspond to link three branch side chains, and one amino residue of the main chain links one side chain, and the structure is expressed as: X-His-His-His, and each His side chain amino group links one X, X is -Arg-His-His-Lys-His-His-Lys-His-His-His-Lys-His-His, and the structure is as follows: 。 3. The polypeptide having transfection activity according to claim 1, wherein The preparation method comprises the following steps: The resin is weighed, swelled in DMF for more than half an hour, the Fmoc deprotection reaction is carried out with a deprotection reagent, 10 min on a shaker, the deprotection reagent is removed, and the resin is washed with DMF for 3 times, then the amino acid condensation reaction is carried out, the corresponding amino acid and condensation reagent are taken, dissolved in the reaction liquid, and then put into the reactor, and the reaction is carried out to obtain a peptide resin after amino acid condensation, and the obtained peptide resin is repeatedly subjected to the above Fmoc deprotection and amino acid condensation reaction steps until the last amino acid is reacted, so that the target peptide is obtained.

4. The polypeptide with transfection activity according to claim 3, wherein, The resin is a chloride ion exchange resin. The deprotection reagent is prepared by dissolving 20wt% piperidine in N,N-dimethylformamide; The condensation reagent is a mixed solution of N,N-diisopropyl carbodiimide and 1-hydroxybenzotriazole.

5. The polypeptide according to any one of claims 1-4 for use in the preparation of a transfection preparation.

6. Use according to claim 5, characterized in that, The preparation is an injection or a smearing agent. The preparation is an injection or a smearing agent.

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