CN112457379B - Cell-penetrating peptide derived from duck circovirus Cap protein and design method and application thereof - Google Patents

Abstract

The application discloses a cell-penetrating peptide derived from Cap protein of duck circovirus and a design method and application thereof. The amino acid sequence of the cell-penetrating peptide is as follows: LRRRFRRRRLRIARPRRRF, respectively; the cell-penetrating peptide is derived from the Capsid structural protein of the duck circovirus, and can obviously improve the film-penetrating efficiency of transferring target objects such as nucleic acid immunopotentiators. It mediates Poly (I: C) and CpG ODN entry into macrophages to induce an increase in IFN- β transcript levels that is approximately 4-fold greater than TAT, 8-fold greater than either immunostimulant alone. The cell-penetrating peptide Du-Cap aa18-37 is safe and efficient, can remarkably improve transmembrane efficiency, further enriches the variety of the cell-penetrating peptide, and can be developed into an ideal conduction vector of a safe and efficient gene drug and a nucleic acid immunostimulant.

Description

Cell-penetrating peptide derived from Cap protein of duck circovirus, and design method and application thereof

Technical Field

The invention relates to a cell-penetrating peptide, in particular to a cell-penetrating peptide derived from a Cap protein of duck circovirus and application thereof.

Background

Immunopotentiators (immunoadjuvants) are a class of immunomodulatory factors, compounds or macromolecular complexes that can increase the immunogenicity of antigens. The immune adjuvant is used for assisting antigen response by stimulating the body's innate immune system. The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) expressed by pathogenic microorganisms through Pattern Recognition Receptors (PRRs) in the body. Toll-like receptors (TLRs) are an important receptor in pattern recognition, and 11 of the most currently found receptors are, for example, TLR3 which mainly recognizes Poly (I: C), TLR9 which recognizes CpG ODN, and TLR4 which recognizes bacterial LPS. Poly (I: C) is a double-stranded RNA analog formed by pairing artificially synthesized Poly-hypoxanthine nucleotide and Poly-cytosine nucleotide, can simulate virus infection, has the function of inducing Interferon (Interferon, IFN) generation, is used as a novel immunologic adjuvant, and is researched in various antiviral immunizations at present. Interferons are important cytokines produced in viral infection or immune-stimulatory responses that interfere with the synthesis of viral RNA and proteins and thereby inhibit viral replication. Interferons can be divided into 2 types, and IFN-alpha and IFN-beta which play a broad-spectrum antiviral role are type I interferons.

After the body pattern recognition receptor recognizes Poly (I: C), 2 tyrosine residues on TLR3 positioned in cytoplasm are phosphorylated, a signal transduction pathway in innate immunity is further activated, and antiviral cytokines such as interferon are activated, so that the replication of viruses is inhibited. CpG refers to a nucleic acid sequence composed of mostly oligomers based on unmethylated cytosine and guanine nucleotides, also called immunostimulatory DNA sequences, and like bacterial DNA, unmethylated CpG enters cells by endocytosis to bind to TLR9 in the cytoplasm and activate signaling to perform a cascade reaction. Accelerating the proliferation and maturation of macrophages and dendritic cells, improving the production level of cytokines, directly activating B lymphocytes, and further enhancing cytotoxic reaction and natural killing reaction under the assistance of the cytokines to complete the removal of pathogenic microorganisms. Since CpG has a good immunostimulating effect, it can be used as an immunoadjuvant in animal vaccines. The existing research shows that after the CpG DNA recombinant plasmid is matched with commercial pig foot-and-mouth disease inactivated vaccine to inoculate a pig body, the titer of the specific antibody induced by the enhanced antigen can reach more than 4 times of that of the standard vaccine.

Although Poly (I: C) and CpG ODN have good immunostimulation effect, pattern recognition receptors TLR3 and TLR9 for recognizing the two immunostimulators are positioned in cytoplasm, and Poly (I: C) and CpG ODN must enter target cells to reach the action site. In addition, these 2 nucleic acid immunostimulants are nucleic acids in nature, rich in negative charges, and the cell membrane is also negatively charged. Because the Poly (I: C) and the CpG ODN carry the same charges, the Poly (I: C) and the CpG ODN can not actively and efficiently enter target cells, and can only rely on endocytosis to stimulate the generation of cascade reaction.

Therefore, how to efficiently mediate the two nucleic acid immunostimulants to enter target cells, especially macrophages, becomes a key problem for the application of Poly (I: C) and CpG ODN as ideal immunologic adjuvants. Macrophages are an important immune cell in the body of an animal and play an important role in both innate immunity and adaptive immunity. When the animal body is infected by pathogenic microorganisms (bacteria or viruses), macrophages are the first defense mechanisms.

Disclosure of Invention

The application provides a novel cell-penetrating peptide, which remarkably improves the penetrating efficiency of a transfer target object.

Although Poly (I: C) and CpG ODN have good immunostimulation effect, the pattern recognition receptors TLR3 and TLR9 for recognizing the two immunostimulators are located in cytoplasm, and Poly (I: C) and CpG ODN must enter target cells to reach the action site. In addition, the two nucleic acid immunostimulants are nucleic acids in nature, and are themselves rich in negative charges, while the cell membrane is also negatively charged. Because the Poly (I: C) and the CpG ODN carry the same charges, the Poly (I: C) and the CpG ODN can not actively and efficiently enter target cells, and can only rely on endocytosis to stimulate the generation of cascade reaction. The newly discovered and identified cell-penetrating peptide Du-Cap aa18-37 is rich in positive charges, can realize electrostatic binding with Poly (I: C) and CpG ODN, and can quickly mediate the cell-penetrating peptide to enter target cells to generate cascade reaction to play a role of immune stimulation.

The present application provides a cell-penetrating peptide comprising the amino acid sequence as set forth in SEQ ID NO: 1; the cell-penetrating peptide is rich in arginine residues and a plurality of alpha-helical structures; further, the amino acid sequence of the cell-penetrating peptide is shown as SEQ ID NO: 1 is shown. Specifically, the amino acid sequence of the cell-penetrating peptide is as follows: LRRRFRRRRLRIARPRRRF are provided.

The cell-penetrating peptide is derived from a Capsid structural protein of a duck circovirus; used as gene medicine or nucleic acid immunopotentiator conducting carrier.

Thus, the present application also provides a genetic drug or nucleic acid immunopotentiator transmembrane conductance vector comprising an amino acid sequence as shown in SEQ ID NO 1; further, the amino acid sequence is shown as SEQ ID NO 1. Such nucleic acid immunopotentiators include, but are not limited to, Poly (I: C) and CpG ODN.

The application also provides a design method of the cell-penetrating peptide used as a gene medicine or nucleic acid immunopotentiator conduction vector, and the amino acid sequence of the cell-penetrating peptide is LRRRFRRRRLRIARPRRRF; the cell-penetrating peptide is derived from a Capsid structural protein of the duck circovirus.

Sequence analysis shows that the N-terminal (aa18-37) of the Capsid protein of the duck circovirus has strong conservation, is rich in arginine with positive charges and has a spatial structure similar to TAT short peptide. After bioinformatics analysis, Du-Cap aa18-37 has the potential to be developed into cell penetrating peptides.

The application also provides application of the cell-penetrating peptide with the amino acid sequence shown as SEQ ID NO 1 as a gene medicine or nucleic acid immunopotentiator conduction carrier.

The application provides an application of cell-penetrating peptide with an amino acid sequence shown as SEQ ID NO 1 in preparation of gene drugs or nucleic acid immunopotentiator conduction vectors.

Alternatively, the nucleic acid immunopotentiator includes, but is not limited to, Poly (I: C) and CpG ODN.

Optionally, the target cell of the genetic drug or nucleic acid immunopotentiator is a macrophage.

The application also provides a polypeptide with an amino acid sequence shown as SEQ ID NO: 1 in the preparation of an animal vaccine containing a nucleic acid immunopotentiator.

Alternatively, the nucleic acid immunopotentiator in the animal vaccine includes, but is not limited to, Poly (I: C) and CpG ODN.

Optionally, the animal vaccine includes, but is not limited to, inactivated vaccine of swine foot and mouth disease, inactivated vaccine of swine delta coronavirus, inactivated vaccine of piglet escherichia coli, attenuated vaccine of swine fever, attenuated vaccine of porcine reproductive and respiratory syndrome, vaccine of porcine pseudorabies virus, recombinant subunit vaccine of avian leukemia virus, and vaccine of avian influenza virus (currently, vaccine applied by other people in combination with CpG alone and poly (ic)), and the like.

Alternatively, the gene drugs include, but are not limited to, apoptin and therapeutic siRNA.

Drawings

FIG. 1 is a diagram showing alignment of sequences of the N-terminal (aa18-37) of Capsid of different reference strains of duck circovirus and comparison of spatial structures of the sequences and TAT short peptides;

FIG. 2 is a diagram of the comparison of cell penetrating function of Du-Cap aa18-37 and TAT by confocal laser microscopy;

FIG. 3 is a comparison graph of the transmembrane efficiency of Du-Cap and TAT short peptides detected by flow cytometry;

FIG. 4 is a graph showing the safety results of the cell pair Du-Cap assay using MTT;

FIG. 5 is a diagram showing the results of gel retardation electrophoresis and transfection experiments after the combination of Du-Cap and plasmid DNA;

FIG. 6 is a graph showing the results of gel retardation electrophoresis of Du-Cap bound to poly (I: C) and the change in IFN-. beta.transcription level;

FIG. 7 is a diagram showing the results of agarose gel electrophoresis detection of Du-Cap binding to CpG ODN and the results of IFN-. beta.transcript level changes.

Detailed Description

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the present application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

Cell Penetrating Peptides (CPPs) are a class of short peptides consisting of 5-30 positively charged basic amino acids. It may be a polypeptide of natural or synthetic origin. The cell membrane can efficiently penetrate through a cell membrane to enter cells, and can efficiently carry different kinds of exogenous macromolecules such as DNA, RNA, protein, peptidoglycan, quantum dots and the like to enter cells of different species. The short peptide has the characteristics of low biological toxicity, high conduction efficiency, avoidance of generation of immune response of an excited organism and wide target of cell penetrating through a membrane, and is a gene drug conduction carrier with wide application prospect in recent years.

The membrane-penetrating peptide can be classified into cationic type, hydrophobic type and amphiphilic type according to the physicochemical characteristics of the composition of the membrane-penetrating peptide. And the cationic TAT short peptide is one of the hottest and most widely used cell-penetrating peptides in the current research.

The nucleic acid immunopotentiators Poly (I: C) and CpG ODN have good immunostimulation effect, but the pattern recognition receptors TLR3 and TLR9 for recognizing the two immunostimulators are positioned in cytoplasm, and Poly (I: C) and CpG ODN must enter target cells to reach the action site. In addition, the two nucleic acid immunostimulants are nucleic acids in nature, and are themselves rich in negative charges, while the cell membrane is also negatively charged. Because the Poly (I: C) and the CpG ODN carry the same charges, the Poly (I: C) and the CpG ODN can not actively and efficiently enter target cells, and can only rely on endocytosis to stimulate the generation of cascade reaction.

Sequence analysis shows that the N-terminal (aa18-37) of the Capsid protein of the duck circovirus has strong conservation, is rich in arginine with positive charges and has a spatial structure similar to TAT short peptide. After bioinformatics analysis, Du-Cap aa18-37 has the potential to be developed into cell penetrating peptides. The newly discovered and identified cell-penetrating peptide Du-Cap aa18-37 is rich in positive charges, can realize electrostatic binding with Poly (I: C) and CpG ODN, and can quickly mediate the cell-penetrating peptide to enter target cells to generate cascade reaction to play a role of immune stimulation.

Du-Cap aa18-37 was artificially synthesized after FITC modification and co-incubated with Hela, HD11 (chicken macrophages) and 3D4/21 (porcine alveolar macrophages), respectively. The result shows that the cell can be penetrated in 20min when the concentration is 5 mu M, and the membrane penetrating efficiency is obviously higher than that of TAT short peptide under the same concentration condition. It is worth mentioning that Du-Cap aa18-37 has a 2.6-fold higher ability to mediate plasmid DNA into cells than TAT, and mediates Poly (I: C) and CpG ODN into macrophages to induce IFN-. beta.transcriptional level to be increased by approximately 4-fold over TAT, which is 8-fold higher than that of separately used immunostimulants.

Therefore, the application discovers and identifies a novel cell-penetrating peptide which is derived from duck circovirus Du-Cap aa18-37, is safe and efficient, can obviously improve the transmembrane efficiency, further enriches the types of the cell-penetrating peptide, and can be developed into an ideal conduction vector of a safe and efficient gene medicament and a nucleic acid immunostimulant.

The following is a description of specific examples:

example 1 bioinformatics analysis and Synthesis of Duck circovirus Capsid aa18-37 candidate cell-penetrating peptides

Alignment analysis of capsids of different duck circovirus reference strains in GenBank was performed using the online server Multalin (http:// www.sacs.ucsf.edu/cgi-bin/Multalin. py), which was found to be highly conserved at the N-terminal sequence (aa18-37) and enriched in positively charged arginine (R), marked with black borders, as shown in fig. 1.

Using an online server PEP-FOLD 3(https://bioserv.rpbs.univ-paris-diderot.

fr/services/PEP-FOLD3/) the three-dimensional structure of the N-terminal sequence (aa18-37) was simulated, and as shown in FIG. 1, it was found to have similar alpha-helical structural features to the commonly used cell-penetrating peptide TAT.

Specifically, in fig. 1: a: comparing sequences of the N ends of different duck circovirus reference strains; consenssus: the consistency of different sequence alignments shows that the amino acid conservation of the position is strong by capital letters and strong by lowercase letters. B: the spatial structure of the Du-Cap aa18-37 short peptide is simulated and compared with that of the TAT short peptide.

From FIG. 1, it can be seen that Du-Cap aa18-37 is not only highly conserved, but also has multiple arginine residues and α -helix structure similar to that of the cell-penetrating peptide TAT.

By combining the two characteristics similar to TAT, the sequence (LRRRFRRRRLRIARPRRRF, named Du-Cap aa18-37, Du-Cap for short) obtained after alignment is artificially synthesized as a candidate sequence of cell-penetrating peptide, and the TAT short peptide commonly used at present is used as a positive control.

The sequence information to be synthesized is therefore as follows: Du-Cap aa 18-37: LRRRFRRRRLRIARPRRRF (SEQ ID NO: 1); TAT: YGRKKRRQRRR (SEQ ID NO: 2), all short peptides were FITC-modified and synthesized by Shanghai Biotech Ltd.

Example 2 confocal laser confocal microscopy of Du-Cap aa18-37 cell penetrating function and characteristics

(1) Sterile cell crawl plates were placed in 12-well cell culture plates, and 5X 10⁵HeLa cells, HD11 cells and 3D4/21 cells were seeded into cell culture plates.

(2) After overnight incubation, the synthetic short peptide (Du-Cap aa18-37) was diluted to 5. mu.M and mixed with 500. mu.L of serum-free medium and added to the wells, TAT as a positive control and FITC dye as a negative controlAnd (4) performing sexual control. The cell mixture was incubated at 37 ℃ with 5% CO₂The incubator of (2) was incubated for 20 min. After incubation, washing was performed 3 times with 1mL of PBS each time, and short peptides that did not enter the cells were removed.

(3) Staining was performed for 5min using the nuclear marker dye Hoechst 33342 and washing was performed with PBS.

(4) The cell slide was taken out, inverted on a slide glass, mounted with clear nail polish, and scanned and photographed using a TCS SP8STED laser confocal microscope at different wavelengths (488nm and 405 nm).

As shown in FIG. 2, it can be seen from FIG. 2 that Du-Cap successfully carried FITC into Hela, HD11 and 3D4/21 cells at 5. mu.M. The cell nuclei labeled by Hochest33342 appear as circular gray, and the FITC-short peptides are labeled as white bright gray.

Example 3 flow cytometry detection of different concentrations of Du-Cap aa18-37 compared to TAT transmembrane efficiency

(1) Will be 5X 10⁵Hela cells were seeded in 12-well plates, and after overnight culture, washed 1 time with PBS.

(2) The short peptides were added to the cells at different concentrations (0.1. mu.M/L, 1. mu.M/L, and 10. mu.M/L) in 500. mu.L of Opti-MEM medium, respectively, without the addition of the short peptide as a negative control, at 37 ℃ and 5% CO₂Under the condition, the mixture is incubated for 20 min.

(3) PBS wash 3 times, each for 1 min. Add 200. mu.L pancreatin and 100. mu.L PBS and mix well to digest the cells for 3 min. The digestion was stopped by adding fresh serum-containing cell culture medium and the cell suspension was collected in a 1.5mL centrifuge tube and centrifuged at 400 Xg for 2 min.

(4) The supernatant was discarded. Adding 1mL of PBS, and washing once; cleaning is repeated for one time; add 400. mu.L PBS to resuspend the cells, mix well, filter each tube of cells separately in a flow tube using a cell screen. 10000 cells were measured using a CyAn ADP7 flow cytometer. Each set of samples was replicated 3 times.

The results of the membrane penetration efficiency of Du-Cap and TAT short peptides measured by flow cytometry are shown in FIG. 3, in which: A. detecting a portal graph of the transmembrane efficiency of Du-Cap and TAT short peptides at different concentrations by flow cytometry; the X-axis represents the fluorescence intensity of FITC; the Y-axis represents cell number; p3-representing the region with FITC fluorescence signal. B. Performing comparative statistical analysis on the membrane penetration efficiency of Du-Cap and TAT short peptides with different concentrations; the X axis represents different concentrations of Du-Cap and TAT short peptides; the Y-axis represents the positive cell FITC fluorescence intensity.

As can be seen from the figure, the membrane penetration efficiency of Du-Cap and TAT short peptide is improved along with the increase of concentration, and under the condition of the same concentration, the membrane penetration efficiency of Du-Cap is obviously higher than that of TAT short peptide, the membrane penetration efficiency of Du-Cap is 3 times of TAT at 1 mu M, and the membrane penetration efficiency of Du-Cap is 1.7 times of TAT at 10 mu M.

Example 4 MTT assay Du-Cap safety on cells

(1) Will be 5X 10³And (3) inoculating the Hela cells into a 96-well plate, and respectively adding short peptides with different concentrations after the cells are attached to the wall for 12 hours, wherein each concentration is provided with 3 multiple wells.

(2)37℃，5％CO₂After further incubation for 12h, 24h, 48h, 20. mu.L of thiazole blue solution (MTT, 5mg/mL) was added to each well and incubation was continued for 3 h. Cell supernatants were aspirated.

(3) Dimethyl sulfoxide (DMSO, 100. mu.L/well) was added. After standing at room temperature for 30min, the absorbance (OD490) was measured at 490nm using a microplate reader, and the Cell viability (Cell viability) of each experimental group was calculated using cells without short peptide treatment as a negative control.

(4) Cell survival (%). cell absorbance value of experimental group/cell absorbance value of control group × 100%.

The results are shown in FIG. 4, where the X-axis represents different concentrations of Du-Cap; the Y-axis represents the viability of the cells. As can be seen from FIG. 4, Du-Cap had no effect on the activity of the cells at 5-40. mu.M, and even at 40. mu.M, the activity of the cells after 48 hours of culture was around 100%.

Example 5 gel electrophoresis and transfection experiments to detect Du-Cap binding to plasmid DNA and mediate its endocytosis

Gel blocking electrophoresis:

(1) mu.M of the Du-Cap short peptide and 1. mu.g of pCDNA3.1-RFP (expressing red fluorescent protein) plasmid DNA were added to 20. mu.L of 20mM HEPES buffer, and incubated at room temperature for 40min, after electrophoresis through 1% agarose gel, ethidium bromide was stained, and the binding of the Du-Cap short peptide to the plasmid DNA was determined by observing the migration delay of the plasmid DNApCDNA3.1-RFP electrophoresis.

Transfection experiments:

(2) mu.M of Du-Cap short peptide and 1. mu.g of pCDNA3.1-RFP plasmid DNA were added to 100. mu.L of Opti-MEM and incubated at room temperature for 40 min. This mixture was then made up to 200. mu.L with Opti-MEM and the suspension was added to the cell wells. After 3h, complete medium was added at 37 ℃ with 5% CO₂The culture was continued in the incubator for 48 hours, after which photographing observation was performed using a fluorescence microscope. The positive cells expressing the red fluorescent protein were statistically analyzed by flow cytometry.

FIG. 5 is a diagram of the structure of gel-block electrophoresis and transfection experiments for detecting the binding of Du-Cap to plasmid DNA and mediating its endocytosis, in which A: agarose gel electrophoresis picture after Du-Cap binds to pCDNA3.1-RFP; lane 1: Du-Cap/pCDNA3.1-RFP complex; lane 2: naked pCDNA3.1-RFP; b, after the Du-Cap/pCDNA3.1-RFP and TAT/pCDNA3.1-RFP complexes were co-incubated with the cells, the expression of the red fluorescent protein RFP was observed by a fluorescence microscope (white bright spots are shown in the example figures); flow cytometry is used to detect the efficiency of expressing red fluorescent protein after Du-Cap/pCDNA3.1-RFP and TAT/pCDNA3.1-RFP incubation of cells (the expression of red fluorescent protein in cells is shown as white bright spots). As can be seen from FIG. 5, Du-Cap can bind to the plasmid DNApCDNA3.1-RFP in vitro by electrostatic interaction, and hysteresis occurs in electrophoresis. Through transfection experiments, Du-Cap, like TAT, can mediate plasmid pCDNA3.1-RFP into cells and does not affect the expression of Red Fluorescent Protein (RFP) (white bright spot marker). But the efficiency of Du-Cap mediated transfer of pCDNA3.1-RFP was 2.6 times that of TAT.

Example 6 gel retardation electrophoresis detection of Du-Cap binding to poly (I: C) and Real-time PCR detection of IFN-. beta.transcriptional level changes

Gel blocking electrophoresis:

(1) mu.M of Du-Cap and 1. mu.g of poly (I: C) were added to a total volume of 20. mu.L of PBS, mixed, and incubated at 37 ℃ for 45 min.

(2) mu.L of the incubated product was subjected to 0.5% agarose gel electrophoresis to analyze the binding of Du-Cap to poly (I: C) in vitro.

Real-time PCR detection of changes in IFN- β transcript levels induced by Du-Cap mediated poly (IC) entry into HD11 cells:

(1) Du-Cap/poly (I: C), TAT/poly (I: C) complex and poly (I: C) were added to HD11 cells, with blank HD11 as a control.

(3)37℃，5％CO₂After incubation in the incubator for 6h, the cells were washed 3 times with PBS, trypsinized and collected.

(4) Total RNA from cells was extracted using a total RNA miniprep kit (from Axygen) according to the instructions.

(5) Taking 1 ug of total RNA to carry out reverse transcription, firstly eliminating the reaction of genome DNA, 5 XgDNA Eraser Bubber 2 uL, gDNA Eraser 1 uL, total RNA 1 ug, RNase Free H₂The content of O is 10 mu L. Acting at 42 deg.C for 2 min.

(6) Reverse transcription reaction: 10 μ L of the above reaction product, RNase Free H₂Taking 4 mu L of O, and taking 4 mu L of 5 XPrime Script buffer 2; taking 1 mu L of RT primer Mix; 1 mu L of Prime Script RT Enzyme is taken; a total of 20. mu.L.

(7) Acting at 37 deg.C for 15 min; the reverse transcription product was used as template at 85 ℃ for 5S.

(8) Fluorescent quantitative PCR detects the change of IFN-beta transcription level. The template and the upstream and downstream primers (upstream primer: F5'-CGGGGTACCGCCTCCAGTACAGCCACCACA-3' (SEQ ID NO: 3); downstream primer 5 '-GGGAAGCTTGTTTGGGGTGTTGCAGTGAGC-3-3' (SEQ ID NO: 4)) for IFN-. beta. (Genbank: GenBank: Y14969) were diluted 25-fold. The system is as follows: 10 μ L of SYBR Green, 0.4 μ L of ROX, 2 μ L of upstream primer, 2 μ L of downstream primer, 5.6 μ L of template, and 20 μ L of total system.

(9) Reaction conditions are as follows: at 95 ℃ for 30S; 95 ℃ and 5S; 60 ℃, 34S; 95 ℃ and 15S; at 60 ℃ for 1 min; for a total of 40 cycles.

(10) Results use relative quantitation 2^–ΔΔCtAnd performing calculation analysis, taking beta-actin as an internal reference gene, analyzing the data difference significance by using a t test, and expressing the statistical result by using the average value +/-standard error.

The results of gel block electrophoresis for detecting the binding of Du-Cap and poly (I: C) and Real-time PCR for detecting the change of IFN-beta transcription level are shown in FIG. 6, in which A: agarose gel electrophoresis patterns of the Du-Cap/poly (I: C) complex and poly (I: C); lane 1: the Du-Cap/poly (I: C) complex; lane 2: naked poly (I: C); and B, detecting the change of the IFN-beta transcription level in different groups of cells by fluorescent quantitative PCR.

As can be seen from FIG. 6, Du-Cap binds to poly (I: C) in vitro, resulting in hysteresis in the electrophoresis of poly (I: C). The Du-Cap/poly (I: C) complex induced a 3.8-fold increase in the transcriptional level of IFN- β in HD11 cells as compared to TAT/poly (I: C).

Example 7 agarose gel electrophoresis detection of Du-Cap binding to CpG ODN and Real-time PCR detection of changes in IFN-. beta.transcript levels

Gel blocking electrophoresis:

(1) mu.M of Du-Cap and 1. mu.g of CpG ODN (synthesized by Jiangsu Hongxn Biotech Co., Ltd.) were added to PBS in a total volume of 20. mu.L, and incubated at 37 ℃ for 45 min.

(2) mu.L of the incubated product was analyzed for Du-Cap binding to CpG ODN in vitro using 0.5% agarose gel electrophoresis.

Real-time PCR detection Du-Cap mediated CpG ODN entering 3D4/21 cells induced IFN-beta transcript level change:

(1) Du-Cap/CpG ODN, TAT/CpG ODN Complex and CpG ODN were added to 3D4/21 (5X 10)⁵one/mL) cells, blank 3D4/21 was used as a control.

(3)37℃，5％CO₂After incubation in the incubator for 24h, total cellular RNA was extracted and cDNA was synthesized as described above. Using this as a template and IFN-. beta.s (Genbank: NM-001003923) upstream and downstream primers (upstream primer: F5'-GCAGTATTGATTATCCACGAGA-3' (SEQ ID NO: 5); downstream primer 5'-TCTGCCCATCAAGTTCCAC-3' (SEQ ID NO: 6)) were referred to the above system. Real-time PCR detects changes in IFN- β transcript levels.

The results of agarose gel electrophoresis for detecting the binding of Du-Cap to CpG ODN and Real-time PCR for detecting the change in IFN- β transcript level are shown in FIG. 7, in which A: agarose gel electrophoresis of the Du-Cap/CpG ODN complex and CpG ODN; lane 1: the Du-Cap/CpG ODN complex; lane 2: naked CpG ODN; and B, detecting the change of the IFN-beta transcription level in different groups of cells by fluorescent quantitative PCR.

As can be seen from FIG. 7, Du-Cap can achieve binding to CpG ODN in vitro, resulting in hysteresis of CpG ODN upon electrophoresis. The Du-Cap/CpG ODN complex induced a 2.7-fold increase in IFN- β transcript levels in 3D4/21 cells as compared to TAT/CpG ODN.

The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

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Claims (6)

1. A cell-penetrating peptide, having an amino acid sequence as set forth in SEQ ID NO: 1 is shown.

2. The amino acid sequence is shown as SEQ ID NO: 1 in the preparation of gene drugs or nucleic acid immunopotentiator conduction carriers.

3. The use of claim 2, wherein the nucleic acid immunopotentiator comprises Poly (I: C) and/or CpG ODN.

4. The use of claim 2, wherein the target cell of the genetic drug or nucleic acid immunopotentiator is a macrophage.

5. The amino acid sequence is shown as SEQ ID NO: 1 in the preparation of an animal vaccine containing a nucleic acid immunopotentiator.

6. The use of claim 5, wherein the nucleic acid immunopotentiator comprises Poly (I: C) and/or CpG ODN.

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