Composite nucleic acid immunological enhancement substance with efficient delivery capacity as well as preparation method and application of composite nucleic acid immunological enhancement substance

Abstract

The invention discloses a compound nucleic acid immunological enhancement substance with efficient delivery capacity as well as a preparation method and application thereof, the compound nucleic acid immunological enhancement substance comprises a VP1-N2 aa 23-43 polypeptide derived from chicken infectious anemia virus and an exogenous active molecule, and the amino acid sequence of the VP1-N2 aa 23-43 polypeptide is shown as SEQ ID NO. 1. The efficient cell-penetrating peptide VP1-N2 aa 23-43 polypeptide is designed, the polypeptide can carry CpG to penetrate into different cells within 30 min, an immune system is activated by simulating a CpG motif in bacterial DNA, a TLR9 signal channel is activated, and immune response is enhanced. Therefore, the VP1-aa 23-43 polypeptide compound nucleic acid immunological enhancement substance with high-efficiency delivery capacity is used for enhancing the immune response of the body to the vaccine antigen and improving the protection effect of the vaccine.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of cell penetrating peptides, and particularly relates to a complex nucleic acid immunopotentiating substance with high delivery capacity and a preparation method and application thereof. BACKGROUND

[0002] Due to the unique lipid bilayer structure of the cell membrane with selective permeability, many potential functional biological macromolecules (such as nucleic acids, polypeptides, proteins) or drugs are blocked outside the cell. These biological macromolecules are difficult to reach a sufficient effective concentration in target tissue cells and organs. Therefore, the application of these functional biological molecules in the fields of biology and pharmacy has been greatly limited. With the development of science and technology, although there are many ways to introduce biological macromolecules into cells at present, for example: electroporation, microinjection, perforin protein method, nanoparticle wrapping, etc. However, these methods still have the characteristics of low delivery efficiency, great harm to cells, lack of tissue targeting, easy to trigger immune response in vivo, and complex operation. Therefore, how to efficiently deliver biological macromolecules into target cells and tissues is still a big problem we are facing.

[0003] Cell penetrating peptides (CPPs) are a class of short peptides that can carry biological macromolecules into cells, generally 5-30 basic amino acids rich in positive charges. It not only can efficiently cross the cell membrane into the cell itself, but also can carry different types of exogenous molecules such as fluorescein, DNA, RNA, protein, quantum dots, etc. into cells of different species. Because this new type of short peptide not only has the characteristics of high transmission efficiency, low biological toxicity, and avoiding triggering immune response in vivo, but also has the characteristics of wide target membrane penetration cells and flexible application, it is a new type of exogenous gene or drug transmission carrier that has developed rapidly in the past 20 years.

[0004] CpG and its nucleic acid derivatives have been proven to be effective immunopotentiating agents. Artificially synthesized oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs can trigger TRL9 (including human plasmacytoid dendritic cells and B cells) signaling pathways, initiate innate immune responses characterized by Th1 and pro-inflammatory cytokine production. When CpG ODNs are used as vaccine adjuvants, they can improve the function of professional antigen-presenting cells and promote the production of humoral and cellular vaccine-specific immune responses. The adjuvant properties of CpG ODNs can be observed when administered systemically or mucosally, and persist in immunodeficient hosts. Therefore, it has high value and significance to develop a complex nucleic acid immunopotentiating substance with high delivery capacity. SUMMARY

[0005] Invention purposes: In view of the deficiencies in the prior art, the present application provides a composite nucleic acid immunopotentiating substance with high delivery capacity, which uses cell-penetrating peptide CVP1 with high cell-penetrating function in combination with CpG to develop a composite nucleic acid immunopotentiating substance with high delivery capacity, so as to realize efficient entry of vaccine effective targets into cells to induce stronger immune response, and effectively solve the problems of low delivery efficiency, great damage to cells, and complex and time-consuming operation in the delivery of bioactive macromolecules.

[0006] The application also provides a preparation method and application of the composite nucleic acid immunopotentiating substance with high delivery capacity.

[0007] Technical scheme: In order to achieve the above-mentioned purpose, the composite nucleic acid immunopotentiating substance with high delivery capacity comprises a polypeptide derived from chicken infectious anemia virus VP1-N2 aa 23-43 and an exogenous active molecule.

[0008] The polypeptide derived from chicken infectious anemia virus VP1-N2 aa 23-43 serves as a high-efficiency cell-penetrating peptide to mediate the exogenous active molecule in the form of a carrier.

[0009] The polypeptide derived from chicken infectious anemia virus VP1-N2 aa 23-43 carries the exogenous active molecule into different cells in a concentration-dependent manner without damaging the cells.

[0010] The cells are chicken liver cancer cells (LMH), human kidney epithelial cells (293T), chicken embryo fibroblasts (DF-1), or chicken lymphoma cells (DT40).

[0011] The exogenous active molecule is CpG nucleic acid or a derivative thereof, wherein the nucleic acid sequence of the CpG nucleic acid is shown in SEQ ID NO. 2, and the derivative is a nucleic acid sequence with the same function as the CpG nucleic acid and a homology of more than 80%.

[0012] The preparation method of the composite nucleic acid immunopotentiating substance with high delivery capacity comprises the following steps: mixing the VP1-N2 aa 23-43 polypeptide and the exogenous active molecule in equal amounts at room temperature.

[0013] The composite nucleic acid immunopotentiating substance with high delivery capacity can be used in the preparation of a medicine or reagent for improving the immune potency of an animal vaccine.

[0014] The application discloses a polypeptide derived from chicken infectious anemia virus VP1-N2 aa 23-43, and application of the polypeptide as a carrier for mediating an exogenous active molecule into cells through a cell-penetrating peptide.

[0015] The polypeptide derived from chicken infectious anemia virus VP1-N2 aa 23-43 carries the exogenous active molecule into different cells in a concentration-dependent manner and without damaging the cells.

[0016] The exogenous active molecule is CpG nucleic acid or a derivative thereof, wherein the nucleic acid sequence of the CpG nucleic acid is shown as SEQ ID NO. 2.

[0017] The application analyzes a sequence region (aa 1-60) rich in arginine at the N terminus of chicken infectious anemia virus VP1 protein, designs a polypeptide (aa 23-43) with potential cell-penetrating function, and artificially synthesizes the polypeptide after FITC labeling. The application artificially synthesizes CpG after Cy3 labeling according to the sequence of CpG. The synthesized polypeptide (aa 23-43) is co-incubated with CpG, and laser confocal microscopy is used to verify the ability of the polypeptide in mediating the exogenous active molecule into cells. Data show that the polypeptide can mediate CpG into cells as a carrier and play a role. Data show that the polypeptide can carry CpG to penetrate into different cells within 30 minutes, and has a concentration-dependent effect. CCK8 shows that the CpG adjuvant based on the cell-penetrating peptide has no obvious cytotoxicity at a low concentration.

[0018] Further, the preparation of the complex nucleic acid immunopotentiating substance with high delivery capacity and delivery into cells comprises the following steps:

[0019] (1) Synthesis of chicken infectious anemia virus VP1-N2 aa 23-43 polypeptide and CpG

[0020] The sequence region (aa 1-60) rich in arginine at the N terminus of CAV VP1 of different reference strains is aligned and analyzed by combining UniProt database and an online server MultiAlin. A cell-penetrating peptide sequence (aa 23-43) is designed and generated according to the characteristics (rich in 5-30 positive charge amino acids) of the cell-penetrating peptide. The cell-penetrating peptide sequence is artificially synthesized after FITC labeling. CpG is labeled with Cy3 and artificially synthesized according to the sequence (tcg cga acg ttc gcc gcg ttc gaa cgc gg).

[0021] (2) The chicken infectious anemia virus VP1-N2 aa 23-43 polypeptide can mediate CpG into cells

[0022] The chicken infectious anemia virus VP1-N2 aa 23-43 polypeptide and CpG-Cy3 are co-incubated at a concentration of 5 muM for 30 min, and laser confocal microscope imaging technology and fluorescence microscope technology are used for detection.

[0023] (3) The chicken infectious anemia virus VP1-N2 aa 23-43 polypeptide carries the CpG transmembrane property

[0024] The chicken infectious anemia virus VP1-N2 aa 23-43 polypeptide carrying 5 muM CpG at different concentrations is co-incubated with LMH cells, and the concentration dependence of the polypeptide is evaluated by fluorescence microscope imaging.

[0025] (4) The safety of the VP1-N2 aa 23-43 polypeptide and CpG to cells is detected by cytotoxicity test (CCK8).

[0026] CpG at different concentrations is incubated with LMH cells, and after co-culturing for 12 h, 24 h and 48 h, the safety of CpG to cells is detected by cytotoxicity test.

[0027] CpG at different concentrations and VP1-N2 aa 23-43 polypeptide at the same concentration are incubated with LMH cells, and after co-culturing for 12 h, 24 h and 48 h, the safety of CpG to cells is detected by cytotoxicity test.

[0028] (5) The CpG based on the chicken infectious anemia virus VP1-N2 aa 23-43 polypeptide can improve the immune potency of animal vaccines.

[0029] The application designs a high-efficiency cell transmembrane peptide derived from the chicken infectious anemia virus VP1-N2 aa 23-43 polypeptide, which can carry CpG to penetrate into different cells within 30 min. CpG is a CpG oligodeoxynucleotide (CpG ODN) based on the CpG motif in bacterial DNA, which can activate the immune system by simulating the CpG motif in bacterial DNA, activate the TLR9 signaling pathway, and enhance the immune response. Therefore, the application obtains a complex nucleic acid immunopotentiating substance with high-efficiency delivery capacity based on the chicken infectious anemia virus VP1-aa 23-43 polypeptide.

[0030] The design principle of the application is based on the efficient delivery function of the functional biological macromolecule ability of the transmembrane peptide. Nucleic acid is a kind of cargo molecule that is easy to carry by the transmembrane peptide. Because nucleic acid itself is negatively charged, it can be directly combined with the complex cationic cell transmembrane peptide through electrostatic interaction. The transmembrane peptide can directly wrap the nucleic acid and compress it into a certain volume of particle transmembrane by directly mixing the transmembrane peptide and the nucleic acid in a certain proportion. The application first carries the nucleic acid adjuvant CpG into cells with VP1-N2 aa 23-43 polypeptide, so as to target the delivery in cells and tissue organs, and can enhance the function of improving the antibody level of the vaccine itself. In order to carry out comparison, the polypeptide (TRRDDVKYSSDHQNRW) of aa 235-250 on CAV-VP1 is synthesized as a control, and it is found that it does not have the function of penetrating the membrane. Further, other fragments on VP1-N2 aa are used as controls, and it is found that the delivery ability will be obviously reduced. And if the arginine contained in VP1-N2 aa is mutated into uncharged glycine (G) or non-polar alanine (A) to synthesize the corresponding polypeptide (VP1-M: LKRLGAGYKFAHGAGQGYGAG), the transmembrane activity will also be lost.

[0031] Advantages: Compared with the prior art, the application has the following advantages:

[0032] The application first carries the nucleic acid adjuvant CpG into cells with VP1-N2 aa 23-43 polypeptide and plays a role, which can effectively improve the antibody level of the vaccine.

[0033] The VP1-N2 aa 23-43 polypeptide of a specific length used in the application has higher transmembrane efficiency than the mainstream transmembrane peptide TAT at present. The application carries the nucleic acid adjuvant CpG into cells with VP1-N2 aa 23-43 polypeptide and plays a role, which is the innovative first discovery of the high-efficiency transmembrane peptide carrying nucleic acid adjuvant on CAV. The transmembrane efficiency is higher and does not affect the function of the biological macromolecule. The cell damage is small, which is more convenient, simple, safe and the like. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Positioning detection of VP1-N2 aa 23-43 polypeptide carrying CpG in LMH cells.

[0035] Figure 2 Positioning detection of VP1-N2 aa 23-43 polypeptide carrying CpG in 293T cells.

[0036] Figure 3 Positioning detection of VP1-N2 aa 23-43 polypeptide carrying CpG in DF-1 cells.

[0037] Figure 4Detection of the transmembrane property of the CpG carried by the VP1-N2 aa 23-43 polypeptide.

[0038] Figure 5 Detection of the transmembrane property of the CpG carried by the VP1-N2 aa 23-43 polypeptide.

[0039] Figure 6 Detection of the safety of the CpG to cells by CCK8.

[0040] Figure 7 Detection of the safety of the combination of the CpG and CVP1-N2 to cells by CCK8.

[0041] Figure 8 Animal pre-experiment of the complex CVP1-CpG of CVP1-N2 and CpG.

[0042] Figure 9 Animal pre-experiment of the complex CVP1-CpG of CVP1-N2 and CpG. DETAILED DESCRIPTION

[0043] The application is further described below in conjunction with the accompanying drawings and examples.

[0044] The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified. The experimental methods not specifically indicated in the examples are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer.

[0045] The raw materials such as vectors and antibodies used in the application are known materials or prepared by known methods.

[0046] DF-1 cells are ATCC CRL-12203, LMH cells are ATCC CRL-2117, 293T cells are ATCC CRL-3216, and DT40 cells are ATCC CRL-2111.

[0047] Inactivated vaccine FAdV4-VP3 (GPV), the construction is described in detail in Chinese patent (application number): 2025106658287. The specific process is as follows:

[0048] 1. Preparation of viral genome: Extract using Genomic Extraction Kit from Tian Gen Company (MAT: 4991108), take 200 uL of goose parvovirus supernatant in a 1.5 mL pipette, add 200 uL of Proteinase K solution, mix well. Add 200 uL of buffer GB, mix well by inverting, 70°C water bath for 10 min, add 200 uL of anhydrous ethanol after short centrifugation, mix well by shaking for 15 sec. Add the above solution to an adsorption column CB3 (the adsorption column is placed in a collection tube), centrifuge at 12000 rpm for 30 sec, discard the waste, and put the adsorption column CB3 back into the collection tube. Add 500 uL of buffer GD to the adsorption column CB3, centrifuge at 12000 rpm for 30 sec, discard the waste, and put the adsorption column CB3 back into the collection tube. Add 600 uL of rinse PW to the adsorption column CB3, centrifuge at 12000 rpm for 30 sec, discard the waste, and put the adsorption column CB3 back into the collection tube. Repeat the addition of 600 uL of rinse PW, centrifuge at 12000 rpm for 30 sec, discard the waste, and put the adsorption column CB3 back into the collection tube. Centrifuge at 12000 rpm for 2 min, discard the waste, and let the adsorption material dry by placing the adsorption column CB3 at room temperature for a few minutes. Transfer the adsorption column CB3 to a 1.5 mL pipette, add 200 ul of elution buffer TE to the middle of the adsorption membrane, and let it stand at room temperature for 2 min. Centrifuge at 12000 rpm for 2 min, collect the solution in the centrifuge tube, and the solution is the viral genome.

[0049] 2. Construction of sgRNA expression vector: According to the Fiber-2 gene sequence of FAdV-4, the sgRNA is designed and screened. The designed sgRNA is cloned into the lentiCRISPR v2 plasmid, and the sgRNA expression vector is verified by sequencing. The specific sgRNA sequence is shown in Table 1, which is synthesized by Nanjing Qikexing Biological Technology Co., Ltd.

[0050] Table 1 sgRNA sequence for Fiber-2 gene

[0051]

[0052]

[0053] The specific process of obtaining double-stranded sgRNA, enzyme cutting of vector plasmid, and construction of knockout vector is as follows:

[0054] (1) Double-stranded sgRNA: sgRNA-F and sgRNA-R (SEQ ID NO. 4-5) were dissolved with ddH2O to a final concentration of 100 μM, and the following annealing system was used: 1 μL of sgRNA-F and sgRNA-R, 1 μL of 10×PCR buffer, 7 μL of ddw, and annealing program of 5°C / min gradient cooling to 25°C to obtain double-stranded DNA.

[0055] (2) Enzymatic digestion of vector plasmid: lentiCRISPR v2 vector was digested with BsmBI restriction enzyme, and the enzyme digestion system was as follows: 1 μL of BsmBI restriction enzyme, 1 μg of lentiCRISPR v2 plasmid, 0.5 μL of 0.1M DTT, 5 μL of NEB buffer 3.1, and the rest was supplemented with ddH2O to 50 μL, and the enzyme digestion conditions were 55°C for 15 min, and the lentiCRISPR v2 vector plasmid containing sticky ends was obtained by gel recovery.

[0056] (3) Construction of knockout plasmid: the double-stranded sgRNA obtained by annealing was diluted 100 times, and the enzyme-digested vector was diluted to 50 ng / μL, and then the following system was used for ligation: 1 μL of vector plasmid, 1 μL of double-stranded sgRNA, 1 μL of T4 ligase, 1 μL of 10×T4 buffer, and 6 μL of ddH2O, and ligation was performed at 16°C overnight, and then transformed into Stbl3 competent cells, and the colonies were picked and expanded for culture, and the plasmid was extracted and sequenced to identify the correct sgRNA expression vector.

[0057] 3. Construction of donor plasmid: a fragment with two LoxP sequences was synthesized by Nanjing Qikexing Biotechnology Co., Ltd., and two LoxP were inserted into the RFP expression cassette at both ends by PCR. The constructed pUC-57-LoxP-RFP-LoxP vector was provided by Yangzhou University.

[0058] The pUC-57-LoxP-RFP-LoxP vector was used as a template to design primers (SEQ ID NO. 6-7) to amplify the RFP expression cassette with LoxP sequences; the pMD19-T vector was linearized as a template; the avian adenovirus genome of serotype 4 was used as a template to design primers (SEQ ID NO. 8-9) to amplify the Fiber-2 gene (SEQ ID NO. 14) and its left and right homologous arms HR1 and HR2, which were cloned into the pMD19-T vector; the goose parvovirus genome was used as a template to design primers (SEQ ID NO. 10-11) to amplify the coding gene (SEQ ID NO. 15) of the VP3 protein of the goose parvovirus GPVSJ20190420 strain. After agarose gel electrophoresis, the gel was recovered, and the HR1-Fiber-2-RFP-HR2 sequence was assembled on the pMD19-T vector by homologous recombination. The final donor plasmid was sent to Nanjing Qikexin Biotechnology Co., Ltd. for sequencing. The primer sequences used for constructing the donor plasmid are shown in Table 2.

[0059] Table 2 PCR primers used for constructing the donor plasmid

[0060]

[0061] 4. Rescue of red fluorescent recombinant virus: LMH cells were plated in a 6-well plate. The next day, 3ug of sgRNA expression vector, 3ug of donor plasmid, and 6uL of transfection reagent Mirus were added to 200uL of Opti-MEM and incubated at room temperature for 45min. Then, they were added to the LMH cells, and the cells were replaced with cell growth medium 6h after transfection. After 12h of transfection, the cell growth medium was discarded, and the LMH cells were infected with 0.1 MOI of FA4-EGFP. After 2h of infection, the cells were replaced with cell maintenance medium. After 3 days of virus infection, the supernatant was centrifuged at 12000rpm for 10min, and then the LMH cells in a new 96-well plate were blind-transfected. Red fluorescent clusters were observed daily under a fluorescence microscope. The appearance of red fluorescent clusters indicated the successful construction of the recombinant virus, which was named FAdV4-VP3(GPV)-RFP. The LMH cells were successfully infected with the recombinant virus containing RFP, and red fluorescence was observed under a fluorescence microscope. The RFP tag was subsequently removed by CRE enzyme.

[0062] 5. Purification and identification of red fluorescent recombinant virus: The rescued red fluorescent recombinant virus was purified using the plaque assay and limiting dilution method to obtain purified red fluorescent recombinant virus. The purity was identified by PCR with primers (SEQ ID NO. 12-13) as shown in Table 3, and pure recombinant virus was successfully obtained.

[0063] Table 3 PCR primers for identifying recombinant virus

[0064]

[0065] 6. Deletion of RFP expression cassette: LMH cells overexpressing Cre recombinase were infected with 0.1 MOI of red fluorescent recombinant virus, and 2 h after infection, the cells were replaced with cell maintenance solution. No red fluorescence was observed in the virus-infected cells, but CPE was observed in the LMH cells. The cells were picked and subjected to limiting dilution until no red fluorescence was observed in the cells in the wells. The final cells were all infected with the recombinant virus for deleting the RFP expression cassette, and the recombinant virus for deleting the RFP expression cassette was obtained. The recombinant virus for deleting the RFP expression cassette was identified by PCR and IFA, and the recombinant virus FAdV4-VP3(GPV) was successfully obtained, i.e., the inactivated vaccine FAdV4-VP3(GPV).

[0066] Example 1

[0067] Preparation and synthesis of chicken infectious anemia virus VP1-N2 aa 23-43 polypeptide and CpG nucleic acid

[0068] The sequences of the arginine-rich region (aa 1-60) of different reference strains of CAV VP1 N-terminal were aligned and analyzed in combination with the UniProt database and the online server MultiAlin. According to the characteristics of the cell-penetrating peptide (rich in 5-30 positively charged amino acids) and the enrichment of arginine, a pseudo-cell-penetrating peptide sequence (aa 23-43) was designed and generated, which was named VP1-N2 aa 23-43. After FITC modification, it was artificially synthesized, and CpG was artificially synthesized after Cy3 modification.

[0069] Therefore, the sequences of the synthesized short peptides are as follows:

[0070] VP1-N2 aa 23-43 (SEQ ID NO. 1): LKRLRRRYKFRHRRRQRYRRR;

[0071] VP1-N2 aa 23-43-FITC: FITC-LKRLRRRYKFRHRRRQRYRRR

[0072] CPG (SEQ ID NO. 2): tcg cga acg ttc gcc gcg ttc gaa cgc gg

[0073] CPG-CY3: CY3-tcg cga acg ttc gcc gcg ttc gaa cgc gg

[0074] Peptide 2 (SEQ ID NO. 3) from CAV-VP1 polypeptide (aa 235-250): TRRDDVKYSSDHQNRW

[0075] Synthesized by Sheng Gong Biotechnology Co., Ltd.

[0076] Example 2

[0077] CVP1-N2 mediates CpG into LMH cells, 293T cells, DF-1 cells

[0078] 1) Inoculate LMH cells, 293T cells, DF-1 cells in cell culture dishes, respectively, continue to culture at 37°C, 5% CO2 for 12h.

[0079] 2) When the cell density reaches 70%, discard the original culture medium, add 500uL Opti-MEM, add 5uM CVP1-N2-FITC and 5uM CpG-Cy3 to the dish, incubate for 30min in a 37°C, 5% CO2 incubator, at the same time set up a negative control group, add 5uM CpG-Cy3 and another polypeptide peptide2 (aa 235-250) from CAV-VP1, after incubation, wash with PBS three times; wash away the short peptides that do not enter the cells.

[0080] 3) After staining with Hoechst 33342 (10ug / mL) for 10min, discard the supernatant and wash with PBS three times.

[0081] 4) Resuspend with 500uL Opti-MEM.

[0082] 5) TCS SP8STED laser confocal scanning microscope scans and takes pictures under different wavelengths (405nm<488nm<561nm) and multi-channel conditions, as shown in Figure 1 、 2 , 3 shows that VP1-N2 aa 23-43 can carry CpG into LMH cells, 293T cells, DF-1 cells and the effect is significant, while the polypeptide of the control group cannot carry CpG into the cells.

[0083] Example 3

[0084] CVP1-N2 mediates CpG into DT40 cells

[0085] 1) Inoculate DT40 cells in a 12-well plate, continue to culture at 37°C, 5% CO2 for 12h.

[0086] 2) Add 5x10 5Cells were transferred into 1.5 mL centrifuge tubes, centrifuged at 800 x g for 5 min, the supernatant was discarded, and the cells were resuspended with 500 uL Opti-MEM, 10 uM CVP1-N2-FITC and 10 uM CpG-CY3 were added to the cells, and a negative control group was set up, 10 uM CpG-Cy3 and another peptide peptide2 (aa 235-250) were added, and incubated at 37°C for 30 min.

[0087] 3) The mixture was transferred to a 1.5 mL centrifuge tube and centrifuged at 800 x g for 5 min. The supernatant was discarded and washed with PBS three times.

[0088] 4) Resuspend the cells with 500 uL Opti-MEM, then add the cells to a new 12-well plate, and observe and take pictures under a fluorescence microscope, as shown in Figure 4 VP1-N2 aa 23-43 can carry CpG into DT40 cells, while the control peptide cannot carry CpG into cells.

[0089] Example 4

[0090] Detection of the membrane penetration property of chicken infectious anemia virus VP1-N2 aa 23-43 peptide carrying CpG by fluorescence microscopy

[0091] 1) 2 x 10 5 LMH cells were inoculated into a 24-well cell culture plate.

[0092] 2) After 12 h of cell inoculation, synthetic short peptides (VP1-N2 aa 23-43-FITC) were set at different concentrations (1 uM, 5 uM, 10 uM, 20 uM), and CpG-Cy3 was set at the same concentration of 5 uM, mixed with 500 uL Opti-MEM and added to the cells, and incubated at 37°C, 5% CO2 for 30 min, then washed with PBS for 3 min each time, and washed away the short peptides that did not enter the cells.

[0093] 3) After staining with Hoechst 33342 (10 ug / mL) for 10 min, the supernatant was discarded, and the cells were washed with PBS, then 500 uL Opti-MEM was added, and observed and photographed under a fluorescence microscope, as shown in Figure 5 As the concentration of VP1-N2 aa 23-43-FITC increased on LMH, the ability to carry CpG into cells increased.

[0094] Example 5

[0095] CCK8 experiment

[0096] 1) 1 x 10 5LMH cells were inoculated into 96-well plates, and after the cells adhered for 12 h, different concentrations of CpG-Cy3 and different concentrations of CpG-Cy3 and the same concentration of VP1-N2 aa 23-43 polypeptide were added, respectively, and 3 replicate wells were set for each concentration.

[0097] 2) After 12 h, 24 h, and 48 h of continuous culture at 37°C and 5% CO2, 10 μL of CCK8 solution was added to each well, and the cells were further cultured at 37°C for 3 h. Then the cell suspension was discarded. The absorbance (OD450) was detected at a wavelength of 450 nm by an enzyme-labeled instrument, and the cells without CpG treatment were used as a negative control, and the cell viability of each experimental group was calculated.

[0098] 3) Cell viability (%) = (absorbance value of cells in the experimental group - absorbance value of cells in the blank group) / (absorbance value of cells in the control group - absorbance value of cells in the blank group) x 100%, and the results are shown in Figure 6 、 7 VP1-N2 aa 23-43 has good safety to cells, and CpG itself has toxicity to cells, and is safe to cells below a concentration of 10 μM.

[0099] Example 6

[0100] Animal experiment after combination of CVP1-N2 and CpG

[0101] 1) One-day-old goslings were selected for immunization experiments.

[0102] 2) Three groups were set up, including a single inactivated vaccine immunization group, an inactivated vaccine + N2-CPG immunization group (CVP1-N2 and CpG were both 50 μg per gosling), and a blank control group. The FAdV4-VP3 (GPV) inactivated vaccine injected in the first two groups was 1 x 10 6 TCID 50 The antigen amount, and the blank group used serum-free DMEM as a control. Three groups were injected subcutaneously, 500 μL per gosling. The neck venous blood was collected at 0 d and 7 d after immunization, and the serum of each gosling was collected to detect the antibody levels of GPV VP3 and FAdV-4 Fiber-2 protein in the serum.

[0103] 3) The antibody levels were detected at 0 d and 7 d, as shown in Figure 8 、 9As shown, the GPVVP3 and FAdV-4 Fiber-2 protein antibody levels of the inactivated vaccine + N2-CPG immunoprotected group were higher than those of the single inactivated vaccine immunoprotected group. It is illustrated that the CpG based on the chicken infectious anemia virus VP1-N2 aa 23-43 polypeptide of the application can improve the animal immune titer, not only has high cell delivery capacity, but also can be used as an immunopotentiating substance in the preparation of a drug or reagent for improving the animal vaccine immune titer.

Claims

1. A complex nucleic acid-based immune enhancer with highly efficient delivery capability, characterized in that, It includes a polypeptide derived from chicken infectious anemia virus VP1-N2 aa 23-43 and an exogenous active molecule, wherein the amino acid sequence of the polypeptide derived from chicken infectious anemia virus VP1-N2 aa 23-43 is shown in SEQ ID NO.

1.

2. The composite nucleic acid immune enhancer with high delivery capability according to claim 1, characterized in that, The polypeptide derived from chicken infectious anemia virus VP1-N2 aa 23-43 serves as a highly efficient cell-penetrating peptide, mediating exogenous active molecules in the form of a carrier.

3. The composite nucleic acid immune enhancer with high delivery capability according to claim 1, characterized in that, The polypeptide derived from chicken infectious anemia virus VP1-N2 aa 23-43 carries exogenous active molecules into different cells, exhibiting concentration dependence and causing no damage to the cells.

4. The composite nucleic acid immune enhancer with high delivery capability according to claim 3, characterized in that, The cells are chicken liver cancer cells (LMH), human kidney epithelial cells (293T), chicken embryo fibroblasts (DF-1), or chicken lymphoma cells (DT40).

5. The composite nucleic acid immune enhancer with high delivery capability according to any one of claims 1-4, characterized in that, The exogenous active molecule is preferably CpG nucleic acid or its derivative, wherein the nucleic acid sequence of the CpG nucleic acid is shown in SEQ ID NO.2, and the derivative is a nucleic acid sequence with the same function as the CpG nucleic acid and more than 80% homology.

6. A method for preparing a complex nucleic acid-based immune-enhancing substance with high delivery capability as described in claim 1, characterized in that, The process includes the following steps: mixing equal amounts of VP1-N2 aa 23-43 peptide and exogenous active molecules at room temperature before use.

7. The use of the complex nucleic acid immune enhancer with high delivery capability as described in claim 1 in the preparation of drugs or reagents for improving the immune titer of animal vaccines.

8. The application of a polypeptide derived from chicken infectious anemia virus VP1-N2 aa 23-43 as a carrier in the form of a highly efficient cell-penetrating peptide to mediate the entry of exogenous active molecules into cells, wherein the amino acid sequence of the polypeptide derived from chicken infectious anemia virus VP1-N2 aa 23-43 is shown in SEQ ID NO.

1.

9. The application according to claim 8, characterized in that, The polypeptide derived from chicken infectious anemia virus VP1-N2 aa23-43 carries exogenous active molecules into different cells, exhibiting concentration dependence and causing no damage to the cells.

10. The application according to claim 8, characterized in that, The exogenous active molecule is a CpG nucleic acid or its derivative, wherein the nucleic acid sequence of the CpG nucleic acid is shown in SEQ ID NO.2.

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