Protein peptide carrier-based medicine and preparation method thereof

Abstract

The invention relates to a medicine based on a protein peptide carrier and a preparation method thereof, the medicine comprises the protein peptide carrier, probiotic DNA and a targeting substance, and the protein peptide carrier has the following amino acid sequence: Cys-Trp-Pro-Trp-Arg8-Pro-Cys-Arg8-Pro-Cys-Arg8-Cys; meanwhile, the invention discloses a preparation method of the medicine. The drug provided by the invention can edit immune cells in a body into immunomodulatory cells, can recognize antigen-specific Th2 cells to enable the cells to be apoptotic, can effectively treat allergic diseases, and has the advantages of high drug carrier carrying capacity, simple drug preparation process, good stability and repeatability, and good biocompatibility, functionality and biodegradability.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a drug based on a protein peptide carrier and a preparation method thereof. Background Art

[0002] Gene therapy is the delivery of genes with specific genetic information to target cells by artificial means. The expressed target protein has the effect of regulating, treating or even curing diseases caused by congenital or acquired genetic defects. Nucleic acids and cell membranes are both negatively charged. Naked nucleic acids are difficult to be directly introduced into cells, and are easily degraded by nucleic acid degrading enzymes in the cytoplasm, which cannot achieve the effects of gene introduction and gene therapy. Therefore, gene delivery must be achieved with the help of external forces or carriers. At present, many vectors, including viral and non-viral vectors, have been developed for nucleic acid delivery. However, viral vectors have the disadvantages of low loading capacity, inflammatory / immunogenic reactions, and difficulty in large-scale manufacturing. In recent years, non-viral vectors used for nucleic acid delivery mainly include organic polymers, inorganic nanoparticles, etc. Although these nucleic acid delivery vectors have been widely studied, they still have many disadvantages, such as poor targeting of organic polymers, poor biodegradability of inorganic nanoparticles, and easy accumulation in the body leading to biological toxicity.

[0003] As for allergic diseases, the current incidence rate of allergic diseases is as high as 30%. Clinically, the only specific treatment method for allergic diseases is desensitization therapy; the course of this method is as long as 3 years; and the efficacy needs to be improved. Summary of the invention

[0004] In view of the existing deficiencies, the present invention provides a drug based on a protein peptide carrier and a preparation method thereof.

[0005] The technical solution adopted by the present invention to solve the technical problem is: a drug based on a protein peptide carrier, comprising a protein peptide carrier, probiotic DNA and a targeting substance, wherein the protein peptide carrier has the following amino acid sequence: Cys-Trp-Pro-Trp-Arg8-Pro-Cys-Arg8-Pro-Cys-Arg8-Cys

[0006] Preferably, the targeting substance comprises ovalbumin, major histocompatibility antigen II and / or monoclonal antibody.

[0007] Preferably, the weight ratio of the protein peptide carrier, the probiotic DNA and the targeting substance is 3-6:1:3-6, and the weight ratio of ovalbumin and major histocompatibility antigen II or monoclonal antibody in the targeting substance is 1:1.

[0008] Preferably, the probiotic DNA is DNA extracted from Lactobacillus rhamnosus.

[0009] A method for preparing a drug based on a protein peptide carrier, comprising the following steps: obtaining a protein peptide carrier with an amino acid sequence of Cys-Trp-Pro-Trp-Arg8-Pro-Cys-Arg8-Pro-Cys-Arg8-Cys; preparing probiotic DNA; and mixing the protein peptide carrier, the probiotic DNA and a targeting substance to obtain a mixture; wherein the targeting substance comprises ovalbumin, major histocompatibility antigen II or a monoclonal antibody.

[0010] Preferably, the probiotic DNA is DNA extracted from Lactobacillus rhamnosus.

[0011] Preferably, the weight ratio of the protein peptide carrier, the probiotic DNA and the targeting substance is 3-6:1:3-6, and the weight ratio of ovalbumin and major histocompatibility antigen II or monoclonal antibody in the targeting substance is 1:1.

[0012] Preferably, the concentration of the protein peptide carrier is 200±20 μg / ml, the concentration of ovalbumin is 100±10 μg / ml, and the concentration of major histocompatibility antigen II or monoclonal antibody is 100±10 μg / ml.

[0013] The beneficial effects of the present invention are as follows: the invention utilizes a chemical method to load a targeting substance into a protein peptide carrier to prepare a drug, the drug can edit immune cells in the body into immune regulatory cells, and can identify antigen-specific Th2 cells that are the root cause of allergic diseases, and cause them to undergo apoptosis, and can effectively treat allergic diseases. The protein peptide carrier has a high loading capacity and can be hydrolyzed by proteases after entering the body, thereby releasing the drug, meeting the requirements for the degradability of the drug carrier. The preparation process is simple, and the stability and repeatability are good, and it has good biocompatibility, functionality and biodegradability. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the characteristic diagram of the peptide Yiminxiao of the present invention;

[0015] Figure 2 This is an experimental data diagram of the specific binding of Moes to antigen-specific CD4+T cells of the present invention;

[0016] Figure 3 This is a graph showing the expression of PD-L1 in Meos induced by the peptide Yiminxiao of the present invention;

[0017] Figure 4 This is a graph showing the expression of PD1 in Meos-induced antigen-specific CD4+T cells of the present invention;

[0018] Figure 5 This is a graph showing experimental data of Meos inducing antigen-specific CD4+T cell apoptosis in the present invention;

[0019] Figure 6 This is a graph of experimental data after administration of the experimental AR mouse model of the present invention;

[0020] Figure 7 This is a graph showing experimental data of reducing antigen-specific CD4+T cells in the airways of mice with airway allergy by administering the drug of the present invention. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with the examples, and a clear and complete description will be given. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention. The implementation conditions used in the examples can be further adjusted according to the conditions of the specific manufacturer, and the unspecified implementation conditions are generally the conditions in conventional experiments.

[0022] The control peptide Yiminxiao in the present invention refers to an experiment in which a preparation containing no effective drug is used instead of the peptide Yiminxiao.

[0023] The present invention provides a drug based on a protein peptide carrier and a preparation method thereof, comprising a protein peptide carrier, probiotic DNA and a targeting substance, wherein the protein peptide carrier has the following amino acid sequence: Cys-Trp-Pro-Trp-Arg8-Pro-Cys-Arg8-Pro-Cys-Arg8-Cys; the targeting substance comprises ovalbumin, major histocompatibility antigen II and / or a monoclonal antibody; and the specific steps are as follows:

[0024] A protein peptide carrier with an amino acid sequence of Cys-Trp-Pro-Trp-Arg8-Pro-Cys-Arg8-Pro-Cys-Arg8-Cys is obtained; proline in the carrier is an amino acid with adhesion properties, and protein molecules rich in proline have mutual adhesion properties, which can significantly increase the adhesion function of the carrier and make it have better loading capacity. The protein peptide carrier is synthesized by any existing polypeptide synthesis method; after entering the body, the carrier can be hydrolyzed by protease to release the drug.

[0025] Preparation of probiotic DNA: Lactobacillus rhamnosus GG (ATCC) was cultured in LB medium, and then the probiotic DNA was extracted using the QIAamp Rapid DNA Mini Kit (Qiagen), which can be called LgDNA. Finally, the quantity and purity of LgDNA were determined by spectrometry (MultiskanTMGO, Thermo Fisher Scientific).

[0026] The protein peptide carrier, probiotic DNA and targeting substance are mixed to prepare a mixture; wherein the targeting substance includes ovalbumin (OVA), major histocompatibility antigen II (MHC II) or monoclonal antibody (Siglecf antibody); such as the weight ratio of the protein peptide carrier, probiotic DNA and targeting substance is 3-6:1:3-6, such as 3.5-5:1:3.5:5, wherein the weight ratio of ovalbumin to major histocompatibility antigen II or monoclonal antibody is 1:1; such as mixing the same volume of 200 μg / ml peptide carrier solution, 100 μg / ml OVA, 100 μg / ml MHC II protein and 50 μg LgDNA, vibrating at 300 rpm for 5 minutes, and then incubating at room temperature for 2 hours for disulfide cross-linking reaction to obtain the final product, that is, the target drug, which can be called peptide Yiminxiao, and the disulfide cross-linking reaction conditions are: the temperature is increased from 4°C to 95°C, and then reduced to 4°C. The diameter and size of Cedit (peptide Yiminxiao nanoparticles) were determined by dynamic light scattering (DLS).

[0027] Stability determination of peptide Yiminxiao: add peptide Yiminxiao to PBS (pH 7.2), or SDS solution, or urea solution at a concentration of 1 μg / ml; take samples after 24, 48, and 96 hours respectively; use enzyme-linked immunosorbent assay and DNA spectroscopy to quantitatively determine the components of peptide Yiminxiao.

[0028] like Figure 1 Characterization of peptide Yiminxiao, A, Electron micrographs of cedit (original magnification: ×5K) NPs (nanoparticles). B, Size distribution of peptide Yiminxiao NPs after treatment with PBS, SDS (0.05 mM), or urea (0.06 mM). NP size was determined by dynamic light scattering. CF, Peptide Yiminxiao was diluted at 1 μg / ml in PBS, pH 7.2. Samples were collected at the indicated time points on the X-axis. The bars represent the number of molecules shown in the peptide Yiminxiao NPs. GH, Eosinophils from Airway tissues were isolated from mice and exposed to FITC-peptide Yiminxiao NPs or cultured in PBS for 30 min. Flow cytometry graphs show FITC+eosinophils. Bar graphs show FITC+eosinophil counts. Column data are presented as mean±SD. Each point in the bar graph represents one sample. Statistics: ANOVA+Bonferroni test (CF) or Student's t test (H). ns: not significant. ****p<0.0001.

[0029] like Figure 2As shown in the figure, Moes specifically binds to antigen-specific CD4+ T cells. AD, CD3+CD4+ T cells were isolated from the spleen of DO11.10 mice and BALB / c mice. Cells were exposed to eosinophils (Meos) primed with FITC-peptide Iminxiao (or control peptide Iminxiao) and cultured for 30 minutes. A, Confocal image (×630) shows Meos (green) binding to DO11.10 CD4+ T cells (red). B, Dead cells were excluded. C, Adherent cells were gated out. D, CD4+FITC+ cell gate. EF, DO11.10 mice and BALB / c mice received nasal gavage (containing FITC-peptide Iminxiao). Three hours later, mice were sacrificed. Single cells were prepared from airway tissues and analyzed by flow cytometry. The gated image is FITC+ cells (cell type is indicated by arrows). The bar graph is the count of FITC+ cells. The group labels of F are the same as those of E, and the column data are expressed as mean±SD. Each point in the bar graph represents one sample. Statistics: ANOVA + Dunnett's test (C) and Student's t-test (F). ****p<0.0001. ViD: a reactive amine fluorescent dye used to stain dead cells. Cedit.a, Cedit.b, and Cedit.c are all control peptides, which correspond to peptides lacking OVA, peptides lacking MHC II, and peptides lacking LgDNA.

[0030] like Figure 3 As shown in the figure, peptide EDIT induced the expression of PD-L1 in Meos, and Meos were prepared and analyzed by ChIP, ELISA, and RT-qPCR. A, Pdl1 mRNA quantity. B, PD-L1 protein quantity. C, Kdm5a mRNA quantity. D, KDM5A protein quantity. E, KDM5A protein quantity in Pdl1 promoter. F, Pol II quantity in Pdl1 promoter. G, H3K9me3 quantity in Pdl1 promoter. H, demethylated Pdl1 promoter quantity. Bar data are expressed as mean±SD. Each point in the bar graph represents one sample. Statistics: ANOVA followed by Bonferroni test. **p<0.01; ***p<0.001; ****p<0.0001. Cedit.c and Cedit.d are both control peptide EDIT, peptide EDIT lacking Ab and LgDNA, respectively.

[0031] like Figure 4As shown in the figure, Meos induces the expression of PD1 in antigen-specific CD4+T cells. AC, Meos were prepared with the peptide Yiminxiao indicated on the X-axis of the bar graph. Meos were co-cultured with DO11.10 CD4+T cells for 24 h, and cellular RNA and protein were extracted and analyzed by RT-qPCR and ELISA. The bar graph shows the number of indicated molecules in CD4+T cells (purified at the end of the experiment). DF, DO11.10 mice were intranasally administered with the peptide Yiminxiao (the type of peptide Yiminxiao is indicated on the X-axis) every day for 5 consecutive days. CD4+T cells were isolated from airway tissues and analyzed by RT-qPCR and ELISA. The bar graph shows the number of indicated molecules in CD4+T cells. Bar data are expressed as mean±SD. Each point in the bar graph represents one sample. Statistics: ANOVA followed by Bonferroni test. *p<0.05; **p<0.01; ***p<0.001. Cedit.a, Cedit.b and Cedit.c are all control peptides, peptides lacking OVA, MHC II and LgDNA, respectively.

[0032] like Figure 5 As shown in the figure, Meos induces apoptosis of antigen-specific CD4+ T cells. AB, The cell types in the coculture environment are indicated above each flow cytometer panel. The gated image is apoptotic cells. The bar graph is the apoptotic cell count. CF, DO11.10 mice and BALB / c mice were given drugs by intranasal gavage every day for 5 days. Single cells were prepared from airway tissues and analyzed by flow cytometry. The gated image is apoptotic cells. The bar graph is the apoptotic cell count. The group labels of the bar graphs are the same as those of the flow cytometer graphs on the left. The column data are expressed as the mean ± SD of 6 samples per group. Statistics: ANOVA+Bonferroni test. ****p<0.0001. ns: not significant. Cedit.a, Cedit.b, and Cedit.c are all control peptide Yiminxiao, lacking OVA, MHC II, and LgDNA of peptide Yiminxiao components, respectively. PD1 / PD-L1 inhibitor: PD1 / PD-L1-in-3 (100nM for nasal instillation).

[0033] like Figure 6As shown in , an experimental AR mouse model was established. Mice were administered drugs by intranasal gavage every day for 5 consecutive days starting on the first day after sensitization. The bar graphs show the number of allergic symptoms (A, B) and the indicator molecules of NLF (CH). The bar data are expressed as the mean ± standard deviation of 6 samples in each group. Each point in the bar graph represents a sample. Statistics: ANOVA + Bonferroni test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Cedit.a, Cedit.b and Cedit.c are all control peptide Yiminxiao, peptide Yiminxiao lacking OVA, MHC II or LgDNA, respectively. Allergic rhinitis. NC: control.

[0034] like Figure 7 As shown in , administration can reduce antigen-specific CD4+T cells in the airways of mice with airway allergy. AR mouse model was established. Mice were given drugs by nasal gavage every day starting from the first day after sensitization for 5 consecutive days. Single cells were prepared from airway tissues and analyzed by flow cytometry. AB, gated plots show CD3+CD4+T cells. Bar graphs show CD3+CD4+T cell counts. CF, gated plots show CD44+ cells in CD3+CD4+T cells. Bar graphs show CD44+ cell counts. The group markers in the bar graphs are the same as those in the flow cytometry graph on the left. Bar data are expressed as mean±SD. Each point in the bar graph represents a sample. Statistics: Student's t-test (B) and ANOVA+Bonferroni test (D, F). Cedit.a, Cedit.b, Cedit.c and Cedit.d are control peptide Yiminxiao, peptide Yiminxiao lacking OVA, MHCII, siglecf Ab and LgDNA, respectively. Allergic rhinitis. NC: control.

[0035] From this, it can be concluded that the peptide Yiminxiao can edit a variety of target cells in vivo, and has successfully edited eosinophils in mice into cells with immune regulatory functions; it can also recognize antigen-specific Th2 cells, effectively cause antigen-specific Th2 cell apoptosis and effectively inhibit experimental respiratory allergic reactions; it can also edit other immune cells in vivo into targeted immune regulatory cells, including CD8+T cells, mast cells, NK cells, etc.

[0036] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A drug based on a protein peptide carrier, characterized in that: The invention comprises a protein peptide carrier, probiotic DNA and a targeting substance. The protein peptide carrier has the following amino acid sequence: Cys-Trp-Pro-Trp-Arg8-Pro-Cys-Arg8-Pro-Cys-Arg8-Cys.

2. The drug based on protein peptide carrier according to claim 1, characterized in that :The targeting substance includes ovalbumin, major histocompatibility antigen II or monoclonal antibody.

3. The drug based on protein peptide carrier according to claim 2, characterized in that :The weight ratio of the protein peptide carrier, probiotic DNA and targeting substance is 3-6:1:3-6, and the weight ratio of ovalbumin and major histocompatibility antigen II or monoclonal antibody in the targeting substance is 1:

1.

4. The drug based on protein peptide carrier according to claim 1, characterized in that :The probiotic DNA is DNA extracted from Lactobacillus rhamnosus.

5. A method for preparing a drug based on a protein peptide carrier, characterized in that: The steps are as follows: obtaining a protein peptide carrier with an amino acid sequence of Cys-Trp-Pro-Trp-Arg8-Pro-Cys-Arg8-Pro-Cys-Arg8-Cys; preparing probiotic DNA; mixing the protein peptide carrier, the probiotic DNA and a targeting substance to obtain a mixture; wherein the targeting substance includes ovalbumin, major histocompatibility antigen II or a monoclonal antibody.

6. The method for preparing a drug based on a protein peptide carrier according to claim 5, characterized in that :The probiotic DNA is DNA extracted from Lactobacillus rhamnosus.

7. The method for preparing a drug based on a protein peptide carrier according to claim 5, characterized in that : The weight ratio of the protein peptide carrier, probiotic DNA and targeting substance is 3-6:1:3-6, and the weight ratio of ovalbumin and major histocompatibility antigen II or monoclonal antibody in the targeting substance is 1:

1.

8. The method for preparing a drug based on a protein peptide carrier according to claim 7, characterized in that : The concentration of the protein peptide carrier is 200±20μg / ml, the concentration of ovalbumin is 100±10μg / ml, and the concentration of major histocompatibility antigen II or monoclonal antibody is 100±10μg / ml.

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