CN104293815A - Nanometer gene vaccine as well as preparation method and application thereof - Google Patents

Abstract

The invention relates to a nanometer gene vaccine as well as a preparation method and an application thereof, belonging to the technical field of biology. In order to overcome the defects such as high preparation time consumption, complicated steps and high cost existing in recombinant proteins, the invention provides a nanometer gene vaccine. According to the nanometer gene vaccine, based on a genetic engineering technology, a signal peptide sequence, two identical NKG2D extracellular sequences and a sequence of cell factors IL-15 needed by eukaryotic expression are introduced, and an expression vector of fused proteins is constructed and is prepared into the nanometer gene vaccine by using a biocompatible chitosan material. Because the nanometer gene vaccine carries positive charges, the vaccine can be actively devoured by tumor cells or muscle cells, and proteins dsNKG2D-IL-15 linked between the tumor cells and lymphocytes are expressed in the eukaryocyte. Therefore, natural killer cells (NK) or cytotoxic T cells (CTL) in vivo are activated, the specific immune response in vivo can be enhanced, and an antitumor drug is prepared.

Description

A kind of nano gene vaccine and its preparation method and application

technical field

The invention relates to a novel nano-gene vaccine with anti-tumor and immune-enhancing activities and a preparation method thereof, belonging to the field of biotechnology.

Background technique

Tumor cells reduce the expression of HLA class I molecules and co-stimulatory molecules, escape the monitoring of T lymphocytes in the body, but activate the activity of NK cells. If the activating signal transmitted by activating receptors on the surface of NK cells is stronger than the inhibitory signal transmitted by inhibitory receptors, NK cells will be activated, secrete cytokines and directly kill tumor cells, known as the body's first anti-tumor line of defense. On the other hand, the fact that tumors escape the monitoring function of NK cells and a large number of experimental evidences prove that the number and function of NK cells in tumor tissues are lower than those in normal tissues. Therefore, how to promote NK cell proliferation and improve the biological activity of NK cells in vivo or in vitro determines the effect of NK cell-based tumor immunotherapy.

NK cells are mainly activated through the following four recognition pathways: 1) Non-self recognition, which is activated by recognizing some pathogen-related molecular patterns through pattern recognition receptors. 2) Stress-induced recognition, that is, recognition of stress molecules on the surface of tumor cells or virus-infected cells, such as MICA or ULBP protein, etc., which bind to the activating receptor NKg2D to activate NK cells. 3) Loss of self-recognition, manifested as tumor cells or virus-infected cells losing or down-regulating the expression of classic HLAI class molecules, causing the weakening of inhibitory signals and activating NK cells. 4) Cytokine-mediated activation is also the most effective stimulator of NK cell activation. For example, IL-15 derived from DC cells is a key cytokine that promotes NK cell proliferation, activation, and cytotoxic effects.

IL-15 is known as a growth factor of NK cells and a key cytokine in the process of NK cell development and differentiation. IL-15 is similar to IL-2 in spatial structure, contains 4 α-helices, and has a molecular weight of 14-15KD. In addition to its specific alpha receptor, IL-15 shares a beta receptor with IL-2 and a gamma receptor with IL-2, IL-4, IL-7, IL-9 and IL-21. Compared with IL-2, IL-15 can effectively prolong the life cycle of NK cells, promote the long-term survival of memory CD8+ T cells in vivo, and maintain immune memory. Although IL-2 has been approved by the FDA for the treatment of metastatic kidney cancer and malignant melanoma, the characteristic of IL-2 to induce tumor antigen-restricted T cell apoptosis would reduce its antitumor effect. In addition, IL-2 has the characteristics of inhibiting the survival of memory T cells and maintaining CD4+CD25+ regulatory T cells, suggesting that using IL-15 instead of IL-2 for tumor therapy will produce better results.

Experiments on IL-15 and IL-15Rα gene knockout mice have proved that IL-15 is mainly trans-presented by cells to exert its effect, that is, after IL-15 is secreted by dendritic cells or other stromal cells, it immediately binds to the cells on the surface of these cells. Receptor α binds (Ka=10-11), and then activates adjacent cells expressing β and γ receptors (such as NK cells, CD8+T cells). Moreover, Lucas et al. prepared a mouse model that could inducibly eliminate CD11high DC in vivo, and found that DC cells in the lymph nodes or spleen trans-presented IL-15, which caused NK cell sensitization (reserve granzyme and interferon in the cytoplasm) essential factor. In addition, Kobayashi et al. transfected IL-15Rα into highly metastatic colon adenocarcinoma cell line MC38 in mice, transplanted normal mice can inhibit tumor metastasis, and transplanted IL-15 gene knockout mice, tumors metastasized to lung tissue in a short time resulted in Mice die. These phenomena suggest that if the mode of action of trans-presenting IL-15 by tumor cells is established, NK cells will not only efficiently expand and activate, but also have the characteristics of targeted anti-tumor.

For tumor cells to trans-present IL-15, the primary method is to transfect tumor cells with IL-15Rα, but how to make tumor cells in vivo specifically take up exogenous gene carriers is still difficult with the current technical means. Another relatively simple strategy is to prepare a certain fusion protein, which not only binds specifically to tumor cells at its amino terminus, but also carries IL-15 molecules at its carboxyl terminus, so as to have the effect of activating NK cells. Therefore, it is very critical to select a suitable molecule that "bridges" between tumor cells and IL-15. It is possible to prepare a monoclonal antibody to a certain tumor antigen, or to select a receptor for the antigen molecule.

MHC class I chain-related antigen A (MICA) is a glycoprotein expressed on the cell surface after the body is stressed, and is widely expressed on the surface of most epithelial tumor cells and virus-infected cells , and only slightly expressed in normal intestinal epithelial tissue, it has become a good candidate target for tumor therapy. NKg2D is the receptor of MICA molecule, which belongs to the C-type lectin family member. One MICA molecule can bind to two homologous NKg2D molecules, and the affinity between them is relatively high (Kd is 8×10-7 ~4×10-9M). Therefore, NKG2D molecules prepared in vitro theoretically have the activity of targeting and recognizing tumors in vivo.

The last authorized patent obtained by our research group (an active factor that enhances lymphocyte targeting and killing tumors and its preparation method, ZL201010533669.9) prepared a recombinant dsNKG2D-IL-15 protein based on prokaryotic expression technology, which has the ability to recognize MICA antigen on the tumor surface and the activity of activating NK cells, and has the function of inhibiting the growth of colon cancer in mice. However, the process of induced expression, in vitro purification and renaturation of the recombinant protein has the disadvantages of long time-consuming, cumbersome steps and high cost, which is not conducive to widespread clinical use. However, when genetic vaccines are applied in vivo, they often have the disadvantages of rapid metabolism and low efficiency of active cellular uptake.

Nano-drugs have become an important direction of modern drug research and development because of their good stability, small irritation to the gastrointestinal tract, low toxicity and side effects, high bioavailability, good targeting and slow-release functions. "Nanogel" is a nano-sized hydrogel particle that can be dispersed in an aqueous solution. Bioactive molecules can be embedded into a cross-linked network structure through ionic bonds, hydrogen bonds, and hydrophobic interactions. Polyelectrolyte nanogels can also be combined with oppositely charged small molecule drugs, nucleic acid drugs, and proteins to deliver the corresponding drugs for the treatment of diseases. There have been a large number of reports at home and abroad on the use of chitosan-modified materials to carry plasmid DNA and siRNA to prepare genetic vaccines.

Therefore, the present invention mainly firstly modifies chitosan into water-soluble hydroxypropyltrimethylammonium chloride chitosan, which can carry the characteristics of positive charge under the condition of pH7.0, thereby adsorbing negatively charged DNA for use in Preparation of nanogels for transporting recombinant gene carriers with anti-tumor activity is expected to become one of the vaccines for tumor treatment.

Contents of the invention

In order to solve the deficiencies of the prior art, the invention provides a nano-gene vaccine and a preparation method thereof. The nano-gene vaccine has immune enhancing and anti-tumor activities.

The method of the present invention can prepare a large amount of nano gene vaccine loaded with pcDNA3.1-sig-dsNKG2D-IL-15 fusion protein expression vector in vitro, and the nano gene vaccine can be taken up by eukaryotic cells to express dsNKg2D-IL-15 protein, And activate the function of NK and CD8+T cells. When applied in vivo, the nano-gene vaccine can up-regulate the function of lymphocytes, and has obvious activity of inhibiting tumor growth.

The scheme of the present invention is based on the previous invention patent (ZL201010533669.9), introducing the signal peptide sequence of mouse β-2 microglobulin upstream of the fusion gene dsNKG2D-IL-15 gene, and preparing the fusion gene ATG-sig -dsNKG2D-IL-15, its sequence is shown in SEQ ID No: 1, and its encoded amino acid sequence is shown in SEQ ID NO: 19.

The fusion gene dsNKG2D-IL-15 of the present invention can be expressed in eukaryotic cells. It is the fusion protein dsNKG2D-IL-15 of dual soluble NK cell activating receptor NKG2D and cytokine IL-15, and the sequence of the fusion gene is shown in SEQ ID NO:3.

The fusion gene ATG-sig-dsNKG2D-IL-15 was inserted between the multiple cloning sites BamH I and Hind III in the pcDNA3.1(-) vector, and was named pcDNA3.1-sig-dsNKg2D-IL-15.

The obtained pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vector is sequenced, and its gene sequence is shown in SEQ ID NO: 2; wherein the nucleotide residue position 10-69 is the mouse β2-microglobulin leader The sequence of the peptide gene, the nucleotide residue position 70-486 is the gene sequence of the first NKG2D extracellular region; 553-969 is the gene sequence of the second NKG2D extracellular region; the nucleotide residue 1036-1380 is Gene sequence of IL-15; nucleotide residues 487-552, 970-1035 are gene sequences of flexible fragments.

A nano-gene vaccine is composed of chitosan-encapsulated fusion protein expression vector pcDNA3.1-sig-dsNKg2D-IL-15.

Said nano gene vaccine, said chitosan is hydroxypropyltrimethylammonium chloride chitosan.

The nano-gene vaccine expresses biologically active factor dsNKG2D-IL-15 through chitosan. Using the characteristic of chitosan with a large number of positive charges, a nano-gene vaccine is prepared, loaded with a large number of negatively charged fusion protein expression vectors, the nano-gene vaccine can secrete soluble dsNKG2D after being phagocytized by eukaryotic cells and expressed by the fusion gene - IL-15 protein. The purpose of not only recognizing tumor cells, but also trans-presenting IL-15 and effectively inhibiting tumors is achieved.

The present invention also provides the preparation method of nano gene vaccine, and its steps are as follows:

1) Construction of pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vector: Using the prokaryotic expression vector pQE31-dsNKG2D-IL-15 as the backbone, PCR was used to amplify the signal peptide of mouse β2-microglobulin and human The fusion fragment SEQ ID NO: 4 of the first extracellular region of the NKG2D receptor, and the fusion fragment SEQ ID NO: 5 of the second extracellular region of NKG2D and IL-15 are inserted into pcDNA3.1 successively. (-) carrier to obtain a fusion protein expression vector.

2) Preparation of nano-gene vaccine: a large amount of endotoxin-free pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vector was extracted using a large plasmid kit. The nano-gene vaccine was prepared by chitosan in vitro swelling method, and after the encapsulation efficiency, particle size and Zeta potential were determined, it was directly used to transfect cells, and the dsNKG2D-IL-15 protein could be secreted.

In the preparation method of the nanometer gene vaccine, the chitosan: fusion protein expression carrier mass ratio is 1:2-2:1.

For the preparation method of the above-mentioned nano gene vaccine, the large plasmid kit used is the large plasmid kit (No.12362) of Qiagen Company.

In the preparation method of the nano-gene vaccine, the chitosan is hydroxypropyltrimethylammonium chloride chitosan.

The preparation method of the above-mentioned nano-gene vaccine, the chitosan in vitro swelling method to prepare the nano-gene vaccine, also includes the following steps:

1) Add the fusion protein expression carrier solution dropwise into chitosan, shake at room temperature for 30 minutes at 300 rpm, and mix well;

2) Centrifuge at 4°C for 20-25 minutes to completely separate free DNA and nanogene vaccine.

The invention also discloses the application of the nanometer gene vaccine in the preparation of medicines for enhancing immunity or suppressing tumors.

The above-mentioned pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vector was transfected into colon cancer cell CT-26 by liposomes, and flow cytometry and tissue immunofluorescence confirmed the expression of NKG2D and IL-15 . The encapsulation efficiency of chitosan material for pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vector can exceed 90%. Between 6.56.

The concentration of dsNKG2D-IL-15 in the cell culture supernatant can be significantly increased after the nanogene vaccine loaded with fusion protein expression vector is co-incubated with tumor cells in vitro. However, the biotoxicity of nanogene vaccines to tumor cells did not change significantly. After the nanogene vaccine is injected into normal mice in vivo, it has the ability to activate NK and CD8+ T cell activation. Tumor-bearing experiments in mice have confirmed that intramuscular injection of nano-gene vaccines can significantly inhibit tumor growth and prolong the survival of tumor patients.

Description of drawings

Figure 1. Schematic diagram of nanogene vaccine particles.

Figure 2. Construction strategy diagram of ATG-sig-dsNKG2D-IL-15 fusion gene fragment.

Figure 3. pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vector identified by double enzyme digestion, product electrophoresis (1.DNA ladder, 2.pcDNA3.1-dsNKG2D-IL-15, 3.pcDNA3.1 -dsNKG2D-IL-15 was digested with BamH I/Kpn I, 4.pcDNA3.1-dsNKG2D-IL-15 was digested with BamH I/Hind III, 5.pcDNA3.1-dsNKG2D-IL-15 was digested with Hind III/ Kpn I digestion).

Figure 4. Flow chromatogram of pcDNA 3.1-sig-dsNKg2D-IL-15 fusion protein expression vector encapsulated in liposomes, transfected into colon cancer cell line CT-26, and labeled with NKG2D antibody.

Figure 5. After CT-26 cells were transfected with liposome-encapsulated expression vectors, the comparison of intracellular IL-15 detected by immunofluorescence method, the expression vector in figure a is pcDNA3.1; the expression vector in figure b is pcDNA3.1-sig -dsNKG2D-IL-15.

Figure 6. The detection of chitosan on the encapsulation efficiency of the fusion protein expression carrier, the electrophoresis results of the supernatant after encapsulation of the fusion protein expression carrier by chitosan (1. chitosan: fusion protein expression carrier is 1:2 , 2. Chitosan: fusion protein expression vector is 1:1, 3. Chitosan: fusion protein expression vector is 2:1, 4.marker).

Figure 7. The size of nano-gene vaccine particles detected by electron microscopy

Fig. 8. Diagram of Zeta potential detection results of nano gene vaccine.

Figure 9. The nanogene vaccine loaded with pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vector was directly incubated with tumor cells, the cell culture supernatant was collected, and the concentration of dsNKG2D-IL-15 in the supernatant was detected by ELISA , The picture in a shows the tumor cell B16BL6, and the picture in b shows the tumor cell RAW264.7.

Figure 10. The results of in vitro tumor cell biotoxicity detection (MTS/PMS method detection) of the nano gene vaccine, the picture a is the tumor cell B16BL6, and the picture b is the tumor cell RAW264.7.

Figure 11. Detection results of NK and CD8+T cells activated in normal mice injected with nano-gene vaccines. Figure a shows CD69+ NK cells, and picture b shows NKG2D+CD8+ T cells.

Figure 12. Curves of the effect of nanogene vaccines on tumor growth and survival in tumor-bearing mice after in vivo use. Figure a shows the growth curve of tumors in vivo, and picture b shows the survival curve of tumor-bearing mice.

Figure 13. The frequency and activation of NK and CD8+ T cells in the spleen of tumor-bearing mice after the use of nanogene vaccine: a shows the frequency and activation of NK cells in the empty chitosan group; b shows the nanogene The frequency and activation of NKG2D on the surface of NK cells in the vaccine intramuscular injection group; c is the frequency and activation of CD8+T cells in the empty chitosan group; d is the frequency and activation of CD8+NKG2D+T cells in the nanogene vaccine intramuscular injection group Its activation; e picture shows the frequency and activation of CD8+CD44+T cells in the nano gene vaccine intramuscular injection group.

Detailed ways

1. Construction of pQE31-dsNKG2D-IL-15 prokaryotic expression vector

1.1 Primer sequences for PCR amplification: Using bioinformatics methods, design corresponding primers respectively, and use the cDNA of human peripheral blood mononuclear cells as a template (see Table 1) to amplify sNKG2D (a) and sNKG2D (b) Gene fragment: The gene fragment of IL-15 was amplified with the pORF hIL-15 vector (from Invivogen) as a template. The reverse complementary sequence of (Gly4Ser)4 (shown in the box in Table 1) was added to the downstream primer sequences of sNKG2D(a) and sNKG2D(b).

Table 1. Primer sequences for sNKG2D (a), sNKG2D (b) and IL-15 gene amplification

1.2 The PCR amplification product sequence of sNKG2D(a) is shown in SEQ ID NO: 6, wherein the 1st-6th base is the sequence of the BamH I restriction site; the 7th base is randomly selected to avoid frameshift mutation of NKG2D Nucleotides added; No. 8-425 is the gene sequence of NKG2D extracellular region; No. 426-482 is the gene sequence of the flexible fragment; No. 483-488 is the sequence of the Stu I restriction site.

1.3 The PCR amplification product sequence of sNKG2D (b) is shown in SEQ ID NO: 7, wherein bases 1-6 are the Stu I restriction site sequence; 7-424 is the gene sequence of the extracellular region of NKG2D; 425- 484 is the gene sequence of the flexible fragment; 485-490 is the sequence of the Pst I restriction site.

1.4 The sequence of the PCR amplification product of IL-15 is shown in SEQ ID NO: 8, wherein bases 1-6 are the sequence of the Pst I restriction site; 7-348 is the gene sequence of IL-15; 349-351 is the gene sequence of Stop codon sequence; 352-357 is Hind III restriction site sequence.

1.5 Construction of the pQE31-dsNKG2D-IL-15 recombinant vector: the above three PCR products were digested with BamH I/Stu I, Stu I/Pst I, Pst I and Hind III respectively, connected by T4 ligase, successively Insert vector pQE31 (pQE31 comes from Qiagen).

2. Construction of pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein eukaryotic expression vector:

2.1 Primer sequences for PCR amplification: by means of bioinformatics, corresponding primers were designed (see Table 2) to amplify the signal peptides-sNKG2D(a), sNKG2D(b) from the pQE31-dsNKG2D-IL-15 prokaryotic expression vector )-gene fragment of IL-15. The downstream primer of the signal peptide-sNKG2D(a) plus the reverse complementary sequence of (Gly4Ser)4 (shown in the box in the table). Figure 2 shows the construction strategy of ATG-sig-dsNKG2D-IL-15 fusion gene fragment.

Table 2. Primer sequences of signal peptide-sNKG2D(a), sNKG2D(b)-IL-15 gene fragment

The PCR amplification product sequence of the signal peptide-sNKG2D(a) is shown in SEQ ID NO: 4, the 1st-6th base is the sequence of the BamH I restriction site; Randomly added nucleotides; No. 8-68 is the gene sequence of mouse β2-microglobulin signal peptide; No. 69-485 is the gene sequence of NKG2D extracellular region; No. 486-543 is the gene sequence of flexible fragment; 544-550 It is the Kpn I restriction site sequence.

The PCR amplification product sequence of sNKG2D(b)-IL-15 is shown in SEQ ID NO: 5, wherein bases 1-6 are the Kpn I restriction site sequence; 7-424 are the gene sequence of the extracellular region of NKG2D ; 425-484 is the gene sequence of the flexible fragment; 485-490 is the Pst I restriction site sequence; 491-830 is the gene sequence of IL-15; 831-833 is the stop codon sequence; 834-840 is the Hind III enzyme cutting site sequence.

2.2 Enzyme digestion identification of pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vector: the above two PCR products were digested with BamH I/Kpn I and Kpn I/Hind III respectively, and connected by T4 ligase , and successively inserted into the pcDNA3.1(-) vector (from Clontech Company) to obtain pcDNA3.1-sig-dsNKg2D-IL-15. Figure 3 shows the electrophoresis results of the pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vector after double digestion with BamH I/Kpn I, Kpn I/Hind III, and BamH I/Hind III. The results show that the recombinant gene fragment has been Was stably inserted into the pcDNA3.1(-) vector. The pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vector was sequenced for all inserts, and compared with the expected sequence by DNA star software, the homology was 99%.

3. Mass preparation and expression identification of pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vector:

3.1 Mass preparation of pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vector: On the basis of correctly obtaining the fusion protein expression vector, use the large extraction plasmid kit (Cat.No.12362) provided by Qiagen to extract Endotoxin-free fusion protein expression vector.

The experimental procedure is as follows:

a) Pick a single clone, culture in LB (2-5mL) containing Amp+ resistance for 8h, 37°C, 300rmp

b) Take 100μL or 50μL for transfer

i. [High-copy fusion protein expression vector in 25mL or 100mL, low-copy fusion protein expression vector in 100mL, transfer (100-200μL)]

ii.500mL LB, transfer (250-500μL), 37℃, 240rpm, 12-16h (shaker)

c) Centrifuge, 6500rpm 20min/7300rpm 15min, 4°C, discard the supernatant

d) Resuspend with 10mL Buffer P1 (full pipetting with pipette tip)

e) Add 10mL Buffer P2, quickly and completely turn it upside down 4-6 times, (turn blue), and let it stand at room temperature for 5 minutes at 15-25°C

f) During the standing process, screw the QIA filter Cartridge cap to the QIA filter Cartridge and put it into a suitable tube

g) Add 10mL of pre-cooled Buffer P3, quickly invert 4-6 times

h) Add the lysate to the QIA filter Maxi Cartridge, let stand at room temperature for 10min, do not insert the QIA filter Plungers, move the QIA filter Cartridge Cap, gently insert the Plunger into the QIA filter Cartridge, and filter the lysate into a 50mL test tube ( new test tube)

i) Add 2.5mL Buffer ER to the filtrate, invert the test tube 10 times, put it on ice, 30min

j) Equilibrate the QIAGEN-tip with 10mL Buffer QBT, empty by gravity flow

k) Put the filtrate of step i into the QIAGEN-tip, flow down

l) Wash QIAGEN-tip twice with Buffer QC, 30mL each time

m) Elute DNA with 15mL Buffer QN to a 30mL endotoxin-free test tube

n) Add 10.5mL of isopropanol at room temperature to the eluted DNA, mix well, and precipitate

o) 15000g, 30min, 4°C [7000rmp, 130min], discard the supernatant

p) Gently blow with 5mL 70% ethanol, centrifuge at 15000g, 10min [7000rpm, 1h], discard the supernatant

q) Let it stand at room temperature for 5-10 minutes, redissolve the DNA with an appropriate amount (200 μL) of Buffer TE, resuspend the obtained DNA, and place it in an ultra-clean bench.

3.2 Expression identification of pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vector in CT-26 cells (from ATCC): In order to clarify whether the fusion protein expression vector is transfected into eukaryotic cells, whether the corresponding protein is expressed, The fusion protein expression vector was encapsulated with liposome and transfected into colon cancer cell CT-26. The transfected cells were detected by flow cytometry for the expression of NKG2D ( FIG. 4 ), and the expression of IL-15 by tissue immunofluorescence ( FIG. 5 ). The results showed that the fusion protein expression vector could express the fusion protein of NKG2D and IL-15.

4. Preparation and identification of nano gene vaccine loaded with pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vector

4.1 Preparation of hydroxypropyltrimethylammonium chloride chitosan

Chitosan (deacetylation degree 85%, Mv=200000) was from Zhejiang Yuhuan Marine Biology Co., Ltd. Weigh 2 grams of chitosan and dissolve it in 2% acetic acid solution, stir and dissolve, add NaOH solution drop by drop to adjust the pH value to 9.0, at this time, a large amount of white flocculent precipitates precipitate, continue soaking for 12 hours, filter and wash Precipitate until the filtrate is neutral. The resulting white precipitate and 50 ml of isopropanol were added into a three-necked flask, stirred magnetically and heated to 80° C., and 2,3-epoxypropyltrimethylammonium chloride was added dropwise. After the reaction is finished, the reaction solution is precipitated in ethanol, and after the precipitate is separated, washed and dried, 2-hydroxypropyltrimethylammonium chloride chitosan can be obtained.

4.2 The preparation of the nano gene vaccine shown in Figure 1:

According to the following experimental process, the fusion protein expression carrier 1 is swollen with water-soluble chitosan to be a nano-sol 2, wherein chitosan: when the mass ratio of the fusion protein expression carrier 1 is 1:2, the average encapsulation efficiency is 40.3 ± 4.52, 1 :1: 83.8±0.46, 2:1: 92.8±2.37. The electrophoresis results of the supernatant after encapsulation are shown in Figure 6. The size of the nano-gene vaccine measured by a laser dynamic light flash instrument is between 200 and 400 nm, and the particle size is uniform (Fig. 7). Zeta potential was 53.8±6.56 (FIG. 8).

experiment process:

1. Using a large number of plasmid extraction kits from Qiagen, measure the concentration and total amount of fusion protein expression vector 1, and adjust the concentration to 1 mg/ml.

2. Weigh the weight of chitosan in ultra-clean bench, dilute to 1mg/ml with ultra-pure water. Wherein chitosan (degree of deacetylation 85%, Mv=200000) comes from Zhejiang Yuhuan Marine Biological Co., Ltd., and is modified as hydroxypropyltrimethylammonium chloride chitosan, N-[(2-hydroxy-3-trimethylammonium) ].

3. According to the mass ratio of 1:0.5, 1:1, and 1:2, the total volume is expected to be 1ml. Add the fusion protein expression vector 1 solution dropwise into the chitosan, shake at 300 rpm for 30 minutes at room temperature, and mix well.

Centrifuge at the maximum speed of 4.4°C for 20-25 minutes to completely separate free DNA and nano-gene vaccines.

5. The concentration and total amount of DNA in the supernatant were detected by an ultraviolet spectrophotometer, and the encapsulation efficiency was calculated. And use 1% agarose gel electrophoresis to observe the DNA residue in the supernatant.

6. The size of the nano-gene vaccine is detected by the laser dynamic light scintillation instrument and the transmission electron microscope, and the potential is detected by the Zeta potential instrument.

4.3 Identification of the expression of the nano-gene vaccine after being ingested by tumor cells: Dilute the nano-gene vaccine to 10% and 20% concentrations in cell culture medium, respectively, and co-incubate with tumor cells RAW264.7 and B16BL6 cells (from ATCC) for 3 days. The cell culture supernatants were collected and tested with an IL-15 ELISA kit, and it was found that the concentrations of IL-15 in the culture supernatants of the two types of cells were significantly increased ( FIG. 9 ).

4.4 Identification of the biological toxicity of nano-gene vaccines: In order to determine the biocompatibility of nano-gene vaccines to tumor cells, 2.5, 5, and 10 μg/ml nano-gene vaccines were co-cultured with RAW264.7 and B16BL6 cells. After 96 hours, the number of living cells in each culture well was detected by MTS/PMS. The results showed that neither the unloaded chitosan nor the chitosan encapsulating the fusion protein expression vector 1 had any obvious toxicity to living cells ( FIG. 10 ).

5. Activity identification of nanogene vaccine

5.1 The effect of nanogene vaccine injection on normal mice on NK and CD8+T cells: In order to observe whether the nanogene vaccine has the ability to activate NK and CD8+T cells after in vivo injection, inject the nanogene vaccine into normal mice intramuscularly, continuously On day 3, mice were sacrificed to separate spleen single cell suspension, and the expression of activating receptor CD69 on the surface of NK cells and NKG2D, an activating receptor on the surface of CD8+T cells, were observed. It was found that, compared with the chitosan injection group alone, the frequency of NK1.1+CD69+ cells and CD8+NKG2D+ T cells in the nanogene vaccine group was significantly increased (Figure 11), indicating that the nanogene vaccine can stimulate normal mice. NK and CD8+ T cell activation.

5.2 Effect of nanogene vaccine injection on tumor growth and survival period of tumor-bearing mice: Firstly, after 5 days of subcutaneous tumor formation on the back of the mice, intramuscular injection of chitosan 100 μg, intratumoral injection of nanogene vaccine 100 μg, intramuscular injection Inject 100 μg of nanogene vaccine and intraperitoneally inject 60 μg of recombinant dsNKG2D-IL-15 protein refolded and purified in vitro, once a day, and record the tumor growth and survival status of mice. The results showed that, compared with the empty chitosan, the intramuscular injection of the nano-gene vaccine could obviously inhibit the growth of the tumor and prolong the survival period of the tumor-bearing mice (Fig. 12). The spleens of tumor-bearing mice were isolated, and it was found that the frequency of NK cells in the spleens of the nanogene vaccine group was increased, and the expression of NKG2D on the surface of NK cells was significantly higher than that of the empty chitosan group; the frequency of CD8+ T cells was not However, the frequency of CD8+NKG2D+T cells and CD8+CD44+T cells in the nanogene vaccine intramuscular injection group increased significantly (Figure 13), suggesting that the nanogene vaccine can inhibit tumor growth by enhancing the activation of lymphocytes.

Claims (8)

1. a fusion gene, is characterized in that, introduced the signal peptide sequence of mouse beta-2 microglobulin by the upstream of fusion gene dsNKG2D-IL-15 gene, the fusion gene ATG-sig-dsNKG2D-IL-15 obtained, its sequence is as shown in SEQ ID NO:1.

2. a fusion protein expression vector pcDNA3.1-sig-dsNKg2D-IL-15, is characterized in that, its gene order is as shown in SEQ ID NO:2.

3. a nano gene vaccine, is characterized in that, encapsulates pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vector form by chitosan.

4. nano gene vaccine as claimed in claim 3, it is characterized in that, described chitosan is HACC.

5. prepare a method for nano gene vaccine according to claim 3, it is characterized in that, step is as follows:

1) structure of pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vector: with prokaryotic expression carrier pQE31-dsNKG2D-IL-15 for skeleton, PCR is utilized to increase respectively the fusion fragment SEQ ID NO:4 of the signal peptide of mouse B2M and first extracellular region of people NKG2D acceptor, with the 2nd extracellular region of NKG2D and the fusion fragment SEQ ID NO:5 of IL-15, these two fragments are successively inserted pcDNA3.1 (-) carrier, obtains fusion protein expression vector;

2) preparation of nano gene vaccine: utilize and carry plasmid kit greatly, extract without intracellular toxin pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vector in a large number, utilize chitosan in vitro swelling method to be prepared into nano gene vaccine.

6. the preparation method of nano gene vaccine as claimed in claim 5, it is characterized in that, described chitosan is HACC.

7. the preparation method of nano gene vaccine as claimed in claim 5, it is characterized in that, chitosan in vitro swelling method prepares nano gene vaccine, comprises the steps:

1) be added dropwise in chitosan by fusion protein expression vector solution, 300rpm, shaken at room temperature 30 minutes, mixes;

2) 4 DEG C of centrifuge 20-25 minute, thorough separated free DNA and nano gene vaccine.

8. the application in the medicine strengthening immunity or Tumor suppression prepared by nano gene vaccine according to claim 3.