CN104894151A - Nano gene drug capable of stimulating lymphocyte activation and preparation method thereof - Google Patents

Abstract

The invention relates to a novel nano gene drug capable of stimulating lymphocyte activation and a preparation method thereof. The invention discloses a fusion gene; and a mouse beta-2 microglobulin signal peptide is introduced from the upstream of the fusion gene dsNKG2D-IL-21 gene to obtain the fusion gene ATG-sig-dsNKG2D-IL-21. The invention also discloses a fusion gene expression carrier pcDNA3.1-sig-dsNKg2D-IL-21. The invention also discloses a nano gene vaccine drug which is formed by encapsulating the pcDNA3.1-sig-dsNKg2D-IL-21 fusion protein expression vector with chitosan. The invention also discloses a preparation method of a nano gene vaccine. The nano drug is used for carrying a nano gel of the recombinant dsNKG2D-IL-21 gene vector capable of stimulating lymphocyte activation and in-vivo targeted distribution, and is hopeful to become a drug for resisting tumors and infectious diseases.

Description

A kind of nano gene medicine stimulating lymphocyte activation and preparation method thereof

Technical field

The present invention relates to a kind of novel nano genomic medicine stimulating lymphocyte activation and preparation method thereof, belong to biological technical field.

Background technology

To physical exertion, anti-infective and antineoplastic function has vital role in lymphocytic activation.But in lasting chronic infection or tumour generating process, the immunologic function of body is suppressed, and is in low activity state.The effective medicine stimulating lymphocyte activation of preparation, to effectively treatment chronic infectious disease and tumour have very important significance.Lymphocytic growth and activate the main dependent cells factor, as IL-2, IL-7, IL-15, IL-21 etc., therefore based on gene and the protein sequence of cytokine, external preparation corresponding gene and protein thing, have the function well promoting lymphopoiesis and stimulate lymphocyte activation in theory.

IL-21 is primarily of the T cell secretion of activation, and its acceptor is mainly expressed in T cell, NK cell, NKT cell, gamma delta T cells, B cell, also can be expressed in scavenger cell, dendritic cell and part surface epithelial cell.After IL-21 and its receptors bind, JAK1, JAK3 tyrosine phosphorylation of primary activation coupled receptors, then STAT3 is activated, thus start the expression of the target gene granzyme A of IL-21 regulation and control, granzyme B, IFN-γ, anti-apoptotic genes expression bcl-3 and IL-21 and IL-21 acceptor, efficiently stimulate the lymphocytic activation of T and NK.In addition, in recent years, IL-21 was considered in the conversion of the classification of IgG antibody-like, play keying action from IgM to promotion B cell.

MHC I class related antigen A (MHC class I chain-related antigen A, MICA) be a kind of glycoprotein of expressing after body is subject to stress reaction at cell surface, in the cell surface wide expression of most of epithelium tumor cell and virus infection, and only trace expression in normal bowel epithelium, become the good candidate targets of oncotherapy.NKg2D is the acceptor of MICA molecule, belongs to C-type lectin family member, and 1 MICA molecule can be combined with the NKg2D molecule of two homologys, the higher (K of avidity therebetween _dbe 8 × 10 ^-7~ 4 × 10 ^-9m).Therefore the NKG2D molecule of external preparation possesses the activity of target recognition of tumor cell and virus infected cell in body in theory.

In simple DNA genomic medicine body during application, initiatively ingestion efficiency efficiency that is low, transfectional cell is not high for cell, and the situation of expression in vivo relevant gene product is subject to the impact of injecting pathway, immunizing dose.In addition, the expression product of DNA genomic medicine generally cannot be gathered in tumour or local infection sites in vivo, thus causes often not reaching desirable effect to the response to treatment of tumour or infectious diseases.

Nano medication because having good stability, little to gastrointestinal irritability, toxic side effect is little, bioavailability is high, the feature such as good targeting and slow-release function, becomes an important directions of modern medicines researchdevelopment." nanogel " (Nanogel) can disperse in aqueous and have the hydrogel particle of nano-scale, is fitted in crosslinked network structure by bioactive molecules by ionic linkage, hydrogen bond and hydrophobic interaction etc.Polyelectrolyte nanogel also can, in conjunction with the small-molecule drug of opposite charges, nucleic acid drug and protein, be used for transporting the treatment of relative medicine for disease.

Summary of the invention

In order to solve the deficiencies in the prior art, the invention provides a kind of nano gene medicine with identification abnormal cells and immune activation effect, i.e. nano gene vaccine, and preparation method thereof, the LS that this nano gene medicine is very strong is active.

Method of the present invention can prepare the nano gene medicine of load pcDNA3.1-sig-dsNKG2D-IL-21 fusion protein expression vector in vitro in a large number, and this nano gene medicine can be absorbed by eukaryotic cell and express dsNKg2D-IL-21 albumen, and activate the function of NK and CD8+T cell.In body during application, this nano gene medicine can assemble in position, tumor tissues place.

The solution of the present invention first in pcDNA3.1 (-) carrier, inserts fusion gene ATG-sig-dsNKG2D-IL-21, and its sequence is as shown in SEQ ID No:1, and the aminoacid sequence of its coding is as shown in SEQ ID NO:13.

The said fusion gene dsNKG2D-IL-IL-21 of the present invention, can at eukaryotic cell expression.Refer to the dsNKG2D-IL-21 formed after 2 NKG2D receptor extracellular region gene orders are connected with cytokine IL-21 gene order, the sequence of this fusion gene is as shown in SEQ ID NO:2.

Fusion gene ATG-sig-dsNKG2D-IL-21 inserts in pcDNA3.1 (-) carrier between multiple clone site BamH I and Hind III, called after pcDNA3.1-sig-dsNKg2D-IL-21.

The pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vector obtained is through order-checking, and its gene order is as shown in SEQ ID NO:3; Wherein nucleotide residue position 10-69 is the sequence of mouse B2M leading peptide gene, and nucleotide residue position 70-486 is the gene order of first NKG2D extracellular region; 553-969 is the gene order of second NKG2D extracellular region; Nucleotide residue 1036-1380 is the gene order of IL-21; Nucleotide residue 487-552,970-1035 are the gene order of soft segment.

A kind of nano gene vaccine, encapsulates integrative gene expression vector pcDNA3.1-sig-dsNKg2D-IL-21 by chitosan and forms.

Above-mentioned nano gene vaccine, described chitosan is HACC.

This nano gene medicine is transported by chitosan and expresses biologically active factors dsNKG2D-IL-21.Utilize chitosan self with the feature of a large amount of positive charge, be prepared into nano gene medicine, the fusion protein expression vector that load is electronegative in a large number, this nano gene medicine is engulfed through eukaryotic cell, after track fusion, can secrete solubility dsNKG2D-IL-21 albumen.Reach the cell of both tumor cell or virus infection, again significant stimulation body endolymph cell, make lymphocyte obviously strengthen function that is antitumor or that infect.

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

1) structure of pcDNA3.1-sig-dsNKg2D-IL-21 integrative gene expression vector: utilize PCR to increase containing the gene fragment SEQ ID NO:6 of the 2nd extracellular domain fragment SEQ ID NO:5 and IL-21 of fusion fragment SEQ ID NO:4 and NKG2D of the signal peptide of mouse B2M and first extracellular region of NKG2D acceptor respectively, successively these 3 fragments are successively inserted pcDNA3.1 (-) carrier, obtain 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-21 fusion protein expression vector in a large number.Utilize chitosan in vitro swelling method to be prepared into nano gene vaccine, after determining its encapsulation rate, size and Zeta potential, be directly used in transfectional cell, can secrete dsNKG2D-IL-21 albumen.

The preparation method of above-mentioned nano gene vaccine, chitosan: fusion protein expression vector mass ratio 1:2 ~ 2:1.

The preparation method of above-mentioned nano gene vaccine, the plasmid kit of carrying greatly of use carries greatly plasmid kit (No.12362) for Qiagen company.

The preparation method of above-mentioned nano gene vaccine, described chitosan is HACC.

The preparation method of above-mentioned nano gene vaccine, chitosan in vitro swelling method prepares nano gene vaccine, also 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.

The invention also discloses nano gene vaccine and prepare the application in the medicine strengthening immunity or Tumor suppression.

Above-mentioned pcDNA3.1-sig-dsNKg2D-IL-21 fusion protein expression vector, after liposome mediated transfection colon cancer cell CT-26, flow cytometer confirms that it expresses NKG2D.ELISA method confirms that cells and supernatant contains IL-21.Chitosan material can more than 90% to the encapsulation rate of pcDNA3.1-sig-dsNKg2D-IL-21 fusion protein expression vector, and its size distribution is between 200 ~ 400nm, and size is homogeneous, and Zeta potential scope is between 53.8 ± 6.56.

After the nano gene vitro Drug of load fusion protein expression vector and tumour cell are hatched altogether, cells and supernatant has activation NK and CD8 ⁺the ability of T cell activation.Tumor-bearing mice experiment in vivo confirms, nano gene medicine is in being obviously gathered in tumor tissues distribution.

Therefore, first the present invention mainly utilizes DNA recombinant technology, the extracellular domain sequence of 2 NKG2D genes and IL-21 gene order are inserted pcDNA3.1 expression vector, then using chitosan-modified is water soluble hydroxypropyl trimethyl ammonium chloride chitosan, the feature of positive charge can be carried under pH7.0 condition, thus the recombinant plasmid dna of adsorption zone negative charge.This Nano medication is used for transporting the nanogel of restructuring dsNKG2D-IL-21 genophore having and stimulate targeting distribution in lymph activation and body, is not reported both at home and abroad, is expected to become one of antitumor medicine with infectious diseases.

Nano medication of the present invention will have following advantage:

1) exogenous supplementary IL-21 protein drug has been proved and has had good anti-tumor activity, but this medicine transformation period is in vivo very short, and within 4 hours, analytic metabolism completes.Therefore the protein molecule that the dsNKG2D-IL-21 gene that we prepare is expressed in vivo, first can overcome transformation period short shortcoming.

2) the dsNKG2D-IL-21 genophore of recombinating expects the dsNKG2D-IL-21 albumen of expressing in vivo, the cell (mainly the cell of tumour cell and virus infection) that identifiable design NKG2D ligand positive is expressed, has good local superiority Distribution Effect in theory.

3) utilize chitosan nano particle to transport recombination, chitosan nano particle not only can be utilized to be easy to the feature of distribution tumor tissues, further proliferating cancer targeting, also can reach the object of local slow release gene, prolong drug transformation period in vivo as far as possible, thus medicament curative effect enhancement.

Accompanying drawing explanation

Fig. 1. nano gene drug particles schematic diagram (1. plasmid DNA; 2. chitosan nano particle).

The construction strategy figure of Fig. 2 .ATG-sig-dsNKG2D-IL-21 fusion gene fragment.

Fig. 3 .pcDNA3.1-sig-dsNKG2D-IL-21 integrative gene expression vector is identified through double digestion, and product electrophorogram (cut by 1.pcDNA3.1-dsNKG2D-IL-21 Xba I/Kpn I enzyme; 2.pcDNA3.1-dsNKG2D-IL-21 cut with Xho I/Kpn I enzyme; 3.pcDNA3.1-dsNKG2D-IL-21 cut with Xba I/Xho I enzyme; 4.DNA ladder).

Fig. 4 .pcDNA 3.1-sig-dsNKg2D-IL-21 integrative gene expression vector, through liposome, after transfection colon cancer cell line CT-26, marks Flow cytometry figure (the A. flow cytometer detection result after NKG2D antibody; B. statistical result).

Fig. 5 .CT-26 cell is after the expression vector transfection of liposome, and ELISA method detects the secretion of cells and supernatant IL-21.

Fig. 6. chitosan is to the detection of integrative gene expression vector encapsulation rate, electrophoresis result figure (the A.M.DNA ladder of supernatant is got after chitosan encapsulating integrative gene expression vector, 1. chitosan: integrative gene expression vector is 2:1,2. chitosan: integrative gene expression vector is 1:1,3. chitosan: integrative gene expression vector is 2:1,4.pcDNA3.1-dsNKG2D-IL-21; B. the calculation result of encapsulation rate).

Fig. 7. detect size (the A. Electronic Speculum detection of nano gene particle; B. dynamic optical shine instrument detect).

Fig. 8. the Zeta potential of nano gene particle detects.

Fig. 9. after hatching with the nano gene particle of load pcDNA3.1-sig-dsNKg2D-IL-21 integrative gene expression vector and tumour cell, spend the night with mouse spleen single cell suspension Dual culture, NK and CD8 ⁺detection (the detection of A.NK cell expresses activation acceptor CD69 of T cell activation; B.CD8 ⁺t cell expresses the detection of Activating receptor CD44).

Figure 10. nano gene medicine after tumor-bearing mice, dynamically observes the distribution of medicine in Mice Body in body after tail vein injection.

Embodiment

The structure of 1.pcDNA3.1-sig-dsNKG2D-IL-21 fusion gene carrier for expression of eukaryon:

1.1PCR the primer sequence of amplification: the means utilizing information biology, design corresponding primer (see table 1) respectively, amplify the gene fragment of signal peptide-sNKG2D (1), sNKG2D (2), IL-21 from mouse spleen single cell suspension.The downstream primer of the downstream primer in signal peptide-sNKG2D (1), sNKG2D (2) all adds the reverse complementary sequence of (Gly4Ser) 4 (showing in square frame in table).Fig. 2 shows the construction strategy of ATG-sig-dsNKG2D-IL-21 fusion gene fragment.

The primer sequence of table 1. signal peptide, NKG2D, IL-21 gene fragment amplification

The pcr amplification product sequence of signal peptide-sNKG2D (1) is as shown in SEQ ID NO:4, and 1-6 bit base is the sequence of Xba I restriction enzyme site; 7th Nucleotide for avoiding NKG2D generation phase shift mutation to add at random; 8-68 is the gene order of mouse B2M signal peptide; 69-485 is the gene order of NKG2D extracellular region; 486-544 is the gene order of soft segment; 545-551 is Xho I restriction enzyme site sequence.

The pcr amplification product sequence of sNKG2D (2) is as shown in SEQ ID NO:5, and wherein base 1-6 is Xho I restriction enzyme site sequence; 7-424 is the gene order of NKG2D extracellular region; 425-484 is the gene order of soft segment; 485-490 is Pst I restriction enzyme site sequence.

The pcr amplification product sequence of IL-21 is as shown in SEQ ID NO:6, and wherein base 1-6 is Pst I restriction enzyme site sequence; 7-376 is IL-21 gene order; 376-381 is Kpn I restriction enzyme site sequence; 373-375 is terminator codon.

2.2pcDNA3.1-sig-dsNKg2D-IL-21 the enzyme of integrative gene expression vector cuts qualification: above-mentioned 2 sections of PCR primer are separately respectively after Xba I/Xho I, Xho I/Pst I, Pst I/Kpn I double digestion, connected by T4 ligase enzyme, successively insert pcDNA3.1 (-) carrier (deriving from Clontech company) and obtain pcDNA3.1-sig-dsNKg2D-IL-21.Fig. 3 shows the electrophoresis result of pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vector after Xba I/Xho I, Xho I/Kpn I, Xba I/Kpn I double digestion, and result display recombination fragment is by stable insertion pcDNA3.1 (-) carrier.PcDNA3.1-sig-dsNKg2D-IL-21 integrative gene expression vector is carried out to whole order-checkings of Insert Fragment, through DNAstar software and expected sequence comparison, homology is 99%.

A large amount of preparation of 2.pcDNA3.1-sig-dsNKg2D-IL-21 integrative gene expression vector and expression identification:

A large amount of preparations of 2.1pcDNA3.1-sig-dsNKg2D-IL-21 integrative gene expression vector: on the correct basis obtaining fusion protein expression vector, what utilize Qiagen company to provide carries greatly plasmid kit (Cat.No.12362), extracts without endotoxic fusion protein expression vector.

Experiment flow is as follows:

A) choose mono-clonal, cultivate 8h, 37 DEG C, 300rmp in the LB (2-5mL) containing Amp+ resistance

B) switching gets 100 μ L or 50 μ L transfer

I. high copy integrative gene expression vector in 25mL or 100mL, low copy integrative gene expression vector in 100mL, switching (100-200 μ L)

Ii.500mL LB, switching (250-500 μ L), 37 DEG C, 240rpm, 12-16h (shaking table)

C) centrifugal, 6500rpm 20min/7300rpm 15min, abandons supernatant by 4 DEG C

D) with 10mL Buffer P1 resuspended (abundant with the piping and druming of rifle head)

E) add 10mL Buffer P2, fully put upside down 4-6 time rapidly, (becoming blue), 15-25 DEG C of room temperature leaves standstill 5min

F) in standing process, QIA filter Cartridge cap is screwed to QIA filter Cartridge, puts in suitable pipe

G) add the Buffer P3 that 10mL shifts to an earlier date precooling, put upside down 4-6 time rapidly

H) lysate is added in QIA filter Maxi Cartridge, room temperature leaves standstill 10min, do not insert QIA filter Plungers, mobile QIA filter Cartridge Cap, insert Plunger gently in QIA filter Cartridge, and filter lysate to 50mL test tube (new test tube)

I) add 2.5mL Buffer ER to filtrate, put upside down test tube 10 times, as on ice, 30min

J) balance QIAGEN-tip with 10mL Buffer QBT, run by gravity stream is empty

K) by the filtrate of the i-th step to QIAGEN-tip, flow down

L) QIAGEN-tip twice is washed with Buffer QC, each 30mL

M) endotoxic test tube is removed with 15mL Buffer QN eluted dna to 30mL

N) add the DNA of Virahol to elution of 10.5mL room temperature placement, mixing, separates out precipitation

O) 15000g, 30min, 4 DEG C [7000rmp, 130min], abandons supernatant

P) with the featheriness of 5mL 70% ethanol, centrifugal 15000g, 10min [7000rpm, 1h], abandon supernatant

Q) room temperature leaves standstill 5-10min, and with appropriate (200 μ L) Buffer TE dissolving DNA again, resuspended obtaining is placed on super clean bench

The expression identification of 2.2pcDNA3.1-sig-dsNKg2D-IL-21 fusion gene carrier in CT-26 cell (from ATCC): after clear and definite integrative gene expression vector transfecting eukaryotic cells cell, whether give expression to corresponding protein, by integrative gene expression vector liposome, transfection colon cancer cell CT-26.Cell after transfection uses the expression (Fig. 4) of flow cytomery NKG2D respectively, and ELISA method detects the expression (Fig. 5) of IL-21 in cell cultures.Result shows that this integrative gene expression vector can give expression to the fusion rotein of NKG2D and IL-21.

3. the preparation of the nano gene vaccine of load pcDNA3.1-sig-dsNKg2D-IL-21 fusion gene carrier and qualification

The preparation of 3.1 HACCs

Chitosan (deacetylation 85%, Mv=200000) is from Zhejiang Yuhuan Marine Bio Co., Ltd..Taking 2 grams of chitosans is dissolved in the acetic acid solution of 2%, and dropwise adding NaOH solution price modification pH value after stirring and dissolving is 9.0, now has a large amount of white flock precipitate to separate out, after continuing to invade bubble 12h, and suction filtration washing precipitation to filtrate is neutrality.Be added in there-necked flask by gained white depositions and 50ml Virahol, magnetic agitation is also warming up to 80 DEG C, dropwise adds 2,3-epoxypropyl trimethylammonium chloride ammonia.After reaction terminates, reaction solution precipitates in ethanol, and after precipitation separation, washing is dry, 2-HACC.

3.2 the preparation of the drug particles of nano gene shown in Fig. 1:

Using water-soluble chitosan swelling integrative gene expression vector 1 by following experiment flow is Nano sol 2, wherein chitosan: integrative gene expression vector 1 mass ratio is when 1:2, be 91.6 ± 1.96 when being 95.3 ± 0.65,2:1 when encapsulation rate mean value is 72.5 ± 4.65,1:1.After encapsulating, the electrophoresis result of supernatant is shown in Fig. 6.Instrument is penetrated in dynamic Laser color break-up and Electronic Speculum measures nano gene vaccine size between 200 ~ 400nm, and size homogeneous (Fig. 7).Zeta potential is 41.0 ± 0.56 (Fig. 8).

Experiment flow:

1. utilize a large amount of plasmid extraction kits of Qiagen company, measure concentration and the total amount of integrative gene expression vector, adjustment concentration is 1mg/ml.

2. weighing the weight of chitosan in Bechtop, is 1mg/ml with ultrapure water dilution.Wherein chitosan (deacetylation 85%, Mv=200000) is from Zhejiang Yuhuan Marine Bio Co., Ltd., is modified to HACC, N-[(2-hydroxy-3-trimethylammonium)].

3. press 1:0.5,1:1,1:2 mass ratio, cumulative volume expection is set to 1ml altogether.Be added dropwise in chitosan by fusion protein expression vector 1 solution, 300rpm, shaken at room temperature 30 minutes, mixes.

4.4 DEG C of whizzer maximum velocity centrifugation 20-25 minute, thorough separated free DNA and nano gene vaccines.

5. DNA concentration and total amount in UV spectrophotometer measuring supernatant, computational envelope rate.And remaining with DNA in 1% agarose gel electrophoresis observation supernatant.

6. instrument and transmission electron microscope detection nano gene vaccine size are penetrated in dynamic Laser color break-up, and zeta potential instrument detects current potential size.

After 4 analyzed in vitro tumour cell picked-up Nano medications, stimulate the activity identification of the situation nano gene vaccine of lymphocyte activation

Nano gene vaccine is become 10% with cell culture medium respectively, 20% concentration, hatch 6 hours altogether, washed cell with mouse colonic cell CT-26 (from ATCC), add mouse spleen single cell suspension Dual culture and spend the night.Next day, collecting cell, carried out DX5/CD69, CD8/CD44 antibody labeling, the frequency of Flow cytometry positive cell.After result confirms that the nano gene medicine of load dsNKG2D-IL-21 is absorbed by tumour cell, can obvious stimulation NK cell expressing CD69, CD8 ⁺t cell expresses CD44 (Fig. 9).

5. the distribution character of nano gene medicine in tumor-bearing mice body

For clearly this chitosan nano medicine distribution in vivo, further Cy5.5 fluorescence dye modification (Shanghai Kai Jing biotech firm completes) is carried out to chitosan nano-material.The subcutaneous dorsal injection 5 × 10 of C57BL6 mouse ⁶b16-RAE melanoma cell, the fluorescently-labeled Nano medication of tail vein injection after 7 days, small animal living body imaging system observes Nano medication DYNAMIC DISTRIBUTION in vivo.Result display (Figure 10), injectable drug 4 ~ 8 hours, chitosan medicine obviously can be gathered in position, tumor tissues place, and can maintain the time of 20 ~ 24 hours in vivo.Nano medication had both reached taxis and had been distributed in tumor tissues, again the object of extension body intracellular metabolite time.

Claims (10)

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-21 gene, the fusion gene ATG-sig-dsNKG2D-IL-21 obtained, its sequence is as shown in SEQ ID No:1.

2. an integrative gene expression vector pcDNA3.1-sig-dsNKg2D-IL-21, 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-21 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-21 integrative gene expression vector: to increase respectively the fusion fragment SEQ ID NO:4 of the signal peptide of mouse B2M and first extracellular region of mouse NKG2D acceptor to utilize PCR, with the 2nd the extracellular region SEQ ID NO:5 of NKG2D, and the fusion fragment SEQ ID NO:6 of IL-21, these three 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-21 integrative gene 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, is characterized in that, chitosan: fusion protein expression vector mass ratio is 1:2 ~ 2:1.

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

8. the preparation method of nano gene vaccine as claimed in claim 5, is characterized in that, the plasmid kit of carrying greatly of use carries plasmid kit greatly for Qiagen company.

9. 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 medicine.

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