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 present invention relates to and a kind of novel there is antitumor and immune-enhancing activity nano gene vaccine and preparation method thereof, belong to biological technical field.

Background technology

Tumour cell reduces the expression of HLA I quasi-molecule and costimulatory molecules, and in escape body, the lymphocytic supervision of T, but have activated the activity of NK cell.If the reactivity signal of NK cell surface activation acceptor transmission is better than the inhibition signal that Inhibitory receptor transmits, NK cell will be activated, secrete cytokines and direct killing tumour cell, and being described as is the antineoplastic the first line of defence of body.On the other hand, the fact of tumor escape NK cell surveillance function and great many of experiments are according to proving, in tumor tissues, the number of NK cell and function are all lower than healthy tissues.Therefore, how in vivo or external promotion NK cell proliferation improve the biologic activity of NK cell, the effect of the immunotherapy of tumors based on NK cell is determined.

NK cell activates mainly through four identification approach below: 1) non-oneself identify, show as by the relevant molecular pattern of some cause of diseases of pattern recognition receptors identification and activated.2) identification of stress-induced, namely tumor cell or virus infected cell surface stress molecule, as MICA or ULBP albumen etc., be combined with Activating receptor NKg2D and activate NK cell.3) identification of disappearance oneself, shows as the cell loss of tumour cell or virus infection or has lowered the expression of classical HLAI quasi-molecule, cause inhibition signal weakening and make NK cell activation.4) cytokine mediated activation is also the most effective stimulus agent of NK cell activation, and the such as IL-15 of DC cell derived is the key cytokines promoting NK cell proliferation, activation and play cellulotoxic effect.

It is the somatomedin of NK cell that IL-15 is described as, and is also the key cytokines in NK cell development atomization.IL-15 and IL-2 space structure is similar, containing 4 α spirals, and molecular weight 14 ~ 15KD.IL-15, except its specificity α is by external, has beta receptor with IL-2, has γ acceptor with IL-2, IL-4, IL-7, IL-9 and IL-21.Compared with IL-2, IL-15 can effectively extend NK cell life cycle, promotes memory CD8+T cell long-term surviving in vivo, maintains immunological memory.Although IL-2 has been used for the treatment of metastatic renal cell carcinoma and malignant melanoma by FDA approval, the feature of the antigen restricted t cell proliferation of IL-2 induced tumor can reduce its anti-tumour effect.IL-2 has the advantages that to suppress memory t cell survival, maintain CD4+CD25+ regulatory T cells in addition, points out and replaces IL-2 for oncotherapy with IL-15, will produce better effect.

The experiment of IL-15 and IL-15R α knock out mice proves that IL-15 is mainly played effect by the trans submission of cell, namely IL-15 is by after dendritic cell or the secretion of other stroma cells, be combined (Ka=10-11) with the receptor alpha of these cell surfaces immediately, reactivation expresses the adjacent cells (as NK cell, CD8+T cell) of β and γ acceptor.And Lucas etc. prepares the mouse model of CD11chigh DC in inducibility purged body, finding the trans submission IL-15 of DC cell in lymphoglandula or spleen, is the necessary factor causing NK cell sensitization (in cytoplasm storage granules enzyme and Interferon, rabbit).In addition, Kobayashi etc. are by IL-15R α transfected height transitivity colon adenocarcinoma cell strain MC38, and transplanting common mouse can shift by Tumor suppression, and transplants the deratization of IL-15 clpp gene, and in the short period of time, metastases causes dead mouse to lung tissue.If the mode of action of the trans submission IL-15 of tumour cell is set up in the prompting of these phenomenons, NK cell is efficient amplification and activation not only, also possesses the feature of targeting anti-tumor.

To the trans submission IL-15 of tumour cell, primary method is to tumor cell transfection IL-15R α, but how to allow interior tumor cell specificity absorb ectogenic genophore, and current technique means is still difficult.Another kind of comparatively easy strategy is certain fusion rotein of preparation, has both made its aminoterminal be combined with tumor cell specific, and has carried IL-15 molecule again at carboxyl terminal, thus possesses the effect activating NK cell.Therefore, select the suitable molecule of " bridging " between tumour cell and IL-15 very crucial, both can prepare the monoclonal antibody of a certain tumour antigen, also can choose the acceptor of this antigen molecule.

MHC I class associated molecule 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, avidity therebetween relatively high (Kd is 8 × 10-7 ~ 4 × 10-9M).Therefore the NKG2D molecule of external preparation possesses the activity of targets identification tumour in body in theory.

The upper granted patent that this seminar obtains (a kind ofly strengthens active factor of lymphocyte target killing tumour and preparation method thereof, ZL201010533669.9), restructuring dsNKG2D-IL-15 albumen has been prepared based on prokaryotic expression technology, this albumen has the activity identifying tumor surface MICA antigen and activate NK cell, and has the function suppressing mouse junction cancer growth.But there is very long, shortcoming that complex steps, cost are higher consuming time from processes such as abduction delivering, purification and renaturation in this recombinant protein, is unfavorable for clinical widely using.And in gene vaccine body during application, often there is the shortcoming that metabolism is fast, cell active ingestion efficiency is low.

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.Gene vaccine is prepared for carrier plasmid DNA, siRNA, both at home and abroad existing a large amount of report about the material after chitosan-modified.

Therefore, the present invention mainly first by chitosan-modified be water soluble hydroxypropyl trimethyl ammonium chloride chitosan, the feature of positive charge can be carried under pH7.0 condition, thus the DNA of adsorption zone negative charge, be used for preparing the nanogel transporting and there is the recombination carrier of anti-tumor activity, be expected to one of vaccine becoming oncotherapy.

Summary of the invention

In order to solve the deficiencies in the prior art, the invention provides a kind of nano gene vaccine and preparation method thereof, this nano gene vaccine has immunostimulant and anti-tumor activity.

Method of the present invention can prepare the nano gene vaccine of load pcDNA3.1-sig-dsNKG2D-IL-15 fusion protein expression vector in vitro in a large number, and this nano gene vaccine can be absorbed by eukaryotic cell and express dsNKg2D-IL-15 albumen, and activate the function of NK and CD8+T cell.In body during application, this nano gene vaccine can raise lymphocytic function, has the activity of obvious Tumor suppression growth.

The solution of the present invention is on the basis of a upper patent of invention (ZL201010533669.9), the signal peptide sequence of mouse beta-2 microglobulin is introduced in the upstream of fusion gene dsNKG2D-IL-15 gene, prepare fusion gene ATG-sig-dsNKG2D-IL-15, its sequence is as shown in SEQ ID No:1, and the aminoacid sequence of its coding is as shown in SEQ ID NO:19.

The said fusion gene dsNKG2D-IL-15 of the present invention, can at eukaryotic cell expression.Be two solubility NK cell activation acceptor NKG2D and cytokine IL-15 fusion rotein dsNKG2D-IL-15, the sequence of this fusion gene is as shown in SEQ ID NO:3.

Fusion gene ATG-sig-dsNKG2D-IL-15 inserts in pcDNA3.1 (-) carrier between multiple clone site BamH I and Hind III, called after pcDNA3.1-sig-dsNKg2D-IL-15.

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:2; 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-15; Nucleotide residue 487-552,970-1035 are the gene order of soft segment.

A kind of nano gene vaccine, encapsulates fusion protein expression vector pcDNA3.1-sig-dsNKg2D-IL-15 by chitosan and forms.

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

This nano gene vaccine is transported by chitosan and expresses biologically active factors dsNKG2D-IL-15.Utilize chitosan self with the feature of a large amount of positive charge, be prepared into nano gene vaccine, the fusion protein expression vector that load is electronegative in a large number, this nano gene vaccine is engulfed through eukaryotic cell, after track fusion, can secrete solubility dsNKG2D-IL-15 albumen.Reach both tumor cells, again trans submission IL-15, the object of effective Tumor suppression.

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-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, 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-15 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-15 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-15 fusion protein expression vector, after liposome mediated transfection colon cancer cell CT-26, flow cytometer and histogenic immunity fluorescent method confirm that it expresses NKG2D and IL-15.Chitosan material can more than 90% to the encapsulation rate of pcDNA3.1-sig-dsNKg2D-IL-15 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.

The nano gene vaccine of load fusion protein expression vector is external hatch altogether with tumour cell after, in cells and supernatant, the concentration of dsNKG2D-IL-15 can significantly improve.And the bio-toxicity of nano gene vaccine to tumour cell does not have considerable change.After injecting normal mouse in nano gene vaccine body, there is the ability activating NK and CD8+T cell activation.In Mice Body, the experiment of lotus knurl confirms, can the growth of obvious Tumor suppression after the intramuscular injection of nano gene vaccine, and the lifetime of prolongation tumour patient.

Accompanying drawing explanation

Fig. 1. nano gene vaccination particles schematic diagram.

The construction strategy figure of Fig. 2 .ATG-sig-dsNKG2D-IL-15 fusion gene fragment.

Fig. 3 .pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vector is identified through double digestion, product electrophorogram (1.DNA ladder, 2.pcDNA3.1-dsNKG2D-IL-15,3.pcDNA3.1-dsNKG2D-IL-15 cut with BamH I/Kpn I enzyme, 4.pcDNA3.1-dsNKG2D-IL-15 BamH I/Hind III digestion, 5.pcDNA3.1-dsNKG2D-IL-15 Hind III/Kpn I enzyme is cut).

Fig. 4 .pcDNA 3.1-sig-dsNKg2D-IL-15 fusion protein expression vector, through liposome, after transfection colon cancer cell line CT-26, marks the streaming figure after NKG2D antibody.

Fig. 5 .CT-26 cell after the expression vector transfection of liposome, IL-15 comparison diagram in immuno-fluorescence assay born of the same parents, a graph expression carrier is pcDNA3.1; B graph expression carrier is pcDNA3.1-sig-dsNKG2D-IL-15.

Fig. 6. chitosan is to the detection of fusion protein expression vector encapsulation rate, electrophoresis result figure (the 1. chitosan: fusion protein expression vector is 1:2 of supernatant is got after chitosan encapsulating fusion protein expression vector, 2. chitosan: fusion protein expression vector is 1:1,3. chitosan: fusion protein expression vector is 2:1,4.marker).

Fig. 7. Electronic Speculum detects the size of nano gene vaccination particles

Fig. 8. the Zeta potential detected result figure of nano gene vaccine.

Fig. 9. with the nano gene vaccine of load pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vector directly and tumour cell hatch, collecting cell culture supernatant, ELISA detects the concentration results figure of dsNKG2D-IL-15 in supernatant, a figure is tumour cell B16BL6, b figure is tumour cell RAW264.7.

Figure 10. tumor cell in vitro bio-toxicity detection (detection of MTS/PMS method) result figure, a figure of nano gene vaccine is tumour cell B16BL6, b figure is tumour cell RAW264.7.

Figure 11. in nano gene vaccine body, inject normal mouse activate NK and CD8+T cell detection results figure, a figure is CD69+NK cell, and b figure is NKG2D+CD8+T cell.

Figure 12. on the graphic representation that tumor-bearing mice tumor growth and lifetime affect after using in nano gene vaccine body, a figure is the growth curve of in-vivo tumour, and b figure is the survival curve of tumor-bearing mice.

Figure 13. after using nano gene vaccine, the frequency of NK and CD8+T cell and Activation thereof in tumor-bearing mice spleen: a figure is unloaded chitosan group NK cell frequencies and Activation thereof; B figure is nano gene vaccine intramuscular injection group NK cell surface NKG2D frequency and Activation thereof; C figure is unloaded chitosan group CD8+T cell frequencies and Activation thereof; D figure is nano gene vaccine intramuscular injection group CD8+NKG2D+T cell frequencies and Activation thereof; E figure is nano gene vaccine intramuscular injection group CD8+CD44+T cell frequencies and Activation thereof.

Embodiment

The structure of 1.pQE31-dsNKG2D-IL-15 prokaryotic expression carrier

The primer sequence of 1.1 pcr amplifications: the means utilizing information biology, design corresponding primer respectively, with the cDNA of human peripheral blood single nucleus cell for template (see table 1) amplifies the gene fragment of sNKG2D (a), sNKG2D (b); With pORF hIL-15 carrier (from Invivogen company) for template, amplify the gene fragment of IL-15.The downstream primer sequence of sNKG2D (a) and sNKG2D (b) adds the reverse complementary sequence (showing in square frame in table 1) of (Gly4Ser) 4 respectively.

Show the primer sequence of 1.sNKG2D (a), sNKG2D (b) and IL-15 gene amplification

The pcr amplification product sequence of 1.2 sNKG2D (a) is as shown in SEQ ID NO:6, and wherein 1-6 bit base is the sequence of BamH I restriction enzyme site; 7th Nucleotide for avoiding NKG2D generation phase shift mutation to add at random; 8-425 is the gene order of NKG2D extracellular region; 426-482 is the gene order of soft segment; 483-488 is Stu I restriction enzyme site sequence.

1.3 the pcr amplification product sequence of sNKG2D (b) is as shown in SEQ ID NO:7, wherein base 1-6 is Stu 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.

1.4 the pcr amplification product sequence of IL-15 is as shown in SEQ ID NO:8, wherein base 1-6 is Pst I restriction enzyme site sequence; 7-348 is the gene order of IL-15; 349-351 is termination codon subsequence; 352-357 is Hind III digestion site sequence.

The structure of 1.5 pQE31-dsNKG2D-IL-15 recombinant vectorss: above-mentioned 3 sections of PCR primer are separately respectively after BamH I/Stu I, Stu I/Pst I, Pst I and Hind III double digestion, connected by T4 ligase enzyme, successively insertion vector pQE31 (pQE31 derives from Qiagen company).

The structure of 2.pcDNA3.1-sig-dsNKg2D-IL-15 fusion rotein carrier for expression of eukaryon:

The primer sequence of 2.1 pcr amplifications: the means utilizing information biology, designs corresponding primer (see table 2) amplifies signal peptide-sNKG2D (a), sNKG2D (b)-IL-15 gene fragment from pQE31-dsNKG2D-IL-15 prokaryotic expression carrier respectively.The reverse complementary sequence (showing in square frame in table) of (Gly4Ser) 4 is added at the downstream primer of signal peptide-sNKG2D (a).Fig. 2 shows the construction strategy of ATG-sig-dsNKG2D-IL-15 fusion gene fragment.

The primer sequence of table 2. signal peptide-sNKG2D (a), sNKG2D (b)-IL-15 gene fragment

The pcr amplification product sequence of signal peptide-sNKG2D (a) is as shown in SEQ ID NO:4, and 1-6 bit base is the sequence of BamH 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; 486-543 is the gene order of soft segment; 544-550 is Kpn I restriction enzyme site sequence.

The pcr amplification product sequence of sNKG2D (b)-IL-15 is as shown in SEQ ID NO:5, and wherein base 1-6 is Kpn 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; 491-830 is the gene order of IL-15; 831-833 is termination codon subsequence; 834-840 is Hind III digestion site sequence.

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

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

A large amount of preparations of 3.1 pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vectors: 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 fusion protein expression vector in 25mL or 100mL, low copy fusion protein 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.

3.2 the expression identification of pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vector in CT-26 cell (from ATCC): after clear and definite fusion protein expression vector transfecting eukaryotic cells cell, whether give expression to corresponding protein, by fusion protein expression vector liposome, transfection colon cancer cell CT-26.Cell after transfection uses the expression (Fig. 4) of flow cytomery NKG2D respectively, the expression (Fig. 5) of histogenic immunity Fluorometric assay IL-15.Result shows that this fusion protein expression vector can give expression to the fusion rotein of NKG2D and IL-15.

4. the preparation of the nano gene vaccine of load pcDNA3.1-sig-dsNKg2D-IL-15 fusion protein expression vector and qualification

The preparation of 4.1 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.

The preparation of nano gene vaccine shown in 4.2 Fig. 1:

Using water-soluble chitosan swelling fusion protein expression vector 1 by following experiment flow is Nano sol 2, wherein chitosan: fusion protein expression vector 1 mass ratio is when 1:2, be 92.8 ± 2.37 when being 83.8 ± 0.46,2:1 when encapsulation rate mean value is 40.3 ± 4.52,1:1.After encapsulating, the electrophoresis result of supernatant is shown in Fig. 6.Dynamic Laser color break-up is penetrated instrument and is measured nano gene vaccine size between 200 ~ 400nm, and size homogeneous (Fig. 7).Zeta potential is 53.8 ± 6.56 (Fig. 8).

Experiment flow:

1. utilize a large amount of plasmid extraction kits of Qiagen company, measure concentration and the total amount of fusion protein expression vector 1, 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.

4.3 nano gene vaccines are by the qualification expressed after tumour cell picked-up: nano gene vaccine is become 10% with cell culture medium respectively, 20% concentration, hatch 3 days altogether with tumour cell RAW264.7, B16BL6 cell (from ATCC).Collecting cell culture supernatant, detects by the ELISA kit of IL-15, finds that the concentration of IL-15 in the culture supernatant of 2 kinds of cells obviously increases (Fig. 9).

The qualification of 4.4 nano gene vaccine biology toxicities: for determining the biocompatibility of nano gene vaccine to tumour cell, use 2.5 respectively, 5, the nano gene vaccine of 10 μ g/ml and RAW264.7 and B16BL6 co-culture of cells.Detect the number of viable cell in each culture hole with MTS/PMS after 96 hours.Result shows, and no matter is unloaded chitosan or the chitosan wrapping up fusion protein expression vector 1, does not all have overt toxicity (Figure 10) to viable cell.

5. the activity identification of nano gene vaccine

5.1 nano gene vaccination normal mouses are on the impact of NK and CD8+T cell: for observing the ability whether possessing activation NK and CD8+T cell in this nano gene vaccine body after injection, by nano gene vaccine intramuscular injection normal mouse, for three days on end, put to death mouse separating spleen single cell suspension, observe the expression of NK cell surface activation acceptor CD69, CD8+T cell surface activation acceptor NKG2D.Found that, compared with chitosan injection group separately, the frequency of nano gene vaccine group NK1.1+CD69+ cell and CD8+NKG2D+T cell significantly raises (Figure 11), illustrates that nano gene vaccine can stimulate NK and CD8+T cell activation in normal mouse body.

On tumor-bearing mice tumor growth and the impact of lifetime after 5.2 nano gene vaccinations: first give the subcutaneous one-tenth knurl of mouse back after 5 days, to the restructuring dsNKG2D-IL-15 protein 60 μ g of mouse respectively intramuscular injection chitosan 100 μ g, intratumor injection nano gene vaccine 100 μ g, intramuscular injection nano gene vaccine 100 μ g and abdominal injection renaturation in vitro purifying, every day 1 time, record mice tumors grew situation and existing state.Result shows, compared with unloaded chitosan, intramuscular injection nano gene vaccine can the growth of obvious Tumor suppression, extends the lifetime (Figure 12) of tumor-bearing mice.Be separated each tumor-bearing mice spleen, in the mouse spleen of discovery nano gene vaccine intramuscular injection group, the frequency of NK cell increases, and the expression of NK cell surface NKG2D is apparently higher than unloaded chitosan group; The frequency of CD8+T cell is without considerable change, but nano gene vaccine intramuscular injection group CD8+NKG2D+T cell, CD8+CD44+T cell frequencies obviously raise (Figure 13), by strengthening lymphocytic activation, Tumor suppression grows prompting nano gene vaccine.

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.