CN105727283A

CN105727283A - Genetic engineering vaccine for treating breast cancer and preparation method thereof

Info

Publication number: CN105727283A
Application number: CN201610087798.7A
Authority: CN
Inventors: 杨廷稳
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-02-16
Filing date: 2016-02-16
Publication date: 2016-07-06

Abstract

The invention discloses a genetic engineering vaccine for treating a breast cancer and a preparation method thereof. The vaccine includes a GM-CSF gene modified human breast cancer cell and an adjuvant. A preparation method of the GM-CSF gene modified human breast cancer cell comprises the steps that the human breast cancer cell is infected with recombinant sendai virus supernate containing the GM-CSF gene to obtain the GM-CSF gene modified human breast cancer cell; the GM-CSF gene modified human breast cancer cell is inactivated and is subjected to heat shock processing. A preparation method of the adjuvant comprises the steps that a human breast cancer cell in logarithmic phase is inoculated to a RPMI 1640 cell culture medium containing 10% by volume of FBS, and a 15-25 micro-mol/mL compound (I) is added for cultivation for 36-48 hours to obtain a cell culture fluid; the cell culture fluid is centrifuged to remove supernate, a precipitate suspends in physiological saline and then is put into liquid nitrogen for 5-10 minutes, then the frozen substance is taken out and is put into warm water at 37 DEG C for thawing, and the freezing-thawing operation is repeated in this way for 3-5 times. The adjuvant can remarkably improve the killability of the GM-CSF gene modified human breast cancer cell to breast cancer cells, and the adjuvant and the GM-CSF gene modified human breast cancer cell has a remarkable synergistic action.

Description

A kind of for recombinant vaccine treating breast carcinoma and preparation method thereof

Technical field

The invention belongs to genetic engineering field, relate to recombinant vaccine, be specifically related to a kind of for recombinant vaccine treating breast carcinoma and preparation method thereof.

Background technology

Whole world breast cancer incidence starts the most in rising trend from late 1970s.8 women of the U.S. the most just have 1 people and suffer from breast cancer.China is not the country occurred frequently of breast carcinoma, but unsuitable optimistic, and the growth rate of China's breast cancer incidence but exceeds national 1～2 percentage point occurred frequently in recent years.Show according to pathogenesis of breast carcinoma data in 2009 of National Cancer Center and Ministry of Public Health prevention and control of diseases office announcement in 2012: tumor registration area, whole nation breast cancer incidence occupies the 1st of female malignant, female mammary gland cancer morbidity (rough and careless) whole nation adds up to 42.55/10 ten thousand, city is 51.91/10 ten thousand, and rural area is 23.12/10 ten thousand.

At present, tumor vaccine is mainly just like Types Below.

Tumour-cell vaccine.Traditional tumor vaccine is by autologous or allogeneic tumor cell or its crude extract, after physics, chemistry or biological method process, suppresses its energy for growth, keeps and strengthen its immunogenicity.But the immunogenicity of this vaccine is weak, it is difficult to cause immunne response.Later, tumor cell was added adjuvant such as bacillus calmette-guerin vaccine (BCG), coryne bacterium parvum or Freund adjuvant etc. to strengthen its immunogenicity, but effect was the most undesirable.And the preparation of this vaccine is the longest, it is impossible to meet the needs of tachysynthesis.In recent years full Cell vaccine is improved, attempted with build strain tumor cell line alternatively thing.Zoopery confirms, what use processed build strain tumor cell has the advantage that the multiple antigen of (a) tumor cells expression as full Cell vaccine, it is to avoid separates, identify the loaded down with trivial details of antigen；The MHC of b tumor cells expression that () is used as vaccine can not mate with patient, can excite immunne response as alloantigen；C () prepares vaccine with building strain tumor cell, simple, substantially reduces patient from making a definite diagnosis the vaccinated time.

Oncogene engineered vaccine.Oncogene engineered vaccine is a kind of vaccine by gene recombination technology, genes of interest being imported recipient cell and prepare.Along with the development that immune-base is theoretical, the development of various vaccines is also maked rapid progress.Chinese scholars is own imports recipient cell by different genes of interest, improves its immunogenicity or the ability of activating effect cell.This vaccine mainly realizes its Antineoplastic effect from the following aspects: (a) improves body anti-tumor ability；B () strengthens immunogenicity of tumor；(c) gene outcome direct killing oncocyte.Focus about the research of oncogene engineered vaccine was concentrated mainly on GM-CSF gene vaccine, polygene combined modification vaccine, gene Modified Dendritic Cells and structure dendritic cell vaccine in recent years, and the combining of immune modification gene and suicide gene.As by B7-l and IL-4, B7-l and IL-7, IFN-Y, GM-CSF and B7-l, the multiple cytokine gene such as IL-12, B7-l and tumor antigen carries out combined modification.Cytokine profiles can be maximized favourable factors and minimized unfavourable ones the most collaborative with the co-transfection of suicide gene, has more preferable immunological effect.

Dendritic cell (DC) vaccine.Current tumor antigen peptide, tumor cell lysate, tumor cell RNA ESW DC or proceed in DC by TAA gene, be regarded as the most promising tumor vaccine and prepare scheme.But various ESW means all have half-life short shortcoming, to induce high-caliber lasting anti tumor immune response, need repeated multiple times internal feedback sensitization DC.Another can solve antigen by the method for tumor cDNA or mRNA ESW DC and lose or the problem of antigenic variation, and turn avoid the difficulty separating massive tumor antigenic peptides.Modern molecular biology technique oneself can expand the most in a large number tumor mRNA and build tumor cDNA library, this is to we provide huge tumor antigen storehouse, also for application cDNA, mRNA ESW DC cell provide raw material.External use tumor cDNA or mRNA impact DC cell need not determine tumor antigen, and the DC cell after impacting theoretically can express any possible tumor associated antigen, thus used by some researchs and achieve good effect.

Peptide vaccine.Angtigen presentation process and the research of Immune discrimination, oneself confirms that tumor antigen must be degraded to small peptide in APC intracellular, and eventually forming peptide-MHC-TCR complex could be T cell identification, and excites ctl response, and this is that the research of peptide vaccine provides theoretical foundation.Tumor-antigen peptide used can be divided into two classes: artificial synthetic polypeptide and natural source polypeptide.Synthesis polypeptide vaccine is as what the determination of tumor antigen epi-position grew up.Synthesizing corresponding polypeptide according to tumor antigen epi-position, then together with adjuvant or individually immunity, can induce the anti tumor immune response for immunogenic epitopes.The advantage of synthesis polypeptide vaccine is: induce immunoreactive specificity higher；Can synthesize in a large number；Antigen purity is high；Various tectotypes are widely used；Only need a small amount of patient's tumor tissues (for determining the expression of antigen)；CD+4T and CD+ST cell can be activated.These advantages of synthetic peptide vaccine make a kind of important vaccination.

Nucleic acid vaccine.Nucleic acid vaccine is to be formed by the antigen gene fragment and vector construction thereof that can cause protective immunological reaction.Including DNA vaccination and RNA vaccine, current most study is DNA vaccination.Compared with other vaccines, DNA vaccination has many advantages: inoculation gene can be with long-term expression, the immunoreation persistent high efficiency of induction；Preparation is simple, easily builds, and economy saves time；Compared with recombiant protein, gene vaccine is closer to natural infection state；Immunoreation is strong, special, extensively, comprehensively, and can produce Cross immunogenicity；Nucleic acid vaccine energy excitating organism cellular immunization and humoral immune reaction simultaneously, can induce the CTL effect that many subunit vaccines can not be induced；Good stability, easily preserves；Immature immune system can be induced to produce T cell immunne response；Dangerous without infection duplication, back mutation will not be produced；Available identical carrier preparation, for the gene vaccine of multiple disease, develops polyvalent vaccine.

GM-CSF (granulocyte macrophage colony simulating factor) is a kind of cytokine mainly produced by macrophage and activating cell, it by promoting the antigen-presenting cell differentiation such as differentiation and maturation dendritic cell, macrophage, ripe and activation and then can promote that Th, Tc, NK identify tumor associated antigen, infiltrate at tumor locus, systemic antitumor is caused to react, may additionally facilitate cell CD45T cell differentiation and maturation and the expression of adhesion molecule B7/BB1 while killing tumor, improve its antigen presentation capability.The most also can increase the expression of macrophage MHC II (MHCII), improve its antigen presentation capability.The tumor cell that GM-CSF gene is modified, by internal injection past after radiological inactivation, can increase local inflammatory response, a large amount of apocytes, macrophage and DC infiltration, promote tumor antigen presentation, it is possible to induce strong anti tumor immune response.

Summary of the invention

It is an object of the invention to provide a kind of novel mastocarcinoma gene engineered vaccine, this recombinant vaccine includes human breast cancer cell and the adjuvant that GM-CSF gene modifies；Wherein, the human breast cancer cell that GM-CSF gene is modified can stablize expression of GM-CSF, killing tumor cell；Adjuvant can be obviously enhanced the human breast cancer cell of the GM-CSF gene modification lethality to breast cancer cell, and the two has significant synergism.

The above-mentioned purpose of the present invention is achieved by techniques below scheme:

A kind of recombinant vaccine for treating breast carcinoma, the human breast cancer cell modified including GM-CSF gene and adjuvant；

The human breast cancer cell that described GM-CSF gene is modified is made by the steps:

Step S1, infects human breast cancer cell with the recombinant sendai virus supernatant containing GM-CSF gene, obtains the human breast cancer cell that GM-CSF gene is modified；

Step S2, the human breast cancer cell that the GM-CSF gene obtaining step S1 is modified inactivates and carries out heat shock process；

Described adjuvant is made by the steps:

Step S1, the human breast cancer cell of trophophase of taking the logarithm is inoculated in RPMI 1640 cell culture fluid containing percent by volume 10%FBS, and the compound (I) of the following structural formula adding 15～25 μm ol/mL is cultivated 36～48h and obtained cell culture fluid,

Step S2, by centrifugal for cell culture fluid supernatant of abandoning, precipitation is suspended in normal saline, places in liquid nitrogen freezing 5～10 minutes, then is taken out by frozen material, puts into 37 DEG C of warm water and melt, such multigelation 3～5 times, described adjuvant.

Further, described adjuvant is made by the steps: step S1, the human breast cancer cell 5 × 10 of trophophase of taking the logarithm⁷Individual be inoculated in 1ml containing percent by volume 10%FBS RPMI 1640 cell culture fluid in, add 20 μm ol/mL described compound (I) cultivate 42h obtain cell culture fluid；Step S2, by centrifugal for cell culture fluid supernatant of abandoning, precipitation is suspended in 1ml normal saline, places in liquid nitrogen freezing 5～10 minutes, then is taken out by frozen material, puts into 37 DEG C of warm water and melt, such multigelation 3～5 times, described adjuvant.

Further, described human breast cancer cell is SKBR3 MCF-7.

Further, described ablation method is for using⁶⁰Co irradiates inactivation.

Further, described heat shock processing method is: temperature bath 0.5h～2h under the conditions of human breast cancer cell is placed in 42 DEG C～65 DEG C.

The application in the medicine of preparation prevention or treatment breast carcinoma of the described mastocarcinoma gene engineered vaccine.

Advantages of the present invention:

The recombinant vaccine that the present invention provides includes human breast cancer cell and the adjuvant that GM-CSF gene modifies；Wherein, the human breast cancer cell that GM-CSF gene is modified can stablize expression of GM-CSF, killing tumor cell；Adjuvant can be obviously enhanced the human breast cancer cell of the GM-CSF gene modification lethality to breast cancer cell, the two has significant synergism, can work in coordination with prevention and treatment breast carcinoma, the most repeated multiple times inoculation can improve prevention or the effect for the treatment of tumor further.

Detailed description of the invention

Further illustrate the essentiality content of the present invention below in conjunction with embodiment, but do not limit scope with this.Although the present invention being explained in detail with reference to preferred embodiment, it will be understood by those within the art that, technical scheme can be modified or equivalent, without deviating from the spirit and scope of technical solution of the present invention.The test method not described in detail is this area routine test operation, and the instrument emphasized the most especially and reagent are this area conventional instrument and reagent.

The preparation of the human breast cancer cell of embodiment 1:GM-CSF genetic modification

The first step: the preparation of the granulocyte macrophage colony simulating factor cDNA containing signal peptide

1, the preparation of granulocyte macrophage colony simulating factor mRNA:

(1) human peripheral lymphocyte separation liquid isolates mononuclearcell, is total to about 1 × 10⁶Cell, stimulates 30 hours through induction, makes cytokine gene expression, in case extracting mRNA.

(2) extracting of mRNA: centrifugal collecting cell, is suspended in 1.5ml RNA extraction buffer, repeatedly lashes homogenate cell with syringe, adds 3ml extraction buffer the most again, be sufficiently mixed, centrifugal 1000cpm 5 minutes, collects supernatant.Taking Oligo (dT)-Cell μ lose Spun Column, mixed content thing, 350g is centrifuged two minutes, discards liquid in post, takes 4ml supernatant upper prop, mixing, discards liquid.Then washing three times with high-salt buffer, low salt buffer washes two times, finally with the elution buffer eluting three times through 65 DEG C of preheatings, every time with 0.25ml, collects eluent, then ethanol precipitation.

2, the synthesis of granulocyte macrophage colony simulating factor cDNA:

(1) design of primer and synthesis: design pair of primers according to human granulocyte macrophage colony stimulus factor in GenBank (GM-CSF) gene order, forward primer:

Upstream: 5 '-GCTCAAGCTTCTGGAGGATGTGGCTGC-3 ' (include Hind III site)；

Downstream: 5 '-GGACGAATTCACTCCTCGAATGGCTCCC-3 '.

(2) synthesis of cDNA: mRNA is dissolved in 20 μ l ddH20,65 DEG C 10 minutes, be placed in the most standby.In Article 1 chain reaction mixed liquor, add 1ml DTT and the RNA of thermal denaturation, 37 DEG C of moisturizings 1 hour, finally add 1 μ l Klenow enzyme, 37 DEG C 30 minutes, 65 DEG C after 10 minutes again with powder and chloroform ,-20 DEG C of preservations after Sephacryl S-300 column purification.

3, PCR amplification GM-CSF gene:

Take the PBL cDNA 10 μ l prepared, add 5 ' and 3 ' amplimer 1 μ l, 10 × reaction buffer 5 μ l, dNTP 4 μ l, add water to 50 μ l, 94 DEG C of degeneration in 5 minutes.

4, the clone of GM-CSF cDNA:

In order to identify the accuracy of PCR primer, first product cloning must be entered PUC18 or pBluescriptDNA, through determined dna sequence confirm as GM-CSFcDNA errorless after again this fragment is reclaimed, and with expression vector recombinate.Therefore PCR primer is dissolved in TE, after suitable Rastriction enzyme, with as the carrier DNA restructuring of enzyme hydrolysis, convert after TG1 and be applied to LB/AP flat board, 37 DEG C of overnight incubation.

5, plasmid DNA extracting in a small amount:

One single bacterium colony is got involved in the 3ml LB containing AP (50 μ g/ml), 37 DEG C of overnight incubation.Bacterium solution being transferred in 1.58ml centrifuge tube, 5000rpm is centrifuged 2 minutes, precipitates thalline.With 100 μ l solution I suspension thalline, then add 200 μ l solution II, gentle reverse mixing, it is placed on ice, after solution is clarified, adds 150 μ l solution III, fully mix.15000rpm is centrifuged for 10 minutes, collects supernatant, with equal-volume phenol/chloroform/isoamyl alcohol extraction once, adds 2.5 times of ethanol mixings, precipitation.Centrifugal 15000rpm 10 minutes, washes once by precipitation with 70% ethanol, and water pump is drained, and is finally dissolved in 20 μ l TE, processes 1 hour with 200 μ g/ml RNase, deposits for-20 DEG C.

6, a large amount of extractings of plasmid DNA:

(1) inoculated and cultured: a single colony inoculation is cultivated to late log phase (OD600 ≈ 0.6) in 3ml LB (containing AP), take 500 μ l and inoculate in the 50ml LB containing antibiotics, cultivate to late log phase equally, take 15ml again and inoculate in the 1.5LLB containing corresponding antibiotics, overnight incubation.

(2) plasmid extraction: 1.5L culture is centrifuged 5500rpm 10 minutes, collects thalline.With absorbent paper, residual liquid on centrifugal tube wall is dried.Thalline is suspended in 30ml solution I, puts in room temperature 10 minutes.Add liquid II 60ml, rearmounted 10 minutes on ice of mix homogeneously.Adding solution III 45ml, after putting ice bath 10 minutes, 15000rpm is centrifuged 10 minutes.Taking supernatant after filtered through gauze, add equal-volume isopropanol precipitating, 15000rpm is centrifuged 10 minutes, washes once with 70% ethanol, vacuum drying.Precipitation being dissolved in 20ml TE, adds equal-volume 5M LiCl and precipitate macromole RNA, place 10 minutes in a water bath, 4 DEG C of 15000rpm are centrifuged 10 minutes, take supernatant, use equal-volume isopropanol precipitating.Centrifugal, be dried, be dissolved in 5ml TE, add RNase to 200 μ g/ml, 37 DEG C 30 minutes.Adding equal-volume 13%PEG (8000)/1.5M NaCl and precipitate DNA, ice bath puts 30 minutes.Centrifugation, dried with TE 5ml dissolving, respectively extract once with phenol, phenol/chloroform/isoamyl alcohol, chloroform/isoamyl alcohol.Finally add 1/10 volume 3M NaAc and 2 times of ethanol precipitations.-20 DEG C of preservations.Used time centrifugation again, is dissolved in TE.

(3) CsCl ultracentrifugation purification DNA: the DNA extracted such as above-mentioned alkaline process, through LiCl after purification, use CsCl ultracentrifugation, by the consumption of 1g/ml, add solid CsCl, 30 DEG C of dissolvings.Every 10mlDNA adds 0.8ml ethidium bromide solution (10mg/ml), and the final densities of CsC solution is 1.55g/ml, refractive index 1.3860, and ethidium bromide concentration is about 704 μ g/ml.Room temperature 8000rpm5 minute, floating on solution is ethidium bromide and the complex of protein formation.Clear solution syringe under scum silica frost is sucked in the centrifuge tube being used for ultrafiltration core, fill remainder with light paraffin oil, and seal.45000 revs/min centrifugal 24-48 hour (20 DEG C).Centrifugal complete, visible two region of DNA bands under normal light shines, bottom zone is made up of closed circular form plasmid DNA, this band is inserted with syringe needle, sucking-off, in 1.5ml pipe, extracts through water saturated n-butyl alcohol repeatedly with isopyknic, until pink is from aqueous phase and organic facies hour.Aqueous phase is added 3 times of TE dilutions, adds 2.5 times of dehydrated alcohol precipitations.Wash once with 70% ethanol after Li Xin, after draining, be dissolved in TE ,-20 DEG C of preservations.

7, restriction enzyme reaction:

The buffer matched is selected according to different restricted enzyme.Typically take 0.5-1 μ g DNA, restricted enzyme 5U, reaction volume 20 μ l, 37 DEG C of moisturizings 1-2 hour, identify enzyme action result with 1.2% agarose gel electrophoresis (containing ethidium bromide 0.5-1 μ g/ml).

8, freeze-thaw method recovery endonuclease bamhi:

Under uviol lamp, DNA fragmentation is scaled off from agarose gel, add 100 μ l TE, at dry ice: freeze in ethanol rearmounted 37 DEG C 15 minutes, repeatedly for three times, 15000rpm be centrifuged 10 minutes after sucking-off supernatant, add 1/10 volume NaAc and 2 times of volume ethanol precipitation, centrifugal collection, precipitation, after water pump is drained, is dissolved in TE, can be attached reaction.

9, DNA connects:

To wait the DNA to be connected mixing of mole number, and add 5 × T4 ligase buffer 4 μ l, add water to 20 μ l, and add T4DNA ligase 2U, 12 DEG C connect overnight, i.e. can be used for converting.

10, the conversion of recombinant DNA:

(1) prepared by competent cell: be inoculated in 50 μ l LB by the recipient bacterium 250 μ l of overnight incubation, 37 DEG C 1.5-3 hour to mid-log phase, bacterium solution to ice bath 10 minutes, 5000rpm is centrifuged 5 minutes, the precipitation thalline pre-cooled 100mMMgCl of 1/2 volume₂Suspending, ice bath 20 minutes, 4 DEG C of 5000rpm are centrifuged 5 minutes, and thalline is suspended in the 100mMCaCl of 1/15-1/10 volume₂In, ice bath is put 60 minutes, i.e. can be used for converting.

(2) conversion of recombinant DNA: add 100 μ l competent cells by connecting DNA overnight, mixing, ice bath is placed 40 minutes, or mix gently, 42 DEG C 2 minutes, coat on the LB flat board containing antibiotics, 37 DEG C of overnight incubation.

11, double-stranded DNA order-checking:

(1) denatured double stranded: sample DNA about 1.5-2 μ g, it is dissolved in 8 μ l ddH₂After 0, add 2M NaOH 2 μ l, to room temperature 10 minutes, add 3 μ l 3MNaAc and neutralize, then add 7 μ l ddH₂After 0, add 60 μ l ethanol precipitations, centrifugal 1500rpm 10 minutes, after draining, be dissolved in 10 μ l ddH₂0。

(2) annealing reaction: 10 μ l degeneration templates, adds 2 μ l primers, 2 μ l annealing buffers, 65 DEG C 5 minutes, be slowly cooled to room temperature.

(3) extension: the reactant liquor after annealing is added 1 μ l ddH₂0 label 3 μ l, T7 polymerase 2 μ l (3U) and the dATP of 0.5 μ l isotope (α-32P) labelling, room temperature reaction 5 minutes.

(4) reaction is terminated: take above-mentioned reactant liquor 4.5 μ l and be marked with in the termination mix of G, A, T, C (each 2.5 μ l) respectively to 4 pipes that subpackage in advance is good, mixing, 37 DEG C 5 minutes, add 5 μ l stop buffers, before electrophoresis loading, 75-80 DEG C of degeneration in 2 minutes put by sample.

Second step: containing the establishment of GM-CSF recombinant sendai virus vector

Will with EcoRI and BamHI enzyme action, with through as the GM-CSF of enzyme action be connected, recombinant vector is properly inserted fragment, named GM-CSF-SV after enzyme action is identified.

3rd step: produce the foundation of the incasing cells containing granulocyte macrophage colony simulating factor recombinant sendai virus

Sendai virus vector is a kind of effectively gene transfer system.Replicate sendai virus vector packaged cell packaging, the virion of high titre, and energy efficient infection target cell can be produced, obtain expression steady in a long-term by being integrated into host cell gene.Sendai virus vector GM-CSF-SV containing GM-CSF gene is packed by the incasing cells 293T that this research uses safety higher, produces and has high titre and do not have the recombinant sendai virus of replication capacity.

1, virus titer measures

Virus titer measures with NIN313/TK for instruction target cell.1×10⁵Target cell was cultivated after 1 day, add through dilution containing vial supernatant, and add Polybrene (8 μ g/ml).37 DEG C, 5%CO2 cultivated after 3 days, changed culture fluid, and added G418 (0.5mg/ml) and screen.Visible anti-G418 cell clonal formation after 10-14 days, after Giemsa dyeing, calculates clone's number and determines virus titer.

2, the preservation of viral supernatants

Select virus titer more than 1 × 10⁴The 293T clone of CFU/ml carries out amplification cultivation, collects 24 hours fresh supernatants, frozen standby in-70 DEG C.

4th step: the foundation of granulocyte macrophage colony simulating factor genetic modification human breast cancer cell Cell bank

1, recombinant sendai virus infects:

When cell produces into 80% fusion, add GM-CSF-SV recombinant sendai virus supernatant (titre 1 × 10⁶CFU/ml), and add Polybrene to final concentration of 8 μ g/ml, put 37 DEG C, 5%CO₂Cultivating 24 hours, add the G418 of final concentration of 0.5mg/ml after passing on by 1:2 or 1:4, screening and culturing is after about two weeks, it is seen that G418 resisting cell is that cloning produces.Picking is single is cloned in 24 orifice plates continuation amplification cultivation, sets up cloning cell line.

2, the extracting of genomic DNA

Respectively by 1 × 10⁶Tumor cell PBS washes secondary, 0.25% trypsinization, 800 leave the heart 6 minutes, add 0.9ml cell pyrolysis liquid (STE 20 μ l, 20%SDS 15 μ l, E.C. 3.4.21.64 10mg/ml), after 37 DEG C of incubated overnight, phenol: chloroform: isoamyl alcohol (25:24:1) extracting secondary, chloroform: isoamyl alcohol 24:1 extracts once, adding 5M NaCl by 1/10 volume, obtain DNA precipitation after adding dehydrated alcohol mixing by 2 times of volumes, 70% ethanol is washed TE after once and is dissolved standby.

3, cell total rna extracting

By guanidinium isothiocyanate-phenol chloroform one step extraction process extracting cell total rna.Take various cell 1 × 10 respectively⁷, PBS washes secondary, divides equally in two 1.5ml EP pipes of immigration after adding PBS 1ml, 1200rpm is centrifugal abandon supernatant after, cell adds solution D 500 μ l and inhales rapidly to beat and make cell cracking with suppression RNase activity.Adding 50 μ l 2M NaAc (pH4.0), the water saturated fresh re-distilled phenol of 500 μ l, DEPC, 200 μ l chloroforms: isoamyl alcohol (49:1), mix rearmounted ice bath 15 minutes, 12000rpm is centrifuged 15 minutes.Sucking-off aqueous phase Han RNA, adds 1 times of volume isopropanol mixing, and-20 DEG C overnight.The 12000rpm coloured glaze heart 15 minutes, after precipitation adds 150 μ l solution D dissolvings, adds equal-volume isopropanol, places more than 1 hour for-20 DEG C, and 12000rpm is centrifuged 15 minutes.Precipitating and wash the most afterwards with 50 μ l DEPC water dissolutioies with 75% ethanol, 65 DEG C of water-baths 10 minutes ,-70 DEG C frozen standby.

4、PCR

First 94 DEG C 5 minutes, connect 60 DEG C 5 minutes, then carry out 35 circular response, last 72 DEG C 10 minutes.Reaction system is 30 μ l (10 × reaction buffer 3 μ l, 5 ' and 3 ' primers each 1 μ l (25pmol), 1.25mmol dNTP 2 μ l, sample DNA 1 μ l, ddH₂0 to 30 μ l).Reaction terminates rear 1.5-2% gel level or 6%PAGE vertical gel electrophoresis is identified.

5、RT-PCR

Reverse transcription reaction: take each 5 μ l of each cell total rna (1 μ g/ μ l), add RNase inhibitor 0.5 μ l, random primer 2 μ l, RNase inhibitor 0.5 μ l is added on 65 DEG C of 5 minutes rearmounted ice baths, 5 × Buffer 4 μ l, dNTP 7 μ l, Mo-MuLV reverse transcriptase 1 μ l rearmounted 37 DEG C 1.5 hours.Add after stop buffer standby.

PCR reacts: respectively taking above-mentioned reverse transcription reaction liquid 2 μ l as template, expand by above-mentioned PCR method, β-actin primer is as internal standard.

6, Southern blot hybridization

(1) DNA enzymatic solution: take each 30 μ g of each cell genomic dna, be dissolved in 17 μ l water, adds 2 μ l SacI enzyme cutting buffering liquid (10 times), adds 2 μ l SacI (30U/ μ l), 37 DEG C of incubated overnight after mixing.

(2) electrophoresis and transfer: after above-mentioned enzymolysis solution 5 DEG C adds 2 DEG C of bromjophenol blue sample-loading buffers, upper 1% agarose gel electrophoresis, 70V, 30mA electrophoresis 2-3 hour.EB dyeing sees whether that enzymolysis is complete.If enzymolysis is incomplete, then adds 1 μ l SacI continuation insulation and within 2-3 hour, make it be completely dissolved.Then carrying out electrophoresis by above-mentioned condition, electrophoresis terminates rear glue and is soaked in degeneration liquid (1.5M NaCl, 0.5M NaOH) 40 minutes, soaks 30 minutes in neutralizer (1M Tris.C1,1.5M NaCl) the most again.It is transferred to nylon membrane method carry out by molecular cloning handbook.

(3) prepared by probe: with the reversion rate viral vector plasmid of factor-containing gene as template, prepare the DNA probe of Neo and GM-CSF by above-mentioned PCR method, and PCR DIG labeling mix (No.1585550) of dNTP Bao Lingman substitutes.

Hybridization and detection: use Bao Manling business recommendations method to carry out.

7, granulocyte macrophage colony simulating factor genetic modification human breast cancer cell seed cell is frozen

After 0.25% trypsinization granulocyte macrophage colony simulating factor genetic modification human breast cancer cell seed cell, move in 5ml culture fluid, be centrifuged 5 minutes with 800rpm, supernatant is exhausted, by 1 × 10⁶Cell adds 1ml frozen stock solution (containing 30% calf serum and 10%DMSO), moves in cryopreservation tube.It is firstly placed on 4 DEG C to lower the temperature 30 minutes, then moves into and the liquid nitrogen of gaseous state continues cooling 30 minutes, finally cryopreservation tube is put into the Liquid nitrogen storage of liquid nitrogen biological container.

5th step: the inactivation of tumor vaccine

1, cell is collected and washing

The granulocyte macrophage colony simulating factor genetic modification human breast cancer cell that conventional amplification is cultivated, after washing twice with PBS, with 0.25% trypsinization, collects 1 × 10⁸Cell, in 400ml centrifuge bottle, includes 400ml pH 7.4 without calcium, magnesium phosphate buffer (PBS).With 1000rpm be centrifuged 10 minutes clear, clean altogether three times, finally PBS supernatant exhausted, by 1 × 10⁷Cell adds 1ml frozen stock solution, moves in cryopreservation tube.

2、⁶⁰Co irradiates granulocyte macrophage colony simulating factor genetic modification human breast cancer cell tumor vaccine cancerous cell

?⁶⁰Under Co therapeutic instrument (THERATRON780-C), it is irradiated respectively by 40G, 60G, 100G radiant intensity, the cell named granulocyte macrophage colony simulating factor genetic modification human breast cancer cell tumor vaccine (SKBR3/GM-CSF) after irradiation.

3, SKBR3/GM-CSF is frozen

It is firstly placed on 4 DEG C to lower the temperature 30 minutes, then moves into and the liquid nitrogen of gaseous state continues cooling 30 minutes, finally cryopreservation tube is put into and preserve in the liquid nitrogen of liquid nitrogen biological container.

4, SKBR3/GM-CSF cell survival rate measures

By the SKBR3/GM-CSF after recovery, suspend with 5ml complete culture solution.Take 4 to dye 2-3 minute with 0.1% phenol orchid (being dissolved in 0.9% normal saline) on microscope slide, observed result.Having deep blue particles infiltration to be dead cell in cell, bright is then living cells.4 each visuals field that take at random of sample count 100 cells, and total cell number × survival rate is i.e. obtained total viable count.

6th step: the heat shock of tumor vaccine

Tumor vaccine is put into 55 DEG C of temperature bath 1h, and heat shock processes human breast cancer cell tumor vaccine.

The present invention using the SKBR3 breast carcinoma cell strain of GM-CSF gene modified as experiment tumor vaccine, verified its in vivo can continuous release corresponding cell GM-CSF, improving the immunogenicity of body, the immune system activating body carries out tumor-killing, prophylaxis of tumours recurrence and transfer.The gene of coding GM-CSF is cloned in sendai virus vector SV/GM-CSF by the present invention, transcribes incasing cells PA317 with this carrier, it is thus achieved that virion.Transduce breast carcinoma cell strain (SKBR3/-A2 positive) with the recombinant sendai virus containing GM-CSF gene, and carry out molecular biological analysis, determine transducer cell energy secrete GM-CSF.Experiment tumor vaccine warp⁶⁰After Co irradiates, transducer cell still can continuous release cytokine be up to 2～3 weeks, but oncogenicity disappears, and the replication capacity without Sendai virus.

Embodiment 2: the preparation of adjuvant

The preparation of adjuvant comprises the steps:

Step S1, the SKBR3 human breast cancer cell 5 × 10 of trophophase of taking the logarithm⁷Individual be inoculated in 1ml containing percent by volume 10%FBS RPMI 1640 cell culture fluid in, add 20 μm ol/mL following structural compounds (I) cultivate 42h obtain cell culture fluid；

Step S2, by centrifugal for cell culture fluid supernatant of abandoning, precipitation is suspended in 1ml normal saline, places in liquid nitrogen freezing 8 minutes, then is taken out by frozen material, puts into 37 DEG C of warm water and melt, such multigelation 4 times, described adjuvant.

Embodiment 3: the preparation of compound (I) and structural identification

Reagent source: ethanol, petroleum ether, ethyl acetate, n-butyl alcohol, dichloromethane are analytical pure, purchased from Shanghai Ling Feng chemical reagent company limited, methanol, analytical pure, purchased from Jiangsu Han Bang chemical reagent company limited.

Preparation method:

A the dried leaves (8kg) of Fructus psidii guajavae immaturus is pulverized by (), (30L × 3 time) are extracted with 80% alcohol heat reflux, united extraction liquid, it is concentrated into without alcohol taste (6L), successively with petroleum ether (6L × 3 time), ethyl acetate (6L × 3 time) and water saturated n-butyl alcohol (6L × 3 time) extraction, respectively obtain petroleum ether extract, acetic acid ethyl ester extract (375g) and n-butyl alcohol extract；Acetic acid ethyl ester extract D101 type macroporous resin remove impurity in (b) step (a), first with 6 column volumes of 5% ethanol elution, again with 8 column volumes of 75% ethanol elution, collecting 75% eluent, concentrating under reduced pressure obtains 75% ethanol elution concentrate (129g)；C in () step (b), 75% ethanol elution concentrate purification on normal-phase silica gel separates, successively with volume ratio be 65:1 (8 column volumes), the methylene chloride-methanol gradient elution of 30:1 (8 column volumes), 15:1 (8 column volumes) and 8:1 (10 column volumes) obtain 4 components；D in () step (c), component 4 (27g) separates further by purification on normal-phase silica gel, successively with volume ratio be 12:1 (8 column volumes), the methylene chloride-methanol gradient elution of 8:1 (10 column volumes) and 5:1 (8 column volumes) obtain 3 components；E reverse phase silica gel that in () step (d), component 2 (15g) is bonded by octadecylsilane separates, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 75%, collecting 8～12 column volume eluents, eluent is concentrated under reduced pressure to give pure compound (I) (340mg).

Structural identification:

Yellow solid；The quasi-molecular ion peak m/z 247.1276 [M+H] that HR-TOF-MS is given⁺, show that compound molecule formula is C₁₅H₁₈O₃, degree of unsaturation is 7.According to¹H-NMR composes (CDCl₃, 600MHz) analyze, H-1 (3.58, d, J=4.3Hz), H-2 (3.62, d, J=4.3Hz), H-4 (2.47, q, J=6.2Hz), H-6a (1.48, dd, J=12.1, 4.6Hz), H-6b (1.22, dd, J=12.1, 13.2Hz), H-7 (2.65, dd, J=13.2, 4.6Hz), H-9 (6.08, s), H-12a (5.03, d, J=12.3Hz), H-12b (4.98, d, J=12.3Hz), H-13 (1.87, s), H-14 (1.32, s), H-15 (1.13, d, J=6.3Hz)；¹³C-NMR composes (CDCl₃, 150MHz) in, show 15 carbon signals, C-1 (86.2, CH), C-2 (65.9, CH), C-3 (202.3, C), C-4 (42.5, CH), C-5 (30.3, C), C-6 (39.2, CH₂), C-7 (49.6, CH), C-8 (199.2, C), C-9 (117.9, CH), C-10 (175.5, C), C-11 (145.1, C), C-12 (111.8, CH₂), C-13 (21.2, CH₃), C-14 (15.2, CH₃), C-15 (9.9, CH₃)；Carbon atom labelling sees following formula.IR spectrogram shows at 1655cm-¹There is absorption at place, illustrates to there is conjugation carbonyl functional group.¹H-NMR stave is bright conjugated alkene proton signal δ H6.08 (1H, s, H-9)；Two olefinic proton signals δ H5.03 (1H, d, J=12.3Hz, H-12a) and 4.98 (1H, d, J=12.3Hz, H-12b)；Three methyl proton signals δ H1.87 (3H, s, H-13), 1.32 (3H, s, H-14) and 1.13 (3H, d, J=6.3Hz, H-15)；Two companiesies oxygen methine proton signal δ H3.58 (1H, d, J=4.3Hz, H-1), 3.62 (1H, d, J=4.3Hz, H-2).¹³C-NMR spectrum combines in DEPT stave this structure bright three methyl carbon signals, two mesomethylene carbon signals, five methine carbon signals (two saturated carbon, two company's oxygen carbon and an alkene carbon), five quaternary carbon signals (two carbonyl carbon, a saturated carbon and two alkene carbon).There is coherent signal by HMBC analysis of spectrum, H-7 and C-6, C-8 and C-11, illustrate that C-7 is connected with the C-11 position of isopropenyl；Me-14 with C-5 dependency shows that the methyl of C-14 is connected with C-5 position；The dependency of Me-15 with C-4 and H-4 with C-15 shows that the methyl of C-15 is connected with C-4 position.In ROESY spectrum, with the dependency of H-7, Me-14 and Me-15, Me-14 show that Me-14, Me-15 and H-7 are beta comfiguration.Comprehensive hydrogen spectrum, carbon spectrum, HMBC spectrum and ROESY spectrum, and document is about correlation type nuclear magnetic data, can substantially determine that this compound is shown below, spatial configuration is determined by ECD test further, and theoretical value is basically identical with experiment value；

Embodiment 4: to mouse tumor model preventive effect

6～8 week old homology BALB/c mouse are randomly divided into 5 groups, often group 20.

Each group processing mode is as follows:

Test group one: take and irradiate the primary SKBR3 tumor vaccine of inactivation by 5 × 10⁵/ Mus inoculates in subcutaneous abdomen；

Test group two: learn from else's experience and irradiate " the SKBR3/GM-CSF tumor vaccine " of inactivation by 5 × 10⁵/ Mus inoculates in subcutaneous abdomen；

Test group three: 0.2ml/ dorsal sc injection pressed by the adjuvant of Example 2 preparation；

Test group four: irradiation of first learning from else's experience inactivates " SKBR3/GM-CSF tumor vaccine " by 5 × 10⁵/ Mus inoculates in subcutaneous abdomen, then 0.2ml/ dorsal sc injection pressed by the adjuvant of Example 2 preparation；

Test group five: irradiation of first learning from else's experience inactivates " SKBR3/GM-CSF tumor vaccine " by 5 × 10⁵/ Mus inoculates in subcutaneous abdomen, then takes adjuvant (preparation method with embodiment 2, but without compound (I)) and press 0.2ml/ dorsal sc injection；

Above-mentioned each test group took 1 × 10 after 7 days⁴Primary SKBR3 tumor vaccine measures gross tumor volume 1 time after opposite side abdominal part hypodermic, lotus tumor every other day.

Observation period is 150 days.

The experimental result of each test group see table:

Embodiment 5: the therapeutical effect to mice primary tumo(u)r

Take 1 × 10³The primary SKBR3 tumor cell lived is inoculated to BALB/c mouse side subcutaneous abdomen, and stochastic averagina is divided into 6 groups, often group 10.Each group processing mode is as follows:

Test group one: " the SKBR3/GM-CSF tumor vaccine " (1 × 10 of the irradiated inactivation of subcutaneous vaccination in the 3rd day after kind tumor⁴/ Mus)；

Test group two: the adjuvant (0.2ml/ Mus) of dorsal sc injection embodiment 2 preparation in the 3rd day after kind tumor；

Test group three: " the SKBR3/GM-CSF tumor vaccine " (1 × 10 of the irradiated inactivation of first subcutaneous vaccination in the 3rd day after kind tumor⁴/ Mus), then the adjuvant (0.2ml/ Mus) of dorsal sc injection embodiment 2 preparation；

Test group four: " the SKBR3/GM-CSF tumor vaccine " (1 × 10 of the irradiated inactivation of first subcutaneous vaccination in the 3rd day after kind tumor⁴/ Mus), then dorsal sc injection adjuvant (0.2ml/ Mus, preparation method with embodiment 2, but without compound (I))；

Test group five: " the SKBR3/GM-CSF tumor vaccine " (1 × 10 of the most first irradiated inactivation of subcutaneous vaccination in the 3rd, 7,11 days after kind tumor⁴/ Mus), then the adjuvant (0.2ml/ Mus) of dorsal sc injection embodiment 2 preparation；

Test group six: after planting tumor, the primary SKBR3 tumor vaccine (1 × 10 of inactivation is irradiated in subcutaneous vaccination in the 3rd day⁴/ Mus).

Observation period is 50 days.

The experimental result of each test group see table:

Result shows, the adjuvant of " the SKBR3/GM-CSF tumor vaccine " of irradiated inactivation and embodiment 2 preparation can work in coordination with prevention and treatment tumor, and the most repeated multiple times inoculation can improve prevention or the effect for the treatment of tumor further.

The effect of above-described embodiment indicates that the essentiality content of the present invention, but does not limit protection scope of the present invention with this.It will be understood by those within the art that, technical scheme can be modified or equivalent, without deviating from essence and the protection domain of technical solution of the present invention.

Claims

1. for treating the recombinant vaccine of breast carcinoma, it is characterised in that include the human breast carcinoma that GM-CSF gene is modified Cell and adjuvant；

Step S1, infects human breast cancer cell with the recombinant sendai virus supernatant containing GM-CSF gene, obtains GM-CSF The human breast cancer cell of genetic modification；

Described adjuvant is made by the steps:

Step S1, the RPMI 1640 that the human breast cancer cell of trophophase of taking the logarithm is inoculated in containing percent by volume 10%FBS is thin In born of the same parents' culture fluid, the compound (I) of the following structural formula adding 15～25 μm ol/mL is cultivated 36～48h and is obtained cell culture fluid,

Step S2, abandons supernatant by centrifugal for cell culture fluid, and precipitation is suspended in normal saline, places into freezing 5～10 in liquid nitrogen Minute, then frozen material is taken out, puts into 37 DEG C of warm water and melt, such multigelation 3～5 times, described adjuvant.

Recombinant vaccine for treating breast carcinoma the most according to claim 1, it is characterised in that described adjuvant passes through Prepared by following steps: step S1, the human breast cancer cell 5 × 10 of trophophase of taking the logarithm⁷The individual 1ml that is inoculated in is containing percent by volume 10% In RPMI 1640 cell culture fluid of FBS, described compound (I) the cultivation 42h adding 20 μm ol/mL obtains cell cultivation Liquid；Step S2, abandons supernatant by centrifugal for cell culture fluid, and precipitation is suspended in 1ml normal saline, places in liquid nitrogen freezing 5～10 minutes, then frozen material is taken out, put into 37 DEG C of warm water and melt, such multigelation 3～5 times, described adjuvant.

Recombinant vaccine for treating breast carcinoma the most according to claim 1 and 2, it is characterised in that: described human milk Adenocarcinoma cell is SKBR3 MCF-7.

Recombinant vaccine for treating breast carcinoma the most according to claim 1 and 2, it is characterised in that: described inactivation Method is for using⁶⁰Co irradiates inactivation.

Recombinant vaccine for treating breast carcinoma the most according to claim 1 and 2, it is characterised in that described heat is stopped Gram processing method is: temperature bath 0.5h～2h under the conditions of human breast cancer cell is placed in 42 DEG C～65 DEG C.

6. the recombinant vaccine for treating breast carcinoma described in claim 1 is at preparation prevention or the medicine for the treatment of breast carcinoma In application.