CN104117060A - Tumor vaccine and preparation method thereof - Google Patents
Tumor vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention provides a tumor vaccine and a preparation method thereof. The preparation method comprises: extracting mRNA from human peripheral blood monouclear cells, synthesizing cDNA in vitro, employing polymerase chain reaction technology to clone granulocyte macrophage colony-stimulating factor (GM-CSF) cDNA containing a signal peptide, recombining with sendai virus vector, so as to construct a recombinant sendai virus vector GM-CSF-SV containing granulocyte macrophage colony-stimulating factor cDNA; and employing a packaging cell 293T with relatively high safety to package the recombinant sendai virus vector GM-CSF-SV containing GM-CSF gene, so as to generate he recombinant sendai virus vector which is high in titer and does not have duplication capability; and infecting human lung cancer cell strain A549, so as to establish a gene engineering human lung cancer cell with GM-CSF expression activity, and employing <60>Co to irradiate and inactivate a tumor vaccine and performing heat shock processing, and preparing the GM-CSF gene modified human lung cancer cell tumor vaccine after different irradiation amounts are explored.
Description
Technical field
The present invention relates to genetic engineering field, more specifically, the invention provides a kind of tumor vaccine and preparation method thereof.
Background technology
Pulmonary carcinoma is one of malignant tumor that whole world M & M is the highest.The medical patients with lung cancer of China belongs to middle and advanced stage mostly at present, and Resection Rate is only 60% left and right, and within postoperative 5 years, survival rate only has an appointment 50%.As the 4th kind of method of oncotherapy after operation, radiotherapy, chemotherapy, Biotherapeutics is rapid at developed recently.Immunization therapy is the key component of Biotherapeutics.Tumor vaccine is the active immunity treatment of tumour-specific, the body specificity active immunity of its induction is replied, acting in animal experiment of enhancing body anti-tumor capacity obtained certainly, and many tumor vaccines have entered clinical and experimental study, demonstrate good prospect.
Lung carcinoma cell tumor vaccine is utilizing its antigenicity induction body to produce anticarcinogenic effect after the deactivation of lung carcinoma cell lonizing radiation.Exist immunogenicity weak, have shortcomings such as oncogenicity.Reduce, eliminate tumor cell oncogenicity, preserve its antigenicity is the emphasis of research as far as possible.Autologous lung carcinoma cell tumor vaccine has autologous special tumor antigen, more safe and effective than allogeneic tumor Cell vaccine, but is not suitable for extensive clinical practice.Allogeneic tumor Cell vaccine is applicable to extensive clinical practice, but less immunogenic generally adds immunological adjuvant in the preparation, as freund adjuvant, and virus, antibacterial, cytokine and oligodeoxynucleotide etc.The clinical III phase that enters of some tumor vaccine is studied.Wish in the near future, can see that the standard care scheme of some tumor vaccine is come out.
In the therapy of tumor of carrying out at present, taking the research of tumor immune gene therapy for maximum.The Cytokine gene therapy of tumor cell targeting is taking active immunity treatment as basis, and by the continuous release of cytokine, thereby excitating organism produces anti tumor immune response, reaches the object for the treatment of tumor.
GM-CSF (grain giant cell colony stimulating factor, granulocyte macrophage colony-stimulating factor) be a kind of cytokine mainly being produced by macrophage and activating cell, it can be by promoting differentiation and maturation dendritic cell (dendritic cell, DC), the antigen-presenting cell differentiation such as macrophage, maturation and activation and then promotion Th, Tc, NK identifies tumor associated antigen, infiltrate at tumor locus, cause systemic antitumor reaction, when killing tumor cells, also can promote the expression of cell CD45 T cell differentiation maturation and adhesion molecule B7/BB1, improve its antigen presentation ability.Also can increase in addition the expression of macrophage ajor histocompatibility Complex II (MHC II), improve its antigen presentation ability.It is an immunological adjuvant that has potentiality that these functions are all pointed out GM-CSF.
The tumor cell of GM-CSF genetic modification is injected after by lonizing radiation deactivation in body, can increase local inflammatory response, and a large amount of apocytes, macrophage and DC infiltrate, and promotes tumor antigen to present, and can induce strong anti tumor immune response.
Summary of the invention
The invention provides tumor vaccine of a kind of GM-CSF genetic modification and preparation method thereof.
For solving the problems of the technologies described above, technical scheme of the present invention is:
A kind of tumor vaccine and preparation method thereof, comprise the infection of the clone of synthetic, pcr amplification GM-CSF gene, the GM-CSF cDNA of the extracting of mRNA and cDNA, establishment containing GM-CSF recombinant sendai virus vector, the preparation of grain giant cell colony stimulating factor recombinant sendai virus incasing cells, human lung carcinoma cell line A549, the foundation of genetic engineering human lung carcinoma cell and the deactivation of tumor vaccine of grain giant cell colony stimulating factor expression activity.Concrete steps are: with infecting A549 containing the recombinant sendai virus supernatant of grain giant cell colony stimulating factor, obtain a giant cell colony stimulating factor genetic modification human lung carcinoma cell, application
60co irradiates and makes its deactivation and carry out heat shock processing.This giant cell colony stimulating factor genetic modification human lung carcinoma cell tumor vaccine can be used for the treatment of pulmonary carcinoma.
The invention has the beneficial effects as follows:
The sendai virus vector marking protein efficiency that this project is used is high, has substantially exceeded existing various carrier.As viral vector of new generation, sendai virus vector has been developed treatment preparation and the AIDS vaccine of the diseases such as treatment serious symptom limb ischemia, coronary heart disease, cancer.
The present invention uses Sendai virus to prepare a kind of tumor vaccine and preparation method thereof, for the treatment of cancer provides a kind of new treatment approach.Research shows, a grain giant cell colony stimulating factor genetic modification human lung carcinoma cell tumor vaccine treatment tumor has not only overcome the clinical direct application cell factor in the past to a certain extent needs the repeated multiple times and obvious shortcoming of side effect, and has obtained good curative effect.
Detailed description of the invention
The first step: contain the preparation of the grain giant cell colony stimulating factor cDNA of signal peptide.
1. the preparation of a giant cell colony stimulating factor mRNA:
(1) human peripheral lymphocyte separating medium is isolated mononuclearcell, is total to approximately 1 × 10
6cell, stimulates 30 hours through induction, makes cytokine gene expression, in order to extracting mRNA.
(2) extracting of mRNA: centrifugal collecting cell, be suspended in 1.5mlRNA extraction buffer, repeatedly lash homogenate cell with syringe, and then add 3ml extraction buffer, fully mix, centrifugal 1000cpm 5 minutes, collects supernatant.Get Oligo (dT)-Cellulose Spun Column, mixed content thing, centrifugal two minutes of 350g, discards liquid in post, gets 4ml supernatant upper prop, mixes, and discards liquid.Then wash three times with high-salt buffer, low salt buffer is washed two times, finally uses through the elution buffer eluting of 65 DEG C of preheatings three times, uses 0.25ml at every turn, collects eluent, then ethanol precipitation.
2. a giant cell colony stimulating factor cDNA's is synthetic:
(1) design of primer is with synthetic: according to one M-CSF of human granular leukocyte in GenBank (GM-CSF) gene order design pair of primers, forward primer: upstream: 5 '-GCTCAAGCTTCTGGAGGATGTGGCTGC-3 ' (including Hind III site); Downstream: 5 '-GGACGAATTCACTCCTCGAATGGCTCCC-3 '.
(2) cDNA's is synthetic: mRNA is dissolved in to 20 μ l ddH
2in O, 65 DEG C 10 minutes, be placed in for subsequent use on ice.In Article 1 chain reaction mixed liquor, add the RNA of 1mlDTT and thermal denaturation, 37 DEG C of moisturizings 1 hour, finally add 1 μ l Klenow enzyme, 37 DEG C 30 minutes, use again powder and chloroform extracting ,-20 DEG C of preservations after Sephacryl S-300 column purification after 10 minutes for 65 DEG C.
3.PCR amplification GM-CSF gene: get the PBL cDNA10 μ l preparing, add 5 ' and 3 ' amplimer, 1 μ l, 10 × reaction buffer, 5 μ l, dNTP4 μ l, add water to 50 μ l, 94 DEG C of degeneration in 5 minutes.
The clone of 4.GM-CSF cDNA: in order to identify the accuracy of PCR product, must first product cloning be entered to PUC18 or pBluescript DNA, confirm as GM-CSF cDNA through determined dna sequence and again this fragment is reclaimed after errorless, and recombinate with expression vector.Therefore PCR product is dissolved in to TE, with after suitable Rastriction enzyme, the carrier DNA restructuring with same enzyme hydrolysis, is applied to LB/AP flat board, 37 DEG C of overnight incubation after conversion TG1.
5. plasmid DNA extracting in a small amount: a single bacterium colony is got involved containing in the 3ml LB of AP (50 μ g/ml) to 37 DEG C of overnight incubation.Bacterium liquid is transferred in 1.58ml centrifuge tube to centrifugal 2 minutes of 5000rpm, precipitation thalline.With 100 μ l solution I suspension thalline, then add 200 μ l solution II, gentleness is put upside down and is mixed, and is placed on ice, after solution clarification, adds 150 μ l solution III, fully mixes.15000rpm 10 minutes is centrifugal, collects supernatant, with the extracting of equal-volume phenol/chloroform/isoamyl alcohol once, add 2.5 times of ethanol and mix, precipitation.Centrifugal 15000rpm10 minute, washes precipitation once with 70% ethanol, and water pump is drained, and is finally dissolved in 20 μ l TE, with 200 μ g/ml RNase processing 1 hour, deposits for-20 DEG C.
6. a large amount of extractings of plasmid DNA:
(1) inoculated and cultured: a single colony inoculation is cultured to logarithm late period (OD600 ≈ 0.6) in 3ml LB (containing AP), get 500 μ l and inoculate 50ml containing in the LB of antibiotics, be cultured to equally logarithm late period, get again 15ml and inoculate containing in the 1.5L LB of corresponding antibiotics, overnight incubation.
(2) plasmid extraction: by centrifugal 1.5L culture 5500rpm 10 minutes, collect thalline.Residual liquid on centrifugal tube wall is dried with absorbent paper.Thalline is suspended in 30ml solution I, puts in room temperature 10 minutes.Add liquid II60ml, mix homogeneously postposition 10 minutes on ice.Add solution III45ml, put ice bath after 10 minutes, centrifugal 10 minutes of 15000rpm.After filtered through gauze, get supernatant, add equal-volume isopropanol precipitating, centrifugal 10 minutes of 15000rpm, washes once vacuum drying with 70% ethanol.Precipitation is dissolved in to 20mlTE, adds equal-volume 5M LiCl precipitation macromole RNA, place 10 minutes in water-bath, centrifugal 10 minutes of 4 DEG C of 15000rpm, get supernatant, use equal-volume isopropanol precipitating.Centrifugal, dry, be dissolved in 5mlTE, add RNase to 200 μ g/ml, 37 DEG C 30 minutes.Add equal-volume 13%PEG (8000)/1.5M NaCl and precipitate DNA, ice bath is put 30 minutes.Centrifugation, dissolves with TE5ml after being dried, and uses phenol, phenol/chloroform/isoamyl alcohol, the each extracting of chloroform/isoamyl alcohol once.Finally add 1/10 volume 3M NaAc and 2 times of ethanol precipitations.-20 DEG C of preservations.Used time is centrifugation again, is dissolved in TE.
(3) CsCl ultracentrifugation purify DNA: as the DNA of above-mentioned alkaline process extracting, after LiCl 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 the complex of ethidium bromide and Protein formation.Clear solution under scum silica frost is sucked for the centrifuge tube of ultrafiltration core with syringe, fill remainder with pumice wax oil, and seal.45000 revs/min centrifugal 24-48 hour (20 DEG C).Centrifugal complete, visible Liang Tiao DNA district band under normal optical is shone, lower region band is made up of closed loop plasmid DNA, insert this band with syringe needle, sucking-off to 1.5ml pipe, with isopyknic through the extracting repeatedly of water saturated n-butyl alcohol, until pink is from water and organic facies hour.Water is added to 3 times of TE dilutions, add 2.5 times of dehydrated alcohol precipitations.Centrifugal rear use 70% ethanol is washed once, after draining, is dissolved in TE ,-20 DEG C of preservations.
7. restriction enzyme reaction: select the buffer that matches according to different restricted enzyme.Generally get 0.5-1 μ g DNA, restricted enzyme 5U, reaction volume 20 μ l, 37 DEG C of moisturizing 1-2 hour, by 1.2% agarose gel electrophoresis (containing ethidium bromide 0.5-1 μ g/ml) qualification enzyme action result.
8. freeze-thaw method reclaims endonuclease bamhi: under uviol lamp, DNA fragmentation is scaled off from agarose gel, add 100 μ l TE, at dry ice: in ethanol, freeze rearmounted 37 DEG C 15 minutes, three times repeatedly, 15000rpm is sucking-off supernatant after centrifugal 10 minutes, adds 1/10 volume NaAc and 2 times of volume ethanol precipitations, centrifugal collection, precipitation, after water pump is drained, is dissolved in TE, can carry out coupled reaction.
9.DNA connects: the DNA to be connected that waits mole number is mixed, add 5 × T4 ligase buffer, 4 μ l, add water to 20 μ l, add T4 DNA ligase 2U, 12 DEG C of connections are spent the night, and can be used for transforming.
10. the conversion of recombinant DNA:
(1) competent cell preparation: the recipient bacterium of overnight incubation 250 μ l are inoculated in 50 μ lLB, 37 DEG C 1.5-3 hour to mid-log phase, bacterium liquid is to ice bath 10 minutes, centrifugal 5 minutes of 5000rpm, precipitation thalline with 1/2 volume through the 100mM of pre-cooling MgCl
2suspend, ice bath 20 minutes, centrifugal 5 minutes of 4 DEG C of 5000rpm, are suspended in thalline the 100mM CaCl of 1/15-1/10 volume
2in, in ice bath, put 60 minutes, can be used for transforming.
(2) conversion of recombinant DNA: the DNA that connection is spent the night adds 100ul competent cell, mixes, in ice bath, place 40 minutes, or mix gently, 42 DEG C 2 minutes, coat on the LB flat board containing antibiotics 37 DEG C of overnight incubation.
10. double-stranded DNA order-checking:
(1) double-stranded degeneration: the about 1.5-2ug of sample DNA, be dissolved in after 8ulddH2O, add 2MNaOH2ul, to room temperature 10 minutes, add 3ul3MNaAc neutralization, then add after 7ulddH20, add 60ul ethanol precipitation, centrifugal 1500rpm10 minute, is dissolved in 10ulddH20 after draining.
(2) annealing reaction: 10ul degeneration template, add 2ul primer, 2ul annealing buffer, 65 DEG C 5 minutes, slowly cool to room temperature.
(3) extension: the reactant liquor after annealing is added to the dATP of 1ulddH20 label 3ul, T7 polymerase 2ul (3U) and 0.5ul isotope (α-32P) labelling, room temperature reaction 5 minutes.
(4) cessation reaction: get above-mentioned reactant liquor 4.5ul to (each 2.5ul) in the termination mix that divides in advance 4 pipes that install to be marked with respectively G, A, T, C, mix, 37 DEG C 5 minutes, add 5ul stop buffer, before electrophoresis loading, sample is put 75-80 DEG C of degeneration in 2 minutes.
Second step: containing the establishment of GM-CSF recombinant sendai virus vector.
To, with EcoRI and BamHI enzyme action, be connected with the GM-CSF through same enzyme action, recombinant vector has correct Insert Fragment, called after GM-CSF-SV after enzyme action qualification.
The 3rd step: produce the foundation containing the incasing cells of grain giant cell colony stimulating factor recombinant sendai virus.
Sendai virus vector is a kind of effectively gene transfer system.Copy sendai virus vector and pack through incasing cells, can produce the virion of high titre, and the efficient target cell infection of energy, steady in a long-term expression obtained by being integrated into host cell gene.This research adopts the higher incasing cells 293T of safety to pack the sendai virus vector GM-CSF-SV containing GM-CSF gene, produces the recombinant sendai virus that has high titre and do not have replication capacity.
1. virus titer is measured
Virus titer is measured a NIN313/TK for instruction target cell.1 × 10
5target cell was cultivated after 1 day, added the viral supernatant that contains through dilution, and added Polybrene (8 μ g/ml).37 DEG C, 5%CO
2cultivate after 3 days, change culture fluid, and add G418 (0.5mg/ml) to screen.10-14 days visible anti-G418 cell clonal formations afterwards, after Giemsa dyeing, calculate clone's number and determine virus titer.
2. the preservation of viral supernatant
Select virus titer to be greater than 1 × 10
4the 293T clone of CFU/ml carries out amplification cultivation, collects 24 hours fresh supernatants, frozen for subsequent use in-70 DEG C.
The 4th step: the foundation of grain giant cell colony stimulating factor genetic modification human lung carcinoma cell Cell bank
1. recombinant sendai virus infects:
In the time that cells produce becomes 80% fusion, add GM-CSF-SV recombinant sendai virus supernatant (titre 1 × 10
6cFU/ml), and add Polybrene to final concentration be 8 μ g/ml, put 37 DEG C, 5%CO2 cultivates 24 hours, after going down to posterity by 1: 2 or 1: 4, adding final concentration is the G418 of 0.5mg/ml, and screening and culturing is after approximately two weeks, and visible G418 resisting cell is cloning and produces.Single being cloned in 24 orifice plates of picking continued amplification cultivation, sets up cloning cell line.
2. the extracting of genomic DNA:
Respectively by 1 × 10
6tumor cell is washed secondary with PBS, 0.25% trypsinization, 800 leave the heart 6 minutes, add 0.9ml cell pyrolysis liquid (STE20 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 extracting in 24: 1 once, add 5M NaCl by 1/10 volume, after adding dehydrated alcohol to mix by 2 times of volumes DNA precipitation, after 70% ethanol is washed once, TE dissolves for subsequent use.
3. cell total rna extracting:
Press guanidinium isothiocyanate-phenol chloroform one step extraction process extracting cell total rna.Get respectively various cells 1 × 10
7, PBS washes secondary, adds after PBS 1ml and divides equally and move in two 1.5ml Eppendorf pipes, and 1200rpm is centrifugal to be abandoned after supernatant, and cell adds solution D500 μ l and inhales rapidly to beat and make lysis with inhibition RNA enzymatic activity.Add 50 μ l 2M NaAc (pH4.0), 500 μ l, the fresh re-distilled phenol that DEPC H20 is saturated, 200 μ l chloroforms: isoamyl alcohol (49: 1), mixes rearmounted ice bath 15 minutes, centrifugal 15 minutes of 12000rpm.Sucking-off, containing RNA water, adds 1 times of volume isopropyl alcohol and mixes, and-20 DEG C are spent the night.The 12000rpm coloured glaze heart 15 minutes, precipitation man 150 μ l solution D add equal-volume isopropyl alcohol after dissolving, and place more than 1 hour centrifugal 15 minutes of 12000rpm for-20 DEG C.Precipitation is washed and is once used afterwards 50 μ l DEPC water dissolutioies with 75% ethanol, 65 DEG C of water-baths 10 minutes, and-70 DEG C are frozen for subsequent use.
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 be 30 μ l (10 × reaction buffer, 3 μ l, 5 ' and the each 1 μ l (25pmol) of 3 ' primer, 1.25mmol dNTP 2 μ l, sample DNA 1 μ, ddH20 to 30 μ l).Reaction finishes rear 1.5-2% gel level or the qualification of 6%PAGE vertical gel electrophoresis.
5.RT-PCR
Reverse transcription reaction: (1 μ g/ μ l) to get the each 5 μ l of each cell total rna, add RNA enzyme inhibitor 0.5 μ l, random primer 2 μ l, 65 DEG C add RNA enzyme inhibitor 0.5 μ l for 5 minutes on rearmounted ice bath, 5 × Ruffer4 μ l, dNTP 7 μ l, Mo-MuLV reverse transcriptase 1 μ l rearmounted 37 DEG C 1.5 hours.Add after stop buffer for subsequent use.
PCR reaction: respectively get above-mentioned reverse transcription reaction liquid 2 μ l as template, increase by above-mentioned PCR method, β-actin primer is as internal standard.
6.Southern blot hybridization
(1) DNA enzymolysis: get the each 30 μ g of each cell genomic dna, be dissolved in 17 μ l water, add 2 μ l Sac I enzyme cutting buffering liquids (10 times), add 2 μ l Sac I (30U/ μ l), 37 DEG C of incubated overnight after mixing.
(2) electrophoresis and transfer: 5 DEG C of above-mentioned enzymolysis solutions add after 2 DEG C of bromjophenol blue sample-loading buffers, upper 1% agarose gel electrophoresis, 70V, 30mA electrophoresis 2-3 hour.EB dyeing observes whether enzymolysis is complete.If enzymolysis is incomplete, adds again 1 μ l Sac I to continue insulation and it was dissolved completely in 2-3 hour.Then carry out electrophoresis by above-mentioned condition, electrophoresis finishes rear glue and is soaked in degeneration liquid (1.5M NaCl, 0.5M NaOH) 40 minutes, and then soaks 30 minutes in neutralizer (1M Tris.C1,1.5M NaCl).Being transferred to nylon membrane method is undertaken by molecular cloning handbook.
(3) probe preparation: taking 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, dNTP substitutes with the PCR DIG labeling mix (No.1585550) of Bao Lingman.Hybridization and detection: adopt Bao Manling company recommend method to carry out.
7. a giant cell colony stimulating factor genetic modification human lung carcinoma cell seed cell is frozen
After 0.25% trypsinization grain giant cell colony stimulating factor genetic modification human lung carcinoma cell seed cell, move in 5ml culture fluid, with 800rpm centrifugal 5 minutes, supernatant is exhausted, by 1 × 10
6cell adds 1ml cryopreserving liquid (containing 30% calf serum and 10%DMSO), moves in cryopreservation tube.First be placed on 4 DEG C of coolings 30 minutes, then move in the liquid nitrogen of gaseous state and continue cooling 30 minutes, finally cryopreservation tube is put into the liquid nitrogen of liquid nitrogen biological container and preserved.
The 5th step: the deactivation of tumor vaccine
1. cell harvesting and washing
The grain giant cell colony stimulating factor genetic modification human lung carcinoma cell of conventional amplification cultivation, with after PBS washed twice, with 0.25% trypsinization, collects 1 × 10
8cell, to 400ml centrifuge bottle, includes 400ml pH7.4 without calcium, magnesium phosphate buffer (PBS).Clear with centrifugal 10 minutes of 1000rpm, clean altogether three times, finally PBS supernatant is exhausted, by 1 × 10
7cell adds 1ml cryopreserving liquid, moves in cryopreservation tube.
2.
60co irradiates grain giant cell colony stimulating factor genetic modification human lung carcinoma cell tumor vaccine cancerous cell
?
60under Co therapeutic instrument (THERATRON 780-C), irradiate respectively postradiation cell called after grain giant cell colony stimulating factor genetic modification human lung carcinoma cell tumor vaccine (A549/GM-CSF) by 40G, 60G, 100G radiant intensity.
3.A549/GM-CSF is frozen
First be placed on 4 DEG C of coolings 30 minutes, then move in the liquid nitrogen of gaseous state and continue cooling 30 minutes, finally cryopreservation tube is put in the liquid nitrogen of liquid nitrogen biological container and preserved.
4.A549/GM-CSF cell survival rate is measured
By the A549/GM-CSF after recovery, with the suspension of 5ml complete culture solution.Get 4 on microscope slide, by 0.1% phenol orchid (being dissolved in 0.9% normal saline) dyeing 2-3 minute, observed result.In cell, have navy blue granule to infiltrate and be dead cell, bright is living cells.4 each 100 cells of visual field counting of getting at random of sample, obtain total viable count by total cell number × survival rate.
The 6th step: the heat shock of tumor vaccine
Tumor vaccine is put into 65 DEG C of temperature and bathes 1h, heat shock handler's lung carcinoma cell tumor vaccine.
The A549 lung cancer cell line that this project has been crossed GM-CSF genetic modification is as experiment tumor vaccine, verified its in vivo can the corresponding cell GM-CSF of continuous release, improve the immunogenicity of body, activate the immune system of body and carry out tumor-killing, the recurrence of prophylaxis of tumours and transfer.There is similar medicine to enter II clinical trial phase abroad, but domesticly there is no quasi-drugs exploitation.This project is cloned in sendai virus vector SV/GM-CSF by the gene clone of coding GM-CSF at present, transcribes incasing cells PA317 with this carrier, obtains virion.With the recombinant sendai virus transduction lung cancer cell line (the A549/-A2 positive) containing GM-CSF gene, and carry out molecular biological analysis, determine transducer cell energy secrete GM-CSF.Experiment tumor vaccine warp
60after Co irradiates, transducer cell still can continuous release cytokine reach 2～3 weeks, but oncogenicity disappears, and not containing the replication capacity of Sendai virus.Be mainly used in Preventive after operation of lung cancer and maybe can not perform the operation and lack the Patients with Advanced Lung Cancer of other Therapeutic Method.
This production technology novelty, technology maturation.This technique reached at present international most advanced level, technique simple, be easy to industrialization, cost reduces greatly, is applicable to Chinese purchasing demand, after launch, will fill up the blank of domestic market.
Specific embodiment 1
Selecting the maximum A549 lung cancer cell line of population of China I class antigen frequency is object.Adopt the clone of synthetic, pcr amplification GM-CSF gene, the GM-CSF cDNA of the extracting of GM-CSF-mRNA and cDNA, structure containing GM-CSF recombinant sendai virus vector, the preparation of GM-CSF recombinant sendai virus incasing cells, human lung carcinoma cell line A549 infection,
60co therapeutic instrument irradiates the technology such as the cytobiologies such as killing tumor cells, molecular biology, virusology, obtain the genetic engineering human lung carcinoma cell tumor vaccine of GM-CSF expression activity, through conventional amplification cultivation, tumor vaccine is put into 42 DEG C～65 DEG C temperature bath 0.5h～2h and heat-treats,
60co therapeutic instrument irradiates deactivation, postradiation cell called after GM-CSF genetic engineering human lung carcinoma cell tumor vaccine (A549/GM-CSF).
Specific embodiment 2
Grain giant cell colony stimulating factor genetic modification human lung carcinoma cell is through various dose
60after Co radiation, all lost in vitro multiplication capacity, and along with the increase of cultivated days, cell survival rate declines, viable count reduces.As shown in table 2, the survival rate of radiation cell after a week is 50%, and zero difference between different radiation dose.Through the cell of 60G and 100G irradiation, in the time being cultured to the 25th day, cell is all dead.The genetic engineering human lung carcinoma cell (HG-1, GM-CSF) that above-mentioned Three doses is irradiated is inoculated in nude mice, does not all produce tumor, illustrates that oncogenicity disappears.
A table 1 giant cell colony stimulating factor genetic modification human lung carcinoma cell
60the postradiation survival rate of CO
Will
60the grain giant cell colony stimulating factor genetic modification human lung carcinoma cell of losing multiplication capacity after Co irradiates is referred to as a giant cell colony stimulating factor genetic modification human lung carcinoma cell tumor vaccine, be called for short HG-1/GM-CSF, the deactivation of this cell preparation, but the ability whether still with secretory granule giant cell colony stimulating factor is its key point as tumor vaccine effect.Warp
60after Co irradiates, 40G, 60G, the equal energy of tri-exposure doses of 100G continuous release grain giant cell colony stimulating factor are more than three weeks.Wherein irradiate latter 4 days secretory volumes and peak, and different radiant intensity is without significant difference.
Specific embodiment 3
" A549/GM-CSF tumor vaccine ", to mouse tumor model preventive effect: 6-8 homology BALB/c mouse in age in week, following cell gets 5 × 10
5/ Mus inoculates in subcutaneous abdomen: the primary A549 tumor vaccine that 1. irradiates deactivation; 2. through irradiating deactivation " A549/GM-CSF tumor vaccine ".After 7 days, get 1 × 10
4primary A549 tumor vaccine, at opposite side abdominal part hypodermic, is measured gross tumor volume 1 time every other day after lotus tumor.Effect of standby tumor vaccine.
Observing after 55 days, " A549/GM-CSF tumor vaccine " group has no tumor growth, and all survives at whole viewing duration (120 days); Other group mice 17 days after kind of tumor is visible tumor ramp and all dead in 46 days.
" A549/GM-CSF tumor vaccine " group uses respectively 1 × 10 in 64,74 and 92 days after kind of tumor
4, 5 × 10
5with 1 × 10
5the A549 tumor vaccine intravenous injection of primary work, result is observed 120 days after being presented at intravenous note tumor, has no tumor growth.
Specific embodiment 4
The therapeutical effect of tumor vaccine to primary tumo(u)r.Get 1 × 10
3the primary A549 tumor cell of living, to the subcutaneous vaccination of BALB/c mouse one flank portion, is equally divided into 3 groups at random.The 1st group after kind of tumor the 3rd day, the tumor vaccine (1 × 10 of subcutaneous vaccination energy secrete GM-CSF
4/ Mus); The tumor vaccine of 1I group the 3rd, 7 days and 11 days subcutaneous vaccination same doses after kind of tumor; 1II group (matched group) subcutaneous vaccination 1 × 10
4irradiate the primary A549 tumor vaccine of deactivation.After planting tumor, observe tumor growth situation, observe altogether 47 days.
The 1st group of mice has 6 mices to have tumor growth (average out to 18mm) for the 16th day after kind of tumor, wherein there are 2 within the 17th day after kind of tumor, to touch less than tumor, after kind of tumor, within 47 days, only have 3 mices to have tumor growth and poor growth, tumor size average out to 688mm.Therefore, inoculate one time tumor vaccine group, 5 mices have been played to protective effect.3 tumor vaccine groups of 1I group inoculation, within 16 days after kind of tumor, there are 4 visible tumor growths of mice (average 5mm), after planting tumor, within 21 days, only has the visible little tumor (2.3mm) of 2 mices, after planting tumor, within 23 days, only have 1 visible tumor of mice slowly to grow, to kind of the about 10mm of size of tumor tumor after 47 days.And control group mice after kind of tumor in 32 days all visible tumor growth and growth rapidly, to 47 days tumor size average out to 2331mm.These data show, subcutaneous vaccination produces the tumour-cell vaccine, particularly repeated multiple times inoculation of GM-CSF, can eliminate the primary tumors of little loading, but tumor vaccine can not stop the tumor growth of large loading.
Specific embodiment 5
The oncogenicity of A549 tumor vaccine system: homology BALB/c mouse subcutaneous abdomen inoculation 1 × 10
2-5 × 10
5after A549 tumor vaccine, visible clear and definite tumor growth, the quantity of initial inoculation oncocyte is depended in the generation of tumor and development.When using through " the A549/GM-CSF tumor vaccine " (1 × 10 of deactivation not
5) when inoculation, all mices are in the ramp of latter 15 days visible tumors of inoculation.The speed of tumor growth is identical with the primary A549 tumor vaccine of row genetic modification not, shows that gene expression after gene transfer and transduction does not directly affect the oncogenicity of A549 tumor vaccine.But " the A549/GM-CSF tumor vaccine " of deactivation inoculated all mices and within latter 25 days, had no tumor growth in inoculation.
Claims (5)
1. tumor vaccine and preparation method thereof, it is characterized in that: this lung cancer cell line is A549, have the following steps and obtain with grain giant cell colony stimulating factor genetic modification human lung carcinoma cell: with infecting A549 containing the recombinant sendai virus supernatant of grain giant cell colony stimulating factor, obtain a giant cell colony stimulating factor genetic modification human lung carcinoma cell, application
60co irradiates and makes its deactivation and carry out heat shock processing.This giant cell colony stimulating factor genetic modification human lung carcinoma cell tumor vaccine can be used for the treatment of pulmonary carcinoma.
2. it is characterized in that as claimed in claim 1: lung cancer cell line is A549.
3. it is characterized in that as claimed in claim 1: application
60co irradiates and makes its deactivation.
4. as claimed in claim 1, it is characterized in that: heat shock handler's lung carcinoma cell tumor vaccine, concrete grammar: tumor vaccine is put into 42 DEG C～65 DEG C temperature and bathes 0.5h～2h.
5. it is characterized in that as claimed in claim 1: this giant cell colony stimulating factor genetic modification human lung carcinoma cell tumor vaccine can be used for the treatment of pulmonary carcinoma.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021184449A1 (en) * | 2020-03-19 | 2021-09-23 | 武汉圣润生物科技有限公司 | Preparation method for and application of genetically engineered antitumor microparticle |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021184449A1 (en) * | 2020-03-19 | 2021-09-23 | 武汉圣润生物科技有限公司 | Preparation method for and application of genetically engineered antitumor microparticle |
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