CN103495158A - Specific chronic myelogenous leukemia epitope vaccine for human leukocyte antigen (HLA) of Chinese population - Google Patents

Abstract

The invention discloses a specific chronic myelogenous leukemia epitope vaccine for a human leukocyte antigen (HLA) of Chinese population. The specific chronic myelogenous leukemia epitope vaccine for the HLA of Chinese population is prepared by the following steps: inserting nucleotide of which a sequence is shown in SEQ ID NO.1 into a shuttle plasmid PRNAiU6.2; and building the shuttle plasmid, and then packaging and purifying lentivirus. A series-connected nucleotide sequence corresponding to each epitope is synthetized by screening immune epitopes of a plurality of specific antigen proteins of chronic myelogenous leukemia (CML) for the HLA of Chinese population; the nucleotide sequence is packaged into a lentivirus over-expression system; a dendritic cell (DC) is infected, so as to prepare the specific chronic myelogenous leukemia epitope vaccine for the HLA of Chinese population. The immunocompetence of the specific chronic myelogenous leukemia epitope vaccine is validated in vitro; a novel method and thinking are provided for immunological therapy of the chronic myelogenous leukemia and removal of minimal residual disease.

Description

The special slow grain leukemia epiposition vaccine of Chinese population HLA

Technical field

The present invention relates to special slow grain leukemia epiposition vaccine of Chinese population HLA and preparation method thereof, belong to the biological medicine technology field.

Background technology

Chronic myelocytic leukemia (CML) is a kind of pernicious myeloproliferative diseases that originates from pluripotential hemopoietic stem cell, occupy all leukemic 15～20%, account for (1.0～1.5)/100,000 at global annual morbidity, usually be divided into clinically chronic phase, accelerated period and acute transformation phase.There is distinctive morphological abnormalities in CML patient more than 95%, and t (9; 22) (q34; Q11) the Ph chromosome or the BCR-ABL fusion gene that form.The coded P210 fusion rotein of BCR-ABL fusion gene is not only the basic reason that causes CML to occur and make progress, and is also the important target spot for the treatment of.The Ph chromosome extensively is present in chronic myelocytic leukemia and a part of acute lymphoblastic leukemia.It is the little G group mark chromosome of normal No. 22 chromosomes of ratio produced by No. 9 chromosomes and No. 22 chromosome translocations, the bcr/abl fusion gene that its transposition produces is positioned on this chromosome, and the coded fusion rotein of this fusion gene has the effect that strengthens tyrosine kinase activity.The generation of this fusion rotein is the leukemic important pathogenesis of Ph chromatin-positive.The Abl proto-oncogene is positioned at chromosome three district's four-tapes No. 9, its breakaway poing near one of this gene 5 ' end 300kb in a big way in, can be at First Exon b(Ib) upstream, First Exon a(Ia) downstream, or between Ia and Ib; The BCR gene is positioned at chromosome one district's one band No. 22, think that at present it has three breakaway poings, M-bcr, m-bcr and u-bcr, its post-rift bcr gene is from beginning to end with translocating to No. 22 ab1 genes on chromosome, forms three kinds of fusion genes, i.e. b2a2/b3a2, e1a2, e19a2, the fusion rotein of three kinds of different molecular weights of its coding, i.e. p190, p210 and p230.Studies confirm that, the P210 fusion rotein is the basic reason that causes the CML morbidity.P210 is a kind of abnormal receptor type tyrosine kinase albumen, with normal tyrosine kinase, compare, P210 has tyrosine kinase (PTK) activity extremely increased, and can cause that the phosphorylation level of the specific substrates protein molecular in signal transduction pathway downstream significantly improves.Protein phosphorylation is a kind of important form of cell signalling, under protein kinase catalysis, occurs, and participates in regulating every physiological function of cell.Under normal circumstances, the activity of tyrosine protein kinase is subject to strict regulation and control.But in CML patient's cell, P210 albumen is after ATP is combined, for a series of substrate proteins provide binding site, tyrosine phosphorylation level is improved greatly, and then activate RAS-MAPK, JAK-STAT, the signal transduction pathways such as PI3K-AKT, change the intragentic expression of core, as c-myc, bcl-2, bcl-x etc., the normal physiological function of interference cell.Above three abnormal signal transduction pathways cause cell proliferation to be accelerated, growth factor dependency weakens, apoptosis is obstructed, the interaction of extracellular matrix and stromal cell is interfered, thereby cause the vicious transformation of cell.The medicine for the treatment of clinically chronic myelocytic leukemia at present is specificity tyrosine protein enzyme inhibitor (TKI).Imatinib is first targeted anticancer medicine for the tumor mechanism manually synthesized.Although traditional chemotherapy means can be improved symptom, mitigate the disease can not stop the conversion of disease, can not fundamentally eliminate malignant clone; Allogeneic Hematopoietic Stem Cell Transplantation is considered to be expected to cure the unique method of CML, but, be subject to the restriction of age and suitable donor, and the transplanting incidence of complications is high, mortality risk is large, thereby only has the fraction patient can do Allogeneic Hematopoietic Stem Cell Transplantation.

In recent years, use the Immuno Suppressive Therapy tumor to obtain certain achievement.9 of the utilization polypeptide vaccines such as Letsch treatments are at least carried out after 3 melanoma excisions once more the patient of recurrence and are carried out long term follow-up, 2 have no recurrence in following up a case by regular visits to, 2 only occur that list is located or recur in early days many places, and 4 patients that stop recurrence have all produced immunne response to vaccine.They confirm to apply the recurrent interval time that polypeptide vaccine has extended malignant melanoma, have confirmed the definite effect [1] of polypeptide vaccine in the treatment malignant tumor.

No matter be traditional chemotherapy or bone marrow transplantation, all exist residual leukemia easily to cause the problem of recurrence, lack at present desirable bone marrow purging measure.Therefore, find a kind of effective method of thoroughly curing slow grain and seem that particular importance is with urgent.Genetic immunization can inducing specific CTL and kill and wound the target cell of expressing tumor antigen, thereby reach the purpose of curing tumor.The BCR-ABL fusion gene forms new aminoacid and the peculiar expression in chronic myelocytic leukemia thereof at the position of fusion place because of it, be speculated as tumor specific antigen, so the immunity of bcr-abl fusion gene is expected to induce the MHC Restricted CTL and for chronic clinical treatment.The Wims1 oncogene (Wilms ' tumor gene, WT1) be positioned 11p13, research finds that nearly all leukemia germinal cell all continued expression WT1 gene, think that the WT1 gene can become a kind of " whiting disorders of blood sign ", for detection of Minimal Residual Disease of Leukemia, WT1 is high expressed closely related with the CML progression of disease in marrow series leukemia cell, its protein polypeptide can cause humoral immunization and cellular immunization, but specificity is eliminated immature leukemia CD34+ CFU-GM, it is another important target spot of CML immunization therapy.

At present, external CML vaccine mostly is the polypeptide vaccine of single epi-position, and mainly for European crowd, its HLA distributes different from Chinese population, and Chinese population HLA gene frequency and spatial distribution present height heterogeneity, therefore, the present invention is intended to development and covers the powerful CML series connection epi-position nucleic acid vaccine of the immune effect for BCR-ABL and WT1 target spot that most of Chinese population HLA distributes, for the immunization therapy of CML provides theoretical foundation and laboratory basis.

Summary of the invention

For above-mentioned prior art, the invention provides special slow grain leukemia epiposition vaccine of a kind of Chinese population HLA and preparation method thereof.

The present invention is achieved by the following technical solutions:

The special slow grain leukemia epiposition vaccine of Chinese population HLA, nucleotide by sequence as shown in SEQ ID NO.1 is inserted in shuttle plasmid PRNAiU6.2, build shuttle plasmid, then carry out packing and the purification of slow virus, obtain the special slow grain leukemia epiposition vaccine of Chinese population HLA.

The preparation method of the special slow grain leukemia epiposition vaccine of described Chinese population HLA comprises the following steps:

(1) by sequence, the nucleotide as shown in SEQ ID NO.1 (there are Sma ICCCGGG and Kpn IGGTACC restriction enzyme site in these nucleotide sequence two ends) is inserted into corresponding SmaI and the KpnI multiple clone site of shuttle plasmid PRNAiU6.2, builds shuttle plasmid;

(2) after successfully building shuttle plasmid, carry out packing and the purification of slow virus, obtain slow virus liquid, be the special slow grain leukemia epiposition vaccine of Chinese population HLA; The packaging system of slow virus is four pUC pUCs, consists of pRsv-REV, pMDlg-pRRE, and pMD2G, this plasmid of PRNAiU6.2-DWH(is that step 1 builds the shuttle plasmid obtained).

Described slow virus packing is as follows with the concrete steps of purification:

1) slow virus incasing cells transfection: with the 293T cell of trypsinization exponential phase, cell density is 0.5 * 10 ⁶/ cm ²the time, being re-seeded into the 15cm Tissue Culture Dish of 25mL, growth medium is DMEM (containing 10%FBS, percentage by volume), 37 ℃, 5%CO ₂in incubator, cultivate;

2) prepare four kinds of plasmid DNA solutions in the slow virus packaging system: get pRsv-REV10 μ g, pMDlg-pRRE15 μ g, pMD2G7.5 μ g and PRNAiU6.2-DWH20 μ g, add sterilized water to be settled to 1800 μ L, then add CaCl ₂(2.5mol/L) solution 200 μ L, mix, and adds 2 * BBS buffer salt solution, 2000 μ L, and room temperature is placed 20～30min, obtains DNA and calcium phosphate mixed liquor, standby;

When 3) cell density in step 1) reaches 60%～70%, carry out transfection: by step 2) DNA and calcium phosphate mixed liquor be transferred in the culture fluid containing cell monolayer, mix, discard the culture fluid that contains transfection mixture after cultivating 12h, add PBS15mL, discard after jog, repeat this step 3 time;

4) add the DMEM cell culture fluid 15mL containing the 100mL/L calf serum in every bottle of cell, continue to cultivate 48h;

5) collect the 293T cell conditioned medium liquid of transfection 72h;

6) the cell conditioned medium liquid of step 5) being collected is in 4 ℃, and the centrifugal 10min of 4000g, collect supernatant;

7 supernatant that step 6) is collected filter with 0.45 μ m filter;

8) supernatant after step 7) is filtered is in 40mL ultracentrifugation pipe, and 4 ℃, the centrifugal 20min of 25000r/min;

9) step 8) centrifugal after, with the resuspended virus precipitation of ice 500ulPBS, in 4 ℃ of dissolvings, spend the night (12h), (through virus titer, measure, virus titer is: 3.05 * 10 to obtain slow virus liquid ⁸tU/ml), be the special slow grain leukemia epiposition vaccine of Chinese population HLA, while specifically applying, can according to circumstances add the adjuvant that vaccine is commonly used.

The present invention is by the immune epitope of screening special CML plurality of antigens albumen for Chinese population HLA, the nucleosides in series acid sequence of synthetic each epitope of correspondence, be packaged into slow virus and cross expression system, infect the DC cell, be prepared into Chinese population HLA specificity chronic myelocytic leukemia epiposition vaccine, and in vitro it carried out to immunocompetence research.For the immunization therapy of chronic myelocytic leukemia provides new direction.Concrete grammar is: by consulting CML epi-position pertinent literature, therefrom choose the immune epitope for the CML antigen protein, after by SYFPEITHIMHC phenotype and Antigen Epitope Prediction system and MAPPP proteasome degraded prognoses system, selected epi-position being carried out to epitope peptide prediction and MAPPP proteasome cutting-epi-position generation forecast, by overlay region amplification gene method and round pcr, synthesize the CML epi-position full-length gene of connecting.Utilize engineered correlation technique that genes of interest is packaged into to slow virus and cross expression system.Recombinant virus infection DC cell, be prepared into the CML epiposition vaccine.The DC cell of blank viral infection and without the DC cell of viral infection as negative control.The PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC) of choosing the K562 cell of BCR/ABLp210 (+) and CML, patient ALL as target cell respectively with DC-BCR/ABL*WT1, DC-GFP and DC co-culture of cells.The expression of the mRNA level in the HEK293 cell of RT-PCR method testing goal gene after slow virus infection.CD3+T lymphocyte in immunomagnetic beads method sorting PBMC, fluidic cell method detects the lymphocytic purity of T of institute's sorting.Identify the morphological change of DC cell under microscope.Lactic acid dehydrogenase (LDH) release test is measured the killing-efficiency of T cell.The ELISPOT method detects the secretion of IFN-γ.Result: predict and consult pertinent literature by SYFPEITHI and MAPPP, choosing 49 immune epitopes, and covering more than 90% of Chinese population HLA phenotype.The synthetic CML series connection of success epi-position full-length gene, and successfully be packaged into slow virus and cross expression system.Viral infection DC cell, RT-PCR confirms genes of interest successful expression in cell.LDH release test validating experiment group T cell killing efficiency is increased significantly than negative control group, and difference has statistical significance (p<0.05).The ELISPOT method shows that the IFN-γ of experimental group CD3+T lymphocyte generation is apparently higher than negative control group.Conclusion: the present invention has successfully built the special chronic myelocytic leukemia epiposition vaccine of Chinese population HLA, and has verified in vitro its immunocompetence, for the immunization therapy of chronic myelocytic leukemia and remove microresidual disease new method and thinking is provided.

The accompanying drawing explanation

Fig. 1: sequencing result and original series are compared schematic diagram.

Fig. 2: observe ripe DC cellular morphology figure under light microscopic.

Fig. 3: the HEK293 cell respectively through the restructuring slow virus and blank viral infection after green fluorescence at intracellular expression schematic diagram, wherein, A is the white light figure of HEK293 cell after recombinant virus infection, and B is the fluorogram of HEK293 cell after recombinant virus infection; C is the white light figure of HEK293 cell after blank viral infection, and D is the fluorogram of HEK293 cell after blank viral infection; E is the white light figure of HEK293 cell after without viral infection, and F is the fluorogram of HEK293 cell after without viral infection.G is the white light figure of DC cell after recombinant virus infection, and F is the fluorogram of DC cell after recombinant virus infection.

Fig. 4: A: the agarose gel electrophoresis figure of recombinant virus plasmid PRNAiU6.2 after XbaI/KpnI restriction enzyme site double digestion; B: the metainfective HEK293 cell of recombinant slow virus is visible purpose band after RT-PCR.

Fig. 5: fluidic cell is learned and is identified the lymphocytic purity of magnetic bead sorting CD3+T, and wherein, A: the cell of irising out is lymphocyte, accounts for 68.1% of PERIPHERAL BLOOD MONONUCLEAR CELL; The B:CD3+T lymphocyte accounts for all lymphocytic 84.1%.

Fig. 6: the LDH method detects the cytotoxicity of T lymphocyte to target cell.

Fig. 7: the ELISPOT method detects the secretion of T lymphocyte IFN-γ, and wherein, A: block diagram shows every 10 ⁵the speckle number (SFC) that the T lymphocyte produces, result shows and can be produced more IFN-γ by the T lymphocyte after the DC-BCR/ABL*WT1 boosting vaccine than other two groups, except WSK; B: show this experimental section speckle figure.

The specific embodiment

Below in conjunction with embodiment, the present invention is further illustrated.Following embodiment is convenient to better understand the present invention, but does not limit the present invention.The experimental technique used in following embodiment if no special instructions, is conventional method.In subordinate embodiment, experiment material used, reagent etc., if no special instructions, all can obtain from commercial channels.

Materials and methods

1. material

1.1 data base used and prognoses system

1.1.1PubMed Literature retrieval

Network address www _.ncbi.com

1.1.2SYFPEITHIMHC phenotype and Antigen Epitope Prediction system

Network address http://www.syfpeithi.de/Scripts/MHCSSever.dll/EpitopePrediction .html;

1.1.3MAPPP proteasome degraded prognoses system

Network address www.mpiib-berlin.mpg.de/MAPPP/expertquery.html

1.1.4DNAMAN software

1.1.5Kozak sequence

1.2 plasmid bacterial strain

Test slow virus shuttle plasmid PRNAiU6.2 used and packaging plasmid pRsv-REV, pMDlg-pRRE, pMD2G all purchased from Shanghai than high biological medicine Science and Technology Ltd.; Incasing cells 293T cell strain is purchased from Shanghai cell institute of the Chinese Academy of Sciences, and coli strain DH5 α is purchased from Invitrogen company.

1.3 cell

The K562 cell strain is preserved by Shandong hospital hematopathy research department.

1.4 object of study

Choose in 2012.1 to 2013.6 in chronic myelocytic leukemia patient 4 examples of Shandong hospital treatment, and acute lymphoblastic leukemia patient 2 examples.Its diagnosis all meets Zhang Zhinan chief editor " diagnosis of hematological diseases and criterion of therapeutical effect " (third edition) diagnostic criteria, as shown in table 1.

Table 1

Patient	Sex	Age	Clinical diagnosis	The BCR/ABL fusion gene
					YGH	The female	36	The chronic myelocytic leukemia chronic phase	P210=284.86%
ZJG	The man	28	Acute lymphoblastic leukemia	P210=93.15%
					GQ	The man	35	The chronic myelocytic leukemia chronic phase	P210=105.04%
FMY	The female	49	The chronic myelocytic leukemia chronic phase	P210=222.29%
					ZSY	The man	68	The chronic myelocytic leukemia chronic phase	P210=421.36%
WSK	The man	46	Acute lymphoblastic leukemia	P210=0%

1.5 main agents

(1) hyclone (FCS), TBD company product.

(2) RPMI1640 culture fluid, Hyclone company product.

(3) dimethyl sulfoxide (DMSO) is Sigma company product

(4) IL-4, IL-2, colony stimulating factor (GM-CSF), Peprotech company product

(5) lymphocyte separation medium (Ficoll, relative density 1.077), Chinese Tianjin Hao Yang company product.

(6) phosphate buffer (PBS) is Suo Laibao biological product company limited product

(7) Trizol reagent, U.S. invitrogen company product.

(8) SYBR Green Realtime PCR Master Mix spins (Shanghai) bio tech ltd purchased from Japan

(9) chloroform, isopropyl alcohol, 75% ethanol, agarose, TAE, EB, Loading buffe etc.

(10) immune sorting magnetic bead, beautiful day Ni company product of Germany

(11) LDH test kit, bio-engineering research institute is built up in Nanjing.Test kit forms and the reagent preparation:

1) 96 hole ELISA Plate is one;

2) substrate buffer 5ml;

3) nadide powder, every powder adds the 1.3ml distilled water and dissolves, and packing is freezing;

4) 2,4 dinitrophenyl hydrazine 5ml;

5) 4mol/NaOH5ml, by 10 times of distilled water dilutions for 4mol/NaOH solution, matching while using;

6) 2mmol/L acetone acid titer 1ml

1.6 key instrument equipment

(1) low speed autobalancing centrifuge (maximum (top) speed 5000r/min) is Beijing Medical Centrifugal Machine Factory's product.

(2) SANYO ultra cold storage freezer (80 ℃) is Japanese SANYO company product.

(3) Haier's medical bio refrigerator (20 ℃) is company of Haier product.

(4) GNP-9270 type water isolation type electro-heating standing-temperature cultivator is the grand experimental facilities company limited of upper Nereid product.

(5) micropipettor of all size is German Eppendorf company product.

(6) Eppendorf pipe is German EppendorfAG company product.

(7) the real-time fluorescence quantitative PCR instrument 7500, are American AB I company product.

(8) Eppendorf centrifuge, model LX-200.

(9) UV2450 uv-spectrophotometric instrument is Shimadzu instrument (Suzhou) company limited product.

(10) HY-5 Clothoid type agitator is Jintan City Zhong great instrument plant product.

2, experimental technique

2.1CMLCTL selection and prediction

Consult and be published in since 2000 PubMed by the document of the research CML epi-position that SCI included, therefrom select CTL epi-position, the Th epi-position of 49 energy for CML multiple protein antigen, cellullar immunologic response and humoral immunoresponse(HI) that can excitating organism.And can with the epi-position of the gene loci stable bond of HLA-I molecule and HLA-II molecule.Utilize SYFPEITHI MHC phenotype and Antigen Epitope Prediction system and MAPPP proteasome degraded prognoses system to carry out epitope peptide prediction and MAPPP proteasome cutting-epi-position generation forecast to selected epi-position.Find out the gene order of multiple BCR-ABL and WT1 finds out the corresponding nucleotide sequence of each epi-position and checks from PubMed Nucleotide, each epi-position nucleotide sequence is sequentially lined up by allelic frequency distribution, conspire to create a nucleotide sequence, the nucleic acid epi-position conspired to create is as shown in SEQ ID NO.2.

2.2 synthesizing ribonucleotide sequence and be packaged into expression system

2.2.1 the front end at above-mentioned tandem nucleotide epitope sequences adds the ACCGGT restriction enzyme site, introduces GCCACC Kozak sequence, ATG is start codon, and end adds that CATCATCATCATCATCAT is 6 his sequence labels, and TGA is termination codon.AgeI and EcoRI, for introducing the restriction enzyme site of T carrier, are carried out the order-checking of the synthetic and nucleotide epitope sequences of nucleotide epitope sequences by Jinan Bo Shang company.

2.2.2 after utilizing restriction enzyme site AgeI and EcoRI to carry out double digestion, the nucleotide epitope sequences of series connection is cut from the T carrier, introduce Smal CCCGGG and Kpnl GGTACC restriction enzyme site (as shown in SEQ ID NO.1) at the sequence two ends, the nucleotide epitope sequences as shown in SEQ ID NO.1 is introduced to corresponding SmaI and the KpnI multiple clone site of shuttle plasmid PRNAiU6.2.

2.2.3, after successfully building shuttle plasmid, carry out packing and the purification of slow virus.The packaging system of slow virus is four pUC pUCs, consists of pRsv-REV, pMDlg-pRRE, and pMD2G, this plasmid of PRNAiU6.2-DWH(is the synthetic plasmid of above-mentioned 2.2.2).

The lentivirus production experimental procedure is as follows:

1) the 293T cell of trypsinization exponential phase for the transfection of slow virus incasing cells, cell density is 0.5 * 10 ⁶the time; Be re-seeded into the 15cm Tissue Culture Dish of 25mL, growth medium is DMEM (containing 10%FBS), 37 ℃, and 5%CO ₂in incubator, cultivate;

2) prepare four kinds of plasmid DNA solutions in the slow virus packaging system: get pRsv-REV10 μ g, pMDlg-pRRE15 μ g, pMD2G7.5 μ g and PRNAiU6.2-DWH20 μ g, add sterilized water to be settled to 1800 μ L, then add CaCl ₂(2.5mol/L) solution 200 μ L, mix, and adds 2 * BBS buffer salt solution, 2000 μ L, and room temperature is placed 20～30min;

3) transfection when cell density reaches 60%～70%.DNA and calcium phosphate mixed liquor are transferred in the DMEM culture fluid containing cell monolayer, mix, after cultivation 12h, discard the culture fluid that contains the transfection mixture, add PBS15mL, discard after jog, repeat this step 3 time;

4) add the cell culture fluid 15mL containing the 100mL/L calf serum in every bottle of cell, continue to cultivate 48h;

5) collect the 293T cell conditioned medium liquid of transfection 72h;

6) by the supernatant collected in 4 ℃, the centrifugal 10min of 4000g, collect supernatant;

7) supernatant is filtered with 0.45 μ m filter;

8) in 40mL ultracentrifugation pipe, 4 ℃, the centrifugal 20min of 25000r/min;

9) then with ice 500ulPBS, heavily revolve virus precipitation, in 4 ℃ of dissolvings spend the night (12h).

The slow virus titer determination:

(1) infect the previous day (Day1), digest the Hela cell and, to its counting, cell spread to 6 orifice plates, each hole paving 2 * 10 ⁵cell.Guarantee that cell has 30-50% full when infecting; 5%CO ₂, 37 ℃ of cell incubated overnight.

(2) infected the same day (Day2), after the slow virus lyolysis is frozen, be made into scope from 10 ^-2to 10 ^-6, concentration differs a series of diluent of 10 times.The slow virus liquid of each concentration is diluted in to complete medium, and final volume is 1ml.Not concussion.

(3) outwell original cell culture medium.Gentle every kind of virus of mixing and culture medium mixed liquor, then add them each hole (each hole 1mL).

(4) each hole adds Polybrene, final concentration 6 μ g/ml.Gently rock six orifice plates to mix.37 ℃, the CO2 incubated overnight.

(5) infect rear first day (Day3), will outwell containing virulent culture medium, change the complete medium .37 ℃ of 2ml into, the CO2 incubated overnight.

(6) infect and within latter the 4th day, detect the GFP cell.

(7) the Hela cell is differed a series of slow virus supernatant of 10 times (from 10 by concentration ^-2to 10 ^-6) infect, wherein need to have not transfected matched group (mock).Count results to GFP positive cell in six orifice plates is:

Mock: without the clone; 10 ^-2dilution: full; Can't count; 10 ^-3dilution: full; Can't count; 10 ^-4dilution: full; Can't count; 10 ^-7dilution: 46.10 ^-8dilution: 5.Can obtain PRNAiU6.2-DWH purpose virus titer according to above method is: 3.05 * 10 ⁸tU/ml.

2.3DC the cultivation of cell

(1) by 2～3 times of aseptic PBS or normal saline dilutions for got peripheral blood.

(2) first in separation test tube (15ml), respectively add 3～4ml lymphocyte separation medium (Ficoll, relative density 1.077), then add gently the blood after dilution along inboard wall of test tube, note making blood be distributed in the upper strata of separating medium, should become obvious layering between the two, avoid the two mixing.Lymphocyte separation medium/blood is about 1/2.

(3) (18 ℃～25 ℃) under room temperature, centrifugal 20 minutes with 2000 rev/mins.

(4) take out separation test tube, now liquid in pipe divides four layers, is followed successively by from top to bottom: blood plasma (transparent pale yellow chromatograph), mononuclearcell (white layer), separating medium (colorless layer), erythrocyte (red color layer).Take out gently mononuclearcell layer (white layer) with capillary pipette, be placed in another 15ml separator tube.Note not drawing blood plasma or separating medium.

(5) with PBS or normal saline, the mononuclearcell of taking-up is done to the equal-volume dilution, under room temperature (18 ℃～25 ℃), 2000 rev/mins centrifugal 10 minutes, abandon supernatant, retain cell precipitation.Twice of PBS washing.

(6) resuspended with complete medium RPMI1640 culture fluid the PERIPHERAL BLOOD MONONUCLEAR CELL of separating, overnight incubation in six orifice plates under 37 ℃ of 5%CO2 conditions.Every Kongzui many 1 * 10 ⁶individual cell.

(7) cultivate second day and suck gently the upper strata culture medium, and not adherent cell.Every hole adds the fresh complete medium of 2ml to continue to cultivate attached cell.And add cytokine: colony stimulating factor (GM-CSF) and interleukin-4 (IL-4), concentration is 1000U/ml.37 ℃ of 5%CO ₂cultivate 5 days.

(8) the immature DC cell of cultivating 5 days is transferred in new six orifice plates and continues to cultivate, every hole adds the fresh complete medium CM-CSF of 2ml, each 1000U/ml of IL-4, and LPS, 37 ℃, 5%CO ₂continue to cultivate 2 days.Form in the 7th day that the cultivates ripe DC cell of micro-Microscopic observation.

2.4 slow virus infection DC cell

1) carry and the previous day the virus liquid needed is taken to 4 degree refrigerators from-80 degree refrigerators and melt

2) cultured DC cell is carried out to cell counting, every hole gets 5 * 10 ⁴individual cell, be inoculated in 24 orifice plates, and every hole culture volume is 300 μ l.

3) take out the virus of 4 ℃ of preservations, use desk centrifuge centrifugal 20 seconds, make virus be suspended from the centrifuge tube bottom fully, according to the MOI(infection multiplicity) value=15, calculate slow virus volume required, the virus liquid that uses pipettor to draw accurate volume joins in culture medium, and in culture medium, adds polybrene8ug/ml to improve the efficiency of infection of viral infection.After mixing, put into CO2 gas incubator (37 ℃, 5%CO ₂) overnight incubation.

4) use the negative control GFP virus liquid of slow virus to infect other one group of DC cell.

5) use and do not add the DC cell of any virus liquid as blank.

6) culture fluid that contains slow virus after infection 24h is replaced with normal culture fluid; After skipping for the first time, if slow virus does not have obvious toxic action to cell, according to normal condition of culture, cultivate and change liquid.Negative control group and blank group liquid carry out cell simultaneously and change liquid.

That 7) infects observes fluorescence on the 6th day under the fluorescence inverted microscope, estimates the efficiency of slow virus infection purpose cell.

2.5 by the expression of RT--PCR testing goal gene

2.5.1RNA extract

(1) collect different disposal group cell, add 1mlTrizol, suction nozzle is blown and beaten into uniform liquid.

(2) add 300 μ l chloroforms, concuss mixes 30s.The centrifugal 15min of 15000rpm.

(3) supernatant is moved in a 1.5mlEp pipe, add the equal-volume isopropyl alcohol, room temperature is placed 5min.Attention: do not draw intermediate layer material, otherwise there will be DNA to pollute.

(4) the centrifugal 10min of 15000rpm, careful abandoning supernatant, prevent RNA precipitation loss.

(5) add 75% ethanol 1ml, do not need piping and druming, the centrifugal 5min of 15000rpm.

(6) careful abandoning supernatant, as far as possible thoroughly siphon away supernatant, prevents that RNA from losing, and drying at room temperature makes the ethanol volatilization.

(7) 30-50 μ lDEPC water dissolution precipitate for precipitation.

(8) ultraviolet spectrometer is measured RNA concentration.

2.5.2RT reaction

Carry out following RT reaction in the 0.5ml centrifuge tube

Of short duration centrifugal mixing, room temperature is placed 10min, reverse transcription reaction condition: 37 ℃ of 15min, 85 ℃ of 5s.2.5.3PCR reaction

Carry out the PCR reaction in the 02ml centrifuge tube.

After being added successively, each component of above reaction system mixes, and of short duration centrifugal.Carry out pcr amplification under following condition: the GAPDH gene, 94 ℃ of denaturation 5min, 94 ℃ of degeneration 45s, 56 ℃ of annealing 45s, 72 ℃ are extended 45smin, totally 30 circulations, last 72 ℃ are extended 5min; Genes of interest, 94 ℃ of denaturation 5min, 94 ℃ of degeneration 45s, 58 ℃ of annealing 45s, 72 ℃ are extended 45smin, totally 30 circulations, last 72 ℃ are extended 5min.

2.6 immunological magnetic bead sorting CD3+T lymphocyte

(1) extract normal person's peripheral blood, with EDTA, manage anticoagulant, separate mononuclearcell (concrete steps are shown in 2.3).

(2) with the resuspended mononuclearcell sub-elected of the special-purpose Buffer of immunological magnetic bead sorting, 20 ℃ of centrifugal 200g, 10-15min, carefully remove supernatant, and repeating step is once.This step is in order to remove residual platelet.

(3) cell counting, get 10 ⁷individual cell, remove supernatant after the centrifugal 10min of 300g, and with meeting, cold Buffer is resuspended to 80ul.

(4) add 20ulCD3 immunomagnetic beads antibody, after mixing, hatch 15min to 4 degree refrigerators;

(5) wash the cell after hatching, centrifugal 300g, 10min with 1-2mlBuffer.Remove supernatant fully.With the resuspended pillar of preparing against of 500ulBuffer, use.

(6) the MS post is placed on to field region, adds 500ulBuffer and clean one time.

(7) 500ul cell suspension is added in the MS post, allow liquid slowly drip.With Buffer, clean 3 times.

(8) add in 1mlBuffer to MS post, the MS post is removed from field region, put into a clean centrifuge tube place, the cell of staying in pillar is released.

(9) centrifugal 1000rmp, 5min, collect cell precipitation

(10) labelling CD3 fluidic cell is learned antibody, the lymphocytic purity of the T that flow cytometer showed sub-elects.

(11) T lymphocyte immunological magnetic bead sorting gone out is put into six orifice plates and is cultivated, and adds IL-2 to maintain growth.

2.7 collect patient's CML PERIPHERAL BLOOD MONONUCLEAR CELL

(1) collect clinically patient's CML full bone marrow, EDTA manages anticoagulant.

(2) separating bone marrow single nuclear cell (concrete steps are shown in 2.3)

(3) by the cell precipitation Eddy diffusion collected in freezing liquid (9ml calf serum+1mlDMSO) (approximately 1 * 10 ¹⁰/ L);

(4) cell suspension is distributed in cryovial to 2ml/ pipe, labelling patient name and frozen time;

(5) cryovial is placed in 4 ℃ of Refrigerator store 30min; Cryovial proceeds to-80 ℃ of Refrigerator store 24h; Cryovial moves into liquid nitrogen container (196 ℃), the position of labelling cryovial.

2.8K562 cell culture

2.8.1 cell recovery

(1) cryovial is proceeded in 40 ℃ of water-baths and (need be worn protective gloves) by liquid nitrogen container (196 ℃) rapidly;

(2) when thawing to after ice-nucleus occurring, take out cryovial, with containing 70% ethanol disinfection cryovial;

(3) cell suspension is all transferred in the centrifuge tube of the RPMI1640 culture fluid (10ml) that does not contain calf serum, repeatedly blown and beaten without measuring pipette;

(4) the centrifugal 10min(of 1000rpm can repeat);

(5) abandon supernatant, resuspended and the piping and druming cell with a small amount of RPMI1640 culture fluid containing the 10-15% calf serum, cell is all transferred in RPMI1640 culture fluid (large bottle 10-14ml, the bottle 6-10ml) culture bottle containing the 10-15% calf serum, and mark cell type and time;

(6) use inverted microscope to detect cell viability and density, if density is too high, with culture fluid, be diluted to suitable concentration;

(7) in 37 ℃, 5%CO ₂in the saturated humidity incubator, cultivate;

(8) after 24h, the activity of observation of cell;

(9) cell suspension is transferred to the 10ml centrifuge tube, the centrifugal 10min of 1000rpm;

(10) abandon supernatant, the resuspended and piping and druming cell with a small amount of RPMI1640 culture fluid containing calf serum, transfer to cell in RPMI1640 culture fluid (large bottle 10-14ml, the bottle 6-10ml) culture bottle containing calf serum;

(11) use inverted microscope to detect cell viability and density;

(12) in 37 ℃, 5%CO ₂cultivate (within later every 2-4 days, changing a culture fluid) in the saturated humidity incubator;

2.8.2 cell culture and going down to posterity

(1) cell suspension is transferred to the 10ml centrifuge tube, the centrifugal 10min of 1000rpm;

(2) abandon supernatant, cell precipitation is resuspended and piping and druming cell with a small amount of RPMI1640 culture fluid containing the 10-15% calf serum, how much be sub-packed in several culture bottles (RPMI1640 that contains the 10-15% calf serum) according to cell, blow and beat gently by aseptic pipette, cell is evenly suspended;

(3) in 37 ℃, 5%CO ₂suspension culture in the saturated humidity incubator;

(4) observation of cell growing state determine whether to continue sub-bottle according to the speed of Growth of Cells and go down to posterity and cultivate and change culture fluid after 24-48h.

2.8.3 frozen cell

(1) collect the cell of exponential phase;

(2) dilute 25 μ l cell suspension, cell counting survival rate with 25 μ l trypan blue solutions > more than 90%;

(3) the centrifugal 10min of cell suspension 1000rpm, remove supernatant;

(4) by the cell Eddy diffusion of precipitation in freezing liquid (9ml calf serum+1mlDMSO) (approximately 1 * 10 ¹⁰/ L);

(5) cell suspension is distributed in cryovial to 2ml/ pipe * 5, labeled cell type and frozen time;

(6) cryovial is placed in 4 ℃ of Refrigerator store 30min;

(7) cryovial proceeds to-80 ℃ of Refrigerator store 24h;

(8) cryovial moves into liquid nitrogen container (196 ℃), the position of labelling cryovial.

2.9 cell killing experiment

2.9.1 after infecting, DC cell and T are lymphocytic, cultivate altogether

Cell counting, after viral infection, DC cell and T lymphocyte are cultivated altogether 3 days with the 10:1 ratio in 24 orifice plates, by the DC cell after blank viral infection and without the DC cell of viral infection, with the T lymphocyte, cultivate altogether respectively, as negative control group simultaneously.

2.9.2 the T lymphocyte be activated and patient's CML leukaemia cultivate altogether

Frozen in advance patient's CML BMNC is cultured to normal condition, as target cell, with 3 groups of T lymphocytes, in 24 orifice plates, cultivate altogether respectively, cell counting, take effect target ratio as 10:1 cultivation 12 hours, after hatching end, every hole is got supernatant 100ul and is carried out the LDH release test.

2.9.3K562 cell and T lymphocyte are cultivated altogether

The K562 cell is cultivated in 24 orifice plates altogether with 3 groups of T lymphocytes respectively, and cell counting, take effect target ratio after 10:1 cultivates 12h, and every hole is got supernatant 100ul and carried out the LDH release test.

2.9.4LDH release test

Determination step following (table 2):

Table 2

Mix, room temperature is placed 5min, and microplate reader is measured absorbance, wavelength 450nm.

2.10ELISPOT test

(1) wrap in advance the activation of slave board: every hole adds the 200uL serum-free medium, after the standing 5-10min of room temperature, it is deducted.

(2) add cell suspension: each experimental group arranges respectively positive control, negative contrast, the negative contrast of background, and experimental port.Positive control hole and negative control wells cell concentration are 1 * 10 ⁵individual/hole, the negative control wells of background adds serum-free medium, and the experimental port cell concentration is 1 * 10 ⁵individual/hole.After cell is resuspended with the 100uL serum-free medium, be inoculated in each hole.The density of cell in hole will be tried one's best evenly.

(3) add stimulus object: the positive control hole adds 10uLPHA, and the negative control wells of negative contrast sky and background adds the 10uL serum-free medium, and experimental port adds K562 cell or CML and patient's ALL PBMC.Cover the plate lid, put into the CO2 incubator, 37 ℃, 5%CO2 cultivates 16-20 hour.

(4) speckle colour developing: topple over hole inner cell and culture medium, the 200uL/ hole adds ice-cold deionized water, and 4 ℃ of ice bath 10min, topple over liquid in hole, and wash 5-7 time in 1 * washingbuffer200uL/ hole.Each 30-60s that stops.The biotin labeled antibody working solution diluted is added to each experimental port, the 100uL/ hole.Hatch 1 hour for 37 ℃.Topple over liquid in hole, 1 * washing buffer200uL/ hole, wash 5 times, stops 30-60s at every turn, in absorbent paper, blots for the last time.The enzyme mark Avidin working solution diluted is added to each experimental port, and the 100uL/ hole, hatch 1 hour for 37 ℃.With 1 * washingbuffer washing 5 times, method is the same.The AEC nitrite ion working solution of now joining is added to each experimental port, the 100uL/ hole.The standing 15-45min of room temperature lucifuge, treat speckle grow into suitable after, topple over liquid in hole, open the plate base, by deionized water wash positive and negative and base 3-5 time, color development stopping process.The plate button is poured in absorbent paper, pats dry the tiny globule, the standing 10-30min of room temperature.

(5) read plate.The ELISPOT plate is placed in and automatically reads, in the plate instrument, to regulate suitable parameter, the speckle counting.

Result

1.HLA specific C ML polypeptide immune epi-position is successfully selected and predicts

Chinese population restricted epitope main in selected epi-position (A2, A24, A1 and A3) is put into to CPF Antigen Epitope Prediction system and predicted, through the CPF system-computed, these epi-positions can reach the crowd coverage rate of Chinese population more than 90%.

We consult and have been published in since nineteen ninety-five PubMed by the document of the research CML epi-position that SCI included, therefrom select 32 CTL epi-positions, and 17 Th epi-positions, these epi-positions can excitating organism cellullar immunologic response and humoral immunoresponse(HI), and can with the epi-position of the gene loci stable bond of HLA-I molecule and HLA-II molecule.And utilize SYFPEITHIMHC phenotype and Antigen Epitope Prediction system and MAPPP proteasome degraded prognoses system to carry out epitope peptide prediction and MAPPP proteasome cutting-epi-position generation forecast to selected epi-position.The SYFPEITHI predictive value of most of selected epi-position is between 15 to 30, point out these epi-positions that higher HLA binding ability is arranged, simultaneously, the value major part of MAPPP prognoses system, higher than 0.5, points out these epi-positions can accurately be cut and submission (table 3) by proteasome in cell.

Table 3

2. nucleotide sequence successfully synthesizes and is packaged into slow virus and crosses expression system

After nucleotide sequence is synthetic, bag proceeds to after slow virus crosses expression system and is checked order, and sequencing result is as follows, and sequencing result and original series are not found gene mutation (Fig. 1) after comparing.For further determining the expression of this sequence in slow virus, to the slow virus recombiant plasmid, PRNAiU6.2-BCR/ABL*WT1 carries out double digestion, and restriction enzyme site is XbaI/KpnI, and the fragment after enzyme action is carried out agarose gel electrophoresis.Can observe the purpose fragment (Fig. 4 A) of 2117bp.

3. light microscopic identifies that ripe DC morphocytology changes

The PBMC of fresh separated is what be dispersed in, ganoid sphaerocyst, after adherent, can by the lymphocyte of suspension with there is the mononuclear cell that sticks feature and separate.Under the inducing of containing GM-CSF and IL-4 cultivating system, adherent monocytic form becomes irregular shape by circle, from cultivate the 3rd day, dendron shape projection appears in a few cell, cell space increases gradually simultaneously, the visible significantly dendron shape projection of the 5th day large part cell is also assembled agglomeratingly, forms colony.After adding LPS to induce the DC maturation, the cell space of most cells further increases, and is the non-adherent cell (Fig. 2) with long dendron shape endochylema projection.

4. virus successfully infects HEK293 cell and DC cell, and recombinant nucleotide sequence successful expression in cell

The HEK293 cell after the slow virus PRNAiU6.2-BCR/ABL*WT1 of restructuring and blank viral PRNAiU6.2-GFP infect 96 hours, is placed in fluorescence microscopy Microscopic observation green fluorescence in intracellular expression respectively.As shown in the figure, all can observe the generation (Fig. 3 A-D) of green fluorescence through the HEK293 cell of PRNAiU6.2-BCR/ABL*WT1 and PRNAiU6.2-GFP infection.(Fig. 3 E-F) appears in the HEK293 cell redgreen fluorescence without viral infection.The efficiency of infection of the metainfective DC cell of recombinant virus PRNAiU6.2-BCR/ABL*WT1 can reach (Fig. 3 G-H) more than 90%.

Further confirm that by the RT-PCR method recombinant nucleotide sequence is in intracellular expression, can observe the genes of interest of 477bp through the HEK293-BCR/ABL*WT1 of recombinant virus infection cell, have no the expression (Fig. 4 B) of genes of interest in the HEK293-GFP of blank viral infection cell and the HEK293 cell without viral infection.

5. fluidic cell is learned and is identified CD3 immunological magnetic bead sorting T lymphocyte purity

Result shows that the T lymphocyte of the CD3+ sub-elected accounts for 84.1%(Fig. 5 A, the B of T cell subsets).

6. cell killing experiment

Measure the kill capability of T lymphocyte to target cell with the LDH release test.The CD3+T lymphocyte sub-elected from the PERIPHERAL BLOOD MONONUCLEAR CELL of Healthy People respectively with DC-BCR/ABL*WT1, DC-GFP and DC cultivate altogether, the T cell can fast and effeciently be increased, and the T lymphocyte after cultivating altogether with DC-BCR/ABL*WT1 has stronger cytotoxicity to K562 cell and CML, patient's ALL PBMC.LDH activity value (M, the 343.50U/L of DC-BCR/ABL*WT1 group; R, 283.77-588.28U/L) than DC-GFP group (M, 304.92U/L; R, 188.89-445.76U/L) and DC group (M312.71U/L; R, 244.86-498.72U/L) be increased significantly (Fig. 6), and reach statistical significance.The P value is respectively 0.001,0.002.Two groups of comparison no difference of science of statistics of DC-GFP and DC.

7.ELISPOT

Detect the generation of CD3+T lymphocyte IFN-γ by the ELISPOT method.By automatically reading the plate device, the speckle number produced in every hole is counted.Resulting SFC value is 10 ⁵the speckle number that the CD3+T lymphocyte produces.In the K562 cell, the SFC value of DC-BCR/ABL*WT1 group is 69, obviously more than DC-GFP group and DC group SFC value (being respectively 6,9).In the patient CML FMY of BCR/ABLP210 (+), the SFC value of three groups is respectively 58,3 and 20.Yet, in the patient ALL WSK of BCR/ABLP210 (-), three groups of SFC values are respectively the SFC value of 15,5,17, DC-BCR/ABL*WT1 group with respect to DC-GFP and the obviously rising (Fig. 7 A, B) of DC group nothing.

Discuss

The mechanism of tumor and seek basic effectively cure method is a difficult problem and the important topic of medical research always.Along with the develop rapidly of molecular biology, biotechnology, people have been deep into molecular level to the understanding of tumor, start to attempt the theory according to tumour immunity, utilize engineered means to prepare tumor vaccine and are applied to oncotherapy.Think that at present the mechanism of tumor is not only relevant with the deactivation of the activation of proto-oncogene and short apoptogene, and reduce relevant with the immune surveillance function of body.The malignant phenotype of some gene is mainly sealed or is knocked out in the gene therapy of tumor by the gene transfer technology, or the immune state of the transfered cell factor and other functioning genes adjustment body, killing tumor cell.

The immunne response that immune system produces cause of disease is divided into two large classes: during the antibody of take is main body mediation and the humoral immune reaction of extracellular pathogen and with the be the theme cell immune response of the infected target cell of specific killing of mediation of cytotoxic T cell (CTL).Wherein cell immune response seems particularly important for self cell of thoroughly having killed pathogen, having removed infected " change ".Cell immune response is mainly that the specific CTL of the auxiliary mediation of Th cell kills and wounds target cell.The lethal effect of CTL is strictly limited by MHC, in killing and wounding antigenic specificity target cell process, can only identify the specific antigen polypeptide interrelated with self MHCI quasi-molecule.Immune system depends on it with the combination of HLA-I quasi-molecule and passes to the CD8+T cell the microorganism of intracellular infection and the immunoreation of tumor, be after antigen is caught by antigen presenting cell (APC), in Cytoplasm by Proteolytic enzyme, the peptide section that hydrolysis forms is transported in endoplasmic reticulum by TAP, submit to the new and MHC-I molecule become by gp96, formation antigenic peptides-MHC-I quasi-molecule complex, then be transported to cell surface, TCR identification for the CD8+ cell, start immunne response.With the antigenic peptides that the HLA-I molecule is combined, usually with 2-3 aminoacid, as anchor point, be inserted in the antigenic peptides engagement groove of HLA-I molecule.The interior antigenic peptides of antigenic peptides engagement groove that can not be inserted into the HLA-I quasi-molecule can not be combined with HLA-I, and the non-anchor point residue of antigenic peptides can not be combined with the HLA-I molecule equally.

Prepare tumor vaccine and be around the anti-tumor immune response of the immunogenicity that how to strengthen tumor and body and how to break tumour immunity and tolerate this key problem and carry out.Desirable tumor vaccine can not only be induced active immunity, and stimulation of host produces immunne response effectively, but also wants safe without toxic side effect, can also prophylaxis of tumours recur the permanent immunity memory function that protectiveness is provided.Tumor-antigen peptide vaccine etc. is conceived to tumor antigen separation, purification, amplification, increases picked-up and the processed of antigen presenting cell to tumor antigen, finally induces and produces the specific for tumour antigen immunne response.

Chronic myelocytic leukemia is most typical leukemia, by mutually the be shifted BCR/ABL fusion gene that forms of the proto-oncogene c-abl in body and bcr gene, it is the root that causes the chronic granulocyte morbidity, the p210 albumen of latter's coding has tyrosine kinase activity and the cell transformation activity of abnormal enhancing, forward approach to by multi-signal, the adhesive function of interference cell, inhibited apoptosis, change the state of immune response of body, cause abnormality proliferation and the differentiation of hematopoietic stem cell, finally cause the generation of chronic myelocytic leukemia.The BCR/ABL fusion gene almost sees all chronic myelocytic leukemiaes, just is being based on above understanding, and BCR/ABL is considered to the target position of slow grain gene and immunization therapy.

In recent years, antigen processing submission, made significant headway in the aspects such as antigen recognition and T cell activation mechanism, for the genetic immunization of carrying out tumor is had laid a good foundation.The restricted antigen of MHC-I quasi-molecule has the important function that causes the specificity antineoplastic immunity killing tumor cell, its mechanism is thought in cell that newborn proteantigen is digested more and is degraded into short polypeptide, then be transported to rough endoplasmic reticulum, at this, with the MHC-I quasi-molecule newly be collectively referred to as, be combined, form the complex co expression in cell surface, by CTL is identified, further mediation CTL is to expressing the target cell specific killing of this antigen, the BCR-ABL fused polypeptide reaches the peculiar expression in slow granulocyte because of it at newborn aminoacid around position of fusion, infer that this fused polypeptide may be tumor specific antigen.

Studies show that, with deriving from solid tumor or leukemic whole oncolysis thing, oncoprotein, RNA or DNA, be carried on the DC that PERIPHERAL BLOOD MONONUCLEAR CELL is originated, induce in vitro special t cell responses, DC costimulatory molecules up-regulated.Produce antitumor CTL effect [32,33].Some scholars apply b3a2 or the autologous DC of b2a2 merging point load patient or Healthy People DC[14], or carry out transfection CML specific peptide section in Healthy People DC[34 with recombinant adenoviral vector], can produce in vitro peptide specific CD8+ and CD4+ Cellular response.This specific polypeptide DC vaccine can produce the antitumor action of targeting for bcr/abl fusion gene and p210 fusion rotein, is a kind of effective specific active immunotherapy.

The PERIPHERAL BLOOD MONONUCLEAR CELL source DC of b2a2 merging point load C ML patient health donor for Crough etc., the donor T co-culture of cells of the latter and enrichment, can stimulate and produce b2a2 specific C D4+T cell, the patient that infusion CD4+T cell recurs after for the CML stem cell transplantation provides new Therapeutic Method [33].Equally, CML specificity b3a2 fusogenic peptide is added to normal donor DC, latter's sensitized donor self T cell forms the effector lymphocyte, the CML patient's of recurring afterwards with transplanting medullary cell is as target cell, the two is cultivated altogether rear result and shows, load fusogenic peptide DC produces powerful cellulotoxic effect to target cell, therefore, above experiment confirmed load the DC cell of specific fusion protein can kill and wound specifically the leukaemia of ph+.

But current vaccine is mostly single epiposition vaccine, be subject to the restricted of HLA, can not be widely used in the crowd.And polyepitope vaccines refers to and utilizes engineered method will carry a plurality of relevant to target antigen and complementary epi-positions resulting vaccine of being connected.With other vaccines, compare, epiposition vaccine has following advantage: epiposition vaccine can overcome the heredity restriction of major histocompatibility complex (MHC) molecule, due to a defined epitope can only by specific MHC molecule in conjunction with and submission to the T cell, so single epiposition vaccine can not cause the immunoreation of expection usually in all individualities, the polyepitope vaccines of elaborate combination can be by MHC molecular recognition the combination of multiple genetic background, thereby obtains efficient submission; Polyepitope vaccines can effectively be dealt with the variation of tumor antigen and some unfavorable factor in immunoreation; And polyepitope vaccines has unique advantage bringing out aspect cellular immunization.The multi-epitope tumor vaccine has obtained research widely in experiment and animal in vitro.Zhao Ping etc. once found out and can and successfully design the polyepitope vaccines for HCMV for CTL epi-position, Th epi-position and the B cell epitope of the multiple protein antigen of HCMV, and had confirmed in vitro its good immune efficacy [35].

In this experiment, we have designed a kind of vaccine of the polypeptide epitope for Chinese population, have comprised BCR/ABL and the WT1 epi-position of multiple HLA phenotype.In vaccine research, the HLA-A2 phenotype, usually used as the restricted epitope in the vaccine preparation process, follows the Caucasia crowd that obvious difference is arranged but the epi-position of Chinese population distributes abroad.Therefore, the CML epiposition vaccine of the applicable Chinese population of preparation seems particularly important.We have chosen 8 kinds of HLA epi-positions, A1, and A2, A3, A24, A68, B8, B61 and C4, through CPF Antigen Epitope Prediction system prediction, the Chinese population coverage rate of these 8 kinds of epi-positions reaches more than 90%.In this vaccine, the DC cell is as antigen presenting cell, and the reorganized slow virus carrier of success infects, and in cell the correction genes of interest.

For further determining the immunology effect of this vaccine, we have carried out experiment in vitro.And choose p210(+) the K562 cell and CML, patient's ALL PBMC as target cell.LDH release test and ELISPOT experiment all confirm that this vaccine all has the immunology effect to selected target cell, and produce killing functions of immunocytes.And ELISPOT experiment confirms that this vaccine is to p210(-) the immunological role of cell than p210(+) the immunological role of cell a little less than.We infer is to be weaker than the immunological role of BCR/ABL due to the immunogenicity of WT1.

The multi-epitope tumor vaccine, as antineoplastic a new generation polypeptide vaccine, has tempting application prospect.We have successfully prepared the HLA specific polypeptide epiposition vaccine for Chinese population, for the preparation of polyepitope vaccines and MRD how more effectively to remove patient CML provide new thinking and method.

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Claims (4)

1. the special slow grain leukemia of Chinese population HLA epiposition vaccine, it is characterized in that: the nucleotide by sequence as shown in SEQ ID NO.1 is inserted in shuttle plasmid PRNAiU6.2, build shuttle plasmid, then carry out packing and the purification of slow virus, obtain the special slow grain leukemia epiposition vaccine of Chinese population HLA.

2. the preparation method of the special slow grain leukemia of Chinese population HLA claimed in claim 1 epiposition vaccine is characterized in that: comprise the following steps:

(1) by sequence, the nucleotide as shown in SEQ ID NO.1 is inserted between the Sma I and Kpn I multiple clone site of shuttle plasmid PRNAiU6.2, builds shuttle plasmid PRNAiU6.2-DWH;

(2) after successfully building shuttle plasmid, carry out packing and the purification of slow virus, obtain slow virus liquid, be the special slow grain leukemia epiposition vaccine of Chinese population HLA; The packaging system of slow virus is four pUC pUCs, consists of pRsv-REV, pMDlg-pRRE, pMD2G, PRNAiU6.2-DWH.

3. preparation method according to claim 2 is characterized in that: described slow virus packing is as follows with the concrete steps of purification:

1) slow virus incasing cells transfection: with the 293T cell of trypsinization exponential phase, cell density is 0.5 * 10 ⁶/ cm ²the time, be re-seeded into Tissue Culture Dish, 37 ℃, 5%CO ₂in incubator, cultivate;

2) prepare four kinds of plasmid DNA solutions in the slow virus packaging system: get pRsv-REV10 μ g, pMDlg-pRRE15 μ g, pMD2G7.5 μ g and PRNAiU6.2-DWH20 μ g, add sterilized water to be settled to 1800 μ L, then add the CaCl of 2.5mol/L ₂solution 200 μ L, mix, and adds 2 * BBS buffer salt solution, 2000 μ L, and room temperature is placed 20～30min, obtains DNA and calcium phosphate mixed liquor, standby;

When 3) cell density in step 1) reaches 60%～70%, carry out transfection: by step 2) DNA and calcium phosphate mixed liquor be transferred in the culture fluid containing cell monolayer, mix, discard the culture fluid that contains transfection mixture after cultivating 12h, add PBS15mL, discard after jog, repeat this step 3 time;

4) add the DMEM cell culture fluid 15mL containing the 100mL/L calf serum in every bottle of cell, continue to cultivate 48h;

5) collect the 293T cell conditioned medium liquid of transfection 72h;

6) the cell conditioned medium liquid of step 5) being collected is in 4 ℃, and the centrifugal 10min of 4000g, collect supernatant;

7 supernatant that step 6) is collected filter with 0.45 μ m filter;

8) supernatant after step 7) is filtered is in 40mL ultracentrifugation pipe, and 4 ℃, the centrifugal 20min of 25000r/min;

9) step 8) centrifugal after, with the resuspended virus precipitation of ice 500ulPBS, in 4 ℃ of dissolvings, spend the night, obtain slow virus liquid, be the special slow grain leukemia epiposition vaccine of Chinese population HLA.

4. preparation method according to claim 3 is characterized in that: in described step 1), the growth medium in Tissue Culture Dish is the DMEM culture medium containing 10%FBS.

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