CN103495158B

CN103495158B - Specific chronic myelogenous leukemia epitope vaccine for human leukocyte antigen (HLA) of Chinese population

Info

Publication number: CN103495158B
Application number: CN201310464205.0A
Authority: CN
Inventors: 纪春岩; 董文灏; 马道新; 章静茹; 邵娜
Original assignee: Qilu Hospital of Shandong University
Current assignee: Qilu Hospital of Shandong University
Priority date: 2013-09-30
Filing date: 2013-09-30
Publication date: 2015-04-08
Anticipated expiration: 2033-09-30
Also published as: CN103495158A

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 chronic granulocytes leukemia epiposition vaccine of Chinese population HLA

Technical field

The present invention relates to special chronic granulocytes leukemia epiposition vaccine of Chinese population HLA and preparation method thereof, belong to biomedicine technical field.

Background technology

Chronic myelocytic leukemia (CML) is a kind of pernicious myeloproliferative diseases originating from pluripotential hemopoietic stem cell, occupy all leukemic 15 ~ 20%, account for (1.0 ~ 1.5)/100,000 at global annual morbidity, be usually divided into chronic phase, accelerated period and acute transformation phase clinically.The morphological abnormalities of CML patient's existing characteristics of more than 95%, i.e. t (9; 22) (q34; Q11) the Ph chromosome formed or BCR-ABL fusion gene.P210 fusion rotein coded by BCR-ABL fusion gene is not only the basic reason causing CML to occur and be in progress, and is also the important target spot for the treatment of.Ph chromosome is extensively present in chronic myelocytic leukemia and a part of acute lymphoblastic leukemia.It is the G group mark chromosome that normal No. 22 chromosomes of ratio that produced by No. 9 chromosomes and No. 22 chromosome translocations are little, the bcr/abl fusion gene that its transposition produces is positioned on this chromosome, and the fusion rotein coded by this fusion gene has the effect strengthening tyrosine kinase activity.The generation of this fusion rotein is the leukemic important pathogenesis of Ph chromatin-positive.Abl proto-oncogene is positioned at No. 9 chromosome three district four bands, its breakaway poing near one of this gene 5 ' end 300kb in a big way in, can at First Exon b(Ib) upstream, First Exon a(Ia) downstream, or between Ia and Ib; BCR gene is positioned at No. 22 chromosome one district one bands, think that it has three breakaway poings at present, M-bcr, m-bcr and u-bcr, its post-rift bcr gene is from beginning to end with the ab1 gene translocated on No. 22 chromosomes, forms three kinds of fusion genes, i.e. b2a2/b3a2, e1a2, e19a2, the fusion rotein of its coding three kinds of different molecular weights, i.e. p190, p210 and p230.Research confirms, P210 fusion rotein is the basic reason causing CML to fall ill.P210 is a kind of abnormal receptor type tyrosine kinase albumen, compare with normal tyrosine kinase, it is active that P210 has the abnormal tyrosine kinase (PTK) increased, and the phosphorylation level of the specific substrates protein molecular in signal transduction pathway downstream can be caused to significantly improve.Protein phosphorylation is a kind of important form of cell signalling, occurs under protein kinase catalysis, participates in the every physiological function regulating cell.Under normal circumstances, the activity of tyrosine protein kinase is subject to strict regulation and control.But in the cell of CML patient, after P210 albumen is combined with ATP, for a series of substrate protein provides binding site, tyrosine phosphorylation level is improved greatly, and then activates 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.The signal transduction pathway of above three exceptions causes that cell proliferation is accelerated, growth factor dependency weakens, apoptosis is obstructed, the interaction of extracellular matrix and stromal cell is interfered, thus causes the vicious transformation of cell.The medicine of current clinical treatment chronic myelocytic leukemia is specific tyrosine protease inhibitor (TKI).Imatinib is the targeted anticancer medicine for tumor mechanism of first man work synthesis.Although traditional chemotherapy means can improve symptom, mitigate the disease, the conversion of disease can not be stoped, fundamentally can not eliminate malignant clone; Allogeneic Hematopoietic Stem Cell Transplantation is considered to the unique method being expected to cure CML, but, be subject to the restriction of age and appropriate donor, and transplanting incidence of complications is high, mortality risk large, thus only has subset of patients to do Allogeneic Hematopoietic Stem Cell Transplantation.

In recent years, Immuno Suppressive Therapy tumor is used to achieve certain achievement.Letsch etc. use polypeptide vaccine treat 9 patients of once more recurring after at least carrying out 3 melanoma excisions and carry out long term follow-up, 2 have no recurrence in following up a case by regular visits to, 2 only occur that single place or many places are recurred in early days, and 4 stop the patient of recurrence all to create immunne response to vaccine.They confirm to apply the recurrent interval time that polypeptide vaccine extends malignant melanoma, confirm the definite effect [1] of polypeptide vaccine in treatment malignant tumor.

No matter be traditional chemotherapy or bone marrow transplantation, all there is the problem that residual leukemia easily causes recurring, lack desirable bone marrow purging measure at present.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, thus reach the object of curing tumor.BCR-ABL fusion gene forms new aminoacid and the peculiar expression in chronic myelocytic leukemia thereof because of it at position of fusion place, be speculated as tumor specific antigen, therefore the immunity of bcr-abl fusion gene is expected to induce MHC Restricted CTL and for chronic clinical treatment.Wims1 oncogene (Wilms ' tumor gene, WT1) 11p13 is positioned, research finds that nearly all leukemia germinal cell all continues process LAN WT1 gene, think that WT1 gene can become a kind of " whiting disorders of blood mark ", for detecting Minimal Residual Disease of Leukemia, WT1 high expressed closely related with CML progression of disease in marrow series leukemia cell, its protein polypeptide can cause humoral immunization and cellular immunization, can eliminate immature leukemia CD34+ CFU-GM by specificity, be 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 develop the CML series connection epi-position nucleic acid vaccine powerful for the immune effect of BCR-ABL and WT1 target spot covering most of Chinese population HLA and distribute, and the immunization therapy for CML provides theoretical foundation and laboratory basis.

Summary of the invention

For above-mentioned prior art, the invention provides special chronic granulocytes 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 chronic granulocytes leukemia epiposition vaccine of Chinese population HLA, the nucleotide of sequence as shown in SEQ ID NO.1 is inserted in shuttle plasmid PRNAiU6.2, build shuttle plasmid, then carry out packaging and the purification of slow virus, namely obtain the special chronic granulocytes leukemia epiposition vaccine of Chinese population HLA.

The preparation method of the special chronic granulocytes leukemia epiposition vaccine of described Chinese population HLA, comprises the following steps:

(1) nucleotide of sequence as shown in SEQ ID NO.1 (there are Sma ICCCGGG and KpnIGGTACC 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 packaging and the purification of slow virus, obtain slow virus liquid, be the special chronic granulocytes leukemia epiposition vaccine of Chinese population HLA; The packaging system of slow virus is four pUC pUCs, consists of pRsv-REV, and this plasmid of pMDlg-pRRE, pMD2G, PRNAiU6.2-DWH(is that step 1 builds the shuttle plasmid obtained).

Described slow virus packaging 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 ²time, be re-seeded into the 15cm Tissue Culture Dish of 25mL, growth medium is DMEM (containing 10%FBS, percentage by volume), 37 DEG C, 5%CO ₂cultivate in incubator;

2) prepare four kinds of plasmid DNA solutions in 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 and be settled to 1800 μ L, then add CaCl ₂(2.5mol/L) solution 200 μ L, mixing, adds 2 × BBS buffer salt solution 2000 μ L, and room temperature places 20 ~ 30min, obtains DNA and calcium phosphate mixed liquor, for subsequent use;

3) when the 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, mixing, the culture fluid containing transfection mixture is discarded after cultivating 12h, add PBS15mL, discard after jog, repeat this step 3 time;

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

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

6) cell conditioned medium liquid step 5) collected is in 4 DEG C, and the centrifugal 10min of 4000g, collects supernatant;

7 supernatant step 6) collected are with 0.45 μm of frit;

8) by step 7) filter after supernatant in 40mL ultracentrifugation pipe, 4 DEG C, the centrifugal 20min of 25000r/min;

9) step 8) centrifugal after, with the resuspended viral pellet of ice 500ulPBS, spend the night (12h) in 4 DEG C of dissolvings, namely obtain slow virus liquid (through virus titer measure, virus titer is: 3.05 × 10 ⁸tU/ml), be the special chronic granulocytes leukemia epiposition vaccine of Chinese population HLA, during embody rule, according to circumstances can add the adjuvant that vaccine is conventional.

The present invention is by screening the immune epitope for the special CML plurality of antigens albumen of Chinese population HLA, synthesize the nucleosides in series acid sequence of an each epitope of correspondence, be packaged into slow virus process LAN system, infect DC cell, be prepared into Chinese population HLA specificity chronic myelocytic leukemia epiposition vaccine, and in vitro immunocompetence research carried out to it.Immunization therapy for chronic myelocytic leukemia provides new direction.Concrete grammar is: by consulting CML epi-position pertinent literature, therefrom choose the immune epitope for CML antigen protein, after epitope peptide prediction and MAPPP proteasome cutting-epi-position generation forecast being carried out to selected epi-position by SYFPEITHIMHC phenotype and Antigen Epitope Prediction system and MAPPP proteasomal degradation prognoses system, to be connected epi-position full-length gene by overlay region amplification gene method and round pcr synthesis CML.Utilize engineered correlation technique that genes of interest is packaged into slow virus process LAN system.Recombinant virus infection DC cell, is prepared into CML epiposition vaccine.The DC cell that Empty virus infects and without the DC cell of viral infection as negative control.Choose the K562 cell of BCR/ABLp210 (+) and the PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC) of CML, ALL patient as target cell respectively with DC-BCR/ABL*WT1, DC-GFP and DC co-culture of cells.The expression of the mRNA level in-site in the HEK293 cell of RT-PCR method testing goal gene after slow virus infection.CD3+T lymphocyte in immunomagnetic beads method sorting PBMC, flow cyctometry method detects the lymphocytic purity of T of institute's sorting.The morphological change of identified under microscope DC cell.Lactic acid dehydrogenase (LDH) release test measures the killing-efficiency of T cell.ELISPOT method detects the secretion of IFN-γ.Result: predicted by SYFPEITHI and MAPPP and consult pertinent literature, choosing 49 immune epitopes, and cover more than 90% of Chinese population HLA phenotype.CML series connection epi-position full-length gene is synthesized in success, and is successfully packaged into slow virus process LAN 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).ELISPOT method shows that IFN-γ that experimental group CD3+T lymphocyte produces is apparently higher than negative control group.Conclusion: the present invention successfully constructs the special chronic myelocytic leukemia epiposition vaccine of Chinese population HLA, and demonstrates its immunocompetence in vitro, for chronic myelocytic leukemia immunization therapy and remove microresidual disease provide new method and thinking.

Accompanying drawing explanation

Fig. 1: sequencing result and original series comparison schematic diagram.

Fig. 2: the ripe DC cellular morphology figure of light Microscopic observation.

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

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

Fig. 5: flow cyctometry qualification magnetic bead sorting CD3+T lymphocytic purity, wherein, A: the cell irised out is lymphocyte, accounts for 68.1% of PERIPHERAL BLOOD MONONUCLEAR CELL; B:CD3+T lymphocyte accounts for all lymphocytic 84.1%.

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

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

Detailed description of the invention

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.Experiment material used in subordinate's embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

Materials and methods

1. material

1.1 data base used and prognoses systems

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 proteasomal degradation prognoses system

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

1.1.4DNAMAN software

1.1.5Kozak sequence

1.2 plasmid-bearing strains

Test slow virus shuttle plasmid PRNAiU6.2 used and packaging plasmid pRsv-REV, pMDlg-pRRE, pMD2G are all purchased from Shanghai Telebio Biomedical Co., 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

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

1.4 object of study

Choose in 2012.1 to 2013.6 in chronic myelocytic leukemia patient 4 example of Shandong hospital treatment, and acute lymphoblastic leukemia patient 2 example.Its diagnosis all meets Zhang Zhinan and edits " 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	BCR/ABL fusion gene
					YGH	Female	36	Chronic myelocytic leukemia chronic phase	P210=284.86%
ZJG	Man	28	Acute lymphoblastic leukemia	P210=93.15%
					GQ	Man	35	Chronic myelocytic leukemia chronic phase	P210=105.04%
FMY	Female	49	Chronic myelocytic leukemia chronic phase	P210=222.29%
					ZSY	Man	68	Chronic myelocytic leukemia chronic phase	P210=421.36%
WSK	Man	46	Acute lymphoblastic leukemia	P210=0%

1.5 main agents

(1) hyclone (FCS), TBD Products.

(2) RPMI1640 culture fluid, Hyclone Products.

(3) dimethyl sulfoxide (DMSO) is Sigma Products

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

(5) lymphocyte separation medium (Ficoll, relative density 1.077), Chinese Tianjin Hao ocean Products.

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

(7) Trizol reagent, U.S. invitrogen Products.

(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, German U.S. sky Ni Products

(11) LDH test kit, Bioengineering Research Institute is built up in Nanjing.Test kit composition and preparation of reagents:

1) 96 hole ELISA Plate one piece;

2) Matrix buffer 5ml;

3) nadide powder, often prop up powder and add the dissolving of 1.3ml distilled water, subpackage is freezing;

4) 2,4 dinitrophenyl hydrazine 5ml;

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

6) 2mmol/L pyruvate standard liquid 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 DEG C) is Japanese SANYO Products.

(3) Haier's medical bio refrigerator (-20 DEG C) is Haier's Products.

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

(5) micropipettor of all size is German Eppendorf Products.

(6) Eppendorf pipe, is German EppendorfAG Products.

(7) real-time fluorescence quantitative PCR instrument 7500 is American AB I Products.

(8) Eppendorf centrifuge, model LX-200.

(9) UV2450 UV detector is Shimadzu instrument (Suzhou) company limited product.

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

2, experimental technique

The selection of 2.1CMLCTL and prediction

Consult from be published in since 2000 PubMed by SCI the document of research CML epi-position of including, therefrom select 49 energy for CTL epi-position, the Th epi-position of CML multiple protein antigen, can the cellullar immunologic response of excitating organism and humoral immunoresponse(HI).And can with the epi-position of the gene loci stable bond of HLA-I molecule and HLA-II molecule.SYFPEITHIMHC phenotype and Antigen Epitope Prediction system and MAPPP proteasomal degradation prognoses system is utilized to carry out epitope peptide prediction and MAPPP proteasome cutting-epi-position generation forecast to selected epi-position.The gene order finding out multiple BCR-ABL and WT1 from PubMed Nucleotide is found out the corresponding nucleotide sequence of each epi-position and checks, each epi-position nucleotide sequence is lined up by allelic frequency distribution order, 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 sequences and be packaged into process LAN system

2.2.1 add ACCGGT restriction enzyme site in the front end of above-mentioned tandem nucleotide epitope sequences, introduce 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 is the restriction enzyme site introducing carrier T, carries out the synthesis of nucleotide epitope sequences and the order-checking 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 carrier T, introduce Smal CCCGGG and Kpnl GGTACC restriction enzyme site (as shown in SEQ ID NO.1) at sequence two ends, the nucleotide epitope sequences such as shown in SEQ ID NO.1 is introduced corresponding SmaI and the KpnI multiple clone site of shuttle plasmid PRNAiU6.2.

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

Lentivirus production experimental procedure is as follows:

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

2) prepare four kinds of plasmid DNA solutions in 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 and be settled to 1800 μ L, then add CaCl ₂(2.5mol/L) solution 200 μ L, mixing, adds 2 × BBS buffer salt solution 2000 μ L, and room temperature places 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, mixing, discard the culture fluid containing transfection mixture after cultivating 12h, add PBS15mL, discard after jog, repeat this step 3 time;

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

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

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

7) by supernatant with 0.45 μm of frit;

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

9) then heavily revolve viral pellet with ice 500ulPBS, spend the night (12h) in 4 DEG C of dissolvings.

Slow virus titer determination:

(1) infect the previous day (Day1), cell also to its counting, spreads to 6 orifice plates by digestion Hela cell, each hole paving 2 × 10 ⁵cell.Guarantee that cell has 30-50% full when infecting; 5%CO ₂, 37 DEG C of cell pellet overnight are cultivated.

(2) infect the same day (Day2), after slow virus lyolysis being 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 complete medium, and final volume is 1ml.Do not shake.

(3) original cell culture medium is outwelled.Virus and culture medium mixed liquor are often planted in gentle mixing, then they are added each hole (each hole 1mL).

(4) each hole adds Polybrene, final concentration 6 μ g/ml.Gently rock six orifice plates with mixing.37 DEG C, CO2 incubated overnight.

(5) first day (Day3) after infecting, will outwell containing virulent culture medium, change the complete medium .37 DEG C of 2ml into, CO2 incubated overnight.

(6) within after infecting the 4th day, GFP cell is detected.

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

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

The cultivation of 2.3DC cell

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

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

(3) (18 DEG C ~ 25 DEG C) under room temperature, centrifugal 20 minutes with 2000 revs/min.

(4) separation test tube is taken out, 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 mononuclearcell layer (white layer) gently 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 equal-volume dilution, under room temperature (18 DEG C ~ 25 DEG C), 2000 revs/min centrifugal 10 minutes, abandons supernatant, retains cell precipitation.PBS washing twice.

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

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

(8) the cultivation immature DC cell of 5 days is transferred in new six orifice plates continues to cultivate, every hole adds 2ml fresh complete medium CM-CSF, each 1000U/ml of IL-4, and LPS, 37 DEG C, 5%CO ₂continue cultivation 2 days.In the form of the ripe DC cell of the 7th day basis of microscopic observation cultivated.

2.4 slow virus infection DC cells

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

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

3) 4 DEG C of viruses of preserving are taken out, use centrifugal 20 seconds of desk centrifuge, virus is suspended from bottom centrifuge tube completely, according to MOI(infection multiplicity) value=15, calculate slow virus volume required, the virus liquid using pipettor to draw exact volume joins in culture medium, and adds polybrene8ug/ml in the medium to improve the efficiency of infection of viral infection.Put into after mixing CO2 gas incubator (37 DEG C, 5%CO ₂) overnight incubation.

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

5) use the DC cell not adding any virus liquid as blank.

6) culture fluid containing slow virus after infecting 24h is replaced with normal culture fluid; First time if slow virus does not have obvious toxic action to cell, cultivates according to normal condition of culture and changes liquid after skipping.Negative control group and blank group liquid carry out cell simultaneously and change liquid.

7) within the 6th day that infects, under fluorescence inverted microscope, observe fluorescence, estimate the efficiency of slow virus infection object 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 blows and beats uniformly liquid.

(2) 300 μ l chloroforms are added, concuss mixing 30s.The centrifugal 15min of 15000rpm.

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

(4) the centrifugal 10min of 15000rpm, careful abandoning supernatant, prevents RNA from precipitating and loses.

(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, prevent RNA from losing, drying at room temperature makes ethanol volatilize.

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

(8) ultraviolet spectrometer measures RNA concentration.

2.5.2RT reaction

Following RT reaction is carried out in 0.5ml centrifuge tube

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

PCR reaction is carried out in 02ml centrifuge tube.

Each for above reaction system component is added rear mixing successively, of short duration centrifugal.Under following condition, carry out pcr amplification: GAPDH gene, 94 DEG C of denaturation 5min, 94 DEG C of degeneration 45s, 56 DEG C of annealing 45s, 72 DEG C extend 45smin, totally 30 circulations, and last 72 DEG C extend 5min; Genes of interest, 94 DEG C of denaturation 5min, 94 DEG C of degeneration 45s, 58 DEG C of annealing 45s, 72 DEG C extend 45smin, totally 30 circulations, and last 72 DEG C extend 5min.

2.6 immunological magnetic bead sorting CD3+T lymphocytes

(1) extract normal human peripheral blood, with the anticoagulant of EDTA pipe, be separated mononuclearcell (concrete steps are shown in 2.3).

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

(3) cell counting, gets 10 ⁷individual cell, removes 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 of refrigerators;

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

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

(7) 500ul cell suspension is added in MS post, allow liquid slowly drip.3 times are cleaned with Buffer.

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

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

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

(11) the T lymphocyte that immunological magnetic bead sorting goes out is put into six orifice plates to cultivate, and add IL-2 maintenance growth.

The PERIPHERAL BLOOD MONONUCLEAR CELL of 2.7 collection patients CML

(1) the full bone marrow of patient CML is collected clinically, the anticoagulant of EDTA pipe.

(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) (about 1 × 10 ¹⁰/ L);

(4) be distributed in cryovial by cell suspension, 2ml/ manages, labelling patient name and frozen time;

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

2.8K562 cell culture

2.8.1 cell recovery

(1) cryovial is proceeded in 40 DEG C of water-baths by liquid nitrogen container (-196 DEG C) rapidly and (need wear protective gloves);

(2) when thawing to after there is ice-nucleus, namely cryovial is taken out, with containing 70% ethanol disinfection cryovial;

(3) cell suspension is all transferred in the centrifuge tube not containing the RPMI1640 culture fluid (10ml) of calf serum, repeatedly blow and beat without measuring pipette;

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

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

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

(7) in 37 DEG C, 5%CO ₂cultivate in saturated humidity incubator;

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

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

(10) abandon supernatant, use a small amount of RPMI1640 culture fluid containing calf serum resuspended and blow and beat cell, cell being transferred in RPMI1640 culture fluid (large bottle 10-14ml, the bottle 6-10ml) culture bottle containing calf serum;

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

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

2.8.2 cell culture and going down to posterity

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

(2) supernatant is abandoned, cell precipitation is used a small amount of RPMI1640 culture fluid containing 10-15% calf serum resuspended and is blown and beaten cell, how much be sub-packed in several culture bottle (containing the RPMI1640 of 10-15% calf serum) according to cell, blow and beat gently with sterile pipettes, make cell even suspension;

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

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

2.8.3 frozen cell

(1) cell of exponential phase is collected;

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

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

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

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

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

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

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

2.9 cell killing experiments

2.9.1 DC cell and the lymphocytic Dual culture of T after infecting

Cell counting, after viral infection, DC cell and T lymphocyte are with 10:1 ratio Dual culture 3 days in 24 orifice plates, simultaneously by metainfective for Empty virus DC cell and without viral infection DC cell respectively with T lymphocyte Dual culture, as negative control group.

2.9.2 the T lymphocyte be activated and leukaemia's Dual culture of patient CML

The BMNC of patient CML frozen is in advance cultured to normal condition, as target cell respectively with 3 groups of T lymphocytes Dual culture in 24 orifice plates, cell counting, with imitate target than for 10:1 cultivate 12 little time, after hatching end, every hole is got supernatant 100ul and is carried out LDH release test.

2.9.3K562 cell and T lymphocyte Dual culture

Using K562 cell as target cell respectively with 3 groups of T lymphocytes Dual culture in 24 orifice plates, cell counting, cultivate after 12h than for 10:1 to imitate target, every hole is got supernatant 100ul and is carried out LDH release test.

2.9.4LDH release test

Determination step following (table 2):

Table 2

Mixing, room temperature places 5min, and microplate reader measures absorbance, wavelength 450nm.

2.10ELISPOT test

(1) activation of pre-bag slave board: every hole adds 200uL serum-free medium, and room temperature is deducted after leaving standstill 5-10min.

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

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

(4) spot development: pouring aperture inner cell and culture medium, 200uL/ hole adds ice-cold deionized water, 4 DEG C of ice bath 10min, liquid in pouring aperture, and 1 × washingbuffer200uL/ hole, washs 5-7 time.Each stop 30-60s.The biotin labeled antibody working solution diluted is added each experimental port, 100uL/ hole.Hatch 1 hour for 37 DEG C.Liquid in pouring aperture, 1 × washingbuffer200uL/ hole, washs 5 times, stops 30-60s at every turn, blot for the last time in absorbent paper.The enzyme mark Avidin working solution diluted is added each experimental port, 100uL/ hole, hatches 1 hour for 37 DEG C.Wash 5 times with 1 × washingbuffer, method is the same.The AEC nitrite ion working solution of now joining is added each experimental port, 100uL/ hole.Room temperature lucifuge leaves standstill 15-45min, treat speckle grow into suitable after, liquid in pouring aperture, opens plate base, with deionized water wash positive and negative and base 3-5 time, color development stopping process.Be poured in absorbent paper by plate button, pat dry the tiny globule, room temperature leaves standstill 10-30min.

(5) plate is read.ELISPOT plate is placed in and automatically reads, in plate instrument, to regulate suitable parameter, spot count.

Result

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

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

We consult from be published in since nineteen ninety-five PubMed by SCI the document of research CML epi-position of including, therefrom select 32 CTL epi-positions, and 17 Th epi-positions, these epi-positions can the cellullar immunologic response of excitating organism 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 proteasomal degradation 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 the selected epi-position of major part is between 15 to 30, these epi-positions are pointed out to have higher HLA binding ability, meanwhile, the value major part of MAPPP prognoses system, higher than 0.5, points out these epi-positions accurately can be cut and submission (table 3) by proteasome in cell.

Table 3

2. nucleotide sequence successfully synthesizes and is packaged into slow virus process LAN system

Check order after after nucleotide sequence synthesis, bag proceeds to slow virus process LAN system, sequencing result is as follows, does not find gene mutation (Fig. 1) after sequencing result and original series comparison.For determining the expression of this sequence in slow virus further, carry out double digestion to slow virus recombiant plasmid PRNAiU6.2-BCR/ABL*WT1, restriction enzyme site is XbaI/KpnI, and the fragment after enzyme action carries out agarose gel electrophoresis.The object fragment (Fig. 4 A) of 2117bp can be observed.

3. light microscopic identifies that ripe DC morphocytology changes

The PBMC of fresh separated is what be dispersed in, ganoid sphaerocyst, after adherent, the lymphocyte of suspension can be separated with the mononuclear cell with adhesive characteristics.Under the induction containing GM-CSF and IL-4 cultivating system, adherent monocytic form becomes irregular shape from circle, from the 3rd day that cultivates, there is dendron shape projection in a few cell, cell space increases gradually simultaneously, the 5th day visible significantly dendron shape projection of most cells is also assembled agglomerating, forms colony.After adding LPS induction DC maturation, the cell space of most cell increases further, in 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

HEK293 cell after the slow virus PRNAiU6.2-BCR/ABL*WT1 of restructuring and Empty virus PRNAiU6.2-GFP infects 96 hours, is placed in fluorescence microscopy Microscopic observation green fluorescence in intracellular expression respectively.As shown in the figure, the HEK293 cell infected through PRNAiU6.2-BCR/ABL*WT1 and PRNAiU6.2-GFP all can observe the generation (Fig. 3 A-D) of green fluorescence.There is (Fig. 3 E-F) 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 more than 90% (Fig. 3 G-H).

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

5. flow cyctometry qualification CD3 immunological magnetic bead sorting T lymphocyte purity

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

6. cell killing experiment

T lymphocyte is measured to the kill capability of target cell with 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 Dual culture, T cell can fast and effeciently be increased, and has stronger cytotoxicity with the PBMC of T lymphocyte to K562 cell and CML, ALL patient after DC-BCR/ABL*WT1 Dual culture.LDH activity value (M, the 343.50U/L of DC-BCR/ABL*WT1 group; R, 283.77-588.28U/L) comparatively 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.P value is respectively 0.001, and 0.002.DC-GFP and DC two groups compares no difference of science of statistics.

7.ELISPOT

The generation of CD3+T lymphocyte IFN-γ is detected by ELISPOT method.By automatic plate reader, the speckle number produced in every hole is counted.The SFC value obtained is 10 ⁵the speckle number that CD3+T lymphocyte produces.In 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.But 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 relative to the obviously rising (Fig. 7 A, B) of DC-GFP and DC group nothing.

Discuss

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

Immune system is divided into two large classes to the immunne response that cause of disease produces: be in main body mediation with antibody and the cell immune response of the humoral immune reaction of extracellular pathogen and the infected target cell of specific killing mediated that is the theme with cytotoxic T cell (CTL).Wherein cell immune response for thoroughly killing pathogen, remove the own cells of infected " change " and seem particularly important.Cell immune response mainly Th cell assists the specific CTL of mediation to kill and wound target cell.The lethal effect of CTL strictly limits by MHC, is killing and wounding the specific antigen polypeptide that can only identify in antigenic specificity target cell process and be associated with self MHCI quasi-molecule.Immune system depends on it to the microorganism of intracellular infection and the immunoreation of tumor and to combine with HLA-I quasi-molecule and to pass to CD8+T cell, namely after antigen is caught by antigen presenting cell (APC), by Proteolytic enzyme in Cytoplasm, the peptide section that hydrolysis is formed is transported in endoplasmic reticulum by TAP, submit to the new and MHC-I molecule become by gp96, formation antigenic peptides-MHC-I MHC molecule complex, then be transported to cell surface, TCR for CD8+ cell identifies, starts immunne response.The antigenic peptides be combined with HLA-I molecule is inserted in the antigenic peptides engagement groove of HLA-I molecule as anchor point with 2-3 aminoacid usually.The antigenic peptides that can not be inserted in the antigenic peptides engagement groove of HLA-I quasi-molecule then can not be combined with HLA-I, and the non-anchor fixed point residue of antigenic peptides can not be combined with HLA-I molecule equally.

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

Chronic myelocytic leukemia is most typical leukemia, the root causing chronic granulocyte to be fallen ill by the proto-oncogene c-abl in body and the bcr gene BCR/ABL fusion gene formed that is mutually shifted, the p210 albumen of the latter's coding has tyrosine kinase activity and the cell transformation activity of abnormal enhancing, approach is forwarded 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.BCR/ABL fusion gene almost sees all chronic myelocytic leukemiaes, and just based on above understanding, BCR/ABL is considered to the target position of slow grain gene and immunization therapy.

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

Research display, with the DC deriving from solid tumor or leukemic whole oncolysis thing, oncoprotein, RNA or DNA are carried on PERIPHERAL BLOOD MONONUCLEAR CELL source, induces specific T cell reaction, DC costimulatory molecules up-regulated in vitro.Produce antitumor CTL effect [32,33].Some scholars apply b3a2 or b2a2 merging point load autologous patient DC or Healthy People DC [14], or carry out transfection CML specific peptide section in Healthy People DC [34] with recombinant adenoviral vector, peptide specific CD8+ and CD4+ Cellular response can be produced in vitro.This specific polypeptide DC vaccine for bcr/abl fusion gene and p210 fusion rotein, can produce the antitumor action of targeting, 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 such as Crough, the donor T co-culture of cells of the latter and enrichment, can stimulate and produce b2a2 specific C D4+T cell, infusion CD4+T cell is that the patient of recurring after CML stem cell transplantation provides new Therapeutic Method [33].Equally, CML specificity b3a2 fusogenic peptide is added normal donors DC, the latter's sensitized donor self T-cell forms effector lymphocyte, with the medullary cell of the CML patient of relapse after transplantation as target cell, result display after the two Dual culture, load fusogenic peptide DC produces powerful cellulotoxic effect to target cell, and therefore, above experiment confirms that the DC cell of load specific fusion protein can kill and wound the leukaemia of ph+ specifically.

But current vaccine is mostly single epiposition vaccine, be subject to the restricted of HLA, can not crowd be widely used in.And polyepitope vaccines refers to and utilizes engineered method to carry out carrying multiple relevant to target antigen and complementary epi-position obtained vaccine of connecting.Compared with other vaccines, epiposition vaccine has following advantage: epiposition vaccine can overcome the heredity restriction of major histocompatibility complex (MHC) molecule, because a defined epitope can only be merged submission to T cell by specific MHC molecular juction, so single epiposition vaccine can not cause the immunoreation of expection usually in all individualities, the polyepitope vaccines of elaborate combination then can be combined by the MHC molecular recognition of multiple genetic background, thus obtains efficient submission; Polyepitope vaccines effectively can deal with some unfavorable factor in the variation of tumor antigen and immunoreation; And polyepitope vaccines has unique advantage bringing out in cellular immunization.Multi-epitope tumor vaccine has been tested in vitro and has been obtained in animal and studied widely.Zhao Ping etc. once found out and for the CTL epi-position of the multiple protein antigen of HCMV, Th epi-position and B cell epi-position and successful design goes out polyepitope vaccines for HCMV, and can confirm its good immune efficacy [35] in vitro.

In this experiment, we devise a kind of polypeptide epitope vaccine for Chinese population, include BCR/ABL and the WT1 epi-position of multiple HLA phenotype.Abroad in vaccine research, HLA-A2 phenotype is usually used as the restricted epitope in vaccine preparation process, but the distribution of the epi-position of Chinese population has obvious difference with Caucasia crowd.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, A2, A3, A24, A68, B8, B61 and C4, and 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, DC cell is as antigen presenting cell, and the reorganized slow virus carrier of success infects, and in cell correction genes of interest.

For determining the immunology effect of this vaccine further, We conducted experiment in vitro.And choose p210(+) K562 cell and the PBMC of CML, ALL patient as target cell.LDH release test and ELISPOT experiment all confirm that this vaccine all has 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 weak.We infer it is because the immunogenicity of WT1 is weaker than the immunological role of BCR/ABL.

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 the MRD of how more effectively removing patient CML provide new thinking and countermeasure.

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Claims

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

Prepared by following steps:

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

(2), after successfully building shuttle plasmid, carry out packaging and the purification of slow virus, obtain slow virus liquid, be the special chronic granulocytes 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.

2. the preparation method of the special chronic granulocytes leukemia epiposition vaccine of the Chinese population HLA described in claim 1, is characterized in that:

Comprise the following steps:

3., according to the preparation method described in claim 2, it is characterized in that: the concrete steps of described slow virus packaging and purification as

Under:

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

2) prepare four kinds of plasmid DNA solution in slow virus packaging system: get pRsv-REV 10 μ g, pMDlg-pRRE 15 μ g,

PMD2G 7.5 μ g and PRNAiU6.2-DWH 20 μ g, adds sterilized water and is settled to 1800 μ L, then add the CaCl of 2.5 mol/L ₂solution 200 μ L, mixing, adds 2 × BBS buffer salt solution 2000 μ L, and room temperature places 20 ~ 30 min, obtains DNA and calcium phosphate mixed liquor, for subsequent use;

3) when the 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, mixing, the culture fluid containing transfection mixture is discarded after cultivating 12 h, add PBS 15 mL, discard after jog, repeat this step 3 time;

4) add DMEM cell culture fluid 15 mL containing 100 mL/L calf serums in every bottle of cell, continue cultivation 48 h;

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

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

7 supernatant step 6) collected are with 0.45 μm of frit;

8) by step 7) filter after supernatant in 40 mL ultracentrifugation pipes, 4 DEG C, centrifugal 20 min of 25000 r/min;

9) step 8) centrifugal after, with the resuspended viral pellet of ice 500ul PBS, spend the night in 4 DEG C of dissolvings, namely obtain slow virus liquid,

Be the special chronic granulocytes leukemia epiposition vaccine of Chinese population HLA.

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