CN104127868A - Tumor vaccine and application thereof - Google Patents
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- CN104127868A CN104127868A CN201410189676.XA CN201410189676A CN104127868A CN 104127868 A CN104127868 A CN 104127868A CN 201410189676 A CN201410189676 A CN 201410189676A CN 104127868 A CN104127868 A CN 104127868A
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Abstract
The invention aims to provide a tumor vaccine and application thereof. The preparation method of the tumor vaccine provided by the invention comprises the following steps in order: (1) introducing the coding gene of a specific fusion protein into an in-vitro tumor cell to obtain a recombinant tumor cell, wherein the specific fusion protein comprising a poly peptide fragment and a B7-1 peptide fragment from the N terminal to the C terminal, with the poly peptide fragment comprising polylysine and polyglutamic acid from the N terminal to the C terminal; and (2) carrying out inactivation on the recombinant tumor cell, thus obtaining the tumor vaccine. Compared with tumor vaccines obtained by direct inactivation of tumor cells, the tumor vaccine provided by the invention has obvious curative effects, and is of great value in treatment and prevention of tumors, especially treatment and prevention of melanoma and lung adenocarcinoma.
Description
Technical field
The present invention relates to a kind of tumor vaccine and application thereof.
Background technology
In recent years, along with developing rapidly of the further investigation to tumor development molecular mechanism and biotechnology, be considered to be the 4th kind of tumor treatment model after operation, radiation and chemotherapy taking immunization therapy as basic tumor biotherapy.Different from traditional Therapeutic Method, tumor biotherapy is under the prerequisite of not damaging with body immune system and function, obtains antineoplastic effect by transferring tumor host defense mechanism or giving the very strong material of specific aim targeting that natural (or genetic engineering) produce.Biotherapy mainly comprises cytokine therapy, immunocyte treatment, gene therapy, molecular targeted therapy, Antybody therapy and tumor vaccine technology etc. at present.Wherein, tumor vaccine therapy has developed into the more effective Therapeutic Method of one, in individual body, injects tumor vaccine, can effectively alleviate conditions of patients and reach the effect of preventing and treating high-risk individuals cancer return.
Because the vaccine therapy of tumor is without obvious toxic-side effects, thereby development in recent years is very fast, and current tumor vaccine mainly contains tumor vaccine taking cell as carrier, monoclonal antibody tumor vaccine, tumor gene vaccine, tumour polypeptide vaccine etc.Wherein, the tumor vaccine that tumor cell is carrier, as a kind of important means of tumor biotherapy, is more and more subject to people and extensively payes attention to.But due to the less immunogenic of tumor cells expression antigen, surperficial MHC molecule, costimulatory molecules are expressed low or lacked, and add the genetic background that tumor itself is complicated, original tumour-cell vaccine often can not be induced very strong immunne response.For a change this deficiency, can adopt molecular modification technology to change immunological characteristic or the genetic background of tumor cell, to improve its immunogenicity, induces stronger immunne response.Thereby the tumor vaccine of developing good effect, applied range has become pressing issues of current treatment and prevention of tumour research.
Summary of the invention
The object of this invention is to provide a kind of tumor vaccine and application thereof.
Tumor vaccine provided by the invention, its preparation method in turn includes the following steps:
(1) encoding gene of special fusion rotein is imported in vitro tumor cell, obtain recombinant tumor cell;
Described special fusion rotein is held to C and is held and comprise successively polypeptide section and B7-1 peptide section from N; Described polypeptide section is held to C and is held and comprise that successively poly-D-lysine (specifically can be for to form poly-D-lysine by 25-35 lysine residue from N; Be preferably by 30 lysine residues and form poly-D-lysine) and polyglutamic acid (can be specifically the polyglutamic acid that formed by 25-35 glutaminic acid residue; Be preferably the polyglutamic acid being formed by 30 glutaminic acid residues);
(2) described recombinant tumor cell is carried out to cell deactivation, obtain tumor vaccine.
For the space structure of avoiding described polypeptide section and described B7-1 peptide section affects each other, between described polypeptide section and described B7-1 peptide section, also can there is connection peptides.Described connection peptides is preferably the connection peptides being made up of 4 alanine residues.
Described special fusion rotein specifically can be as shown in the sequence of sequence table 1.
The encoding gene of described special fusion rotein specifically can be as shown in the sequence of sequence table 2.
In the preparation method of described tumor vaccine, between step (1) and step (2), also can comprise the steps: to get described recombinant tumor cell, process (simulation clinical treatment) with chemotherapeutics.Described chemotherapeutics specifically can be cisplatin (Cisplatin, CDDP).The mode of described " processing with chemotherapeutics " is specific as follows: 1. get the cell suspension of described recombinant tumor cell, adding cisplatin and making its concentration is 0.05mg/ml, cultivates collecting cell after 5 days, with cultivating 30 days after the washing of PBS buffer; 2. completing steps 1. after, collect living cells, with suspending after the washing of PBS buffer, obtain cell suspension; 3. get the cell suspension that 2. step obtains, adding cisplatin and making its concentration is 0.05mg/ml, cultivates collecting cell after 5 days, with cultivating 30 days after the washing of PBS buffer; 4. completing steps 3. after, collect living cells, with suspending after the washing of PBS buffer, obtain cell suspension.
In described step (2), described cell deactivation specifically adopts ametycin to carry out.The actual conditions of described cell deactivation can be: under the ametycin concentration of 100 μ g/ml, cultivate 1 hour.
The preparation method of described tumor vaccine is specific as follows:
The encoding gene of described special fusion rotein is imported in vitro tumor cell by (I), obtains recombinant tumor cell;
(II) with the RPMI-1640 culture fluid described recombinant tumor cell that suspends, obtaining viable cell concentrations is 1 × 10
6the cell suspension of individual/ml;
(III) gets the cell suspension that step (II) obtains, and adding cisplatin and making its concentration is 0.05mg/ml, at 37 DEG C, 5%CO
2environment in leave standstill and cultivate 5 days, then collecting cell with the PBS buffer washing of pH7.4,0.01M, is then cultivated 30 days in RPMI-1640 culture fluid;
After (IV) completing steps (III), collect living cells, with the PBS buffer washing of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining viable cell concentrations is 1 × 10
6the cell suspension of individual/ml;
(V) gets the cell suspension that step (IV) obtains, and adding cisplatin and making its concentration is 0.05mg/ml, at 37 DEG C, 5%CO
2environment in leave standstill and cultivate 5 days, then collecting cell with the PBS buffer washing of pH7.4,0.01M, is then cultivated 30 days in RPMI-1640 culture fluid;
After (VI) completing steps (V), collect living cells, with the PBS buffer washing of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining viable cell concentrations is 1 × 10
6the cell suspension of individual/ml;
(VII) gets the cell suspension that step (VI) obtains, and adding ametycin and making its concentration is 100 μ g/ml, at 37 DEG C, 5%CO
2environment in leave standstill cultivate 1 hour;
After (VIII) completing steps (VII), collect by the cell of ametycin deactivation, with the PBS buffer washing of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining cell concentration is 1 × 10
6the cell suspension of individual/ml, is tumor vaccine.
Described in vitro tumor cell can be melanoma cell or lung adenocarcinoma cell.
Described melanoma cell refers to from melanomatous in vitro tumor cell, can obtain by primary separation, also can obtain by being purchased approach.Melanoma cell specifically can be B16-F10 cell
.
Described lung adenocarcinoma cell refers to the in vitro tumor cell from adenocarcinoma of lung.Described lung adenocarcinoma cell can obtain by primary separation LA795 adenocarcinoma of lung mice with tumor (animal housing provides by Cancer Hospital of Chinese Academy of Medical Sciences, qualified number: SCXK11-00-0005) tumor tissues, also can obtain by being purchased approach.Lung adenocarcinoma cell specifically can be LA795 cell (Shanghai Yan Sheng Industrial Co., Ltd., article No. is YS2359).
Can on the basis of tumor vaccine provided by the invention, can induce stronger immunne response by adding immunological adjuvant.
Described special fusion rotein cross-film is expressed, and poly-D-lysine embeds the cell membrane of tumor cell, and B7-1 peptide section is free in outside the cell membrane of tumor cell.B7-1 peptide section provides costimulatory signal, inducer T lymphocyte propagation and secrete cytokines, thus strengthen the activation of T cell and the specific killing action of tumor.Compared with existing tumor vaccine (tumor vaccine that directly killing tumor cells obtains), the immunogenicity of tumor vaccine provided by the invention is stronger, Graft Versus Tumor is higher, tumor cell for transfer or drug resistance after chemotherapy is more effective, and prevention and treatment to tumor have significant application value.
Compared with existing tumor vaccine (tumor vaccine that directly killing tumor cells obtains), tumor vaccine provided by the invention can effectively prevent and treat kinds of tumors and occur, and has the curative effect of highly significant.The present invention has great value for treatment and the prevention, particularly melanoma of tumor and the treatment of adenocarcinoma of lung and prevention.
Brief description of the drawings
Fig. 1 is the structural representation of carrier pDisplay.
Fig. 2 is the characterization result of reconstitution cell first and reconstitution cell second.
Detailed description of the invention
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.
Carrier pDisplay (structural representation is shown in Fig. 1): Invitrogen company, USA.B16-F10 cell:
.LA795 cell: Shanghai Yan Sheng Industrial Co., Ltd., product article No. is YS2359.RPMI-1640 culture fluid: HyClone, article number is SH30809.01B.Mouse-anti people B7-1 monoclonal antibody: Bioscience company of the U.S..Cisplatin (Cisplatin, CDDP): Sigma company of the U.S., catalog number is P4394.Ametycin (Mitomycin C, MMC): purchased from Sigma, catalog number is M0503.C57BL/J6 mice: Beijing company of dimension tonneau China, male, body weight 18-26g.T739 mice: Cancer Hospital of Chinese Academy of Medical Sciences animal housing, male, body weight 18-26g.
Embodiment 1, prepare tumor vaccine
One, prepare recombinant tumor cell
1, by between the Sma I and SalI restriction enzyme site of the double chain DNA molecule forward insertion vector pDisplay shown in the sequence of sequence table 2, obtain recombiant plasmid.
2, Transfected Recombinant Plasmid B16-F10 cell step 1 being obtained, obtains reconstitution cell first.
3, by carrier pDisplay transfection B16-F10 cell, obtain control cells first.
4, Transfected Recombinant Plasmid LA795 cell step 1 being obtained, obtains reconstitution cell second.
5, by carrier pDisplay transfection LA795 cell, obtain control cells second.
Two, the sign of reconstitution cell
1, the sign of reconstitution cell first
(1) with RPMI-1640 culture fluid suspension reconstitution cell first, control cells first or B16-F10 cell, obtaining cell concentration is 1 × 10
6the cell suspension of individual/ml;
(2) get the cell suspension that 100 μ l step 1 obtain, adding 2 μ L mouse-anti people's B7-1 monoclonal antibodies and making its concentration is 200 μ g/ml, room temperature leaves standstill hatches 1h, then wash and suspend with the PBS buffer of pH7.4,0.01M, then add Goat anti mouse IgG/FITC 4 DEG C of standing 1h of hatching, then wash and suspend with the PBS buffer of pH7.4,0.01M, obtaining 1 × 10
6the cell suspension of individual/ml, 1 cell suspension of fine-still, on microscope slide, carries out immunofluorescence confocal laser scanning microscope.
Fig. 2 is shown in by photo, and A is reconstitution cell first, and B is control cells first.B16-F10 cell is consistent with the phenotype of control cells first.Can observe, reconstitution cell first surface shows fluorescence (having B7-1), and control cells first and B16-F10 cell surface all do not show fluorescence.
2, the sign of reconstitution cell second
(1) with RPMI-1640 culture fluid suspension reconstitution cell second, control cells second or LA795 cell, obtaining cell concentration is 1 × 10
6the cell suspension of individual/ml;
(2) with (2) of step 1.
Fig. 2 is shown in by photo, and A is reconstitution cell second, and B is control cells second, and C is LA795 cell.LA795 cell is consistent with the phenotype of control cells second.Can observe, reconstitution cell second surface shows fluorescence (having B7-1), and control cells second and LA795 cell surface all do not show fluorescence.
Three, prepare tumor vaccine
1, prepare tumor vaccine first-I
(1) by RPMI-1640 culture fluid suspension reconstitution cell first, obtaining viable cell concentrations is 1 × 10
6the cell suspension of individual/ml.
(2) get the cell suspension that step (1) obtains, adding cisplatin and making its concentration is 0.05mg/ml, at 37 DEG C, 5%CO
2environment in leave standstill and cultivate 5 days, then collecting cell with the PBS buffer washing of pH7.4,0.01M, is then cultivated 30 days in RPMI-1640 culture fluid.
(3) after completing steps (2), collect living cells, with the PBS buffer washing of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining viable cell concentrations is 1 × 10
6the cell suspension of individual/ml.
(4) get the cell suspension that step (3) obtains, adding cisplatin and making its concentration is 0.05mg/ml, at 37 DEG C, 5%CO
2environment in leave standstill and cultivate 5 days, then collecting cell with the PBS buffer washing of pH7.4,0.01M, is then cultivated 30 days in RPMI-1640 culture fluid.
(5) after completing steps (4), collect living cells, with the PBS buffer washing of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining viable cell concentrations is 1 × 10
6the cell suspension of individual/ml.
(6) get the cell suspension that step (5) obtains, adding ametycin (effect is as killed cells) and making its concentration is 100 μ g/ml, at 37 DEG C, 5%CO
2environment in leave standstill cultivate 1 hour.
(7) after completing steps (6), collect by the cell of ametycin deactivation, with the PBS buffer washing of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining cell concentration is 1 × 10
6the cell suspension of individual/ml, is tumor vaccine first-I.
2, prepare tumor vaccine first-II
(1) by RPMI-1640 culture fluid suspension reconstitution cell first, obtaining viable cell concentrations is 1 × 10
6the cell suspension of individual/ml.
(2) get the cell suspension that step (1) obtains, adding ametycin (effect is as killed cells) and making its concentration is 100 μ g/ml, at 37 DEG C, 5%CO
2environment in leave standstill cultivate 1 hour.
(3) after completing steps (2), collect by the cell of ametycin deactivation, with the PBS buffer washing of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining cell concentration is 1 × 10
6the cell suspension of individual/ml, is tumor vaccine first-II.
3, prepare tumor vaccine second-I
By reconstitution cell second replacement reconstitution cell first, other obtains tumor vaccine second-I with step 1.
4, prepare tumor vaccine second-II
By reconstitution cell second replacement reconstitution cell first, other obtains tumor vaccine second-II with step 2.
5, prepare reference substance first-I
With control cells methyl, for reconstitution cell first, other obtains reference substance first-I with step 1.
6, prepare reference substance first-II
With control cells methyl, for reconstitution cell first, other obtains reference substance first-II with step 2.
7, prepare reference substance first-III
By B16-F10 cell replacement reconstitution cell first, other obtains reference substance first-III with step 1.
8, prepare reference substance first-IV
By B16-F10 cell replacement reconstitution cell first, other obtains reference substance first-IV with step 2.
9, prepare reference substance second-I
By control cells second replacement reconstitution cell first, other obtains reference substance second-I with step 1.
10, prepare reference substance second-II
By control cells second replacement reconstitution cell first, other obtains reference substance second-II with step 2.
11, prepare reference substance second-III
By LA795 cell replacement reconstitution cell first, other obtains reference substance second-III with step 1.
12, prepare reference substance second-IV
By LA795 cell replacement reconstitution cell first, other obtains reference substance second-IV with step 2.
The immunological effect of embodiment 2, tumor vaccine first
One, promote lymphopoiesis
1, get C57BL/J6 mice, get spleen, separate and obtain lymphocyte.
2, the lymphocyte obtaining by RPMI-1640 culture fluid suspension step 1, obtaining cell concentration is 1 × 10
6the lymphocyte suspension of individual/ml.
3,50 μ l products to be tested are mixed with the lymphocyte suspension that 50 μ l steps 2 obtain, at 37 DEG C, 5%CO
2environment in leave standstill cultivate 3 days, then adopt MTT colorimetric method for determining light absorption value (A570nm), calculate proliferation index (Proliferation Index, PI).Product to be tested is respectively tumor vaccine first-I, tumor vaccine first-II, reference substance first-I, reference substance first-II, reference substance first-III or reference substance first-IV prepared by embodiment 1.The processing that replaces product to be tested with equal-volume RPMI-1640 culture fluid is set, as blank.Every kind of product to be tested arranges 5 reprocessings, and blank arranges 5 reprocessings.
The light absorption value in PI=(light absorption value in light absorption value-blank hole of test hole)/blank hole.
The PI that adds the test group of tumor vaccine first-I is 3.95 ± 0.6.
The PI that adds the test group of tumor vaccine first-II is 3.93 ± 0.3.
The PI that adds the test group of reference substance first-I is 1.71 ± 0.2.
The PI that adds the test group of reference substance first-II is 1.74 ± 0.3.
The PI that adds the test group of reference substance first-III is 1.70 ± 0.4.
The PI that adds the test group of reference substance first-IV is 1.73 ± 0.7.
Result shows: compared with each reference substance, tumor vaccine first-I and tumor vaccine first-II can stimulate lymphopoiesis significantly, and the effect of each reference substance does not have significant difference.
Two, promote lymphocytic emiocytosis cytokine
1, the pretreatment of tumor cell
(1) with RPMI-1640 culture fluid suspension B16-F10 cell, obtaining viable cell concentrations is 1 × 10
6the cell suspension of individual/ml.
(2) get the cell suspension that step (1) obtains, adding cisplatin and making its concentration is 0.05mg/ml, at 37 DEG C, 5%CO
2environment in leave standstill and cultivate 5 days, then collecting cell with the PBS buffer washing of pH7.4,0.01M, is then cultivated 30 days in RPMI-1640 culture fluid.
(3) after completing steps (2), collect living cells, with the PBS buffer washing of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining viable cell concentrations is 1 × 10
6the cell suspension of individual/ml.
(4) get the cell suspension that step (3) obtains, adding cisplatin and making its concentration is 0.05mg/ml, at 37 DEG C, 5%CO
2environment in leave standstill and cultivate 5 days, then collecting cell with the PBS buffer washing of pH7.4,0.01M, is then cultivated 30 days in RPMI-1640 culture fluid.
(5) after completing steps (4), collect living cells, with the PBS buffer washing of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining viable cell concentrations is 1 × 10
6the cell suspension of individual/ml.
2, prepare mouse model
Get C57BL/J6 mice, the cell suspension obtaining in step 1 of right fore subcutaneous injection weekly (is that date of injection is respectively experiment the 1st day, the 8th day and the 15th day; In the cell suspension of each every injected in mice, contain 2 × 10
6individual living cells).Test the 5th day, the 12nd day and the 19th day, the tumor in situ size of mice is followed successively by 1.4cm
3, 2.1cm
3and 3.2cm
3, obtained model mice.
3, experiment the 36th day, gets the spleen of the mice of model mice, separates and obtains lymphocyte.
4, the lymphocyte obtaining by RPMI-1640 culture fluid suspension step 3, obtaining cell concentration is 1 × 10
6the lymphocyte suspension of individual/ml.
5,50 μ l products to be tested are mixed with the lymphocyte suspension that 50 μ l steps 4 obtain, at 37 DEG C, 5%CO
2environment in leave standstill cultivate 48 hours, the then centrifugal 4min of 250g, collect supernatant.Product to be tested is respectively tumor vaccine first-I, tumor vaccine first-II, reference substance first-I, reference substance first-II, reference substance first-III or reference substance first-IV prepared by embodiment 1.The processing that replaces product to be tested with equal-volume RPMI-1640 culture fluid is set, as blank.Every kind of product to be tested arranges 5 reprocessings, and blank arranges 5 reprocessings.
6, the supernatant that detecting step 5 obtains, detects the wherein concentration of IFN-γ and the concentration of IL-2.Be Mouse IFN-gamma Quantikine ELISA Kit (RD company of the U.S., catalog number: MIF00) for detection of the test kit of IFN-γ.Be Mouse IL-2Quantikine ELISA Kit (RD company of the U.S., catalog number: M2000) for detection of the test kit of IL-2.
The results are shown in Table 1.
The testing result (pg/ml) of table 1 mouse boosting cell secretion of gamma-IFN and IL-2
| ? | IFN-γ | IL-2 |
| Blank | 39.0±2.5 | 15.6±0.5 |
| Reference substance first-IV | 46.2±3.5 | 26.9±1.3 |
| Reference substance first-III | 45.6±2.8 | 26.3±1.5 |
| Reference substance first-II | 46.4±3.1 | 26.7±1.2 |
| Reference substance first-I | 45.9±3.3 | 25.9±1.6 |
| Tumor vaccine first-II | 131.6±98 | 101.9±5.9 |
| Tumor vaccine first-I | 132.9±10.2 | 101.3±6.4 |
Three, zoopery
1, the pretreatment of tumor cell
(1) with RPMI-1640 culture fluid suspension B16-F10 cell, obtaining viable cell concentrations is 1 × 10
6the cell suspension of individual/ml.
(2) get the cell suspension that step (1) obtains, adding cisplatin and making its concentration is 0.05mg/ml, at 37 DEG C, 5%CO
2environment in leave standstill and cultivate 5 days, then collecting cell with the PBS buffer washing of pH7.4,0.01M, is then cultivated 30 days in RPMI-1640 culture fluid.
(3) after completing steps (2), collect living cells, with the PBS buffer washing of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining viable cell concentrations is 1 × 10
6the cell suspension of individual/ml.
(4) get the cell suspension that step (3) obtains, adding cisplatin and making its concentration is 0.05mg/ml, at 37 DEG C, 5%CO
2environment in leave standstill and cultivate 5 days, then collecting cell with the PBS buffer washing of pH7.4,0.01M, is then cultivated 30 days in RPMI-1640 culture fluid.
(5) after completing steps (4), collect living cells, with the PBS buffer washing of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining viable cell concentrations is 1 × 10
6the cell suspension of individual/ml.
2, prepare mouse model
Get C57BL/J6 mice, the cell suspension obtaining in step 1 of right fore subcutaneous injection weekly (is that date of injection is respectively experiment the 1st day, the 8th day and the 15th day; In the cell suspension of each every injected in mice, contain 2 × 10
6individual living cells).Test the 5th day, the 12nd day and the 19th day, the tumor in situ size of mice is followed successively by 1.4 ± 0.5cm
3, 2.1 ± 0.1cm
3with 3.2 ± 1.2cm
3, obtained model mice.
3, grouping administration
Get model mice, be divided at random 7 groups, every group 10, give as follows different products to be tested: (be that date of injection is respectively experiment the 22nd day, the 29th day and the 36th day at product to be tested of right fore subcutaneous injection weekly, with step 2 meter natural law continuously), each every injected in mice 0.2ml product to be tested.Product to be tested is respectively tumor vaccine first-I, tumor vaccine first-II, reference substance first-I, reference substance first-II, reference substance first-III or reference substance first-IV prepared by embodiment 1.The processing that replaces product to be tested with equal-volume RPMI-1640 culture fluid is set, as blank.Continue to observe the size variation of tumor in situ, respectively at tumor size and the survival rate of experiment the 26th day, the 33rd day and the 40th day statistics mice, test the survival rate of the 45th day statistics mice.The results are shown in Table 2.When statistics tumor size, only add up survival mice.
Table 2 tumor size and survival results
The immunological effect of embodiment 3, tumor vaccine second
1, the pretreatment of tumor cell
(1) with RPMI-1640 culture fluid suspension LA795 cell, obtaining viable cell concentrations is 1 × 10
6the cell suspension of individual/ml.
(2) get the cell suspension that step (1) obtains, adding cisplatin and making its concentration is 0.05mg/ml, at 37 DEG C, 5%CO
2environment in leave standstill and cultivate 5 days, then collecting cell with the PBS buffer washing of pH7.4,0.01M, is then cultivated 30 days in RPMI-1640 culture fluid.
(3) after completing steps (2), collect living cells, with the PBS buffer washing of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining viable cell concentrations is 1 × 10
6the cell suspension of individual/ml.
(4) get the cell suspension that step (3) obtains, adding cisplatin and making its concentration is 0.05mg/ml, at 37 DEG C, 5%CO
2environment in leave standstill and cultivate 5 days, then collecting cell with the PBS buffer washing of pH7.4,0.01M, is then cultivated 30 days in RPMI-1640 culture fluid.
(5) after completing steps (4), collect living cells, with the PBS buffer washing of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining viable cell concentrations is 1 × 10
6cell suspension.
2, prepare mouse model
Get T739 mice, the cell suspension obtaining in step 1 of right fore subcutaneous injection weekly (is that date of injection is respectively experiment the 1st day, the 8th day and the 15th day; In the cell suspension of each every injected in mice, contain 2 × 10
6individual living cells).Test the 19th day, the metastatic nodules of each mouse lung is 8.5 ± 1.2, and the weight of the tumor in situ (injecting the tumor of the original position generation of tumor cell) of each mice is 3.3 ± 0.9g, has obtained model mice.
3, grouping administration
Get model mice, be divided at random 7 groups, every group 10, give as follows different products to be tested: (be that date of injection is respectively experiment the 22nd day, the 29th day and the 36th day at product to be tested of right fore subcutaneous injection weekly, with step 1 meter natural law continuously), each every injected in mice 0.2ml product to be tested.Product to be tested is respectively tumor vaccine first-I, tumor vaccine first-II, reference substance first-I, reference substance first-II, reference substance first-III or reference substance first-IV prepared by embodiment 1.The processing that replaces product to be tested with equal-volume RPMI-1640 culture fluid is set, as blank.Test the 40th day, the metastatic nodules of adding up each mouse lung, add up each mice tumor in situ (inject tumor cell original position occur tumor) weight, statistics mice survival rate.The results are shown in Table 3.When the metastatic nodules of statistics mouse lung and tumor in situ weight, only add up survival mice.
Metastatic nodules, tumor in situ weight and the survival rate of table 3 mouse lung
Claims (8)
1. a tumor vaccine, its preparation method in turn includes the following steps:
(1) encoding gene of special fusion rotein is imported in vitro tumor cell, obtain recombinant tumor cell;
Described special fusion rotein is held to C and is held and comprise successively polypeptide section and B7-1 peptide section from N; Described polypeptide section is held to C and is held and comprise successively poly-D-lysine and polyglutamic acid from N;
(2) described recombinant tumor cell is carried out to cell deactivation, obtain tumor vaccine.
2. tumor vaccine as claimed in claim 1, is characterized in that: between described polypeptide section and described B7-1 peptide section, also have connection peptides.
3. tumor vaccine as claimed in claim 2, is characterized in that: described connection peptides is the connection peptides being made up of 4 alanine residues.
4. tumor vaccine as claimed in claim 3, is characterized in that: described special fusion rotein is as shown in the sequence 1 of sequence table.
5. tumor vaccine as claimed in claim 4, is characterized in that: the encoding gene of described special fusion rotein is as shown in the sequence 2 of sequence table.
6. as the tumor vaccine as described in arbitrary in claim 1 to 5, it is characterized in that: in the preparation method of described tumor vaccine, between step (1) and step (2), also can comprise the steps: to get described recombinant tumor cell, process with chemotherapeutics.
7. tumor vaccine as claimed in claim 6, is characterized in that: described chemotherapeutics is cisplatin.
8. as the tumor vaccine as described in arbitrary in claim 1 to 7, it is characterized in that: described in vitro tumor cell is melanoma cell or lung adenocarcinoma cell.
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