CN101824434A - Antigen presenting cell, preparation method thereof and application thereof - Google Patents
Antigen presenting cell, preparation method thereof and application thereof Download PDFInfo
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Abstract
The invention discloses an antigen presenting cell, a preparation method thereof and application thereof. The preparation method of the antigen presenting cell comprises the following step of expressing antigenic determinant-humanized beta2 microglobulin fusion protein in isolated antigen presenting cell with the downward-adjusted expression of the TAP to obtain the antigen presenting cell which expresses the antigenic determinant/MHC I compound at the surface of the cell. The method can be used for preparing the cell with various varieties and genes, can specifically present various exogenous antigenic determinants with high level, is particularly used for preparing the humanized professional antigen presenting cell, and can be applied to presenting the exogenous antigenic determinant with lower affinity. The antigen presenting cell prepared by the method can be used for preparing the vaccine, thereby having the profound application prospect in the development of the cell vaccine and the adoptive therapy of the T cell.
Description
Technical field
The present invention relates to antigen presenting cell and preparation method thereof and application.
Background technology
CD8 positive cell poison T cell is resisted the very important effect of performance in virus infection, the antineoplastic immune at body.This T cell is by the acceptor of himself cell surface expression, and the main histocompatibility complex of polypeptide that embeds virus or tumour sudden change of identification target cell surface expression invades or the dissident of sudden change thereby kill and wound, and reaches the purpose of protection body.In view of the vital role of cytotoxic T cell in immunoprotection, how to induce in vivo to produce the key that high-intensity cytotoxic T cell immune response becomes vaccine development.
The stimulation of cytotoxic T cell produces and realizes by the main tissue compatible complex molecule of professional antigen presenting cell surface expression.And the formation of the MHC-I of this chimeric exotic antigen polypeptide is to realize with the cellular activity of the accuracy controlling of presenting by being called antigen processing.Albumen in the born of the same parents at first is degraded into little albumen fragment by a kind of combined enzyme agent that is called proteasome (protesome), these small protein fragments pump into endoplasmic reticulum by the relevant translocator TAP (TAP is made of TAP1 and two subunits of TAP2) of antigen processing then, in endoplasmic reticulum, these small protein fragments are by the further processing of aminoterminal enzyme, form the amino acid whose little peptide of 9-12, the polypeptide be made up of multiple proteins of these little peptides loads mixture and is embedded among the MHC-I then, by the golgi body structure polypeptide epitope is offered to cell surface, stimulate the propagation of special T cell, set up the cell immune response of body.
Overwhelming majority high-affinity antineoplastic specificity cell is eliminated by immunological tolerance in the thymus development process.The antitumor T cell that exists in the body is induced these t cell responses antigen presenting cells based on low-affinity, and the highdensity MHC-I that is embedded with specific polypeptide need be provided.Method commonly used at present is to use the synthetic polypeptide to be loaded into antigen presenting cell, or uses the propagation of molten Jie's product arms tumour stimulation inducing specific T cell of tumour cell.Use the molten Jie's thing arms of polypeptide or tumour antigen presenting cell, the effect of the inaccessible maintaining stimulus T of the instantaneous high-density polypeptide complex cell of a property crossed only can be provided.Another stimulates the method for the antitumor T cell of low-affinity to be to use the TPA epi-position of transformation, strengthens itself and MHC bonded ability, thereby improves in the residence time on antigen presenting cell surface, strengthens the intensity of stimulation T cell.Although these ways strengthen the density that cell surface is mounted with specific polypeptide in various degree, improve the efficient that stimulates the T cell, but, limit more tumour-specific epitope polypeptide and combine with MHC-I because of the MHC-I overwhelming majority that is expressed in cell surface has been mounted with at antigen processing and the antigen from oneself protein of offering to form in the process.
Summary of the invention
The purpose of this invention is to provide a kind of antigen presenting cell and preparation method thereof and application.
The method for preparing antigen presenting cell provided by the present invention, be antigen expressed determinant-people source β2Wei Qiudanbai fusion rotein in the isolated antigen presenting cell of antigen processing dependency transporter protein expression downward modulation, obtain antigen presenting cell at cell surface expression antigenic determinant/MHCI complex body.
Wherein, the method of described antigen expressed determinant-people source β2Wei Qiudanbai fusion rotein is the dna molecular of coding for antigens determinant to be connected with the dna molecular of coding people source β2Wei Qiudanbai obtain fusion gene, and described fusion gene is imported the described fusion rotein of expression in the antigen presenting cell.The antigen presenting cell that described antigen is handled the downward modulation of dependency transporter protein expression is that the rna interference vector that will handle dependency transporter protein gene at antigen imports the antigen presenting cell acquisition.
The antigen presenting cell of described TAP protein expression downward modulation can obtain by existing several different methods, as by inserting inactivation deactivation TAP albumen, perhaps makes the downward modulation of TAP protein expression by prior aries such as transgenation, disappearances.The heterodimer that TAP albumen is made up of TAP1 and TAP2, TAP1 and TAP2 are respectively crossed over endoplasmic reticulum and are formed " hole " spline structure for 6 times altogether, and dependency ATP carries out active transport to the peptide section.The TAP1 deactivation can be made TAP proteins lose activity, perhaps the TAP2 deactivation be made TAP proteins lose activity, also two whole inactivations of composing factor can be reached and make the active purpose of TAP proteins lose.As what the rna interference vector importing antigen presenting cell at the TAP2 gene can be obtained.Described rna interference vector at antigen processing dependency transporter albumen 2 genes is a lentiviral vectors, retroviral vector, adenovirus carrier, vaccinia virus, plasmid vector and little circular DNA carrier.Described lentiviral vectors can obtain by commercial sources, as LK-siTAP2-88, LK-siTAP2-89, LK-siTAP2-90, LK-siTAP2-91 or the LK-siTAP2-92 of Sigma company sale.The antigen presenting cell of described TAP2 protein expression downward modulation also can be the RMA-S cell, the RMA-S cell is the mutant strain of the t cell lymphoma RBL-5 cell of C57BL/6 mouse source, Rauscher virus induction, antigen is handled dependency transporter gene 2 (TAP2) sudden change, cell surface expression is not in conjunction with the MHC I quasi-molecule (unloaded MHC I quasi-molecule) of last antigenic determinant, and this unloaded I quasi-molecule can be directly in conjunction with corresponding exogenous antigen determinant.(Ljunggren，H.G.，Karre，K.，1985.Host?resistancedirected?selectively?againstH-2-deficient?lymphoma?variants.?Analysis?ofthe?mechani?sm.J.Exp.Med.162，1745.)
Described antigen presenting cell is meant and can absorbs, processing treatment antigen, and a para-immunity cell of T, bone-marrow-derived lymphocyte is given in the antigen presentation that will handle.Described antigen presenting cell mainly comprises the target cell etc. of the virus infection of monokaryon-phagocytic cell, dendritic cell, B cell and endotheliocyte, tumour cell, as mouse embryo fibroblasts and mouse lymphoma cell.
Method for preparing antigen presenting cell also belong to protection scope of the present invention.
Experiment showed, the antigen presenting cell energy inducing specific cytotoxic T cell of method for preparing, can be used to prepare vaccine.
Another object of the present invention provides a kind of vaccine.
Vaccine provided by the present invention, its activeconstituents are described antigen presenting cell.
Described antigen presenting cell can also can be mixed with into vaccine with various adjuvants with described antigen presenting cell directly as vaccine.Described adjuvant can be existing multiple adjuvant, as aluminium hydroxide or MF59 etc.
The method that the present invention prepares antigen presenting cell is after utilizing polypeptide to be transferred to endoplasmic reticulum, could with the characteristics of MHC I quasi-molecule assembling, reach and be reduced in antigen presenting cell surface expression self polypeptide/MHC I quasi-molecule by suppressing the TAP function, with the cell of TAP down-regulated expression and antigenic determinant hatches altogether or antigenic determinant is connected in the endoplasmic reticulum assembles with the MHC molecule, and do not need that TAP's is auxiliary, reach and both reduce endogenous self polypeptide MHC/MHC I quasi-molecule, enhancement antigen determinant/MHC I quasi-molecule can induce the specificity antineoplastic T cell of High Fragmentation power to produce in the expression on antigen presenting cell surface again.Method of the present invention can be used to prepare various kinds and genotypic cell, specific high level is offered various exogenous antigen determinants, the full-time antigen presenting cell that particularly prepares the people source as dendritic cell, and is applied to offer the lower exogenous antigen determinant of avidity.The antigen presenting cell of method of the present invention preparation can be used to prepare vaccine, in the development of cell vaccine and T cell are adopted treatment profound application prospect is arranged.
Description of drawings
Fig. 1 is the structural representation of LK-OVA-h β 2m carrier.
Fig. 2 is the clone of screening mTAP2 downward modulation
A: flow cytometer detects each clone surface MHC I (H-2Db) expression level; B: flow cytometer detects RMA-91 surface MHC I (H-2Kb) expression level; C:Western blot detects the TAP2 protein level.
Fig. 3 is screening high expression level OVA
257-264The clone of/MHC I complex body
A: flow cytometer detects the expression of K42-OVA-h β 2m and K42-91-OVA-h β 2m cell surface h β 2m; B: flow cytometer detects the expression of K42-91-OVA-h β 2m cell surface MHC I (H-2Kb); C:Western blot detects h β 2m protein expression level in K42-OVA-h β 2m and the K42-91-OVA-h β 2m born of the same parents.
Fig. 4 is that the intravital knurl that presses down of specific cytotoxic T cells in vitro killing experiments and mouse is tested
A:RMA-91+OVA
257-264With K42-91-OVA-h β 2m inductive specific cytotoxic T cells in vitro killing experiments result; Knurl cubing result in the B:C57BL/6 mouse body; C:C57BL/6 mouse surviving rate.
Embodiment
Among the following embodiment if no special instructions method therefor be ordinary method, agents useful for same all can obtain from commercial channels.
Following embodiment is by making up high expression level OVA
257-264The antigen presenting cell sets forth in detail of/MHC I complex body method for preparing antigen presenting cell of the present invention.
1) makes up the antigen presenting cell that the TAP2 expressing quantity is reduced
LK-siTAP2-88, LK-siTAP2-89, LK-siTAP2-90, LK-siTAP2-91 and LK-siTAP2-92 slow virus expression vector are available from Sigma company, LK-siTAP2-88, LK-siTAP2-89, LK-siTAP2-90, LK-siTAP2-91 and LK-siTAP2-92 load the little stem loop RNA (shRNA) at mouse TAP2 gene different loci respectively, and each target site sequence is seen Sigma product (MISSION
TMShRNA TRCN0000066388, MISSIOM
TMShRNA TRCN0000066389, MISSION
TMShRNA TRCN0000066390, MISSION
TMShRNA TRCN0000066391 and MISSION
TMShRNA TRCN0000066392).
LK-siTAP2-88, LK-siTAP2-89, LK-siTAP2-90, LK-siTAP2-91 and LK-siTAP2-92 slow virus expression vector and pLP1, pLP2, pLP/VSVG (Invitrogen) is packaged into the slow virus particle, with packaged slow virus particle difference called after LK-88, LK-89, LK-90, LK-91 and the LK-92 that contains LK-siTAP2-88, LK-siTAP2-89, LK-siTAP2-90, LK-siTAP2-91 and LK-siTAP2-92.
Packing slow virus particulate method is as follows:
1. transfection is preceding 12-16 hour, shop 5 * 10
6293T cell (ATCC SD3515) adds growth medium and promptly contains 10% foetal calf serum (hereinafter to be referred as FBS in the 10cm Tissue Culture Dish, Invitrogen, Dulbecco ' s modified Eagle ' s medium 16000-044) is (hereinafter to be referred as DMEM, Invitrogen is 12800-017) to 10ml.
2. transfection
(1) mixes plasmid
| PLP1, pLP2 and pLP/VSVG | The packaging plasmid mixture | ??Invitrogen，K4975-00 | ??12μg |
| LK-siTAP2-88 or LK-s iTAP2-89 or LK-s iTAP2-90 or LK-siTAP2-91 or LK-siTAP2-92 | The virus vector plasmid | Sigma is on production number is seen | ??8ug |
To be diluted to 250ul with transfection water (2.5mM Hepes pH value 7.3) behind the above 20ug plasmid mixture, add 250ul 0.5M CaCl
2, mixing;
(2) plasmid that mixes in (1) is slowly dropwise added 500 μ l, 2 * HeBS (0.28M NaCl, 0.05MHEPES 1.5mM Na
2HPO
4PH7.00), outstanding with the top speed whirlpool when dripping;
(3) static 20 minutes; After substratum is abandoned in suction then, add the DMEM 5ml that does not contain serum again;
(4) mixture that obtains in (3) is slowly added on the 293T monolayer cell by ground, leniently vibrate culture dish;
(5) culture dish in (4) is put into 5%CO
237 ℃ of cell culture incubators; 3.5-4 after hour, inhale and abandon supernatant, add the DMEM substratum that 8ml contains 10%FBS;
3. receive poison
After the transfection between 36-48 hour, collect viral supernatant liquid, 4 ℃ of centrifuged supernatant, 2500rpm, 10 minutes, 0.45 μ m filtered, and obtains the slow virus particle, after the packing-80 ℃ frozen.
Slow virus particle LK-88, LK-89, LK-90, LK-91 and LK-92 infect K42 mouse embryo fibroblasts (Nakamura respectively, K.et al. (2001) .Functional specialization ofcalreticulin domains.J.Cell Biol 154,961-972) (Institute of Microorganism, Academia Sinica), concrete grammar is as follows:
Infect preceding 12 hours with 6 orifice plates shops cell, every hole divides 2 * 10
5Individual cell is added RPMI 1640 (Invitrogen, 31800-022) substratum that 3ml contains 10%FBS; Substratum in the hole is abandoned in suction, and every hole adds 5ml slow virus particle LK-88, LK-89, LK-90, LK-91 and LK-92 respectively and add 5 μ l 8mg/ml polybrene that (Sigma H9268) makes its final concentration to 8 μ g/ μ l; Inhale after 6 hours and abandon the slow virus particle, every hole adds RPMI 1640 substratum that 3ml contains 10%FBS; Infect after 48 hours with 6 μ g/ml tetracycline (Amresco, J593) two weeks of screening, drug-fast cell mass obtains monoclonal cell behind limiting dilution, with the stable monoclonal cell difference called after K42-88 that infects LK-siTAP2-88, LK-siTAP2-89, LK-siTAP2-90, LK-siTAP2-91 and LK-siTAP2-92, K42-89, K42-90, K42-91 and K42-92.
Flow cytometer detects each clone surface MHCI (H-2Db) expression level, and method is as follows:
Collect 1 * 10
5Cell, be resuspended in 200ul streaming damping fluid after PBS damping fluid (PH 7.4) is given a baby a bath on the third day after its birth time, (containing 1%FBS, the PBS damping fluid of 0.1% sodium azide) also adds monoclonal antibody (the santa cruz of the anti-mouse MHC of 2ug I (H-2Db) mixture, sc-52541), in placing 30 minutes on ice.Be resuspended in 200ul streaming damping fluid after PBS damping fluid (PH 7.4) is given a baby a bath on the third day after its birth time, and add the 5ul FITC anti-mouse IgG of labelled goat (Epigen) lucifuge and placed on ice 30 minutes.After giving a baby a bath on the third day after its birth time, PBS damping fluid (PH 7.4) is resuspended in fixedly damping fluid (contain the PBS damping fluid of 2% formaldehyde, PH 7.4) upflowing cell instrument detection of 300ul.
The flow cytometer detected result shows K42-88 shown in Fig. 2 A, K42-89, and K42-90, the expression of H-2Db has downward modulation in various degree among K42-91 and the K42-92, wherein the down-regulated expression maximum of H-2Db in the K42-91 cell.
Slow virus particle LK-91 infecting mouse lymphoma cell line RMA (Ljunggren, H.G., Karre, K., 1985.Host resistance directed selectively againstH-2-deficient lymphomavariants.Analysis of the mechanism.J.Exp.Med.162,1745.) (Institute of Microorganism, Academia Sinica) concrete grammar is as follows:
With 6 orifice plates shop cell, 0.5ml 2 * 10 is divided in every hole
5/ ml cell, every hole add 3ml slow virus particle LK-91, add 3.5 μ l 8mg/ml polybrene simultaneously, put to 5%CO after centrifugal 45 minutes in 30 ℃ of 1200rpm
237 ℃ of cell culture incubators.Infect the cell of collecting after 6 hours in each hole, the centrifugal respectively supernatant of abandoning, cell precipitation is resuspended in the RPMI1640 that contains 10%FBS, and (Invitrogen 31800-022) puts back to each hole in 5%CO
2Cultivate in 37 ℃ of cell culture incubators.Infect after 48 hours and screened for two weeks with 6 μ g/ml tetracyclines, drug-fast cell mass obtains monoclonal cell behind limiting dilution, called after RMA-91.
Flow cytometer detects RMA-91 surface MHCI (H-2Kb) expression level, and method is as follows:
Collect 1 * 10
5RMA-91, after giving a baby a bath on the third day after its birth time, PBS damping fluid (pH 7.4) is resuspended in 200ul streaming damping fluid, (containing 1%FBS, the PBS damping fluid of 0.1% sodium azide) also adds the monoclonal antibody (Epigen) of the anti-mouse MHC of 2ug I (H-2Kb) complex body, in placing 30 minutes on ice.Be resuspended in 200ul streaming damping fluid after PBS damping fluid (PH 7.4) is given a baby a bath on the third day after its birth time, and add the 5ul FITC anti-mouse IgG of labelled goat (Epigen) lucifuge and placed on ice 30 minutes.After giving a baby a bath on the third day after its birth time, PBS damping fluid (pH 7.4) is resuspended in fixedly damping fluid (contain the PBS damping fluid of 2% formaldehyde, pH 7.4) upflowing cell instrument detection of 300ul.
The flow cytometer detected result shows the down-regulated expression of H-Kb in the RMA-91 cell shown in Fig. 2 B.Western blot detects TAP2 protein expression level among clone K42-91, RMA-91 and the RMA-S.RMA-S cell (Ljunggren, H.G., Karre, K., 1985.Host resistance directed selectivelyagainst H-2-deficient lymphoma variants.Analysis of the mechanism.J.Exp.Med.162,1745.), be the RMA cell of natural disappearance TAP2.With the expression of Calnexin as confidential reference items.
The used antibody of Western blot:
Detect mouse TAP2: an antibody is the anti-mouse TAP of rabbit antiserum(antisera) (Epigen); Secondary antibody be the anti-rabbit igg of HRP labelled goat (Santa Cruz, C1207);
Detect Calnexin: one-level antibody is the anti-people Calnexin of rabbit antiserum(antisera) (Epigen), this antiserum(antisera) can with mouse Calnexin cross reaction, secondary antibody be the anti-mouse IgG of HRP labelled goat (SantaCruz, D1807).
Western blot detected result shows all obviously downward modulations in clone K42-91 and RMA-91 of TAP2 protein expression level shown in Fig. 2 C.
2) make up high expression level OVA
257-264The antigen presenting cell of/MHC I complex body
LK-OVA-h β 2m slow virus expression vector loads the amalgamation and expression frame that ovum gallinaceum pure albumen (OVA) epi-position and people source β2Wei Qiudanbai (β 2microglobulin, β 2m) connect, and the structure of LK-OVA-h β 2m as shown in Figure 1.
The construction process of LK-OVA-h β 2m slow virus expression vector is as follows:
With the dna fragmentation of gene synthetic method (Invitrogen company is synthetic) acquisition encoding fusion protein OVA-h β 2m, this fragment has the viscosity latter end, and the two ends enzyme is cut to and a little is BamHI and KpnI, and its nucleotide sequence is shown in sequence in the sequence table 1.
The dna fragmentation of encoding fusion protein OVA-h β 2m inserts pLK, and (Sigma SHC001) replaces puromycin resistance gene puroR, called after LK-OVA-h β 2m slow virus expression vector between BamHI and the KpnI restriction enzyme site after the hPGK promotor of carrier.
LK-OVA-h β 2m slow virus expression vector and pLP1, pLP2, pLP/VSVG (Invitrogen) packs according to 1) described in method be packaged into the slow virus particle, called after LK-OVA-h β 2m.
Slow virus particle LK-OVA-h β 2m is according to 1) in method infect K42 mouse embryo fibroblasts and clone K42-91.The stable K42 mouse embryo fibroblasts that infects, clone K42-91 be called after K42-OVA-h β 2m and K42-91-OVA-h β 2m respectively.
Flow cytometer detects K42-OVA-h β 2m, K42-91-OVA-h β 2m surface h β 2m and MHCI (H-2Kb) expression level:
Flow cytometer detects K42-OVA-h β 2m, and the method for the expression level of K42-91-OVA-h β 2m surface h β 2m is as follows:
Collect 1 * 10
5Be resuspended in 200ul streaming damping fluid (containing 1%FBS, the PBS damping fluid of 0.1% sodium azide) after cell, PBS damping fluid (PH 7.4) are given a baby a bath on the third day after its birth time, add the monoclonal antibody (Epigen) of the anti-people's microglobulin of 2ug β 2m simultaneously, on ice in placing 30 minutes.Be resuspended in 200ul streaming damping fluid after PBS damping fluid (PH 7.4) is given a baby a bath on the third day after its birth time, and add the 5ul FITC anti-mouse IgG of labelled goat (Epigen) lucifuge and placed on ice 30 minutes.After giving a baby a bath on the third day after its birth time, PBS damping fluid (pH 7.4) is resuspended in fixedly damping fluid (contain the PBS damping fluid of 2% formaldehyde, pH 7.4) upflowing cell instrument detection of 300ul.
The concrete grammar of the expression level of flow cytometer detection clone surface MHC I (H-2Kb) is as follows:
Collect 1 * 10
5Cells, be resuspended in 200ul streaming damping fluid after PBS damping fluid (pH 7.4) is given a baby a bath on the third day after its birth time and (contain 1%FBS, the PBS damping fluid of 0.1% sodium azide), adds the monoclonal antibody (Epigen) of the anti-mouse MHC of 2ug I (H-2Kb) complex body simultaneously, on ice in placing 30 minutes.Be resuspended in 200ul streaming damping fluid after PBS damping fluid (pH 7.4) is given a baby a bath on the third day after its birth time, and add the 5ul FITC anti-mouse IgG of labelled goat (Epigen) lucifuge and placed on ice 30 minutes.After giving a baby a bath on the third day after its birth time, PBS damping fluid (pH 7.4) is resuspended in fixedly damping fluid (contain the PBS damping fluid of 2% formaldehyde, pH 7.4) upflowing cell instrument detection of 300ul.
The flow cytometer detected result is shown in Fig. 3 A and B, and K42-OVA-h β 2m and K42-91-OVA-h β 2m surface all have h β 2m to express, and K42-91-OVA-h β 2m expresses than K42-OVA-h β 2m height.The MHCI (H-Kb) on K42-91-OVA-h β 2m surface has recovery, illustrates that the MHC I that increases is mainly from the OVA-h β 2m that does not rely on TAP2.
Western blot detects h β 2m protein expression level in K42-OVA-h β 2m and the K42-91-OVA-h β 2m born of the same parents.With the expression of Calnexin as confidential reference items.
The used antibody of Western blot:
Detect h β 2m: one-level antibody is the monoclonal antibody (Epigen) of anti-people's microglobulin β 2m; Secondary antibody be the anti-mouse IgG of HRP labelled goat (Santa Cruz, D1807).
Detect Calnexin: one-level antibody is the anti-people Calnexin of rabbit antiserum(antisera) (Epigen), this antiserum(antisera) can with mouse Calnexin cross reaction, secondary antibody be the anti-mouse IgG of HRP labelled goat (Santa Cruz, D1807).
Western blot detected result is shown in Fig. 3 C, the quite h β 2m protein expression level of level is arranged in K42-OVA-h β 2m and the K42-91-OVA-h β 2m born of the same parents, and it is lower at cell surface K42-OVA-h β 2m than the h β 2m protein expression level of K42-91-OVA-h β 2m, explanation is in K42-OVA-h β 2m cell, and a part of OVA-h β 2m is trapped in cell interior in the little peptide of endoplasmic reticulum and TAP transhipment and the m β 2m of self competition; In K42-91-OVA-h β 2m, because the TAP function is suppressed, OVA-h β 2m can give full expression on cytolemma.
Can detect K42-OVA-h β 2m and K42-91-OVA-h β 2m by the T cell antigen receptor identification at the OVA epi-position.
Concrete experimental technique is as follows:
K42-OVA-h β 2m, K42-91-OVA-h β 2m, EG7, K42-LK are as target cell, and B3Z is the effector cell.
K42-OVA-h β 2m, K42-91-OVA-h β 2m, EG7 cell (the EL4 cell of endogenous expression OVA chicken egg protein) (Moore MW, Carbone FR, Bevan MJ.Introduction of soluble protein intothe class I pathway of antigen processing and presentation.Cell 1988; 54:777-85.) (the K42-LK cell is through the stable monoclonal cell system that screens that infects of slow virus particle LK for (Institute of Microorganism, Academia Sinica) and K42-LK cell, slow virus particle LK is LK carrier (Sigma, SHC001) and pLP1, pLP2 and pLP/VSVG are packaged into) respectively with the RPMI 1640 that contains 10%FBS resuspended to concentration be 1 * 10
6/ ml, 96 orifice plates at the bottom of the U type hole, shop are got 100ul and are spread the 1st hole, and doubling dilution to the 6 holes, every thereafter hole make every hole contain the equal 100ul of cell suspension, and cell count is respectively 1 * 10
5, 2
-1* 10
5, 2
-2* 10
5, 2
-3* 10
5, 2
-4* 10
5, 2
-5* 10
5
OVA
257-264Specific CD8+T cell B3Z (Karttunen J, Sanderson S, Shastri N.Detection of rare antigen-presenting cells by the lacZ T-cell activationassay suggests an expression cloning strategy for T-cell antigens.Proc NatlAcad Sci U S A 1992; 89:6020-4.) (Institute of Microorganism, Academia Sinica) with the RPMI 1640 that contains 10%FBS resuspended to concentration be 1 * 10
6/ ml adds 100ul, mixing in above-mentioned every hole.Then 96 orifice plates are put into 5%CO
237 ℃ of cell culture incubators were hatched 12 hours altogether, centrifugal collecting cell, abandon supernatant, in cell, add 100 μ l contain substrate lysate (the 0.15mM chloroaniline is red-β-D-galactopyranoside (CPRG) (Calbiochem), 0.125%NP40 (EMD Sciences, La Jol la, CA), 9mM MgCl
2With 100mM 2 mercapto ethanol PBS damping fluid), 37 ℃ of lucifuges were placed 4 hours, and every hole adds 50 μ l termination reaction liquid (300mM glycine, 15mM EDTA) back microplate reader at 636nm and 595nm difference reading, and OD636 is light as a setting.
The result is shown in Fig. 3 D, and the K42-LK cell can not be by OVA
257-264Specific CD8+T cell B3Z identification, K42-OVA-h β 2m, K42-91-OVA-h β 2m, the EG7 cell can be by OVA
257-264Specific CD8+T cell B3Z identification.
OVA
257-264Polypeptide is hatched altogether with RMA-91 cell, RMA-S cell and RMA cell respectively, and the cell of acquisition is called after RMA-91+OVA respectively
257-264, RMA-S+OVA
257-264And RMA+OVA
257-264, method is as follows:
26 ℃ of (5%CO in RPMI 1640 substratum that contain 10%FBS
2) cultivating RMA respectively, RMA-91 and RMA-S 24 hours get RMA respectively, and RMA-91 and RMA-S cell are given a baby a bath on the third day after its birth inferior with PBS damping fluid (pH 7.4), and adjusting cell concns with serum-free RPMI 1640 is 5 * 10
6/ ml spreads 24 orifice plates, and every hole 1ml cell suspension adds the OVA that 10 μ l 5mg/ml are dissolved in DMSO simultaneously
257-264Polypeptide (it is synthetic that hundred victory companies are filled in Beijing, and its aminoacid sequence is Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu), making final concentration is 50 μ g/ml, mixing; Placed 4 hours for 26 ℃, placed 4 hours for 37 ℃ again; Centrifugal, obtain RMA-91+OVA respectively
257-264, RMA-S+OVA
257-264And RMA+OVA
257-264Cell.
Flow cytometer detects RMA-91+OVA
257-264, RMA+OVA
257-264, RMA-S+OVA
257-264, K42-OVA-h β 2m and K42-91-OVA-h β 2m, K42 mouse embryo fibroblasts, EG7 cell, EL4 mouse lymphoma cell (available from the female willing biochemical industry QK10063EL4 of company limited in Shanghai (suspension) mouse lymphoma cell) surperficial OVA
257-264/ MHC I expression level, method is as follows:
The flow cytometer detected result shown in Fig. 3 E and F, K42-91-OVA-h β 2m surface OVA
257-264The expression level of/MHC I complex body is apparently higher than K42-OVA-h β 2m or EG7; RMA, RMA-91 and RMA-S cell under the same conditions with polypeptide OVA
257-264After hatching altogether, the RMA-S cell of the natural disappearance of TAP2 and the RMA-91 cell loading polypeptide OVA that reduces TAP2 through siRNA
257-264Ability apparently higher than normal cell RMA.
3) high expression level OVA
257-264The function of the antigen presenting cell of/MHC I complex body
A) at OVA
257-264Specific cytotoxic T cells in vitro killing experiments
The C57BL/6 mouse is divided into 7 groups, and 3 every group, the immune RMA+OVA of difference
257-264, RMA-91+OVA
257-264, RMA-S+OVA
257-264, K42-LK, K42-OVA-h β 2m, K42-91-OVA-h β 2m.RMA+OVA
257-264, RMA-91+OVA
257-264, RMA-S+OVA
257-264Cell is adjusted concentration with the RPMI 1640 that contains 10%FBS before immunity be 5 * 10
6/ ml adds the mitomycin of 10 μ l 1mg/ml with RPMI 1640 configurations simultaneously, and making the mitomycin final concentration is 10 μ g/ml; K42-LK, K42-OVA-h β 2m, K42-91-OVA-h β 2m cell are adjusted concentration with the RPMI 1640 that contains 10%FBS before immunity be 5 * 10
6/ ml adds the mitomycin of 20 μ l 1mg/ml with the RPMI1640 configuration simultaneously, and the final concentration that makes mitomycin is 20ug/ml.Mitomycin is handled after one hour and is washed each cell three times with PBS, abdominal injection immune mouse then, every injected in mice 5 * 10
6Individual cell/500 μ l PBS, immunity in per 7 days once, totally three times; The mouse of only injecting PBS is as the control group that does not have immunity.Collect splenocyte.Last immunity was got the spleen of respectively organizing mouse under the gnotobasis after 10 days, place on the cell filter membrane, spleen and cell filter membrane are put in the 6cm Tissue Culture Dish that 4ml RPMI 1640 is housed, with the syringe sieve front end spleen of milling gently of living, splenocyte is fully discharged, splenocyte is centrifugal, and neat supernatant adds 10ml erythrocyte lysate (139.6mmol/L NH
4Cl, 16.96mmol/L Tris transfers pH to 7.2 with 1mol/L HCl), the static 4-5 of mixing minute, treat that red corpuscle is broken fully, centrifugal 5 minutes of 1500rpm abandons supernatant and removes red corpuscle, obtains the white corpuscle precipitation.With after containing 10%FBS RPMI 1640 and giving a baby a bath on the third day after its birth time, adjusting cell concn is 3 * 10 with cell precipitation
6/ ml spreads 24 porocyte culture plates, and every hole 1ml cell suspension adds the OVA that 20u IL-2 (ChironB.V., Amsterdam, the Netherlands) and 10 μ l 5mg/ml are dissolved in DMSO simultaneously
257-264Polypeptide (it is synthetic that hundred victory companies are filled in Beijing), making final concentration is 50 μ g/ml.Place 5%CO
2, 37 ℃ of cell culture incubators.
Splenocyte is at external use OVA
257-264Polypeptide stimulated after 5 days, extracting spleen cell, and with after containing 10%FBS RPMI 1640 and washing secondary, centrifugal collection splenocyte, 10%FBS RPMI 1640 is resuspended with containing, and the adjustment cell concn is 8 * 10
6/ ml, 96 orifice plates at the bottom of the U type hole, shop are got 100ul and are spread the 1st hole, and doubling dilution to the 4 holes, every thereafter hole make every hole contain the equal 100ul of cell suspension, and cell count is respectively 8 * 10
5, 4 * 10
5, 2 * 10
5, 1 * 10
5Splenocyte action effect cell, EG7 and EL4 are respectively as target cell.
Get 10 respectively
6-10
7EG7 and EL4 are resuspended in 200ul Hank balanced salt solution (HBSS) (8g/L NaCl, 0.4g/L KCl, 1g/L glucose, 60mg/L KH after giving a baby a bath on the third day after its birth time with PBS
2PO
4, 47.5mg/L Na
2HPO
4, pH to 7.2), (Wako, 341-07401) fluorescence dye, CFSE final concentration are 2uM, 37 ℃ of lucifuge water-baths 10 minutes to add CFSE.With containing that 10%FBS RPMI 1640 gives a baby a bath on the third day after its birth time and resuspended, the adjustment cell concn is 10 with in the FBS serum and back
5/ ml adds 100ul according to every hole and joins respectively in above-mentioned 96 orifice plates that are covered with splenocyte, mixing, and other spreads a hole only contains the painted target cell of CFSE and compares group.37 ℃ of (5%CO
2) cultivate the cell mixing of drawing after 6 hours in each hole, with 10 μ g/ml propidium iodide (PI) (Sigma-Aldrich, Poole, England, P4170) dyeing of fluorescence dye room temperature is 3 minutes, and the upflowing cell instrument detects, calculate kill rate, formula is as follows:
The whole CFSE of % specificity death=%
+PI
+The spontaneous CFSE of (death) cell-%
+PI
+(death) cell/(the spontaneous CFSE of 100%-%
+PI
+(death) cell) * 100%;
Whole CFSE in the formula
+PI
+(death) cell is the cell that CFSE and PI dyeing all are positive; Spontaneous CFSE
+PI
+(death) cell is under the same conditions, the cell that CFSE and PI dyeing all is positive in having only the control group of target cell.
The result of kill rate shown in Fig. 4 A and B, high expression level OVA
257-264The clone RMA-91+OVA of/MHC I complex body
257-264, RMA-S+OVA
257-264, K42-91-OVA-h β 2m inductive cytotoxic T cell has specific killing to EG7, and EL4 do not had lethal effect.RMA+OVA
257-264With K42-OVA-h β 2m EG7 there be lower killing and wounding, EL4 is not had lethal effect.
B) the intravital knurl that presses down of mouse is tested
The C57BL/6 mouse is divided into 6 groups, and 10 every group, the immune RMA+OVA of difference
257-264, RMA-91+OVA
257-264, RMA-S+OVA
257-264, K42-OVA-h β 2m and K42-91-OVA-h β 2m, abdominal injection immune mouse, every injected in mice 5 * 10
6Cell/500 μ l PBS, immunity in per 7 days once, totally three times, the mouse of only injecting PBS is as the control group that does not have immunity.After 12 days of last immunity, in the right armpit subcutaneous injection 3 * 10 of mouse
6Individual EG7 cell/200 μ l PBS, the growing state of observation mouse tumor.After treating solid tumor growth, the next day measure knurl volume (V=π ab
2/ 2, a is a major diameter, and b is vertical minor axis) and write down and respectively organize the mouse surviving rate.
Knurl cubing result shown in Fig. 4 C, immune RMA-91+OVA
257-264C57BL/6 mouse, immune RMA-S+OVA
257-264The C57BL/6 mouse of C57BL/6 mouse, immune K42-91-OVA-h β 2m than immune RMA+OVA
257-264C57BL/6 mouse or the long knurl of the C57BL/6 mouse of immune K42-OVA-h β 2m postpone, development is slow, the solid tumor volume is little.K42-91-OVA-h β 2m effect is best.
When the solid tumor volume surpasses 25000mm
3The time, mouse is on the point of dying, and implement to put to death to mouse this moment, puts to death and be designated as the survival fate same day.
Mouse surviving rate result shown in Fig. 4 D, immune RMA-91+OVA
257-264C57BL/6 mouse, immune RMA-S+OVA
257-264The C57BL/6 mouse survival fate of C57BL/6 mouse, immune K42-91-OVA-h β 2m obviously than immune RMA+OVA
257-264C57BL/6 mouse or the fate of the C57BL/6 mouse of immune K42-OVA-h β 2m long.In 50 days that observe, wherein immune RMA-91+OVA
257-264C57BL/6 mouse, immune RMA-S+OVA
257-264The C57BL/6 mouse in the long knurl of 1 mouse is respectively arranged; 2 not long knurls are arranged in the C57BL/6 mouse of immunity K42-OVA-h β 2m.K42-91-OVA-h β 2m effect is best.
In sum, high expression level OVA
257-264The clone RMA-91+OVA of/MHCI complex body
257-264, RMA-S+OVA
257-264, K42-91-OVA-h β 2m can induce special cytotoxic T cell, can be used as vaccine.
Sequence table
＜110〉Institute of Microorganism, Academia Sinica
＜120〉antigen presenting cell and preparation method thereof and application
<130>CGGNARW92125
<160>2
<210>1
<211>435
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>1
atgtctcgct?ccgtggcctt?agctgtgctc?gcgctactct?ctctttctgg?cctcgagggc????60
agtataatca?actttgaaaa?actgggtggc?ggatcgggcg?gaggcggatc?aggaggctca????120
ggtgggtcag?gaggcatcca?gcgtactcca?aagattcagg?tttactcacg?tcatccagca????180
gagaatggaa?agtcaaattt?cctgaattgc?tatgtgtctg?ggtttcatcc?atccgacatt????240
gaagttgact?tactgaagaa?tggagagaga?attgaaaaag?tggagcattc?agacttgtct????300
ttcagcaagg?actggtcttt?ctatctcttg?tactacactg?aattcacccc?cactgaaaaa????360
gatgagtatg?cctgccgtgt?gaaccatgtg?actttgtcac?agcccaagat?agttaagtgg????420
gatcgagaca?tgtaa?????????????????????????????????????????????????????435
<210>2
<211>119
<212>PRT
＜213〉artificial sequence
<220>
<230>
<400>2
Met?Ser?Arg?Ser?Val?Ala?Leu?Ala?Val?Leu?Ala?Leu?Leu?Ser?Leu?Ser
1???????????????5???????????????????10??????????????????15
Gly?Leu?Glu?Gly?Ile?Gln?Arg?Thr?Pro?Lys?Ile?Gln?Val?Tyr?Ser?Arg
20??????????????????25??????????????????30
His?Pro?Ala?Glu?Asn?Gly?Lys?Ser?Asn?Phe?Leu?Asn?Cys?Tyr?Val?Ser
35??????????????????40??????????????????45
Gly?Phe?His?Pro?Ser?Asp?Ile?Glu?Val?Asp?Leu?Leu?Lys?Asn?Gly?Glu
50??????????????????55??????????????????60
Arg?Ile?Glu?Lys?Val?Glu?His?Ser?Asp?Leu?Ser?Phe?Ser?Lys?Asp?Trp
65??????????????????70??????????????????75??????????????????80
Ser?Phe?Tyr?Leu?Leu?Tyr?Tyr?Thr?Glu?Phe?Thr?Pro?Thr?Glu?Lys?Asp
85??????????????????90??????????????????95
Glu?Tyr?Ala?Cys?Arg?Val?Asn?His?Val?Thr?Leu?Ser?Gln?Pro?Lys?Ile
100?????????????????105?????????????????110
Val?Lys?Trp?Asp?Arg?Asp?Met
115
Claims (9)
1. the method for preparing antigen presenting cell, be antigen expressed determinant-people source β2Wei Qiudanbai fusion rotein in the isolated antigen presenting cell of antigen processing dependency transporter protein expression downward modulation, obtain antigen presenting cell at cell surface expression antigenic determinant/MHC I complex body.
2. method according to claim 1, it is characterized in that: the method for described antigen expressed determinant-people source β2Wei Qiudanbai fusion rotein is the dna molecular of coding for antigens determinant to be connected with the dna molecular of coding people source β2Wei Qiudanbai obtain fusion gene, and described fusion gene is imported the described fusion rotein of expression in the antigen presenting cell.
3. method according to claim 1 and 2 is characterized in that: the antigen presenting cell that described antigen is handled the downward modulation of dependency transporter protein expression is that the rna interference vector that will handle dependency transporter protein gene at antigen imports the antigen presenting cell acquisition.
4. method according to claim 3 is characterized in that: described rna interference vector at antigen processing dependency transporter protein gene is a lentiviral vectors, retroviral vector, adenovirus carrier, vaccinia virus, plasmid vector or little circular DNA carrier.
5. method according to claim 4 is characterized in that: described lentiviral vectors is LK-siTAP2-88, LK-siTAP2-89, LK-siTAP2-90, LK-siTAP2-91 or LK-siTAP2-92.
6. method according to claim 5 is characterized in that: the aminoacid sequence of described people source β2Wei Qiudanbai is shown in the sequence in the sequence table 2.
7. the antigen presenting cell of arbitrary described method preparation in the claim 1 to 6.
8. arbitrary described method or the described antigen presenting cell of claim 7 application in the preparation vaccine in the claim 1 to 6.
9. vaccine, its activeconstituents is arbitrary described antigen presenting cell in the claim 1 to 6.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103194477A (en) * | 2013-04-08 | 2013-07-10 | 武汉华美生物工程有限公司 | Fused type prokaryotic expression vector and construction method and application thereof |
| CN110195042A (en) * | 2019-06-11 | 2019-09-03 | 焦顺昌 | A kind of preparation method and application of Dendritic Cells |
-
2009
- 2009-03-04 CN CN2009100788574A patent/CN101824434B/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103194477A (en) * | 2013-04-08 | 2013-07-10 | 武汉华美生物工程有限公司 | Fused type prokaryotic expression vector and construction method and application thereof |
| CN103194477B (en) * | 2013-04-08 | 2015-05-20 | 武汉华美生物工程有限公司 | Fused type prokaryotic expression vector and construction method and application thereof |
| CN110195042A (en) * | 2019-06-11 | 2019-09-03 | 焦顺昌 | A kind of preparation method and application of Dendritic Cells |
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| CN101824434B (en) | 2012-07-04 |
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