CN101948508A - Tumor-resistance CTL (Cytotoxic T Lymphocyte) epitope peptide derived from PIWIL2 and application thereof - Google Patents
Tumor-resistance CTL (Cytotoxic T Lymphocyte) epitope peptide derived from PIWIL2 and application thereof Download PDFInfo
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Abstract
The invention relates to a tumor-resistance CTL epitope peptide derived from PIWIL2, i.e. a nonapeptide, which is characterized in that the sequence of the epitope peptide is a-Leu-Gly-Gly-Glu-Leu-Trp-Gly-Val (wherein a is Lys or Tyr); the sequence of the epitope peptide P724 is Lys-Leu-Gly-Gly-Glu-Leu-Trp-Gly-Val; and the sequence of the tumor-resistance CTL epitope peptide P724-1Y is Tyr-Leu-Gly-Gly-Glu-Leu-Trp-Gly-Val. The invention can ensure that the specificity CTL induced by the tumor-resistance CTL epitope peptide in vitro and in vivo has a certain kill rate on tumor cells MCF-7 and can be used for preparing a tumor therapeutic peptide vaccine.
Description
Technical field
The invention belongs to the polypeptide technical field in the biochemical field, be specifically related to the antitumor CTL epitope peptide and the application in preparation tumor therapeutic polypeptide vaccine thereof in a kind of PIWIL2 source.
Background technology
PIWIL2 belongs to tumor-testis antigen (Cancer-Testis Antigen, CTA) family, in healthy tissues, exist only in the testis tissue, in tumor tissues such as mammary cancer, ovarian cancer, prostate cancer and carcinoma of testis, present high expression level, and in normal galactophore tissue, do not express.The generation of PIWIL2 and tumour, the development closely related, it is the target spot of an extraordinary diagnosing tumor and treatment, the more important thing is because its special organization expression characteristic and take place with mammary cancer, the cognation of development, make it become a very ideal mammary cancer immunotherapy target antigen.
Along with immunologic development of modern times, (Cytotoxic T lymphocyte, the vital role of CTL) bringing into play in tumour more and more comes into one's own cytotoxic T lymphocyte.How to excite the specificity cellular immunity response of CTL mediation effectively, bring into play antitumor usefulness, become an important topic in current tumor therapeutic polypeptide vaccine development field.Previously discovering, cause the CTL immune response, is not complete tumour antigen molecule, but the specific CTL epi-position (Epitope) in antigen source.Behind the antigen presenting cell picked-up tumour antigen, be processed into polypeptide fragment by proteolyzing into 8-10 amino acid length, it is the CTL epi-position, and then combine with major histocompatibility complex I (MHCI) quasi-molecule in the endoplasmic and to form polypeptide-MHC mixture (peptide-MHC complex, pMHC), and the most at last the pMHC submission to TXi Baoshouti (the T cell receptor of cell surface for the CD8+T cell surface, TCR) identification, thereby activation CTL cell causes the CTL immunne response.But immunogenicity existence weak and immunological tolerance is the two big obstacles that the CTL epi-position is used as the tumor therapeutic polypeptide vaccine.Therefore, how to improve the immunogenicity of CTL epi-position and break body becomes the development of tumor therapeutic polypeptide vaccine to the immunological tolerance of CTL epi-position key.
At present, in the enhancing strategy of numerous tumor therapeutic polypeptide vaccines, the CTL epi-position is carried out molecular modification be considered to one of the most promising method.The transformation of CTL epi-position can be passed through to transform epi-position MHC binding site, thereby improve the avidity of epi-position and MHC and the stability of pMHC, to reach the purpose of enhancing immunity originality; Also can pass through to transform epi-position TCR binding site, thereby improve pMHC and TCR bonded stability, to reach the purpose that improves the CTL activity and overcome the CTL tolerance.Recent research shows, though can improve the immunogenicity of vaccine to a certain extent based on the polypeptide vaccine of epi-position MHC binding site transformation, do not reach the ideal tumor killing effect in immunotherapy of tumors; And based on the polypeptide vaccine that epi-position TCR binding site is transformed, be expected to by strengthening the stimulation ability of low-affinity T cell or recruiting the new T cell with cross reactivity of a group and break the tumour immunity tolerance and reach the ideal tumor killing effect.
The HLA-A2.1 molecule mainly combines with nonapeptide, and its two and nine is the main anchored site of MHC.Jorg Ruppert (Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules.Cell.1993,74 (5): 929-37.) find in the advantage amino acid of the main anchored site of research: two are L or M, nine is L, V or I, epi-position and MHC bonded ability can be improved 10～100 times; And to the transformation of less important anchored site, the most frequently used and the most effective a kind of method is exactly to use tyrosine (Tyr) to replace in one amino acid.(A general strategy to enhance immunogenicity of low-affinity HLA-A2.1-associated peptides:implication in the identification of cryptic tumor epitopes.Eur J Immunol.2000,30 (12): 3411-21.) introducing of pointing out a Tyr mainly relies on the hydrogen bond action of the hydrophobic interaction of its side-chain benzene ring and phenolic hydroxyl group to strengthen the interaction of epitope peptide and HLA molecule to Tourdot.
The application adopts method theoretical and that experiment combines, modifies and preliminary evaluation goes out candidate CTL epitope peptide and analogue thereof with the high PIWIL2 source of MHC molecule binding ability.We are by the screening to CT datebase, found a kind of cancer testis antigens c T80 (being PIWIL2) of high expression level in mammary cancer of new identification, its positive expression rate in mammary cancer has surpassed 80%, be a unusual mammary cancer immunotherapy of ideal target antigen in the CT antigen, and identify also there is not the article report about their epi-position at present.According to antigenic primary structure, adopt the immunologic information section of learning to do, utilization SYFPEITHI, BIMAS and Net CTL 1.2 databases have carried out forecast analysis to the HLA-A2.1 restricted CTL epitope of antigen PIWIL2.
Summary of the invention
The object of the invention is to provide a kind of PIWIL2 antitumor CTL epitope peptide with antitumor action with artificial synthetic method, and the preparation method of epitope peptide is provided.
Another purpose of the present invention is to provide the application of this PIWIL2 antitumor CTL epitope peptide in preparation tumor therapeutic polypeptide vaccine.
For achieving the above object, the present invention adopts following technical scheme:
The PIWIL2 antitumor CTL epitope peptide is nonapeptide, and its sequence is: a-Leu-Gly-Gly-Glu-Leu-Trp-Gly-Val; Wherein, a is Lys or Tyr.
When a was Lys, sequence was Lys-Leu-Gly-Gly-Glu-Leu-Trp-Gly-Val (is KLGGELWGV, is designated as P724), and molecular weight is 958.1.
When a was Tyr, sequence was Tyr-Leu-Gly-Gly-Glu-Leu-Trp-Gly-Val (is YLGGELWGV, is designated as P724-1Y), and molecular weight is 993.1.
The preparation method of described PIWIL2 antitumor CTL epitope peptide can adopt solid-phase synthesis to synthesize the CTL epitope peptide.Basic procedure is as follows: at first an amino is connected on the insoluble solid phase carrier Wang resin by the amino acid of Fmoc radical protection, and the protecting group of desamidizate then, first amino acid promptly has been connected on the solid phase carrier; Secondly amino is activated with condensing agent by second amino acid whose carboxyl of Fmoc radical protection; amino acid after the activation forms peptide bond with first the amino acid whose amino reaction that is connected on solid phase carrier again, has just generated a dipeptides that has protecting group this moment on solid phase carrier.Repeat above-mentioned peptide bond and form reaction, peptide chain is grown to the N end from the C end, until reaching needed peptide chain length, cutting at last obtains the purpose peptide.Behind the HPLC purifying, its purity is greater than 90%, and mass spectroscopy also confirms that its molecular weight meets theoretical value.
The application of described PIWIL2 antitumor CTL epitope peptide in preparation tumor therapeutic polypeptide vaccine.
Advantage of the present invention: utilize high expression level in mammary cancer and the antigen PIWIL2 that in human normal tissue, do not express, filter out epitope peptide with anti-tumor activity, the nonapeptide of identifying there is no bibliographical information, for development is provided fundamental basis based on the tumor therapeutic polypeptide vaccine of antigen PIWIL2, and lay the foundation for follow-up polyvalent antigen peptide vaccine construction.
Description of drawings
Fig. 1 is the mass spectroscopy collection of illustrative plates of epitope peptide P724 of the present invention;
Fig. 2 is the mass spectroscopy collection of illustrative plates of epitope peptide P724-1Y of the present invention;
Fig. 3 is the detected result of epitope peptide inductive specific CTL of the present invention to tumour cell MCF-7 lethal effect;
Fig. 4 be epitope peptide of the present invention in the transgenic mice body inductive specific CTL to the detected result of tumour cell MCF-7 lethal effect.
Laboratory apparatus title and model:
Electron spray(ES)-ion trap multi-stage ms instrument BRU KER Esquire-3000 Germany Brooker dalton instrument company
Flow cytometer Calibur U.S. company BD
Embodiment
The present invention is described further by the following examples, but protection scope of the present invention is not limited thereto.
Embodiment 1: the preparation of antitumor CTL epitope peptide P724 (Lys-Leu-Gly-Gly-Glu-Leu-Trp-Gly-Val)
Adopt the Fmoc solid-phase synthesis, prolong one by one from C end → N end.Protect amino acid whose alpha-amino group with fluorenes methoxy carbonyl acyl group (Fmoc), various Fmoc protect amino acid whose Side chain protective group to be respectively Trp (Boc), Glu (OtBu), Lys (Boc) (on behalf of tertbutyloxycarbonyl, OtBu, Boc represent uncle's fourth fat).C with the alpha-amino group protection holds first amino acid earlier, and promptly Fmoc-Val-OH makes condensing agent with N, N '-DIC (DIC), and adds 1-hydroxy benzo triazole (HOBt), and Fmoc-Val-OH is connected on the wang resin; Successively with DMF, anhydrous methanol, methylene dichloride, DMF washing; Use piperidines-DMF mixed solution (V then
Piperidines: V
DMF=1: 4) remove the Fmoc protecting group, successively with DMF, anhydrous methanol, methylene dichloride, DMF washing.Be that Fmoc-Gly-OH makes condensing agent with DIC with second amino acid of C end again, and add HOBt, Fmoc-Gly-OH is connected on the amino of Val, with piperidines-DMF mixed solution (V
Piperidines: V
DMF=1: 4) slough the Fmoc protecting group, successively with DMF, anhydrous methanol, methylene dichloride, DMF washing.Get the Gly-Val-Wang resin.Be connected with Fmoc-Trp (Boc)-OH again, repeat above-mentioned steps, connect successively and go up Leu, Glu, Gly, Gly, Leu, Lys, peptide chain is progressively prolonged, by sequence from C end → N end with piperidines-DMF mixed solution (V
Piperidines: V
DMF=1: 4) slough the Fmoc protecting group, with cutting reagent (V
Trifluoroacetic acid: V
Thioanisole: V
Water: V
Phenol: V
1=82.5: 5: 5: 5: 2.5) P724 is downcut from the Wang resin, and slough Boc, OtBu, Boc protecting group, obtain antitumor CTL epitope peptide P724 crude product.
HPLC separation and purification: use C
18The separation and purification of preparation property HPLC post (Partisi 110 ODS-39.4 * 250mm, Whatman company).Get above-mentioned P724 crude product 30mg and be dissolved among the aqueous solution a (the TFA volume content is 0.1%, and the CAN volume content is 30%) that 3ml contains trifluoroacetic acid (TFA), acetonitrile (CAN), carry out the constant gradient wash-out with a, flow velocity 5ml/min collects main peak, and lyophilize gets smart peptide.Product is carried out mass spectroscopy see Fig. 1, the result confirms that molecular weight is 958.1, is consistent with theoretical value.
Embodiment 2: the preparation of antitumor CTL epitope peptide P724-1Y (Tyr-Leu-Gly-Gly-Glu-Leu-Trp-Gly-Val)
Adopt synthetic method similarly to Example 1, difference only is to substitute Lys with Tyr.
Product is carried out mass spectroscopy, the results are shown in Figure 2, confirm that molecular weight is 993.1, be consistent with theoretical value.
The CTL epitope peptide of above-mentioned preparation can be used for preparing the tumor therapeutic polypeptide vaccine, and its application experiment is as follows:
1, epitope peptide and HLA-A2.1 molecule combining power test
(1) the centrifugal collection of 800rpm HLA-A2.1 expresses the T2 cell (ATCC company) of the positive and endogenous antigen processing treatment anergy, the phosphate buffered saline buffer of PH7.2 (PBS) washing 3 times, serum-free RPMI 1640 substratum (Gibico company) resuspended to cell density be 1 * 10
6/ mL is inoculated in 24 orifice plates.
(2) every hole adds 50 μ g epitope peptides and 0.5 μ g people β
2Microglobulin (Sigma company) is hatched 18h for 37 ℃ altogether;
(3) with 4 ℃ PBA (with the 2g bovine serum albumin, 0.2g sodiumazide is dissolved among the PBS of 100ml PH7.4 and promptly gets PBA) washing 3 times, add the monoclonal antibody BB7.2 (Sigma company) of 100 μ l with the mouse-anti people HLA-A2.1 molecule after 100 times of dilutions of PBS of PH 7.4,4 ℃ of lucifuges are hatched 40min;
(4) with 4 ℃ PBA washing 3 times, add the sheep anti-mouse igg (Beijing Bo Aosen Bioisystech Co., Ltd) of 50 μ l with fluorescein isothiocyanate (FITC) mark after 50 times of dilutions of PBS of PH 7.4,4 ℃ of lucifuges are hatched 30min;
(5) PBA washing back is detected in flow cytometer.The result represents with fluorescence coefficient FI, sees table 1 for details.
The result shows that two epitope peptides all demonstrate bonding force preferably, and transforms peptide P724-1Y far above parent peptide P724.
Table 1 epitope peptide and HLA-A2.1 molecule combining power test result
| Epitope peptide | FI |
| P724 | 0.38 |
| P724-1Y | 1.08 |
Annotate: FI=(epitope peptide average fluorescent strength-background average fluorescent strength)/background average fluorescent strength
FI＞1.5: epitope peptide and HLA-A2.1 have strong bonding force;
0.5＜FI＜1.5: medium bonding force; FI＜0.5: weak bonding force;
2, human peripheral blood mononuclear cell's (PBMCs) separation with induce and LDH detects:
The peripheral blood that extracts healthy donor separates through density gradient centrifugation, obtains PBMCs, adds interleukin II (IL-2, Peprotech company) and people β
2Microglobulin is induced differentiation CTL cell.Method is as follows:
(1) with the peripheral blood 40ml after 40ml PBS (PH 7.2) the dilution anti-freezing processing;
(2) add 4ml Lymphoprep parting liquid (Axis-Shield company) in the centrifuge tube;
(3) add in the 8ml step (1) peripheral blood after the dilution on 4ml Lymphoprep parting liquid liquid level;
(4) 20 ℃ of centrifugal (2000rmp * 20min);
(5) after centrifugal, be divided into four layers, discard the superiors, drawing the second layer with glass pipette is tunica albuginea layer (being rich in PBMCs);
(6) twice of PBS (PH 7.2) centrifuge washing of the tunica albuginea layer of sucking-off;
(7) with 24 orifice plate bed boards, the concentration of cell is 1 * 10
6/ ml, every hole 1ml;
Every hole added 10 μ g people β in (8) second days
2Microglobulin and 10 μ g epitope peptides set up PBS group (substituting epitope peptide with PBS) as negative control group simultaneously;
Every hole added 50u IL-2 in (9) the 3rd days;
Changed liquid in per 2～3 days, carrying out the second wheel load peptide behind the Yu Qitian (is that 50u IL-2,10 μ g people β are added in every hole
2Microglobulin and 10 μ g epitope peptides/PBS), carry out third round lotus peptide behind the fortnight.Behind the third round lotus peptide 3 days, obtain effector cell CTL, carry out the LDH test then and detect cytotoxic activity.
LDH detects: use CytoTox
Non-Radioactive Cytotoxicity Assay (cytotoxicity detection kit, Promega company) carries out the LDH test and detects cytotoxic activity.Step following (seeing the test kit specification sheets for details):
1) sets up check-out console (100 μ l/ hole)
(1) set up experimental group: with tumour cell MCF-7 (ATCC company) as target cell (optimum target cell number: 5000) by the different targets of imitating than 10: 1,20: 1,40: the 1 above-mentioned effector cell CTL of adding
(2) set up the spontaneous release group of effector cell
(3) set up the spontaneous release group of target cell
(4) set up the maximum release group of target cell
(5) set up the volume correction control group
(6) set up the background control group
2) lysis and results supernatant
(1) 37 ℃, 5%CO
2Hatch check-out console (5h)
(2) 45min adds lysate before the results supernatant in maximum release group of target cell and volume correction control group, adds 10 μ l lysates (10 *) in every hole
(3) the centrifugal 4min of 250g, the results supernatant
3) LDH detects
(1) transferase 45 0 μ l supernatant is to another orifice plate
The substrate mixed solution of dilution is added in (2) 50 μ l/ holes rapidly, and the room temperature lucifuge is hatched 30min
(3) add 50 μ l stop baths
(4), detect 490nm absorption value OD in one hour with the bubble removal that contains in the hole.
Cell killing rate calculation formula is as follows:
Kill rate (%)=[(OD
Experimental group-OD
The spontaneous release group of effector cell-OD
The spontaneous release group of target cell)/(OD
The maximum release group of target cell-OD
The spontaneous release group of target cell)] * 100%
(annotate: the mean absorbance that the absorption value of all experimental group, the spontaneous release group of effector cell, the spontaneous release group of target cell all should the subtracting background control group; The maximum release group of target cell absorption value should deduct the mean absorbance of volume correction control group)
The results are shown in Figure 3, as shown in Figure 3, two epitope peptide inductive specific CTLs all demonstrate certain kill rate to tumour cell MCF-7.Thus, the PIWIL2 antitumor CTL epitope peptide can be used in preparation tumor therapeutic polypeptide vaccine as can be known.
3. transgenic mice in vivo test
3.1 induce in the CTL body:
Immunization protocol: picked at random 8～12 week HLA-A2.1/Kb transgenic mices in age (second professor Cao Xuetao of medical university of army is so kind as to give), male and female at random, every group of 4 mouse.With 100 μ g epitope peptides, 140 μ g Th epitope peptide (I-A
bThe Th cell epitope in hepatitis B virus core antigen source, TPPAYRPPNAPIL, can adopt conventional Fmoc solid-phase synthesis preparation or buy the commercially available prod), 50 μ l Freund's incomplete adjuvant (IFA, sigma company) and the immunological reagent that obtains of the PBS mixing and emulsifying of 50 μ l PH7.2 in the subcutaneous injection of mouse tail root, dosage is 100 μ l/, inject respectively at the 1st day, the 6th day, the 11st day, inject altogether 3 times, got the mouse spleen cell on the 12nd day.PBS group (promptly not adding epitope peptide and Th epitope peptide) and Th+PBS group (promptly not adding epitope peptide) is set does negative control.
3.2 effect CTL preparation:
(1) mouse of above-mentioned injection three times was taken off neck in the 12nd day and cause death, after alcohol-pickled 2～3min sterilization, aseptic taking-up mouse spleen in super clean bench;
(2) 200 order steel meshes grind, and PBS (PH7.2) flushing gets splenocyte suspension;
(3) 800rpm is centrifugal, abandons supernatant;
(4) add 5ml erythrocyte cracked liquid (Beijing ancient cooking vessel state company), re-suspended cell is hatched 5min for 4 ℃;
(5) 800rpm is centrifugal, collecting cell, PBS (PH7.2) washing 3 times;
(6) cell be resuspended in RPMI 1640 substratum (Gibico company) that 10ml contains 10% foetal calf serum (Hangzhou folium ilicis chinensis company) cell suspension, then it is cultivated every hole 5ml in 6 orifice plates;
(7) next day, every hole added 250U Recombinant mouse IL-2 (PROSPEC company), 50 μ g epitope peptides;
(8) vitro culture is gathered in the crops effector cell CTL after 6 days, carries out LDH and detects.
LDH detection method the same (see specification sheets: human peripheral blood mononuclear cell's (PBMCs) separation with induce and the LDH test section), wherein experimental group set up be with tumour cell MCF-7 as target cell, by the different targets of imitating than 20: 1,40: 1,80: the 1 above-mentioned effector cell CTL of adding.
The results are shown in Figure 4, as shown in Figure 4, two epitope peptide inductive specific CTLs all demonstrate certain kill rate to tumour cell MCF-7.Thus, prove that further the PIWIL2 antitumor CTL epitope peptide can be used in preparation tumor therapeutic polypeptide vaccine.
Claims (5)
1.PIWIL2 antitumor CTL epitope peptide is characterized in that, described antitumor CTL epitope peptide is a nonapeptide, and its sequence is a-Leu-Gly-Gly-Glu-Leu-Trp-Gly-Val; Wherein a is Lys or Tyr.
2. PIWIL2 antitumor CTL epitope peptide according to claim 1, wherein a is Lys, sequence is Lys-Leu-Gly-Gly-Glu-Leu-Trp-Gly-Val.
3. PIWIL2 antitumor CTL epitope peptide according to claim 1, wherein a is Tyr, sequence is Tyr-Leu-Gly-Gly-Glu-Leu-Trp-Gly-Val.
4. the preparation method of the arbitrary described PIWIL2 antitumor CTL epitope peptide of claim 1 to 3 is characterized in that, adopts solid-phase synthesis to prepare the CTL epitope peptide.
5. the application of the arbitrary described PIWIL2 antitumor CTL epitope peptide of claim 1 to 3 in preparation tumor therapeutic polypeptide vaccine.
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| CN102516363A (en) * | 2012-01-05 | 2012-06-27 | 郑州大学 | CTL (Cytotoxic T Lymphocytes) epitope peptide from PL2L60 source and application thereof |
| CN105949302A (en) * | 2016-05-27 | 2016-09-21 | 郑州大学 | FAP(fibroblast activation protein)-sourced anti-tumor CTL (cytotoxic T lymphocyte) epitope peptide P639 and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1910284A (en) * | 2004-01-20 | 2007-02-07 | 爱知县 | Epitope/peptide recognized by HLA-A2402-restricted Ep-CAM-specific CTL and use of the same |
| CN101137392A (en) * | 2005-02-16 | 2008-03-05 | 锡耶纳技术研究大学 | Poly-epitope peptide derived from thymidylate synthase having immunological and anti-tumour activity |
| CN101508721A (en) * | 2009-03-24 | 2009-08-19 | 中国人民解放军第三军医大学 | Limited simulation CTL epitope of tumor antigen Ran HLA-A*0201 and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1910284A (en) * | 2004-01-20 | 2007-02-07 | 爱知县 | Epitope/peptide recognized by HLA-A2402-restricted Ep-CAM-specific CTL and use of the same |
| CN101137392A (en) * | 2005-02-16 | 2008-03-05 | 锡耶纳技术研究大学 | Poly-epitope peptide derived from thymidylate synthase having immunological and anti-tumour activity |
| CN101508721A (en) * | 2009-03-24 | 2009-08-19 | 中国人民解放军第三军医大学 | Limited simulation CTL epitope of tumor antigen Ran HLA-A*0201 and uses thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102516363A (en) * | 2012-01-05 | 2012-06-27 | 郑州大学 | CTL (Cytotoxic T Lymphocytes) epitope peptide from PL2L60 source and application thereof |
| CN105949302A (en) * | 2016-05-27 | 2016-09-21 | 郑州大学 | FAP(fibroblast activation protein)-sourced anti-tumor CTL (cytotoxic T lymphocyte) epitope peptide P639 and application thereof |
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