CN102258773B - Mesylate protein kinase receptor EphA2 dominant epitope compound and preparation method and application thereof - Google Patents
Mesylate protein kinase receptor EphA2 dominant epitope compound and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a mesylate protein kinase receptor EphA2 dominant epitope compound and a preparation method and application thereof in preparing a therapeutic vaccine for tumor. The compound is prepared from EphA258-66 epitope fusion peptide, EphA2550-558 epitope fusion peptide and EphA2253-261 epitope fusion peptide in mol ratio of 1:1:1 and an LIGHT gene recombinant eukaryotic expression vector; the EphA258-66/550-558/253-261 epitope fusion peptides are respectively prepared from EphA258-66/550-558/253-261 epitopes and a transmembrane sequence HIV-Tat49-57, an endoplasmic reticulum retention signal sequence KDEL and a linker sequence AAY; the combination of the three EphA2 dominant epitopes can effectively overcome the deficiency of a single epitope on immune effect; the three epitope fusion peptides can enter the kytoplasm under the guide action of the transmembrane sequence, target the endoplasmic reticulum under the action of the endoplasmic reticulum retention signal sequence, and efficiently enter an MHC-I (major histocompatibility complex-class I) antigen presenting route; and the LIGHT gene recombinant eukaryotic expression vector can express LIGHT protein in vivoand play a role of a molecular adjuvant to promote the immune response of epitope specific T cells.
Description
Technical field
The invention belongs to biomedicine field, relate to a kind of epitope complex, the particularly advantage epi-position complex of a kind of tyrosine protein kinase receptor EphA2 also relates to the preparation method and application of this advantage epi-position complex.
Background technology
The treatment of malignant tumor has obtained a lot of progress, but MRD still is difficult to thoroughly be removed by schemes such as existing radiotherapy, chemotherapy, operations.Use immunotherapy of tumors excitating organism immune surveillance system, eliminate remaining tumor cell, might thoroughly cure tumor.Therefore, the exploitation of tumor vaccine becomes and studies one of focus now.
Descend at tumor antigen immunogenicity in body, cause specific cellular immunity to activate situation not enough, the periphery immunologic tolerance, the general thought of tumor vaccine design is to use various technology to strengthen immune system to the identification ability of tumor antigen, improve immune microenvironment, cause strong specificity antineoplastic cellular immunization, thereby the prevention tumour progression, and finally eliminate tumor.
CD8
+The T cell is the key component of body immune system, the key effect of performance at immune surveillance, immune defence and the immunization therapy process of pathogenic microbes such as virus, antibacterial and fungus and malignant tumor etc.Cytotoxic T cell (CTL) is activated by the surperficial MHC-I quasi-molecule-antigenic peptide complexes of its TXi Baoshouti (TCR) specific recognition antigen presenting cell (APC), and finally kills target cell.The antigenic peptides of being combined with the MHC-I quasi-molecule (long 8 ~ 10 aminoacid) is defined as the CTL epi-position.Most of MHC-I quasi-molecules are only in conjunction with the endogenous antigen peptide, the exogenous antigen peptide overwhelming majority enters MHC-II class antigen presentation approach after by the APC endocytosis to activate helper T lymphocyte (Th cell), only there is the minority exogenous antigen peptide can be by MHC-I quasi-molecule submission, namely intersects submission.Therefore, how the exogenous antigen peptide being dropped into MHC-I class angtigen presentation approach effectively is the key that excites specific CTL to reply.
Give birth to erythropoietin hepatocyte (erythropoietin producing hepatocellular, Eph) be receptor tyrosine kinase (receptor tyrosine kinases, RTKs) subtribe in the superfamily, EphA2 is one of Eph receptor tyrosine kinase, wide expression in the multiple tissue in human epithelium source or cell, exchanging ganglion cell's growth, migration and vascularization has potential effect.Recent study finds that EphA2 is overexpression in many tumor tissues, and especially expression is higher in the invasive tumor cell of height, comprises colon cancer, breast carcinoma, carcinoma of prostate, pulmonary carcinoma etc., may be a novel targets for the treatment of malignant tumor.
The trans-activator Tat of HIV-1 can initiatively pass cell membrane and enter cell interior, also can be transferred in the cell by it with the covalently bound heterologous protein of Tat, and the normal configuration of cell and function is unaffected.Discover, sequence (the Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg that is formed by Tat 49-57 amino acids residue, RKKRRQRRR) can exercise the function of protein transduction fully, this is the existing membrane property minimal segment of no cytotoxicity again of wearing of Tat.
Endoplasmic reticulum is the most important place of MHC-I quasi-molecule submission antigenic peptides, if exogenous antigen peptide targeting is transported to endoplasmic, is expected to strengthen the efficient that the exogenous antigen peptide is offered by the MHC-I quasi-molecule.(Lys-Asp-Glu-Leu, KDEL) motif is a kind of ER retention signal sequence of classics to lysine-asparagicacid-glutamate-leu.Discover that the KDEL motif that is positioned at the peptide carboxyl terminal can effectively promote the exogenous antigen peptide of coupling with it to enter MHC-I class antigen presentation approach, and this antigen presentation process shows as the relevant non-dependence of transporter/proteasome of antigen processing.
LIGHT(homologous to lymphotoxins, inducible, competes with HSV glycoprotein D for HVEM, expressed by Tlymphocyte) is a newfound tumor necrosis factor (TNF) superfamily member, be a kind of II type transmembrane glycoprotein, how with trimerical form abduction delivering in activated T cell and selective expression on immature dendritic cell (DC), be that a multifunctional multi-effect is answered molecule, have the activity of common stimulation and cell toxicant two aspects concurrently.Discover that LIGHT all plays a significant role in generation, tumour immunity, autoimmune disease, graft-rejection and the antiviral immunity of lymphatic organ.
But up to now, Shang Weijian by the EphA2 epi-position, the relevant report of wearing the complex that film sequence HIV-Tat49-57, ER retention signal sequence KDEL and LIGHT form.
Summary of the invention
In view of this, one of purpose of the present invention is to provide a kind of tyrosine protein kinase receptor EphA2 advantage epi-position complex, two of purpose is to provide the preparation method of described tyrosine protein kinase receptor EphA2 advantage epi-position complex, and three of purpose is to provide the application of described tyrosine protein kinase receptor EphA2 advantage epi-position complex at pharmaceutical field.
For achieving the above object, the invention provides following technical scheme:
1. tyrosine protein kinase receptor EphA2 advantage epi-position complex is made up of EphA2 58-66 epitope fusion peptide, EphA2 550-558 epitope fusion peptide, EphA2 253-261 epitope fusion peptide and LIGHT gene recombinaton carrier for expression of eukaryon; The mol ratio of described EphA2 58-66 epitope fusion peptide, EphA2 550-558 epitope fusion peptide and EphA2 253-261 epitope fusion peptide is 1: 1: 1;
Described EphA2 58-66 epitope fusion peptide by EphA2 58-66 epi-position, wear film sequence HIV-Tat49-57, ER retention signal sequence KDEL and joint sequence AAY forms; Wear the aminoterminal that the film sequence is positioned at fusogenic peptide, the ER retention signal sequence is positioned at the c-terminus of fusogenic peptide, EphA2 58-66 epi-position wear between film sequence and the ER retention signal sequence and respectively by joint sequence with wear the film sequence and be connected with the ER retention signal sequence;
Described EphA2 550-558 epitope fusion peptide by EphA2 550-558 epi-position, wear film sequence HIV-Tat49-57, ER retention signal sequence KDEL and joint sequence AAY forms; Wear the aminoterminal that the film sequence is positioned at fusogenic peptide, the ER retention signal sequence is positioned at the c-terminus of fusogenic peptide, EphA2 550-558 wear between film sequence and the ER retention signal sequence and respectively by joint sequence AAY with wear the film sequence and be connected with the ER retention signal sequence;
Described EphA2 253-261 epitope fusion peptide by EphA2 253-261 epi-position, wear film sequence HIV-Tat49-57, ER retention signal sequence KDEL and joint sequence AAY forms; Wear the aminoterminal that the film sequence is positioned at fusogenic peptide, the ER retention signal sequence is positioned at the c-terminus of fusogenic peptide, EphA2 253-261 epi-position wear between film sequence and the ER retention signal sequence and respectively by joint sequence with wear the film sequence and be connected with the ER retention signal sequence.
Further, described LIGHT gene recombinaton carrier for expression of eukaryon is to insert LIGHT cDNA full length sequence and obtain in carrier for expression of eukaryon PCI-neo.
2. the preparation method of described tyrosine protein kinase receptor EphA2 advantage epi-position complex, may further comprise the steps: under stirring condition, dripping mol ratio in the phosphate buffer that is dissolved with LIGHT dna recombinant expression carrier is the mixed aqueous solution of 1: 1: 1 EphA2 58-66 epitope fusion peptide, EphA2 550-558 epitope fusion peptide and EphA2 253-261 epitope fusion peptide, dropwising the back continues to stir 30 minutes, left standstill again 30 minutes, and namely got cheese chloric acid protein kinase receptor 2 advantage epi-position complex.
3. the application of described tyrosine protein kinase receptor EphA2 advantage epi-position complex in the preparation tumor therapeutic vaccine.
Further, described tumor therapeutic vaccine is the lung cancer therapy vaccine.
Beneficial effect of the present invention is: the advantage epi-position of cheese chloric acid protein kinase receptor EphA2 that the present invention has utilized molecule software on-line prediction, choose the advantage epi-position of score rank front three: EphA2 58-66, EphA2 550-558 and EphA2 253-261, merge and wear film sequence and ER retention signal sequence, three kinds of advantage epitope fusion peptides have been made up respectively, the EphA2 advantage epi-position complex of further having united LIGHT gene recombinaton construction of eukaryotic expression vector again.Three kinds of advantage epi-position associatings can effectively remedy the deficiency of single epi-position immune effect, thereby can more effectively bring out immunne response, obtain desirable immune effect; Three kinds of advantage epitope fusion peptides can enter in the cell under the guide effect of wearing the film sequence, and targeting endoplasmic reticulum under the effect of ER retention signal sequence finally efficiently enters MHC-I class antigen presentation approach again, excites epitope specificity CTL to reply; Simultaneously, LIGHT gene recombinaton carrier for expression of eukaryon is at cell inner expression LIGHT molecule, and the LIGHT molecule can be brought into play the molecule adjuvant effect, effectively regulates the function of CTL, promotes the immunne response of epitope specificity CTL.Experimental result shows that EphA2 advantage epi-position complex of the present invention can effectively excite CTL secretion interferon-and produce cellulotoxic effect, killing tumor cell external; Can suppress tumor growth in vivo, prolong the mean survival time (MST) of tumor animal and improve the The average survival time rate, can be used for preparing for example lung cancer therapy vaccine of tumor therapeutic vaccine.The preparation method of EphA2 advantage epi-position complex of the present invention is simple, in the immunotherapy of tumors field excellent development application prospect is arranged.
Description of drawings
Fig. 1 is that the agarose gel electrophoresis of LIGHT gene is identified figure.
Fig. 2 is the transmission electron microscope picture of EphA2 advantage epi-position complex.
Fig. 3 is the ability that enzyme linked immunological speckle (ELISPOT) method detects EphA2 advantage epi-position complex activated t cell secretion interferon-(IFN-γ).
Fig. 4 is
51The Cr method for releasing detects EphA2 advantage epi-position complex activated t cell to the specificity kill and wounding effect of lung carcinoma cell Lewis.
Fig. 5 is the tumor growth curve that gives or do not give the tumor-bearing mice of EphA2 advantage epi-position complex.
Fig. 6 is the The average survival time rate that gives or do not give the tumor-bearing mice of EphA2 advantage epi-position complex.
The specific embodiment
In order to make the purpose, technical solutions and advantages of the present invention clearer, below in conjunction with accompanying drawing the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in the preferred embodiment, usually according to normal condition, the molecular cloning experiment guide (third edition for example, J. work such as Sa nurse Brooker, Huang Peitang etc. translate, Science Press, 2002) described in condition, or the condition of advising according to manufacturer.
One, the preparation of EphA2 advantage epi-position complex
1, the prediction of EphA2 advantage epi-position
The hyper-base order refers to the family at same HLA, or even the antigenic peptides that the HLA allotype molecule of different families is admitted has the peptide motif that identical with similar anchor the residue formation.The main anchor residues of hyper-base order is the amino acid residue of peptide chain the 2nd (P2) and the 9th (P9), and when P2, P9 amino acids residue were suitable amino acid residue, peptide and HLA-A*0201 had high-affinity.Utilize the online software of SYFPEITHI (http://www.syfpeithi.de/scripts/MHCServer.dll/home.htm) that the Kd restricted CTL epitope of tumor antigen EphA2 is predicted.The result obtains score value greater than 3 EphA2 advantage epi-positions of 25, is respectively EphA2 58-66(IMNDMPIYM), EphA2 550-558(VLAGVGFFI) and EphA2 253-261(WLVPIGQCL).
2, EphA2 advantage epitope fusion peptide is synthetic
3 EphA2 advantage epi-positions that selection dopes: EphA2 58-66, EphA2 550-558 and EphA2 253-261, designed three epitope fusion peptides respectively: EphA2 58-66 epitope fusion peptide, EphA2 550-558 epitope fusion peptide and EphA2 253-261 epitope fusion peptide.Every epitope fusion peptide by corresponding EphA2 advantage epi-position, wear film sequence HIV-Tat49-57(RKKRRQRRR), ER retention signal sequence KDEL and joint sequence AAY form, wear the aminoterminal that the film sequence is positioned at peptide, the ER retention signal sequence is positioned at the c-terminus of peptide, EphA2 advantage epi-position is connected with the two wearing between film sequence and the ER retention signal sequence and by joint sequence, so that EphA2 advantage epi-position keeps independence and effective submission.Namely, the aminoacid sequence of EphA2 58-66 epitope fusion peptide is shown in SEQ ID No.1, the aminoacid sequence of EphA2 550-558 epitope fusion peptide is shown in SEQ ID No.2, and the aminoacid sequence of EphA2 253-261 epitope fusion peptide is shown in SEQ ID No.3.Simultaneously, design ovalbumin (OVA) the 257-264 epitope fusion peptide of aminoacid sequence shown in SEQ ID No.4 peptide in contrast.
Synthesizing at ABI 431A type solid-phase polypeptide synthesizer of epitope fusion peptide carried out.Employing standard fluorenylmethyloxycarbonyl (Fmoc) scheme, arginine adopts twice coupling.The initial hydroxymethyl phenoxy methyl polystyrene resin of 0.125mmol (HMP resin) of selecting for use makes peptide chain extend to aminoterminal one by one from c-terminus according to amino acid sequence of polypeptide, and the consumption of every seed amino acid is 0.5mmol, with the mol ratio of resin be 4:1.Various amino acid whose alpha-amidos are the Fmoc protection, and all the other side chain protected groups are respectively: Lys (Boc), Ser (tBu), Glu (OtBu), Arg (Pmc), His (Trt), Thr (tBu) and Tyr (tBu).First aminoacid is connected on the resin with 4-dimethylamino naphthyridine (DMAP); amino acid whose activation is with I-hydroxybenzotriazole (HOBt) and dicyclohexylcarbodiimide (DCC), after the coupling is 20% piperidines aqueous solution removal Fmoc protecting group with volume fraction.After polypeptide is synthetic; resin-thick peptide product is mixed in 10mL cutting liquid A(by crystallization phenol 0.75g, 1 under condition of ice bath; the 2-dithioglycol is that EDT 0.25mL, thioanisole 0.5mL, deionized water 0.25mL and trifluoroacetic acid are that TFA 10mL forms) in; after liquid temp to be cut rises to room temperature; stirring reaction 2 hours; peptide chain cracking from the resin is got off, remove the kinds of protect group simultaneously.Reaction mixture is filtered to remove resin through the G4 glass sand hourglass, successively use 1mL TFA, 5 ~ 10mL dichloromethane to wash reaction bulb, resin and funnel repeatedly.Filtrate is evaporated to 1 ~ 2mL under room temperature low pressure, adds 50mL pre-cooling ether sedimentation polypeptide, 4 ℃ of placements are spent the night, and the G6 glass sand hourglass filters, and vacuum is drained, and namely gets the polypeptide crude product, and-20 ℃ of preservations are standby.
The polypeptide crude product is made the solution that concentration is 20mg/mL with dimethyl sulfoxide (DMSO) dissolving, after via hole diameter was the filtering with microporous membrane of 0.45 μ m, (Sweden Amerssham Bioscienc company) carried out purification with the SOURCE gel column at AKTA explorer 100 type medium pressure liguid chromatographs.Mobile phase A is that 10% ethanol and volume fraction are that 0.1% TFA forms by volume fraction, and Mobile phase B is that 90% ethanol and volume fraction are that 0.1% TFA forms by volume fraction; Gradient is: earlier with 1.5 column volumes of mobile phase A eluting, use 8 column volumes of mixed liquor eluting (volume fraction that Mobile phase B accounts for mixed liquor increases to 80% gradually by 0% in 8 column volumes) of mobile phase A and Mobile phase B again, use 0.5 column volume of mixed liquor eluting (volume fraction that Mobile phase B accounts for mixed liquor increases to 100% gradually by 80% in 0.5 column volume) of mobile phase A and Mobile phase B again, collect polypeptide solution at the main peak place, lyophilization, namely get the pure product of polypeptide, with the DMSO dissolving ,-20 ℃ of preservations are standby.
The pure product of polypeptide are identified purity with Delta 600 type high pressure liquid chromatographs, adopt Symmetry Shield
TMC18 post, mobile phase are that 10% ~ 60% acetonitrile and volume fraction are that 0.1% TFA forms by volume fraction, and flow velocity is 1mL/min.The result shows that the purity of synthetic polypeptide all reaches more than 90%.Simultaneously, with the pure product of polypeptide API 2000 LC/MS type electro-spray ionization mass spectrograph determining molecular weights.The result shows that the molecular weight of synthetic polypeptide conforms to EphA2 58-66 epitope fusion peptide, EphA2 550-558 epitope fusion peptide, EphA2 253-261 epitope fusion peptide and the theoretical molecular of OVA 257-264 epitope fusion peptide respectively.
3, LIGHT gene recombinaton Construction of eukaryotic
Collector's PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC), use recombinant interleukin 4(rIL-4) and reorganization granulocyte-macrophage colony stimutaing factor (rGM-CSF) induce and be divided into immature DC, with the total RNA of RNA extraction agent box extracting immature DC, after determined by ultraviolet spectrophotometry concentration and the purity ,-70 ℃ of preservations are standby.
Be the complete open reading frame ORF of the LIGHT gene of NM_003807 according to the GenBank accession number, design following PCR primer: forward primer: 5 '-cttgggcatggaggagagtgtcgta-3 ' (SEQ ID No.6), downstream primer: 5 '-tcacaccat-gaaagccccgaagtaag-3 ' (SEQ ID No.7) entrusts precious biological engineering (Dalian) company limited synthetic.
Being cDNA with the total RNA reverse transcription of immature DC, is template with this cDNA again, adopts aforementioned upstream and downstream primer PCR amplification LIGHT cDNA total length, and PCR system cumulative volume is 100 μ L, and PCR cycling condition parameter is: 94 ℃ of pre-degeneration 2 minutes; 30 seconds, 60 ℃ annealing of 94 ℃ of degeneration were extended totally 30 circulations l minute for 30 seconds, 72 ℃ again; Last 72 ℃ were extended 10 minutes.Be that 1% agarose gel electrophoresis identifies that (result as shown in Figure 1 with the PCR product through mass fraction, the M swimming lane is dna molecular amount standard, 1 swimming lane is the PCR product, as seen the PCR product has unique band at the about 720bp of molecular weight place, conform to the expection molecular weight) after, cut glue and reclaim purification, be connected in 16 ℃ under the effect of T4 dna ligase with T carrier pGEM-T Easy again and spend the night, connect product transformed into escherichia coli DH5 α competent cell, with the LB plate screening positive colony that contains ampicillin, picking positive colony list bacterium colony, with extracting plasmid after the LB culture medium incubated overnight that contains ampicillin, carrying out double digestion with restricted enzyme EcoR I and Pst I identifies, and entrust Shanghai Sangon Biological Engineering Technology And Service Co., Ltd to carry out sequence verification, correctly inserted the positive colony plasmid called after LIGHT-pGEM-T Easy of LIGHT cDNA full length sequence (SEQ ID No.5).
Going out LIGHT cDNA total length with EcoR I enzyme action from LIGHT-pGEM-T Easy, is after 1% agarose gel electrophoresis is identified through mass fraction, cuts glue and reclaims purification, determined by ultraviolet spectrophotometry concentration; Simultaneously, PCI-neo makes linearisation with EcoR I enzyme action carrier for expression of eukaryon, is after 1% agarose gel electrophoresis is identified through mass fraction, cuts glue and reclaims purification, determined by ultraviolet spectrophotometry concentration; Again LIGHT cDNA total length and linearizing carrier for expression of eukaryon pCI-neo are connected in 16 ℃ under the effect of T4 dna ligase and spend the night, connect product transformed into escherichia coli DH5 α competent cell, with the LB plate screening positive colony that contains ampicillin, picking positive colony list bacterium colony, with extracting plasmid after the LB culture medium incubated overnight that contains ampicillin, carry out double digestion with EcoR I and Kpn I and identify that gained positive colony plasmid is LIGHT gene recombinaton carrier for expression of eukaryon called after LIGHT-pCI-neo.
4, the preparation of EphA2 advantage epi-position complex
Be 0.1mol/L with LIGHT gene recombinaton carrier for expression of eukaryon LIGHT-pCI-neo concentration, pH is that the solution that concentration is 500 μ g/mL is made in the dissolving of 7.4 phosphate buffer (PBS), get this solution 100 μ L, under the DL state, (be 1: 1: 1 EphA2 58-66 epitope fusion peptide by mol ratio with the speed of 5 μ L/min to wherein splashing into the polypeptide mixed solution that total concentration is 1000 μ g/mL, EphA2 550-558 epitope fusion peptide and EphA2 253-261 epitope fusion peptide are formed) 100 μ L, dropwise the back and continued DL 30 minutes, left standstill again 30 minutes, and namely got EphA2 advantage epi-position complex.
The EphA2 advantage epi-position complex of prepared fresh being dripped on 200 order copper mesh, adsorbed 3 minutes, blot with absorbent paper, dried 30 seconds, is 1% acetic acid uranium aqueous solution negative staining 30 seconds with mass fraction, blots with absorbent paper, dries the 80kV transmission electron microscope observing 30 seconds.The result as shown in Figure 2, gained EphA2 advantage epi-position complex is uniform subcircular granule, most granule major diameters are less than 25nm.
According to above-mentioned same quadrat method, substitute EphA2 58-66 epitope fusion peptide, EphA2 550-558 epitope fusion peptide and EphA2 253-261 epitope fusion peptide with OVA 257-264 epitope fusion peptide, make OVA epi-position complex.
Two, EphA2 advantage epi-position complex promotes the detection of T cells with antigenic specificity immunne response
With 30 6 ~ 8 age in week female C57BL/6 mice be divided into 3 groups at random: experimental group, matched group and blank group, 10 every group; Be that 0.1mol/L, pH are that the solution that concentration is 1mg/mL is made in 7.4 PBS dissolving with concentration respectively with EphA2 advantage epi-position complex and OVA epi-position complex; Experimental group is that the EphA2 advantage epi-position complex solution of 1mg/mL is immunogen with concentration, and matched group is that the OVA epi-position complex solution of 1mg/mL is immunogen with concentration, and blank group is that 0.1mol/L, pH are that 7.4 PBS is immunogen with concentration; Each organizes the subcutaneous injection immunogen in mice dorsal part root of the tail portion, every 100 μ L, and every 1 week of interval afterwards, use with quadrat method booster immunization 1 time, immunity is 3 times altogether; 1 week after the last immunity, disconnected neck is put to death and is respectively organized mice, under aseptic condition, get spleen, mill with 100 eye mesh screens, the collecting cell suspension, separating splenocyte with the liquid-tight degree gradient centrifugation of Ficoll-Hypaque layering, is that to regulate cell density be 1 * 10 for RPMI 1640 culture medium of 10% hyclone with containing mass fraction
6/ mL, the action effect cell.
1, the ELISPOT method detects the ability of EphA2 advantage epi-position complex activated t cell secretion of gamma-IFN
Adopt the ELISPOT detection kit to detect, concrete operations reference reagent box description: every hole adding volume fraction is 70% ethanol 100 μ L, room temperature was placed 10 minutes, the PBS washing, add IFN-γ capture antibody (dilution factor is 1: 100) 100 μ L again, 4 ℃ of overnight incubation of temperature, the PBS washing, add mass fraction again and be 2% defatted milk powder solution 100 μ L, room temperature sealing 2 hours, the PBS washing, add effector lymphocyte 100 μ L again, temperature was hatched 48 hours for 37 ℃, PBST(namely contains the PBS that mass fraction is 0.1% polysorbas20) washing, add biotin labeled anti-IFN-gamma antibodies (dilution factor is 1: 100) 100 μ L again, temperature was hatched 1.5 hours for 37 ℃, the PBST washing, add alkali phosphatase (dilution factor is 1: 5000) the 100 μ L of marked by streptavidin again, temperature was hatched 1 hour for 37 ℃, the PBST washing, pat dry culture plate, add instant BCIP/NBT substrate reactions liquid 100 μ L again, the colour developing of room temperature lucifuge was used the distilled water cessation reaction to speckle formation in 2 ~ 10 minutes, the speckle number is detected in dry back, and each organizes the speckle number with the equal value representation in 3 multiple holes.
The results are shown in Figure 3, the speckle number (247/10 of experimental group
5Cell) (matched group is 39/10 apparently higher than other 2 groups
5Cell, blank group is 11/10
5Cell), show that EphA2 advantage epi-position complex of the present invention has good immunogenicity, effectively the activated t cell secretion of gamma-IFN.
2,
51The Cr method for releasing detects EphA2 advantage epi-position complex activated t cell to the specificity kill and wounding effect of tumor cell
With containing the RPMI 1640 culture medium In vitro culture that mass fraction is 10% hyclone, behind the collecting cell, centrifuge washing 2 times, be that to regulate cell density be 1 * 10 for the RPMI1640 culture medium of 10% hyclone with containing mass fraction with lung carcinoma cell Lewis
6/ mL gets 1mL cell suspension and puts in the 10mL serum bottle, adds 100 μ Ci Na
51CrO
4, temperature was hatched 90 minutes for 37 ℃, and fully washed cell is removed unconjugated
51Cr is that to adjust cell density be 1 * 10 for RPMI 1640 culture medium of 10% hyclone with containing mass fraction again
5/ mL is as target cell.In the U-shaped plate in 96 holes, be to add effector lymphocyte and target cell in 100: 1,50: 1 and 25: 1 by imitating target than (E/T) respectively, imitate the target ratio for every kind and establish 3 multiple holes, establish maximum release group (be the hydrochloric acid substitution effect cell of 2mol/L with concentration) and minimum release group (usefulness contains the RPMI 1640 culture medium substitution effect cells that mass fraction is 10% hyclone) simultaneously; With centrifugal 30 seconds of 96 orifice plate 500r/min, temperature was hatched 4 hours for 37 ℃, 1000r/min is centrifugal 10 minutes again, supernatant 100 μ L are got in each hole, measure per minute radioactivity (cpm value) and calculate kill rate with gamma counter: kill rate (%)=[(experimental group cpm average-minimum release group cpm average)/(maximum release group cpm average-minimum release group cpm average)] * 100%, kill rate is judged to specific killing greater than 15%.
The results are shown in Figure 4, experimental group is imitated target comparison Lewis cell at each all the specific killing effect, and along with imitating target than increasing, the specific killing effect strengthens gradually, when E/T is 100: 1, kill rate is 52.7%, and matched group and blank group do not have the specific killing effect to the Lewis cell, show EphA2 advantage epi-position complex of the present invention effectively the activated t cell generation to the specificity kill and wounding effect of tumor cell.
3, anti-tumor activity detects in the EphA2 advantage epi-position composite body
30 C57BL/6 mices are divided into 3 groups at random: experimental group, matched group and blank group, 10 every group; Be that 0.1mol/L, pH are that the solution that concentration is 1mg/mL is made in 7.4 PBS dissolving with concentration respectively with EphA2 advantage epi-position complex and OVA epi-position complex; Experimental group is that the EphA2 advantage epi-position complex solution of 1mg/mL is immunogen with concentration, and matched group is that the OVA epi-position complex solution of 1mg/mL is immunogen with concentration, and blank group is that 0.1mol/L, pH are that 7.4 PBS is immunogen with concentration; Each organizes the subcutaneous injection immunogen in mice dorsal part root of the tail portion, every 100 μ L, and every 1 week of interval afterwards, use with quadrat method booster immunization 1 time, immunity is 3 times altogether; After the immunity for the first time, subcutaneous injection 1 * 10 after each group mice left dorsal
6Individual Lewis cell is observed survival rate and the gross tumor volume of respectively organizing mice, draws tumor growth curve.
Each is organized the tumor growth curve of mice and sees Fig. 5, and the control group mice gross tumor volume reaches 1300mm in the time of the 30th day
3, and the experimental mice tumor growth is slow, gross tumor volume only is 600mm
3Each is organized the The average survival time rate of mice and sees Fig. 6, and 60 days survival rate of control group mice is 30%, and the experimental mice survival rate is higher, and 60 days survival rate is 50%.Illustrate that EphA2 advantage epi-position complex of the present invention can effectively suppress tumor growth, prolong the mean survival time (MST) of tumor bearing nude mice and improve the The average survival time rate.
Explanation is at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although by invention has been described with reference to the preferred embodiments of the present invention, but those of ordinary skill in the art is to be understood that, can make various changes to it in the form and details, and not depart from the spirit and scope of the present invention that appended claims limits.
＜110〉Fuzhou General Hospital, Nanjing Military Area, PLA
＜120〉cheese chloric acid protein kinase receptor EphA2 advantage epi-position complex and its preparation method and application
<160> 7
<210> 1
<211> 28
<212> PRT
＜213〉artificial sequence
<220>
＜223〉EphA2 58-66 epitope fusion peptide
<400> 1
Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Ala Tyr Ile Met Asn
1 5 10 15
Asp Met Pro Ile Tyr Met Ala Ala Tyr Lys Asp Glu Leu
20 25
<210> 2
<211> 28
<212> PRT
＜213〉artificial sequence
<220>
＜223〉EphA2 550-558 epitope fusion peptide
<400> 2
Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Ala Tyr Val Leu Ala
1 5 10 15
Gly Val Gly Phe Phe Ile Ala Ala Tyr Lys Asp Glu Leu
20 25
<210> 3
<211> 28
<212> PRT
＜213〉artificial sequence
<220>
＜223〉EphA2 253-261 epitope fusion peptide
<400> 3
Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Ala Tyr Trp Leu Val
1 5 10 15
Pro Ile Gly Gln Cys Leu Ala Ala Tyr Lys Asp Glu Leu
20 25
<210> 4
<211> 27
<212> PRT
＜213〉artificial sequence
<220>
＜223〉ovalbumin epitope peptide
<400> 4
Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Ala Tyr Ser Ile Ile
1 5 10 15
Asn Phe Glu Lys Leu Ala Ala Tyr Lys Asp Glu Leu
20 25
<210> 5
<211> 723
<212> DNA
＜213〉homo sapiens (homo sapiens)
<220>
<223> LIGHT cDNA
<400> 5
atggaggaga gtgtcgtacg gccctcagtg tttgtggtgg atggacagac cgacatccca 60
ttcacgaggc tgggacgaag ccaccggaga cagtcgtgca gtgtggcccg ggtgggtctg 120
ggtctcttgc tgttgctgat gggggccggg ctggccgtcc aaggctggtt cctcctgcag 180
ctgcactggc gtctaggaga gatggtcacc cgcctgcctg acggacctgc aggctcctgg 240
gagcagctga tacaagagcg aaggtctcac gaggtcaacc cagcagcgca tctcacaggg 300
gccaactcca gcttgaccgg cagcgggggg ccgctgttat gggagactca gctgggcctg 360
gccttcctga ggggcctcag ctaccacgat ggggcccttg tggtcaccaa agctggctac 420
tactacatct actccaaggt gcagctgggc ggtgtgggct gcccgctggg cctggccagc 480
accatcaccc acggcctcta caagcgcaca ccccgctacc ccgaggagct ggagctgttg 540
gtcagccagc agtcaccctg cggacgggcc accagcagct cccgggtctg gtgggacagc 600
agcttcctgg gtggtgtggt acacctggag gctggggaga aggtggtcgt ccgtgtgctg 660
gatgaacgcc tggttcgact gcgtgatggt acccggtctt acttcggggc tttcatggtg 720
tga 723
<210> 6
<211> 25
<212> DNA
＜213〉artificial sequence
<220>
＜223〉forward primer of pcr amplification LIGHT cDNA total length
<400> 6
cttgggcatg gaggagagtg tcgta 25
<210> 7
<211> 26
<212> DNA
＜213〉artificial sequence
<220>
＜223〉downstream primer of pcr amplification LIGHT cDNA total length
<400> 7
tcacaccatg aaagccccga agtaag 26
Claims (5)
1. cheese chloric acid protein kinase receptor EphA2 advantage epi-position complex is characterized in that: be made up of EphA2 58-66 epitope fusion peptide, EphA2 550-558 epitope fusion peptide, EphA2 253-261 epitope fusion peptide and LIGHT gene recombinaton carrier for expression of eukaryon; The mol ratio of described EphA2 58-66 epitope fusion peptide, EphA2 550-558 epitope fusion peptide and EphA2 253-261 epitope fusion peptide is 1: 1: 1;
Described EphA2 58-66 epitope fusion peptide by EphA2 58-66 epi-position, wear film sequence HIV-Tat49-57, ER retention signal sequence KDEL and joint sequence AAY forms; Wear the aminoterminal that the film sequence is positioned at fusogenic peptide, the ER retention signal sequence is positioned at the c-terminus of fusogenic peptide, EphA2 58-66 epi-position wear between film sequence and the ER retention signal sequence and respectively by joint sequence with wear the film sequence and be connected with the ER retention signal sequence;
Described EphA2 550-558 epitope fusion peptide by EphA2 550-558 epi-position, wear film sequence HIV-Tat49-57, ER retention signal sequence KDEL and joint sequence AAY forms; Wear the aminoterminal that the film sequence is positioned at fusogenic peptide, the ER retention signal sequence is positioned at the c-terminus of fusogenic peptide, EphA2 550-558 wear between film sequence and the ER retention signal sequence and respectively by joint sequence AAY with wear the film sequence and be connected with the ER retention signal sequence;
Described EphA2 253-261 epitope fusion peptide by EphA2 253-261 epi-position, wear film sequence HIV-Tat49-57, ER retention signal sequence KDEL and joint sequence AAY forms; Wear the aminoterminal that the film sequence is positioned at fusogenic peptide, the ER retention signal sequence is positioned at the c-terminus of fusogenic peptide, EphA2 253-261 epi-position wear between film sequence and the ER retention signal sequence and respectively by joint sequence with wear the film sequence and be connected with the ER retention signal sequence.
2. cheese chloric acid protein kinase receptor EphA2 advantage epi-position complex according to claim 1 is characterized in that: described LIGHT gene recombinaton carrier for expression of eukaryon is to insert LIGHT cDNA full length sequence and obtain in carrier for expression of eukaryon PCI-neo.
3. the preparation method of the described cheese chloric acid of claim 1 protein kinase receptor EphA2 advantage epi-position complex, it is characterized in that: under stirring condition, dripping mol ratio in the phosphate buffer that is dissolved with LIGHT dna recombinant expression carrier is the mixed aqueous solution of 1: 1: 1 EphA2 58-66 epitope fusion peptide, EphA2 550-558 epitope fusion peptide and EphA2 253-261 epitope fusion peptide, dropwising the back continues to stir 30 minutes, left standstill again 30 minutes, and namely got cheese chloric acid protein kinase receptor 2 advantage epi-position complex.
4. the application of the described cheese chloric acid of claim 1 protein kinase receptor EphA2 advantage epi-position complex in the preparation tumor therapeutic vaccine.
5. the application of cheese chloric acid protein kinase receptor EphA2 advantage epi-position complex according to claim 4, it is characterized in that: described tumor therapeutic vaccine is the lung cancer therapy vaccine.
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| US20030147810A1 (en) * | 2002-02-01 | 2003-08-07 | University Of Michigan | Compositions and methods for reporting of protease activity within the secretory pathway |
| US20050048550A1 (en) * | 2003-07-30 | 2005-03-03 | University Of Pittsburgh-Of The Commonwealth System Of Higher Education | EphA2 T-cell epitope agonists and uses therefor |
| CN1893972A (en) * | 2003-10-15 | 2007-01-10 | 医学免疫公司 | Listeria-based EphA2 vaccines |
| US7252993B2 (en) * | 2004-03-12 | 2007-08-07 | Battelle Energy Alliance, Llc | Plasmids encoding therapeutic agents |
| CN101490085A (en) * | 2006-06-12 | 2009-07-22 | 特鲁比昂药品公司 | Single-chain multivalent binding proteins with effector function |
| CA2690449A1 (en) * | 2007-06-18 | 2008-12-24 | Medimmune, Llc | Synergistic treatment of cells that express epha2 and erbb2 |
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